BR112013008700B1 - molécula de rna auto-replicante, partícula de replicon de alfavírus, composição, molécula de dna recombinante, uso da molécula de rna auto-replicante - Google Patents

molécula de rna auto-replicante, partícula de replicon de alfavírus, composição, molécula de dna recombinante, uso da molécula de rna auto-replicante Download PDF

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BR112013008700B1
BR112013008700B1 BR112013008700-5A BR112013008700A BR112013008700B1 BR 112013008700 B1 BR112013008700 B1 BR 112013008700B1 BR 112013008700 A BR112013008700 A BR 112013008700A BR 112013008700 B1 BR112013008700 B1 BR 112013008700B1
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Anders Lilja
Rebecca Loomis
Michael Franti
Peter Mason
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Abstract

PLATAFORMAS DE LIBERAÇÃO DE ANTÍGENO Essa revelação fornece plataformas para a liberação de proteínas de herpesvírus às célula, particularmente proteínas que formam complexos in vivo. Em algumas modalidades, essas proteínas e os complexos que formam despertam anticorpos neutralizantes potentes. Dessa forma, a apresentação de proteínas de herpesvírus usando as plataformas reveladas permite a geração de respostas imunes amplas e potentes úteis para o desenvolvimento de vacinas,

Description

PEDIDO RELACIONADO
[0001]Esse pedido reivindica o benefício do Pedido Provisório de Patente U.S. N°:61/391.960, depositado em 11 de outubro de 2010, cujos ensinamentos são aqui incorporados por referência.
FUNDAMENTOS DA INVENÇÃO
[0002]Herpes vírus estão disseminados e causam uma ampla gama de doenças em humanos que, nos piores casos, podem levar a uma morbidade e uma mortalidade substanciais, primariamente em indivíduos imunocomprometidos(por exemplo, receptores de transplante e indivíduos infectados pelo HIV). Os humanos são suscetíveis à infecção por pelo menos oito herpesvírus. O vírus do herpes simples-1 (HSV-1, HHV-1), o vírus do herpes simples-2 (HSV-2, HHV-2) e o vírus varicela zoster (VZV, HHV-3) são vírus da subfamília alfa, citomegalovírus (CMV, HHV-5) e os roseolovírus (HHV-6 e HHV-7) são vírus da subfamília beta, o vírus de Epstein- Barr (EBV, HHV-4) e herpesvírus associado ao sarcoma de Kaposi (KSHV, HHV-8) são vírus da subfamília gama que infectam humanos.
[0003]A infecção pelo CMV leva a uma morbidade e uma mortalidade substanciais em indivíduos imunocomprometidos (por exemplo, receptores de transplante e indivíduos infectados pelo HIV), e a infecção congênita pode resultar em defeitos devastadores no desenvolvimento neurológico em neonatos. As glicoproteínas do envelope do CMV gB, gH, gL, gM e gN representam candidatos a vacinas atraentes, na medida em que são expressas na superfície viral e podem despertar respostas imunes humorais protetoras neutralizantes de vírus. Algumas estratégias para vacinas para o CMV visavam a principal glicoproteína de superfície B (gB), que pode induzir uma resposta de anticorpo dominante (Go e Pollard, JID 197: 1.631-1.633 (2008)). A glicoproteína gB de CMV pode induzir uma resposta de anticorpo neutralizante, e uma grande fração dos anticorpos que neutralizam a infecção de fibroblastos em soros de pacientes CMV-positivos é dirigida contra gB (Britt 1990). Similarmente, foi relatado que gH e gM/gN são alvos da resposta imune à infecção natural (Urban e cols. (1996) J. Gen. Virol. 77 (Parte 7): 1.537-47; Mach e cols. (2000) J. Virol. 74(24): 11.881-92).
[0004] Complexos de proteínas de CMV também são candidatos a vacinas atraentes, pois parecem estar envolvidos em processos importantes no ciclo de vida viral. Por exemplo, o complexo de gH/gL/gO parece ter papeis importantes na entrada tanto em fibroblasto quanto em células epiteliais/endoteliais. O modelo que prevalece sugere que o complexo de gH/gL/gO medeia a infecção de fibroblastos. mutantes hCMV gO-null produzem pequenas placas em fibroblastos e titulação de vírus muito baixa, indicando um papel na entrada (Dunn (2003), Proc. Natl. Acad. Sci. U.S.A. 100: 14.223-28; Hobom (2000) J. Virol. 74: 7.720-29). Estudos recentes sugerem que gO não é incorporado em vírions com gH/gL, mas pode atuar como um chaperone molecular, aumentando a exportação de gH/gL do ER para o aparelho de Golgi e incorporação em vírions (Ryckman (2009) J. Virol. 82: 60-70). Por meio de experimentos de “pulse-chase”, foi demonstrado que pequenas quantidades de gO permanecem ligadas ao gH/gL por períodos de tempo longos, mas a maior parte da gO se dissocia e/ou é degrada do complexo de gH/gL/gO, já que não é encontrada em vírions extracelulares ou secretada por células. Quando gO foi deletada de uma cepa clínica de CMV (TR), aquelas partículas virais tinham quantidades significantemente reduzidas de gH/gL incorporadas no vírion. Adicionalmente, gO deletada do vírus TR também inibiu a entrada em células epiteliais e endoteliais, sugerindo que gH/gL também é necessária para entrada na célula epitelial/endotelial (Wille (2010) J. Virol. 84(5): 2.585-96).
[0005] gH/gL de CMV também pode se associar a UL128, UL130 e UL131A (aqui denominado UL131) e formam um complexo pentamérico que é necessário para entrada em vários tipos de células, incluindo células epiteliais, células endoteliais e células dendríticas (Hahn e cols. (2004) J. Virol. 78(18): 10.023-33; Wang e Shenk (2005) Proc. Natl. Acad. Sci. U.S.A. 102(50): 18.153-8; Gerna e cols. (2005). J. Gen. Virol. 84 (Parte 6): 1.431-6; Ryckman e cols. (2008) J. Virol. 82: 60-70). Em contraste, esse complexo não é necessário para infecção de fibroblastos. Isolados laboratoriais de hCMV carregam mutações no lócus de UL128- UL131, e surgem mutações em isolados clínicos após poucas passagens em fibroblastos cultivados (Akter e cols. (2003) J. Gen. Virol. 84 (Parte 5): 1.117-22). Durante infecção natural, o complexo pentamérico desperta anticorpos que neutralizam a infecção de células epiteliais, células endoteliais (e, provavelmente, qualquer outro tipo de célula em que o complexo pentamérico medeie a entrada viral) com potência muito elevada (Macagno e cols. (2010) J. Virol. 84(2): 1.005-13). Também parece que anticorpos para esse complexo contribuem significantemente para a habilidade de soros humanos para neutralizar a infecção de células epiteliais (Genini e cols. (2011) J. Clin. Virol. 52(2): 113-8).
[0006] US 5.767.250 revela métodos para a produção de certos complexos de proteínas de CMV que contêm gH e gL. Os complexos são produzidos por introdução de uma construção de DNA que codifica gH e uma construção de DNA que codifica gL em uma célula de tal modo que a gH e a gL sejam co- expressas.
[0007] WO 2004/076645 descreve moléculas de DNA recombinante que codificam proteínas de CMV. De acordo com esse documento, combinações de moléculas de DNA distintas que codificam proteínas de CMV diferentes podem ser introduzidas em células para causar co-expressão das proteínas de CMV codificadas. Quando gM e gN foram co- expressas dessa forma, elas formaram um complexo ligado por dissulfeto. Coelhos imunizados com construções de DNA que produziam o complexo de gM/gN ou com uma construção de DNA que codifica gB produziram respostas equivalentes de anticorpo neutralizante.
[0008] Existe necessidade de ácidos nucléicos que codificam duas ou mais proteínas do herpesvírus, de métodos de expressão de duas ou mais proteínas do herpesvírus na mesma célula e de métodos de imunização que produzem respostas imunes melhores.
SUMÁRIO DA INVENÇÃO
[0009] A invenção está relacionada às plataformas para coliberação de duas ou mais proteínas do herpesvírus, por exemplo, proteínas de citomegalovírus (CMV), às células, particularmente proteínas que formam complexos in vivo. Em um aspecto, a invenção consiste em moléculas de ácido nucléico policistrônico recombinante que contêm uma primeira seqüência que codifica uma primeira proteína de herpesvírus (por exemplo, CMV) ou fragmento desta, e uma segunda seqüência que codifica uma segunda proteína de herpesvírus (por exemplo, CMV) ou fragmento desta.
[0010] Por exemplo, a invenção fornece uma molécula de RNA auto-replicante que compreende um polinucleotídeo que compreende: a) uma primeira seqüência de nucleotídeos que codifica uma primeira proteína ou fragmento desta de um herpesvírus; e b) uma segunda seqüência de nucleotídeos que codifica uma segunda proteína, ou fragmento desta, do herpesvírus. A primeira seqüência de nucleotídeos e a segunda seqüência de nucleotídeos estão ligadas operacionalmente a um ou mais elementos de controle de modo que, quando a molécula de RNA auto-replicante é introduzida em uma célula adequada, a primeira e a segunda proteínas do herpesvírus, ou fragmentos destas, são produzidas em uma quantidade suficiente para a formação de um complexo na célula que contém a primeira e a segunda proteínas, ou fragmentos destas. De preferência, a primeira proteína e a segunda proteína não são a mesma proteína ou fragmentos da mesma proteína, a primeira proteína não é um fragmento da segunda proteína e a segunda proteína não é um fragmento da primeira proteína. A primeira seqüência de nucleotídeos pode estar ligada operacionalmente a um primeiro elemento de controle e a segunda seqüência de nucleotídeos pode estar ligada operacionalmente a um segundo elemento de controle.
[0011] A molécula de RNA auto-replicante pode ainda compreender uma terceira seqüência de nucleotídeos que codifica uma terceira proteína, ou fragmento desta, do referido herpesvírus, opcionalmente uma quarta seqüência de nucleotídeos que codifica uma quarta proteína, ou fragmento desta, do referido herpesvírus; e, opcionalmente, uma quinta seqüência de nucleotídeos que codifica uma quinta proteína, ou fragmento desta, do referido herpesvírus. Quando seqüências que codificam proteínas ou fragmentos adicionais de um herpesvírus estão presentes (ou seja, a terceira, quarta e quinta seqüências de nucleotídeos), elas estão ligadas operacionalmente a um ou mais elementos de controle. Em um exemplo de uma construção pentacistrônica, a primeira seqüência de nucleotídeos está ligada operacionalmente a um primeiro elemento de controle, a segunda seqüência de nucleotídeos está ligada operacionalmente a um segundo elemento de controle, a terceira seqüência de nucleotídeos está ligada operacionalmente a um terceiro elemento de controle, a quarta seqüência de nucleotídeos está ligada operacionalmente a um quarto elemento de controle e a quinta seqüência de nucleotídeos está ligada operacionalmente a um quinto elemento de controle. Os elementos de controle presentes na construção (por exemplo, primeiro, segundo, terceiro, quarto e quinto elementos de controle) podem ser selecionados independentemente do grupo que consiste em um promotor subgenômico, um IRES e um sítio 2A viral (por exemplo, FMDV).
[0012]O herpesvírus pode ser HSV-1, 1, HSV-2, VZV, EBV tipo 1, EBV tipo 2, CMV, HHV-6 tipo A, HHV-6 tipo B, HHV-7 e HHV-8. Em algumas modalidades, a molécula de ácido nucléico policistrônico recombinante (por exemplo, RNA auto-replicante) codifica gH ou um fragmento desta e gL ou um fragmento desta de qualquer um desses herpesvírus. Em modalidades mais particulares, o herpesvírus é CMV ou VZV.
[0013]Quandoamoléculadeácidonucléico policistrônico recombinante (por exemplo, RNA auto- replicante) codifica duas ou mais proteínas de VZV, as proteínas podem ser selecionadas do grupo que consiste em gB, gE, gH, gI, gL e um fragmento (por exemplo, de pelo menos 10 aminoácidos) destas. Em algumas modalidades, a molécula de ácido nucléico policistrônico recombinante (por exemplo, RNA auto-replicante) codifica gH de VZV ou um fragmento desta e gL de VZV ou um fragmento desta.
[0014]Em um exemplo particular, a invenção fornece uma molécula de RNA auto-replicante que compreende um polinucleotídeo que compreende: a) uma primeira seqüência que codifica uma primeira proteína de citomegalovírus (CMV) ou fragmento desta; e b) uma segunda seqüência quecodifica uma segunda proteína deCMVou fragmento desta. Aprimeira seqüência easegundaseqüência estãoligadas operacionalmente a um ou mais elementos de controle de tal forma que, quando a molécula de RNA auto-replicante é introduzida em uma célula adequada, a primeira e a segunda proteínas de CMV são produzidas em uma quantidade suficiente para a formação de um complexo na célula que contém a primeira e a segunda proteínas de CMV, ou fragmentos destas.
[0015] A primeira proteína de CMV e a segunda proteína de CMV são selecionadas independentemente do grupo que consiste em gB, gH, gL; gO; gM, gN; UL128, UL130, UL131, e um fragmento de qualquer um dos citados anteriormente. De preferência, a primeira proteína de CMV e a segunda proteína de CMV não são a mesma proteína ou fragmentos da mesma proteína, a primeira proteína de CMV não é um fragmento da segunda proteína de CMV, e a segunda proteína de CMV não é um fragmento da primeira proteína de CMV. Se desejado, a molécula de RNA auto-replicante pode ainda compreender uma terceira seqüência que codifica uma terceira proteína de CMV, em que a terceira seqüência está ligada operacionalmente a um elemento de controle. Similarmente, seqüências adicionais que codificam proteínas de CMV adicionais (por exemplo, uma quarta seqüência que codifica uma quarta proteína de CMV, uma quinta seqüência que codifica uma quinta proteína de CMV) podem ser incluídas. Os elementos de controle podem ser selecionados independentemente do grupo que consiste em um promotor subgenômico, e IRES, e um sítio 2A viral.
[0016] Em algumas modalidades, a molécula de ácido nucléico auto-replicante codifica as proteínas de CMV gH e gL. Em outras modalidades, a molécula de RNA auto- replicante codifica as proteínas de CMV gH, gL e gO. Em outras modalidades, a molécula de RNA auto-replicante codifica as proteínas de CMV gH, gL, UL128, UL130 e UL131.
[0017] As moléculas de RNA auto-replicantes podem ser um replicon de alfavírus. Nesses casos, o replicon de alfavírus pode ser liberado na forma de uma partícula de replicon de alfavírus (VRP). A molécula de RNA auto- replicante também pode estar na forma de uma molécula de RNA “naked”.
[0018] A invenção também está relacionada a uma molécula de DNA recombinante que codifica uma molécula de RNA auto- replicante como aqui descrita. Em algumas modalidades, a molécula de DNA recombinante é um plasmídeo. Em algumas modalidades, a molécula de DNA recombinante inclui um promotor de mamífero que dirige a transcrição da molécula de RNA auto-replicante codificada.
[0019] A invenção também está relacionada às composições que compreendem uma molécula de RNA auto-replicante como aqui descrita e um veículo farmaceuticamente aceitável. A molécula de RNA auto-replicante pode ser “naked”. Em algumas modalidades, a composição compreende uma molécula de RNA auto-replicante que codifica as proteínas de CMV gH e gL. Em outras modalidades, a composição ainda compreende uma molécula de RNA auto-replicante que codifica a proteína de CMV gB. A composição também pode conter um sistema de liberação de RNA como, por exemplo, um lipossomo, uma nanopartícula polimérica, uma nanoemulsão óleo-em-água catiônica, ou combinações destes. Por exemplo, a molécula de RNA auto-replicante pode ser encapsulada em um lipossomo.
[0020] Em certas modalidades, a composição compreende uma VRP que contém a replicon de alfavírus que codifica duas ou mais proteínas de CMV. Em algumas modalidades, a VRP compreende um replicon que codifica gH e gL de CMV. Se desejado, a composição pode ainda compreender uma segunda VRP que contém um replicon que codifica gB de CMV. A composição também pode compreender um adjuvante.
[0021] A invenção também está relacionada aos métodos de formação de um complexo de proteínas de CMV. Em algumas modalidades, um RNA auto-replicante que codifica duas ou mais proteínas de CMV é liberado a uma célula, a célula é mantida sob condições adequadas à expressão das proteínas de CMV, em que um complexo de proteínas de CMV é formado. Em outras modalidades, uma VRP que contém um RNA auto- replicante que codifica duas ou mais proteínas de CMV é liberado a uma célula, a célula é mantida sob condições adequadas à expressão das proteínas de CMV, em que um complexo de proteínas de CMV é formado. O método pode ser usado para formar um complexo de proteínas de CMV em uma célula in vivo.
[0022] A invenção também está relacionada a um método para indução de uma resposta imune em um indivíduo. Em algumas modalidades, um RNA auto-replicante que codifica duas ou mais proteínas de CMV é administrado ao indivíduo. A molécula de RNA auto-replicante pode ser administrada como uma composição que contém um sistema de liberação de RNA, por exemplo, um lipossomo. Em outras modalidades, uma VRP que contém um RNA auto-replicante que codifica duas ou mais proteínas de CMV é administrada ao indivíduo. Em modalidades preferidas, a molécula de RNA auto-replicante codifica proteínas de CMV gH e gL. De preferência, a resposta imune induzida compreende a produção de anticorpos neutralizantes anti-CMV. Mais preferivelmente, os anticorpos neutralizantes são complemento-independentes.
[0023] A invenção também está relacionada a um método de inibição da entrada de CMV em uma célula que compreende o contato da célula com uma molécula de RNA auto-replicante que codifica duas ou mais proteínas de CMV, por exemplo, gH e gL. A célula pode ser selecionada do grupo que consiste em uma célula epitelial, uma célula endotelial, um fibroblasto, e combinações destes. Em algumas modalidades, a célula é colocada em contato com uma VRP que contém um RNA auto-replicante que codifica duas ou mais proteínas de CMV.
[0024] A invenção também está relacionada ao uso de uma molécula de RNA auto-replicante que codifica duas ou mais proteínas de CMV (por exemplo, uma VRP, uma composição que compreende a molécula de RNA auto-replicante e um lipossomo) para formar um complexo de proteínas de CMV em uma célula, para induzir uma resposta imune ou para inibir a entrada de CMV em uma célula.
BREVE DESCRIÇÃO DOS DESENHOS
[0025] A FIG. 1 é uma representação esquemática de CMV que identifica complexos de glicoproteínas conhecidos envolvidos na entrada de CMV em células-alvo. As glicoproteínas do envelope representam candidatos a vacinas atraentes, na medida em que são expressas na superfície viral e podem despertar respostas imunes humorais neutralizantes de vírus protetoras e de longa duração. As glicoproteínas estruturais que medeiam esses processos podem ser divididas em duas classes; aquelas que são conservadas em toda a família dos herpesvírus e aquelas que não são. Entre aquelas que são conservadas estão gB, gH, gL, gM e gN. Muitas dessas glicoproteínas formam complexos entre elas (gH/gL/±gO; gH/gL/UL128/UL130/UL131; gM/gN) para facilitar a localização na superfície viral e para realizar suas funções na adesão viral, entrada e fusão à célula.
[0026] As FIGS. 2A-2F são representações esquemáticas de construções de CMV. FIG. 2A, representação esquemática das construções de gB (“gB FL”, gB de comprimento total; gBs solúveis “gB sol 750” e “gB sol 692”) descritas no Exemplo 1. Duas versões solúveis diferentes de gB foram construídas; gB sol 750 não possui o domínio de expansão transmembrana e o domínio citoplasmático, gB sol 692 também não possui uma região hidrofóbica e é similar à gB sol descrita em Reap e cols. (2007) Clin. Vacc. Immunol. 14: 748-55. FIG. 2B, representação esquemática dos vetores de replicon de gB usados para produzir partículas de replicação viral (VRPs). FIG. 2C, representação esquemática das construções de gH (“gH FL”, gH de comprimento total; gH solúvel “gH sol”) descritas no Exemplo 1. Uma única versão solúvel de gH foi construída que não possuía o domínio de expansão transmembrana. FIG. 2D, representação esquemática dos vetores de replicon de gH usados para produzir VRPs. FIG. 2E, representação esquemática da construção de gL descrita no Exemplo 1. FIG. 2F, representação esquemática do vetor de replicon de gL usado para produzir VRPs. Nas FIGS 2B, 2D e 2F, “NSP1”, “NSP2”, “NSP3” e “NSP4”, são proteínas não estruturais de alfavírus 1-4, respectivamente, necessárias para a replicação do vírus.
[0027] As FIGS. 3A e 3B mostram que camundongos imunizados com VRPs de gB (FL, sol 750, sol 692) ou gH (FL, sol) induziram respostas de anticorpo que eram neutralizantes na presença de complemento de porquinho-da- índia. O ensaio de neutralização foi feito por pré- incubação da cepa do vírus CMV TB40UL32E-GFP (que codifica a proteína fluorescente verde aprimorada-GFP, Sampaio e cols.(2005) J. Virol. 79(5):2.754-67), com soros de camundongo e complemento de porquinho-da-índia antes da infecção de células epiteliais ARPE-19. Cinco dias pós- infecção, o número de células GFP-positivas foi determinado. FIG. 3A, Curva de diluição de soro para todos os soros analisados em células ARPE-19 na presença de complemento. FIG. 3B, titulações de 50% de neutralização para as amostras de soros. Vírus incubados com soros pré- imunes geraram neutralização baixa em diluições baixas (1:40-1:80). Soros de gB (FL, sol 750, sol 692) tiveram atividade neutralizante muito baixa com titulações de 50% de neutralização entre 1:1.800-1:2.100.Todos os camundongos imunizados com gB geraram um perfil de neutralização similar. Soros de gH (FL, sol) tiveram atividade neutralizante com titulações de 50% de neutralização em torno de 1:160. Veja o Exemplo 1.
[0028]A FIG. 4A é uma ilustração esquemática de repliconsmonocistrônicosquecodificamproteína fluorescente verde (GFP) ou proteína fluorescente vermelha (mCherry) e a replicon bicistrônico que codifica GFP e mCherry. “NSP1”,“NSP2”,“NSP3” e “NSP4”, são proteínas não estruturais de alfavírus 1-4, respectivamente. O sistema de replicon de alfavírus policistrônico foi projetado por produção de modificações ao sistema de replicon de alfavírus existente para acomodar múltiplos promotores subgenômicos que dirigem genes de interesse.
[0029]A FIG. 4B são gráficos de fluorescência que mostram análise por FACS de células BHKV infectadas com VRPs contendo RNAs mono- e bicistrônicos. VRPs policistrônicas de alfavírus geram mais células que expressam ambos os genes de interesse em quantidades aproximadamente iguais (GFP e mCherry; 72,48%) do que a coinfecção de VRP de GFP + VRP de mCherry (26,30%). Veja o Exemplo 2.
[0030] A FIG. 5A é uma ilustração esquemática de construção de construções de replicon de alfavírus policistrônico que codificam gH/gL e gH/gL/gO.
[0031] A FIG. 5B mostra que gH/gL formam um complexo in vitro. VRPs contendo replicons que codificam gH, gL, gO, gH/gL ou gH/gL/gO foram produzidas em células BHKV. As VRPs resultantes foram usadas para infectar células ARPE-19 para demonstrar formação de complexo in vitro. As células ARPE- 19 infectadas por alfavírus foram coletadas e analisadas quanto à presença de gH e gL. Células ARPE-19 infectadas com VRPs que codificam gH/gL produziram complexos de gH/gL ligados por dissulfeto (veja na ausência de DTT, calor). gO não altera forma detectável a associação gH/gL. O gráfico à esquerda mostra a expressão da proteína gH. O gráfico à direita mostra a expressão de proteína gL. Marcadores de peso molecular são indicados entre os gráficos. • = gH monomérica, •• = gL monomérica, < = heterodímero (gH + gL), * = dímero de heterodímeros.
[0032] A FIG. 5C mostra imunoprecipitação de gH e complexos de gH/gL de células BHKV infectadas com VRPs. A imunoprecipitação foi realizada usando anticorpos IgG de camundongo como um controle (Raias 2, 4, 7 e 10) ou anticorpos anti-gH de camundongo (Genway) para imunoprecipitar gH (Raias 3, 5, 8 e 11). Foram realizados western blots usando anticorpo anti-gL de coelho e anticorpo anti-gH de coelho reunidos em pool. As Raias 1, 6 e 9 mostram a proteína gH (banda superior aproximadamente 75 kDa) e proteína gL (banda inferior aproximadamente 30 kDa) para referência. As Raias 2 e 3 são lisados infectados com gH-VRP. A Raia 2 mostra que o anticorpo de controle não imunoprecipitou gH. A Raia 3 mostra que o anticorpo anti-gH imunoprecipitou gH. As Raias 4 e 5 são de lisados infectados com gL-VRP apenas. Nenhuma proteína gH foi imunoprecipitada. As Raias 7 e 8 são de lisados infectados com gH/gL-VRP bicistrônica. A Raia 8 mostra que gL foi imunoprecipitada usando o anticorpo de gH. (veja o asterisco). As Raias 10 e 11 são de lisados infectados com gH/gL/gO-VRP tricistrônica. A Raia 11 mostra que gL foi imunoprecipitada usando o anticorpo de gH (veja o asterisco). Marcadores de peso molecular também são mostrados (MW). Veja o Exemplo 3.
[0033]A FIG. 6 mostra que VRPs que afetam a formação de complexo de gH/gL in vitro induzem resposta imune potente para CMV que é qualitativamente e quantitativamente superior à resposta às VRPs de gB. As FIG. 6A e FIG. 6B mostram curvas de diluição de soro para camundongos imunizados com VRP de gH, gL, gO, gH + gL, gH + gL + gO, gH/gL e gH/gL/gO na neutralização de infecção por TB40- UL32-EGFP de células ARPE-19 na presença (FIG. 6A) ou ausência (FIG. 6B) de complemento. Várias diluições de soros foram pré-incubadas com TB40UL32E-GFP na presença ou ausência de complemento de porquinho-da-índia e depois adicionadas às células epiteliais ARPE-19. Após infecção por 5 dias com o vírus, as células GFP-positivas foram contadas. A FIG. 6C é um gráfico que mostra titulações de 50% de neutralização obtidas na presença e ausência de complemento. “3wp3”, três semanas pós-terceira imunização. VRPs que expressam proteínas de CMV únicas (VRPs de gH, gL, gO ou VRPs gH, gL e gO co-administradas) não aumentam a atividade neutralizante além daquela de gH isoladamente. Em contraste, soros de camundongos imunizados com VRPs bicistrônicas gH/gL ou tricistrônicas gH/gL/gO demonstraram respostas neutralizantes potentes. Além disso, as respostas neutralizantes potentes foram similares na presença e ausência de complemento de porquinho-da-índia, mostrando que VRPs policistrônicas induziram com sucesso uma resposta imune complemento-independente. Veja o Exemplo 4.
[0034] A FIG. 7 mostra que VRPs que afetam a formação de complexo de gH/gL in vitro induziram anticorpos que neutralizaram potentemente a infecção de células de fibroblastos MRC-5. A FIG. 7A mostra curvas de diluição de soro para camundongos imunizados com VRP de gH, gL, gO, gH + gL, gH + gL + gO, gH/gL e gH/gL/gO em células MRC-5 na ausência de complemento. Várias diluições de soros foram pré-incubadas com TB40GFP na presença ou ausência de complemento de porquinho-da-índia e depois adicionadas às células de fibroblastos MRC-5. Após infecção por 5 dias com o vírus, as células GFP-positivas foram contadas. A FIG. 7B é um gráfico que mostra titulações de 50% de neutralização obtidas em um modelo de célula de fibroblasto MRC-5 na ausência de complemento. “3wp3”, três semanas pós-terceira imunização. VRPs que expressam proteínas de CMV únicas (VRPs de gH, gL, gO ou VRPs gH, gL e gO co-administradas) não aumentam a atividade neutralizante além daquela de gH isoladamente. Em contraste, soros de camundongos imunizados com VRPs bicistrônicas gH/gL ou tricistrônicas gH/gL/gO demonstraramrespostasneutralizantesextremamente potentes. Veja o Exemplo 4.
[0035]As FIGS. 8A e 8B são gráficos que mostram que os anticorpos neutralizantes induzidos por liberação das VRPs policistrônicas eram anticorpos de neutralização cruzada. Os soros de camundongos imunizados com gH/gL e VRPs de gH/gL/gO foram capazes de neutralizar cepas clínicas de CMV TB40UL32E-GFP e VR1814 tanto em células epiteliais ARPE-19 (FIG. 8A) quanto em células de fibroblastos MRC-5 (FIG. 8B) na ausência de complemento de porquinho-da-índia em um ensaio de neutralização IE-1.
[0036]A FIG. 9 é um gráfico que mostra que os anticorpos neutralizantes desenvolvidos contra gH FL/gL são complemento-independentes e similares à imunidade natural na titulação. Os camundongos foram imunizados com VRPs de gB FL ou gH FL/gL a 1 x 106 IU, 3 vezes, com intervalo de 3 semanas antes da sangria terminal. Os soros foram analisados quanto à habilidade para neutralizar infecção por CMV TB40UL32E-EGFP de células ARPE-19 na presença e ausência de complemento de porquinho-da-índia em um ensaio deneutralização.Diferentementedeanticorpos desenvolvidos por gB, anticorpos desenvolvidos por gH FL/gL são complemento-independentes. Além disso, os anticorpos de gH FL/gL nesses camundongos vacinados eram similares em termos de titulação àqueles encontrados em indivíduos humanos infectados naturalmente.
[0037]AFIG.10mostraummapadeplasmídeoparagH- SGPgL-SGPgO modificado por pVCR.
[0038]AFIG.11mostraummapadeplasmídeoparagH- SGPgL modificado por pVCR.
[0039] A FIG. 12 mostra um mapa de plasmídeo para gH sol-SGPgL modificado por pVCR.
[0040] A FIG. 13 mostra um mapa de plasmídeo para gH sol-SGPgL-SGPgO modificado por pVCR.
[0041] As FIG. 14A-14G mostram a seqüência de nucleotídeos (ID. DE SEQ. N°: 83) do plasmídeo que codifica a molécula de RNA auto-replicante A160 que codifica glicoproteína de superfície de CMV H (gH) e glicoproteína de superfície de CMV L (gL). As seqüências de nucleotídeos que codificam gH e gL estão sublinhadas.
[0042] As FIG. 15A-15H show a seqüência de nucleotídeos (ID. DE SEQ. N°: 84) do plasmídeo que codifica a molécula de RNA auto-replicante A322 que codifica a forma solúvel de glicoproteína de superfície de CMV H (gH sol) e glicoproteína de superfície de CMV L (gL). As seqüências de nucleotídeos que codificam gH sol e gL estão sublinhadas.
[0043] As FIG. 16A-16H mostram a seqüência de nucleotídeos (ID. DE SEQ. N°: 85) do plasmídeo que codifica a molécula de RNA auto-replicante A323 que codifica a glicoproteína de superfície de CMV B (gB). A seqüência de nucleotídeos que codifica gB está sublinhada.
[0044] As FIG. 17A e 17B são histogramas que mostram titulações 50% neutralizantes de soros de camundongos que foram imunizados com VRP ou RNA auto-replicante. A FIG. 17A mostra titulações 50% neutralizantes contra cepa de CMV humano TB40UL32E-EGFP (TB40) em células ARPE-19, e a FIG. 17B mostra titulações 50% neutralizantes contra cepa de CMV humano 8819 em células ARPE-19.
[0045] A FIG. 18 é uma representação esquemática de replicons pentacistrônicos de RNA, A526, A527, A554, A555 e A556, que codificam cinco proteínas de CMV. Promotores subgenômicos são mostrados por setas, outros elementos de controle estão marcados.
[0046]A FIG. 19 é um histograma de fluorescência que mostra que células BHKV transfectadas com o replicon de RNA A527expressamocomplexopentaméricode gH/gL/UL128/UL130/UL131. A coloração de células foi realizada usando anticorpos que se ligam a um epitopo conformacional presente no complexo pentamérico (Macagno (2010) J. Virol. 84(2):1.005-13).
[0047]A FIG. 20 é uma representação esquemática e um gráfico. A representação esquemática mostra replicons de RNA bicistrônico, A160 e A531-A537, que codificam gH e gL de CMV. O gráfico mostra atividade neutralizante de soros imunes de camundongos imunizados com VRPs que continham os replicons.
[0048]A FIG. 21 é um gráfico que mostra a resposta de anticorpo anti-proteína de VZV em soros imunes de camundongos imunizados com replicons monocistrônicos de RNA que codificava proteínas de VZV ou replicons de RNA bicistrônico que codificava VZV gE e gI, ou gH e gL. Os camundongos foram imunizados com 7 g de RNA formulado com uma CNE (veja o Exemplo 7).
[0049]A FIG. 22 é um gráfico que mostra resposta de anticorpo anti-proteína de VZV em soros imunes de camundongos imunizados com replicons monocistrônicos de RNA que codificava proteínas de VZV ou replicons de RNA bicistrônico que codificava VZV gE e gI, ou gH e gL. Os camundongos foram imunizados com 1 μg de RNA formulado com uma CNE (veja o Exemplo 7).
DESCRIÇÃO DETALHADA DA INVENÇÃO
[0050]A invenção fornece plataformas para coliberação de proteínas do herpesvírus, por exemplo, proteínas de citomegalovírus(CMV),às células,particularmente proteínas que formam complexos in vivo. Em algumas modalidades, essas proteínas e os complexos que formam despertam anticorpos neutralizantes potentes. A resposta imune produzida por coliberação de proteínasde herpesvírus (por exemplo, CMV), particularmente aquelas que formam complexos in vivo (por exemplo,gH/gL), podeser superior à resposta imune produzida usando outras abordagens. Por exemplo, uma molécula de RNA (por exemplo, um replicon) que codifica tanto gH quanto gL de CMV pode induzir melhores titulações neutralizantes e/ou imunidade protetora em comparação com uma molécula de RNA que codifica gB, uma molécula de RNA que codifica gH, uma molécula de RNA que codifica gL, ou até mesmo uma mistura de moléculas de RNA que individualmente codificam gH ou gL. Além disso, um replicon que codifica gH/gL/UL128/UL130/UL131 pode fornecer respostas superior em relação àqueles que codificam apenas gH/gL.
[0051]Em um aspecto geral, a invenção está relacionada às plataformas para liberação de duas ou mais proteínas de herpesvírus (por exemplo, CMV) às células. As plataformas compreendem moléculas de ácido nucléico policistrônico recombinante que contêm uma primeira seqüência que codifica uma primeira proteína de herpesvírus (por exemplo, CMV) ou fragmento desta, e uma segunda seqüência que codifica uma segunda proteína de herpesvírus (por exemplo, CMV) ou fragmento desta. Se desejado, uma ou mais seqüências adicionais que codificam proteínas adicionais, por exemplo, uma terceira proteína de herpesvírus (por exemplo, CMV) ou fragmento desta, uma quarta proteína de herpesvírus (por exemplo, CMV) ou fragmento desta, uma quinta proteína de herpesvírus (por exemplo, CMV) ou fragmento desta etc. podem estar presentes na molécula de ácido nucléico policistrônico recombinante. As seqüências que codificam proteínas de herpesvírus (por exemplo, CMV) ou fragmentos destas estão ligadas operacionalmente a um ou mais elementos de controle adequados de modo que as proteínas de herpesvírus (por exemplo, CMV) ou fragmentos sejam produzidos por uma célula que contém o ácido nucléico policistrônico recombinante.
[0052]Nos ácidos nucléicos policistrônicos aqui descritos, a primeira e segunda proteínas do herpesvírus codificadas, ou fragmentos destas, e a terceira, quarta e quinta proteínas do herpesvírus codificadas, ou fragmentos destas, se presentes, geralmente e preferivelmente são do mesmo herpesvírus. Em certos exemplos, todas as proteínas do herpesvírus, ou fragmentos destas, codificadas por um vetor policistrônico são proteínas de CMV ou proteínas de VZV.
[0053]As moléculas de ácido nucléico policistrônico recombinante aqui descritas fornecem a vantagem de liberar seqüências que codificam duas ou mais proteínas de herpesvírus (por exemplo, CMV) a uma célula, e dirigir a expressão das proteínas de herpesvírus (por exemplo, CMV) em níveis suficientes para resultar na formação de um complexo de proteínas que contém as duas ou mais proteínas de herpesvírus (por exemplo, CMV) in vivo. Com o uso dessa abordagem, as duas ou mais proteínas de herpesvírus codificadas (por exemplo, CMV) podem ser expressas em níveis intracelulares suficientes para a formação de complexos de proteínas de herpesvírus (por exemplo, CMV) (por exemplo, gH/gL). Por exemplo, as proteínas de herpesvírus codificadas (por exemplo, CMV), ou fragmentos destas, podem ser expressas substancialmente no mesmo nível ou, se desejado, em níveis diferentes por seleção de seqüências de controle da expressão apropriadas (por exemplo, promotores, IRES, sítio 2A etc.). Isso é significantemente a forma mais eficiente de produzir complexos de proteínas in vivo do que por coliberação de duas ou mais moléculas de DNA individuais que codificam herpesvírus diferentes (por exemplo, CMV) à mesma célula, o que pode ser ineficiente e altamente variável. Veja, por exemplo, WO 2004/076645.
[0054]A molécula de ácido nucléico policistrônico recombinante pode ser baseada em qualquer ácido nucléico desejado como, por exemplo, DNA (por exemplo, plasmídeo ou DNA viral) ou RNA. Qualquer DNA ou RNA adequado pode ser usado como o vetor de ácido nucléico que carrega os quadros de leitura aberta que codificam proteínas de herpesvírus (por exemplo, CMV) ou fragmentos destas. Vetores de ácido nucléico adequados possuem a capacidade de carregar e dirigir a expressão de mais de um gene de proteína. Esses vetores de ácido nucléico são conhecidos na técnica e incluem, por exemplo, plasmídeos, DNA obtido de vírus de DNA como, por exemplo, vetores de vírus da vacínia (por exemplo, NYVAC, veja US 5.494.807) e vetores de poxvírus (por exemplo, vetor do vírus da varíola dos canários ALVAC, Sanofi Pasteur), e RNA obtidos de vírus de RNA adequados como, por exemplo, um alfavírus. Se desejado, a molécula de ácido nucléico policistrônico recombinante pode ser modificada, por exemplo, conter nucleobases e/ou ligações modificadas, como aqui descrito mais adiante. De preferência, a molécula de ácido nucléico policistrônico é uma molécula de RNA.
[0055] Em alguns aspectos, a molécula de ácido nucléico policistrônico recombinante é uma molécula de DNA como, por exemplo, DNA de plasmídeo. Essas moléculas de DNA podem codificar, por exemplo, um replicon policistrônico e conter um promotor de mamífero que dirige a transcrição do replicon. Moléculas de ácido nucléico policistrônico recombinante ou desse tipo podem ser administradas a um mamífero e depois transcritas in situ para produzir um replicon policistrônico que expressa proteínas do herpesvírus.
[0056] Em alguns aspectos, a invenção é uma molécula de ácido nucléico policistrônico que contém uma seqüência que codifica uma gH de herpesvírus ou fragmento desta, e uma gL de herpesvírus ou um fragmento desta. As proteínas gH e gL, ou fragmentos destas, podem ser de qualquer herpesvírus desejado como, por exemplo, HSV-1, HSV-2, VZV, EBV tipo 1, EBV tipo 2, CMV, HHV-6 tipo A, HHV-6 tipo B, HHV-7, KSHV, e semelhantes. De preferência, o herpesvírus é VZV, HSV-2, HSV-1, EBV (tipo 1 ou tipo 2) ou CMV. Mais preferivelmente, o herpesvírus é VZV, HSV-2 ou CMV. Ainda mais preferivelmente, o herpesvírus é CMV. As seqüências de proteínas gH e gL e de ácidos nucléicos que codificam as proteínas desses vírus são bem conhecidas na técnica.seqüências exemplares são identificadas na Tabela 1. A molécula de ácido nucléico policistrônico pode conter uma primeira seqüência que codifica uma proteína gH revelada na Tabela 1, ou um fragmento desta, ou uma seqüência que é pelo menos cerca de 90%,91%,92%,93%,94%,95%,96%,97%),98% ou 99% idêntica a ela. A molécula de ácidonucléico policistrônico também pode conter uma segunda seqüência que codifica uma proteína gL revelada na Tabela 1, ou um fragmento desta, ou uma seqüência que é pelo menos cerca de 90%,91%,92%,93%,94%,95%,96%,97%,98% ou 99%idêntica a ela.Tabela 1
Figure img0001
[0057] Nessa descrição da invenção, para facilitar uma descrição nítida dos ácidos nucléicos, componentes particulares da seqüência são denominados uma “primeira seqüência”, uma “segunda seqüência” etc. Deve ser subentendido que a primeira e segunda seqüências podem aparecer em qualquer ordem ou orientação desejada, e que não se deseja definir uma ordem ou orientação em particular pelas palavras “primeira”, “segunda” etc. Similarmente, complexos de proteínas são citados por listagem das proteínas que estão presentes no complexo, por exemplo, gH/gL. Isso visa descrever o complexo pelas proteínas que estão presentes no complexo, e não indica quantidades relativas das proteínas ou a ordem ou orientação de seqüências que codificam as proteínas em um ácido nucléico recombinante.
[0058] Certas modalidades preferidas, por exemplo, VRP e RNA auto-replicante de alfavírus que contêm seqüências que codificam proteínas de CMV, são ainda aqui descritos. Há a intenção de que as seqüências que codificam proteínas de CMV nessas modalidades preferidas possam ser substituídas com seqüências que codificam proteínas, por exemplo, gH e gL, de outros herpesvírus.
Plataformas de VRP de alfavírus
[0059] Em algumas modalidades, proteínas de CMV são liberadas a uma célula usando partículas de replicon de alfavírus (VRP) que empregam replicons policistrônicos (ou vetores) como descrito abaixo. Como aqui usado, o termo “policistrônico” inclui vetores bicistrônicos, bem como vetores que compreendem três ou mais cístrons. Cístrons em um vetor policistrônico podem codificar proteínas de CMV das mesmas cepas de CMV ou de cepas de CMV diferentes. Os cístrons podem ser orientados em qualquer ordem 5’-3’. Qualquer seqüência de nucleotídeos que codifica uma proteína de CMV pode ser usada para produzir a proteína. Seqüências exemplares úteis para preparação dos ácidos nucléicos policistrônicos que codificam duas ou maisproteínas de CMV ou fragmentos destas são aqui descritas.
[0060]Como aqui usado, o termo “alfavírus” possui seu significado convencional na técnica e inclui várias espécies como, por exemplo, vírus da encefalite eqüina venezuelana (VEE; por exemplo, asno de Trinidad, TC83CR etc.), vírus Semliki Forest (SFV), vírus Sindbis, vírus do rio Ross, vírus da encefalite eqüina ocidental, vírus da encefalite eqüina oriental, vírus Chikungunya, vírus S.A. AR86, vírus Everglades, vírus Mucambo, vírus Barmah Forest, vírus Middelburg, vírus Pixuna, vírus O’nyong-nyong, vírus Getah, vírus Sagiyama, vírus Bebaru, vírus Mayaro, vírus Una, vírus Aura, vírus Whataroa, vírus Banbanki, vírus Kyzylagach, vírus Highlands J., vírus Fort Morgan, vírus Ndumu e vírus Buggy Creek. O termo “alfavírus” também pode incluir alfavírus quiméricos (por exemplo, como descrito por Perri e cols.,(2003) J. Virol. 77(19):10.394-403) que contêm seqüências do genoma de mais de um alfavírus.
[0061]Uma “partícula de replicon de alfavírus” (VRP) ou “partícula de replicon” é um replicon de alfavírus empacotado com proteínas estruturais de alfavírus.
[0062]Um “replicon de alfavírus” (ou “replicon”) é uma molécula de RNA que pode dirigir sua própria amplificação in vivo em uma célula-alvo. O replicon codifica a(s) polimerase(s) que catalisa a amplificação de RNA (nsP1, nsP2, nsP3, nsP4) e contém seqüências de RNA cis necessárias à replicação que são reconhecidas e utilizadas pela(s) polimerase(s) codificada(s). Um replicon de alfavírus tipicamente contém os seguintes elementos ordenados:seqüências virais 5’ necessárias em cis para replicação, seqüências que codificam proteínas não estruturais biologicamente ativas de alfavírus (nsP1, nsP2, nsP3, nsP4), seqüências virais 3’ necessárias em cis para replicação, e um traço de poliadenilato. Um replicon de alfavírus também pode conter um ou mais promotores subgenômicos virais da “região de junção” que dirigem a expressão de seqüências de nucleotídeos heterólogas, que podem, em certas modalidades, serem modificadas a fim de aumentar ou reduzir a transcrição viral do fragmento subgenômico e da seqüência(s) heteróloga(s) a serem expressos. Outros elementos de controle podem ser usados, como descrito abaixo.
[0063] Replicons de alfavírus que codificam proteínas de CMV são usados para produzir VRPs. Esses replicons de alfavírus compreendem seqüências que codificam pelo menos duas proteínas de CMV ou fragmentos destas. Essas seqüências estão ligadas operacionalmente a um ou mais elementos de controle adequados, por exemplo, um promotor subgenômico, um IRES (por exemplo, EMCV, EV71) e um sítio 2A viral, que podem ser iguais ou diferentes. A liberação de componentes desses complexos usando os vetores policistrônicos aqui revelados é uma forma eficiente de fornecer seqüências de ácidos nucléicos que codificam duas ou mais proteínas de CMV em quantidades relativas desejadas; enquanto que se múltiplas construções de alfavírus forem usadas para liberar proteínas de CMV individuais para formação de complexo, a coliberação eficiente de VRPs seria necessária.
[0064] Qualquer combinação de elementos de controle adequados pode ser usada em qualquer ordem. Em um exemplo, um único promotor subgenômico é ligado operacionalmente a duas seqüências que codificam duas proteínas de CMV diferentes, e um IRES é posicionado entre as duas seqüências codificadoras. Em outro exemplo, duas seqüências que codificam duas proteínas de CMV diferentes estão ligadas operacionalmente a promotores separados. Ainda em outro exemplo, as duas seqüências que codificam duas proteínas de CMV diferentes estão ligadas operacionalmente a um único promotor. As duas seqüências que codificam duas proteínas de CMV diferentes estão ligadas entre elas por meio de uma seqüência de nucleotídeos que codifica um sítio 2A viral e, dessa forma, codificam uma única cadeia de aminoácidos que contêm as seqüências de aminoácidos de ambas as proteínas de CMV. O sítio 2A viral nesse contexto é usado para gerar duas proteínas de CMV da poliproteína codificada.
Promotores subgenômicos
[0065] Promotores subgenômicos, também conhecidos como promotores da região de junção, podem ser usados para regular a expressão de proteína. Promotores subgenômicos alfavirais regulam a expressão de proteínas estruturais alfavirais. Veja Strauss e Strauss, “The Alfaviruses: Gene Expression, Replication, and Evolution”, Microbiol. Rev. setembro de 1994; 58(3): 491-562. Um polinucleotídeo policistrônico pode compreender um promotor subgenômico de qualquer alfavírus. Quando dois ou mais promotores subgenômicos estão presentes em um polinucleotídeo policistrônico, os promotores podem ser iguais ou diferentes. Por exemplo, o promotor subgenômico pode ter a seqüência CTCTCTACGGCTAACCTGAATGGA (ID. DE SEQ. N°: 1). Em certas modalidades, os promotores subgenômicos podem sermodificados a fim de aumentar ou reduzir a transcrição viral das proteínas. Veja Patente U.S. N°:6.592.874.
Sítio interno de entrada de ribossomos (IRES)
[0066]Em algumas modalidades, um ou mais elementos de controle são um sítio interno de entrada de ribossomos (IRES). Um IRES permite que sejam feitas múltiplas proteínas a partir de um único transcrito de mRNA, já que os ribossomos se ligam a cada IRES e iniciam a tradução na ausência de a 5’-cap, que é normalmente necessário para iniciar a tradução de proteína em células eucarióticas. Por exemplo, o IRES pode ser EV71 ou EMCV.
Sítio 2A viral
[0067]A proteína de FMDV 2A é um peptídeo curto que serve para separar as proteínas estruturais de FMDV de uma proteína não estrutural (FMDV 2B). Estudo inicial sobre esse peptídeo sugeriu que ele atua como uma protease autocatalítica, mas outro trabalho (por exemplo, Donnelly e cols.,(2001), J. Gen. Virol. 82,1.013-1.025) sugere que essa seqüência curta e o único aminoácido seguinte de FMDV 2B(Gly)atuam como parada-início da tradução. Independentemente do modo de ação preciso, a seqüência pode ser inserida entre dois polipeptídeos, e efetuar a produção de múltiplos polipeptídeos individuais a partir de um único quadro de leitura aberta. No contexto dessa invenção, seqüências de FMDV 2A podem ser inseridas entre as seqüências que codificam pelo menos duas proteínas de CMV, permitindo sua síntese como parte de um único quadro de leitura aberta. Por exemplo, o quadro de leitura aberta pode codificar uma proteína gH e uma proteína gL separadas por uma seqüência que codifica um sítio 2A viral. Então um único mRNA é transcrito durante a etapa de tradução, os peptídeos de gH e gL são produzidos separadamente em função da atividade do sítio 2A viral. Qualquer seqüência de 2A viral adequada pode ser usada. Freqüentemente, um sítio 2A viral compreende a seqüência de consenso Asp-Val/Ile-Glu-X- Asn-Pro-Gly-Pro, em que X é qualquer aminoácido (ID. DE SEQ. N°: 2). Por exemplo, a seqüência de peptídeos de 2A do vírus da febre aftosa 2 é DVESNPGP (ID. DE SEQ. N°:3). Veja Trichas e cols.,“Use of the viral 2A peptide for bicistronic expression in transgenic mice”, BMC Biol. 15 de setembro de 2008; 6: 40, e Halpin e cols., “Self-processing 2A-poliproteins - a system for coordinate expression of multiple proteins in transgenic plants”, Plant J. Fevereiro de 1999;17(4):453-9.
[0068]Em algumas modalidades, um replicon de alfavírus é um replicon quimérico, por exemplo, um replicon quimérico de VEE-Sindbis (VCR) ou um replicon da cepa de VEE TC83 (TC83R) ou um replicon quimérico de TC83-Sindbis (TC83CR). Em algumas modalidades, um VCR contém o sinal de empacotamento e UTR 3’ de um replicon de Sindbis no lugar de seqüências em nsP3 e na extremidade 3’ do replicon de VEE; veja Perri e cols., J. Virol. 77,10.394-403,2003. Em algumas modalidades, um TC83CR contém o sinal de empacotamentoeUTR 3’deum repliconde Sindbis no lugarde seqüênciasem nsP3 enaextremidade3’de um replicon dacepa de VEE TC83.
Produção de VRPs
[0069]Métodos de preparação de VRPs são bem conhecidos na técnica. Em algumas modalidades, um alfavírus é montado em uma VRP usando uma célula de empacotamento. Uma “célula de empacotamento de alfavírus” (ou “célula de empacotamento”) é uma célula que contém um ou mais cassetes de expressão de proteína estrutural de alfavírus e que produz partículas recombinantes de alfavírus após introdução de um replicon de alfavírus, sistema de iniciação de vetor estratificado eucariótico (por exemplo, Patente U.S. 5.814.482) ou partícula recombinante de alfavírus. Os (um ou mais) cassetes diferentes de proteína estrutural de alfavírus servem como “helpers” por fornecimento das proteínas estruturais de alfavírus. Um “cassete de proteína estrutural de alfavírus” é um cassete de expressão que codifica uma ou mais proteínas estruturais de alfavírus e compreende pelo menos um e até cinco cópias (ou seja, 1, 2, 3, 4 ou 5) de uma seqüência de reconhecimento de replicase de alfavírus. Cassetes de expressão de proteína estrutural tipicamente compreendem, de 5’ para 3’, uma seqüência 5’ que inicia a transcrição de RNA de alfavírus, um promotor da região subgenômica de alfavírus opcional, uma seqüência de nucleotídeos que codifica a proteína estrutural de alfavírus, uma região não traduzida 3’ (que também dirige a transcrição de RNA) e um traço de poliA. Veja, por exemplo, WO 2010/019437.
[0070] Em modalidades preferidas, dois cassetes de proteína estrutural de alfavírus diferentes (helpers defeituosos em “split”) são usados em uma célula de empacotamento para minimizar os eventos de recombinação que poderiam produzir um vírus replicação-competente. Em algumas modalidades, um cassete de proteína estrutural de alfavírus codifica a proteína do capsídeo (C), mas nenhuma das glicoproteínas (E2 e E1). Em algumas modalidades, um cassete de proteína estrutural de alfavírus codifica a proteína do capsídeo e as glicoproteínas E1 ou E2 (mas não ambas). Em algumas modalidades, um cassete de proteína estrutural de alfavírus codifica as glicoproteínas E2 e E1, mas não a proteína do capsídeo. Em algumas modalidades, um cassete de proteína estrutural de alfavírus codifica a glicoproteína E1 ou E2 (mas não ambas) e não a proteína do capsídeo.
[0071]Em algumas modalidades, VRPs são produzidas pela introdução simultânea de replicons e RNAs helpers em células de várias fontes. Sob essas condições, por exemplo, células BHKV (1 x 107) são eletroporadas a, por exemplo, 220 volts, 1.000 μF, 2 pulsos manualmente com 10 μg de RNA de replicon:6 μg de RNA de Cap helper defeituoso: 10 μg de RNA de Gly helper defeituoso, e o sobrenadante contendo alfavírusé coletado aproximadamente 24horasmais tarde.Replicons e/ou helpers também podem ser introduzidos em formas deDNA que lançam RNAs adequadosdentro decélulastransfectadas.
[0072]Uma célula de empacotamento pode ser uma célula mamífera ou uma célula não mamífera, por exemplo, uma célula de inseto (por exemplo, SF9) ou aviária (por exemplo, um fibroblasto primário de pinto ou pato ou linhagem de células de fibroblasto). Veja a Patente U.S. 7.445.924. Fontes de células aviárias incluem, sem limitação, células-tronco embrionárias aviárias como, por exemplo, EB66® (VIVALIS); células de galinha, incluindo células-tronco embrionárias de galinha como, por exemplo, células EBx®, fibroblastos embrionários de galinha e células germinativas embrionárias de galinha; células de pato como, por exemplo, as linhagens de células AGE1.CR e AGE1.CR.pIX (ProBioGen)que sãodescritas, por exemplo, em Vaccine 27:4.975-4.982(2009) e WO 2005/042728); e células de ganso. Em algumas modalidades, uma célula de empacotamento é um fibroblasto primário de pato ou linhagem de células retinianas de pato, por exemplo, AGE.CR (PROBIOGEN).
[0073]Fontes de células mamíferas para introdução simultânea de ácido nucléico e/ou células de empacotamento incluem, sem limitação, células humanas ou de primata não humano, incluindo células PerC6 (PER.C6) (CRUCELL N.V.), que são descritas, por exemplo, em WO 01/38362 e WO 02/40665, além das depositadas sob o número de depósito ECACC 96022940); MRC-5 (ATCC CCL-171);WI-38 (ATCC CCL-75); células pulmonares fetais rhesus (ATCC CL-160); células de rim embrionário humano (por exemplo, células 293, tipicamente transformadas por DNA aparado de adenovírus tipo 5); células VERO de rins de macaco); células de cavalo, de vaca (por exemplo, células MDBK), de carneiro, de cão (por exemplo, células MDCK de rins de cães, ATCC CCL34 MDCK (NBL2) ou MDCK 33016, número de depósito DSM ACC 2219, como descrito em WO 97/37001); gato, e células de roedores (por exemplo, células de hamster como, por exemplo, BHK21-F, células HKCC, ou células de ovário de hamster chinês (CHO)), e podem ser obtidas de uma ampla variedade de estágios do desenvolvimento incluindo, por exemplo, adultas, neonatais, fetais e de embriões.
[0074]Emalgumasmodalidades,umacélulade empacotamento é transformada estavelmente com um ou mais cassetes de expressão de proteína estrutural. Cassetes de expressão de proteína estrutural podem ser introduzidos em células usando técnicas-padrão de DNA recombinante, incluindo transferência de DNA mediada por transferrina- policátion, transfecção com ácidos nucléicos naked ou encapsulados, fusão celular mediada por lipossomo, transporte intracelular de glóbulos de látex revestidos com DNA, fusão de protoplasto, infecção viral, eletroporação, métodos de “pistola gênica” e transfecção mediada por DEAE ou fosfato de cálcio. Cassetes de expressão de proteína estrutural tipicamente são introduzidos em uma célula hospedeira como moléculas de DNA, mas também podem ser introduzidos como RNA transcrito in vitro. Cada cassete de expressão pode ser introduzido separadamente ou de forma substancialmente simultânea.
[0075] Em algumas modalidades, linhagens de células de empacotamento de alfavírus estáveis são usadas para produzir partículas recombinantes de alfavírus. Estas são células alfavírus-permissivas que compreendem cassetes de DNA que expressam o RNA helper defeituoso integrado estavelmente em seus genomas. Veja Polo e cols., Proc. Natl. Acad. Sci. U.S.A. 96, 4.598-603, 1999. Os RNAs helper sao expressos constitutivamente, mas as proteínas estruturais de alfavírus não o são, pois os genes estão sob o controle de um promotor subgenômico de alfavírus (Polo e cols., 1999). Após introdução de um replicon de alfavírus no genoma de uma célula de empacotamento por transfecção ou infecção de VRP, são produzidas enzimas replicase que desencadeiam a expressão dos genes do capsídeo e de glicoproteína nos RNAs helper, e VRPs de saída são produzidas. A introdução do replicon pode ser obtida por diversos métodos, incluindo tanto transfecção quanto infecção com um estoque de semeadura de partículas de replicon de alfavírus. A célula de empacotamento é então incubada sob condições e por um tempo suficiente para produzir partículas de replicon de alfavírus empacotadas no sobrenadante da cultura.
[0076] Dessa forma, as células de empacotamento permitem que as VRPs atuem como vírus com autopropagação. Essa tecnologia permite que as VRPs sejam produzidas basicamente da mesma forma e usando o mesmo equipamento que aquele usado para vacinas vivas atenuadas ou outros vetores virais que possuem linhagens de células produtoras disponíveis, por exemplo, vetores de adenovírus replicação-incompetentes desenvolvidos em células que expressam os genes de E1A e E1B de adenovírus.
[0077] Em algumas modalidades, é usado um processo em duas etapas: a primeira etapa compreende a produção de um estoque de semeadura de partículas de replicon de alfavírus por transfecção de uma célula de empacotamento com um RNA de replicon ou replicon à base de DNA de plasmídeo. Um estoque de partículas de replicon bem maior é então produzido em uma segunda etapa, por infecção de uma cultura fresca de células de empacotamento com o estoque de semeadura. Essa infecção pode ser realizada usando várias multiplicidades de infecção (MOI), incluindo uma MOI = 0,00001, 0,00005, 0,0001, 0,0005, 0,001, 0,005, 0,01, 0,05, 0,1, 0,5, 1,0, 3, 5, 10 ou 20. Em algumas modalidades, a infecção é realizada em uma MOI baixa (por exemplo, menos de 1). AO longo do tempo, as partículas de replicon podem ser coletadas de células de empacotamento infectadas com o estoque de semeadura. Em algumas modalidades, partículas de replicon podem então ser passadas em culturas ainda maiores de células de empacotamento virgens por infecção de baixa multiplicidade repetida, resultando em preparações em escala comercial com a mesma titulação elevada.
Plataformas de RNA auto-replicante
[0078] Duas ou mais proteínas de CMV podem ser produzidas por expressão de ácidos nucléicos recombinantes que codificam as proteínas nas células de um indivíduo. De preferência, as moléculas de ácido nucléico recombinante codificam duas ou mais proteínas de CMV, por exemplo, são policistrônicas. Como definido acima, “policistrônica” inclui bicistrônicas. Ácidos nucléicos preferidos que podem ser administrados a um indivíduo para causar a produção de proteínas de CMV são moléculas de RNA auto-replicantes. As moléculas de RNA auto-replicantes da invenção são baseadas no RNA genômico de vírus de RNA, mas não possuem os genes que codificam uma ou mais proteínas estruturais. As moléculas de RNA auto-replicantes são capazes de ser traduzidas para produzir proteínas não estruturais do vírus de RNA e proteínas de CMV codificadas pelo RNA auto- replicante.
[0079] O RNA auto-replicante geralmente contém pelo menos um ou mais genes selecionados do grupo que consiste em replicase viral, proteases virais, helicases virais e outras proteínas virais não estruturais, e também compreendem seqüências de replicação da extremidade 5’ e 3’ cis-ativas, e uma seqüência heteróloga que codifica duas ou mais proteínas de CMV desejadas. Um promotor subgenômico que dirige a expressão da(s) seqüência(s) heteróloga(s) pode ser incluído no RNA auto-replicante. Se desejado, uma seqüência heteróloga pode ser fundida in frame a outras regiões codificadoras no RNA auto-replicante e/ou pode estar sob o controle de um sítio interno de entrada de ribossomos (IRES).
[0080] As moléculas de RNA auto-replicantes da invenção podem ser projetadas de tal modo que a molécula de RNA auto-replicante não possa induzir a produção de partículas virais infecciosas. Isso pode ser obtido, por exemplo, por omissão de um ou mais genes virais que codificam proteínas estruturais que são necessárias para a produção de partículas virais no RNA auto-replicante. Por exemplo, quando a molécula de RNA auto-replicante é baseada em um alfavírus, por exemplo, no vírus Sindbis (SIN), vírus Semliki Forest e vírus da encefalite eqüina venezuelana (VEE), um ou mais genes que codificam proteínas virais estruturais, por exemplo, glicoproteínas do capsídeo e/ou do envelope, podem ser omitidos. Se desejado, as moléculas de RNA auto-replicantes da invenção podem ser projetadas para induzir a produção de partículas virais infecciosas que são atenuadas ou virulentas, ou para produzir partículas virais que são capazes de uma única rodada de infecção subseqüente.
[0081] Uma molécula de RNA auto-replicante pode, quando liberada a uma célula de vertebrado, até mesmo sem nenhuma proteína, levar à produção de múltiplos RNAs-filhos por transcrição dela próprio (ou de uma cópia anti-senso dela própria). O RNA auto-replicante pode ser traduzido diretamente após liberação a uma célula, e essa tradução fornece uma RNA polimerase RNA-dependente que então produz transcritos do RNA liberado. Dessa forma, o RNA liberado leva à produção de múltiplos RNAs-filhos. Esses transcritos são anti-senso em relação ao RNA liberado e podem, eles próprios, serem traduzidos para fornece a expressão in situ da proteína de CMV codificada, ou podem ser transcritos para fornecerem transcritos adicionais com o mesmo sentido que o RNA liberado sendo então traduzidos para fornecer a expressão in situ da(s) proteína(s) de CMV codificada(s).
[0082] Um sistema adequado para obtenção de auto- replicação é o uso de um replicon de RNA baseado em alfavírus, por exemplo, um replicon de alfavírus como aqui descrito. Esses replicons de fita + são traduzidos após liberação a uma célula para produzir uma replicase (ou replicase- transcriptase). A replicase é traduzida como uma poliproteína que se autocliva para fornecer um complexo de replicação que cria cópias genômicas de fita - do RNA de fita + liberado. Esses próprios transcritos de fita - podem ser transcritos para gerar cópias adicionais do RNA de fita + progenitor e também para dar origem a um ou mais transcritos subgenômicos que codificam duas ou mais proteínas de CMV. A tradução do transcrito subgenômico, dessa forma, leva à expressão in situ da(s) proteína(s) de CMV pela célula infectada. Replicons de alfavírus adequados podem usar uma replicase de um vírus Sindbis, de um vírus Semliki Forest, de um vírus da encefalite eqüina oriental, de um vírus da encefalite eqüina venezuelana etc.
[0083] Dessa forma, uma molécula de RNA auto-replicante preferida codifica: (i) uma RNA polimerase RNA-dependente que pode transcrever RNA da molécula de RNA auto-replicante e (ii) duas ou mais proteínas de CMV ou fragmentos destas. A polimerase pode ser uma replicase de alfavírus, por exemplo, que compreende a proteína de alfavírus nsP4. A proteína nsP4 é o componente catalítico crucial da replicase.
[0084]Enquanto os genomas naturais do alfavírus codificam proteínas estruturais de vírion além da poliproteína não estrutural de replicase, prefere-se que uma molécula de RNA auto-replicante baseada em alfavírus da invenção não codifique todas as proteínas estruturais de alfavírus. Dessa forma, o RNA auto-replicante pode levar à produção de cópiasRNA genômico dele próprio em uma célula, mas não à produção de vírions de alfavírus quecontêm RNA. A incapacidade de produzir esses vírions significa que, diferentemente de um alfavírus do tipo selvagem, a molécula de RNA auto-replicante não pode se perpetuar em forma infecciosa. As proteínas estruturais de alfavírus que são necessárias à perpetuação nos vírus do tipo selvagem estão ausentes de RNAs auto-replicantes da invenção e seu lugar é ocupado por gene(s) que codifica o produto gênico desejado (proteína de CMV ou fragmento desta), de tal forma que o transcrito subgenômico codifique o produto gênico desejado em vez das proteínas estruturais do vírion de alfavírus.
[0085]Dessa forma, uma molécula de RNA auto-replicante útil com a invenção possuem duas seqüências que codificam proteínas de CMV diferentes ou fragmentos destas. As seqüências que codificam as proteínas de CMV ou fragmentos podem estar em qualquer orientação desejada, e podem estar operacionalmente ligadas aos mesmos promotores ou a promotores separados. Se desejado, as seqüências que codificam as proteínas de CMV ou fragmentos podem ser parte de um único quadro de leitura aberta. Em algumas modalidades, o RNA pode ter uma ou mais seqüências ou quadros de leitura aberta adicionais (downstream), por exemplo, que codificam outras proteínas de CMV adicionais ou fragmentos destas. Uma molécula de RNA auto-replicante pode ter uma seqüência 5’ que é compatível com a replicase codificada.
[0086]Em um aspecto, a molécula de RNA auto-replicante é derivada ou baseada em um alfavírus, por exemplo, um replicon de alfavírus, como aqui definidos. Em outros aspectos, a molécula de RNA auto-replicante é derivada ou baseada em um vírus diferente de um alfavírus, preferivelmente, um vírus de RNA de tida positiva e, mais preferivelmente, um picornavírus, flavivírus, rubivírus, pestivírus, hepacivírus, calicivírus ou coronavírus. Seqüências de alfavírus do tipo selvagem adequadas são bem conhecidas e estão disponíveis por depositórios de seqüências, por exemplo, a “American Type Culture Collection”, Rockville, Md. Exemplos representativos de alfavírus adequados incluem o vírus Aura (ATCC VR-368), vírus Bebaru (ATCC VR-600, ATCC VR-1240), Cabassou (ATCC VR-922), vírus Chikungunya (ATCC VR-64, ATCC VR-1241), vírus da encefalomielite eqüina oriental (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR- 924), vírus Getah (ATCC VR- 369, ATCC VR-1243), Kyzylagach (ATCC VR-927), vírus Mayaro (ATCCVR-66;ATCC VR-1277), Middleburg (ATCC VR-370), vírus Mucambo (ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371), vírusPixuna (ATCC VR-372,ATCC VR-1245), vírus do rio Ross (ATCC VR-373, ATCC VR-1246), Semliki Forest (ATCC VR-67, ATCC VR-1247), vírus Sindbis (ATCC VR-68, ATCC VR-1248), Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR- 374), encefalomielite eqüina venezuelana (ATCC VR-69, ATCC VR- 923, ATCC VR-1250 ATCC VR-1249, ATCC VR-532), encefalomielite eqüina ocidental (ATCC VR-70, ATCC VR-1251, ATCC VR-622, ATCC VR- 1252), Whataroa (ATCC VR-926) e Y-62- 33 (ATCC VR-375).
[0087] As moléculas de RNA auto-replicantes da invenção podem conter um ou mais nucleotídeos modificados e, portanto, possuem estabilidade aumentada e são resistentes à degradação e depuração in vivo, e outras vantagens. Sem se prender a uma teoria específica, acredita-se que as moléculas de RNA auto-replicantes que contêm nucleotídeos modificados evitam ou reduzem a estimulação de receptores imunes endossômicos e citoplasmáticos quando o RNA auto- replicante é liberado em uma célula. Isso permite que ocorra a auto-replicação, amplificação e expressão de proteína. Isso também reduz as preocupações de segurança em relação ao RNA auto-replicante que não contém nucleotídeos modificados, pois o RNA auto-replicante que contém nucleotídeos modificados reduz a ativação do sistema imune inato e subseqüente conseqüências indesejadas (por exemplo, inflamação no local de injeção, irritação no local de injeção, dor, e semelhantes). Acredita-se também que as moléculas de RNA produzidas em conseqüência da auto- replicação são reconhecidas como ácidos nucléicos estranhos pelos receptores imunes citoplasmáticos. Dessa forma, as moléculas de RNA auto-replicantes que contêm nucleotídeos modificados permitem a amplificação eficiente do RNA em uma célula hospedeira e a expressão de proteínas de CMV, bem como efeitos adjuvantes.
[0088]A seqüência de RNA pode ser modificada com relação ao seu uso de códons, por exemplo, para aumentar a eficácia de tradução e a meia-vida do RNA. Uma cauda de poli A (por exemplo, de cerca de 30 resíduos de adenosina ou mais) pode ser anexada à extremidade 3’ do RNA para aumentar sua meia-vida. A extremidade 5’doRNA pode ser coberta com umribonucleotídeomodificado com aestrutura m7G (5’) ppp (5’) N (estrutura de cap 0) ou um derivado deste, que podeser incorporado durante a síntesede RNA ou pode ser criado enzimaticamente após transcrição de RNA (por exemplo, por utilização da enzima de cobertura do vírus de vacínia (VCE) que consiste em mRNA trifosfatase, guanilil-transferase e guanina-7-metiltransferase, que catalisa a construção de estruturas de cap 0N7- monometiladas). A estrutura de cap 0 pode fornecer estabilidade e eficácia de tradução à molécula de RNA. O cap 5’ da molécula de RNA pode ainda ser modificado por uma 2’-O-Metiltransferase, o que resulta na geração de uma estrutura de cap 1 (m7Gppp [m2’-0]N), que pode aumentar ainda mais a eficácia de tradução. Uma estrutura de cap 1 também pode aumentar a potência in vivo.
[0089]Como aqui usado, o termo “nucleotídeo modificado” se refere a um nucleotídeo que contém uma ou mais modificações químicas (por exemplo, substituições) dentro ou nas bases nitrogenadas do nucleosídeo (por exemplo, citosina (C), timina (T) ou uracil (U), adenina (A) ou guanina (G)). Se desejado, uma molécula de RNA auto- replicante pode conter modificações químicas dentro ou na porção de açúcar do nucleosídeo (por exemplo, ribose, desoxirribose, ribose modificada, desoxirribose modificada, análogo de açúcar de seis membros ou análogo de açúcar de cadeia aberta), ou no fosfato.
[0090] As moléculas de RNA auto-replicantes podem conter pelo menos um nucleotídeo modificado, que preferivelmente não é parte do cap 5’ (por exemplo, além da modificação que é parte do cap 5’). Conseqüentemente, a molécula de RNA auto-replicante pode conter um nucleotídeo modificado em uma única posição, pode conter um nucleotídeo modificado particular (por exemplo, pseudouridina, N6-metiladenosina, 5-metilcitidina, 5-metiluridina) em duas ou mais posições, ou pode conter dois, três, quatro, cinco, seis, sete, oito, nove, dez ou mais nucleotídeos modificados (por exemplo, cada um em uma ou mais posições). De preferência, as moléculas de RNA auto-replicantes compreendem nucleotídeos modificados que contêm uma modificação em ou na base nitrogenada, mas não contêm porções de açúcar ou fosfato modificadas.
[0091] Em alguns exemplos, entre 0,001% e 99% ou 100% dos nucleotídeos em uma molécula de RNA auto-replicante são nucleotídeos modificados. Por exemplo, 0,001%-25%, 0,01%- 25%, 0,1%-25% ou 1%-25% dos nucleotídeos em uma molécula de RNA auto-replicante são nucleotídeos modificados.
[0092] Em outros exemplos, entre 0,001% e 99% ou 100% de um nucleotídeo não modificado em particular em uma molécula de RNA auto-replicante são substituídos com um nucleotídeo modificado. Por exemplo, cerca de 1% dos nucleotídeos na molécula de RNA auto-replicante que contêm uridina podem ser modificados, por exemplo, por substituição de uridina com pseudouridina. Em outros exemplos, a quantidade desejada (percentagem) de dois, três ou quatro nucleotídeos particulares (nucleotídeos que contêm uridina, citidina, guanosina ou adenina) em uma molécula de RNA auto- replicante são nucleotídeos modificados. Por exemplo, 0,001%-25%, 0,01%-25%, 0,1%-25 ou l%-25% de um nucleotídeo particular em uma molécula de RNA auto-replicante são nucleotídeos modificados. Em outros exemplos, 0,001%-20%, 0,001%-15%, 0,001%-10%, 0,01%-20%, 0,01%-15%, 0,1%-25, 0,01%-10%, l%-20%, 1%-15%, 1%-10% ou cerca de 5%, cerca de 10%, cerca de 15%, cerca de 20% de um nucleotídeo particular em uma molécula de RNA auto-replicante são nucleotídeos modificados.
[0093] Prefere-se que menos de 100% dos nucleotídeos em uma molécula de RNA auto-replicante sejam nucleotídeos modificados. Prefere-se também que menos de 100% de um nucleotídeo particular em uma molécula de RNA auto- replicante sejam nucleotídeos modificados. Dessa forma, moléculas de RNA auto-replicantes preferidas compreendem pelo menos alguns nucleotídeos não modificados.
[0094] Há mais de 96 modificações de nucleosídeos de ocorrência natural encontradas no RNA mamífero. Veja, por exemplo, Limbach e cols., Nucleic Acids Research, 22(12): 2.183-2.196 (1994). A preparação de nucleotídeos e nucleotídeos e nucleosídeos modificados é bem conhecida na técnica, por exemplo, pela Patente U.S. Números 4373071, 4458066, 4500707, 4668777, 4973679, 5047524, 5132418, 5153319, 5262530, 5700642 todas aqui incorporadas por referência em sua totalidade, e muitos nucleosídeos modificados e nucleotídeos modificados estão disponíveis comercialmente.
[0095] Nucleobases modificadas que podem ser incorporadas em nucleosídeos e nucleotídeos modificados e estar presentes nas moléculas de RNA incluem:m5C (5- metilcitidina),m5U(5-metiluridina),m6A(N6- metiladenosina),s2U(2-tiouridina),Um(2’-O- metiluridina),m1A(1-metiladenosina);m2A(2- metiladenosina); Am (2-1-O-metiladenosina); ms2m6A (2- metiltio-N6-metiladenosina); i6A (N6-isopenteniladenosina); ms2i6A (2-metiltio-N6isopenteniladenosina); io6A (N6-(cis- hidroxiisopentenil)adenosina); ms2io6A (2-metiltio-N6-(cis- hidroxiisopentenil)adenosina);g6A(N6- glycinilcarbamoiladenosina);t6A(N6-treonil carbamoiladenosina);ms2t6A(2-metiltio-N6-treonil carbamoiladenosina);m6t6A(N6-metil-N6- treonilcarbamoiladenosina);hn6A(N6- hidroxinorvalilcarbamoiladenosina);ms2hn6A (2-metiltio-N6- hidroxinorvalilcarbamoiladenosina);Ar(p)(2’-O- ribosiladenosina(fosfato)); I(inosina); m1I(1- metilinosina); m’Im (1,2’-O-dimetilinosina); m3C (3- metilcitidina);Cm(2T-O-metilcitidina);s2C(2- tiocitidina);ac4C(N4-acetilcitidina);f5C(5- formilcitidina); m5Cm (5,2-O-dimetilcitidina); ac4Cm (N4- acetil-2TO-metilcitidina);k2C(lisidina);m1G(1- metilguanosina);m2G(N2-metilguanosina);m7G(7- metilguanosina); Gm (2’-O-metilguanosina); m22G (N2,N2- dimetilguanosina); m2Gm (N2,2’-O-dimetilguanosina); m22Gm (N2,N2,2’-O-trimetilguanosina);Gr(p)(2’-O- ribosilguanosina(fosfato));yW(wibutosina);o2yW (peroxiwibutosina);OHyW(hidroxiwibutosina);OHyW* (hidroxiwibutosina não modificada); imG (wiosina); mimG (metilguanosina); Q (queuosina); oQ (epoxiqueuosina); galQ (galatactosil-queuosina); manQ (manosil-queuosina); preQo (7-ciano-7-deazaguanosina);preQi(7-aminometil-7- deazaguanosina); G* (arcaeosina); D (diidrouridina); m5Um (5,2’-O-dimetiluridina); s4U (4-tiouridina); m5s2U (5- metil-2-tiouridina); s2Um (2-tio-2’-O-metiluridina); acp3U (3-(3-amino-3-carboxipropil)uridina);ho5U(5- hidroxiuridina); mo5U (5-metoxiuridina); cmo5U (ácido uridina 5-oxiacético); mcmo5U (éster metílico ácido uridina 5-oxiacético); chm5U (5-(carboxihidroximetil)uridina)); mchm5U (éster metílico de 5-(carboxihidroximetil)uridina); mcm5U(5-metoxicarbonilmetiluridina);mcm5Um(S- metoxicarbonilmetil-2-O-metiluridina);mcm5s2U(5- metoxicarbonilmetil-2-tiouridina); nm5s2U (5-aminometil-2- tiouridina); mnm5U (5-metilaminometiluridina); mnm5s2U (5- metilaminometil-2-tiouridina); mnm5se2U (5-metilaminometil- 2-selenouridina); ncm5U (5-carbamoilmetiluridina); ncm5Um (5-carbamoilmetil-2’-O-metiluridina);cmnm5U(5- carboximetilaminometiluridina); cnmm5Um (5-carboximetil aminometil-2-L-O-etiluridina);cmnm5s2U(5- carboximetilaminometil-2-tiouridina);m62A(N6,N6- dimetiladenosina); Tm (2’-O-metilinosina); m4C(N4- metilcitidina); m4Cm (N4,2-O-dimetilcitidina); hm5C (5- hidroximetilcitidina); m3U (3- metiluridina); cm5U (5- carboximetiluridina);m6Am(N6,T-O-dimetiladenosina); rn62Am(N6,N6,O-2-trimetiladenosina);ni27G(N2,7- dimetilguanosina); m2’27G (N2,N2,7-trimetilguanosina); m3Um (3,2T-O-dimetiluridina); m5D (5-metildiidrouridina); f5Cm (5-formil-2’-O-metilcitidina);m1Gm(1,2’-O- dimetilguanosina);m’Am(1,2-O- dimetiladenosina)irinometiluridina);tm5s2U(S- taurinometil-2-tiouridina)); imG-14 (4-demetilguanosina); imG2(isoguanosina);ac6A(N6-acetiladenosina), hipoxantina,inosina,8-oxo-adenina,derivados7- substituídos destes, diidrouracil, pseudouracil, 2- tiouracil,4-tiouracil,5-aminouracil,5-(C1-C6)- alquiluracil, 5-metiluracil, 5-(C2-C6)-alqueniluracil, 5- (C2-C6)-alquiniluracil,5-(hidroximetil)uracil,5- clorouracil,5-fluoruracil,5-bromouracil,5- hidroxicitosina, 5-(C1-C6)-alquilcitosina, 5-metilcitosina, 5-(C2-C6)-alquenilcitosina, 5-(C2-C6)-alquinilcitosina, 5- clorocitosina,5-fluorcitosina,5-bromocitosina,N2- dimetilguanina, 7-deazaguanina, 8-azaguanina, guanina 7- deaza-7-substituída,7-deaza-7-(C2-C6)alquinilguanina, guanina7-deaza-8-substituída,8-hidroxiguanina,6- tioguanina,8-oxoguanina,2-aminopurina,2-amino-6- cloropurina, 2,4-diaminopurina, 2,6-diaminopurina,8- azapurina, 7-deazapurina substituída, purina 7-deaza-7- substituída,purina7-deaza-8-substituída,hidrogênio (resíduo abásico), m5C, m5U, m6A, s2U, W ou 2’-O-metil-U. Qualquer uma ou qualquer combinação dessas nucleobases modificadas pode ser incluída no RNA auto-replicante da invenção. Muitas dessas nucleobases modificadas e seus ribonucleosídeos correspondentes estão disponíveis por fornecedores comerciais.
[0096]Se desejado, a molécula de RNA auto-replicante pode conter ligações fosforamidato, fosforotioato e/ou metilfosfonato.
[0097]As moléculas de RNA auto-replicantes que compreendem pelo menos um nucleotídeo modificado podem ser preparadas usando qualquer método adequado. Vários métodos adequados são conhecidos na técnica para a produção de moléculas de RNA que contêm nucleotídeos modificados. Por exemplo, uma molécula de RNA auto-replicante que contém nucleotídeos modificados pode ser preparada por transcrição (por exemplo, transcrição in vitro) de um DNA que codifica a molécula de RNA auto-replicante usandouma RNA polimerase DNA-dependente adequada, por exemplo, RNA polimerase de fago T7, RNA polimerase de fago SP6, RNA polimerase de fago T3, e semelhantes, ou mutantes dessas polimerases que permitem a incorporação eficiente de nucleotídeos modificados em moléculas de RNA. A reação de transcrição conterá nucleotídeos e nucleotídeos modificados, e ouros componentes que apóiam a atividade da polimerase selecionada como, por exemplo, um tampão adequado, e sais adequados. A incorporação de análogos de nucleotídeos em um RNA auto-replicante pode ser planejada, por exemplo, para alterar a estabilidade dessas moléculas de RNA, para aumentar a resistência contra RNAses, para estabelecer replicação após introdução em células hospedeiras adequadas (“infectividade” do RNA) e/ou para induzir ou reduzir respostas imunes inatas e adaptivas.
[0098]Métodos sintéticos adequados podem ser usados isoladamente, ou em combinação com um ou mais outros métodos (por exemplo, tecnologia de DNA ou RNA recombinante), para produzir uma molécula de RNA auto- replicante que contêm um ou mais nucleotídeos modificados. Métodos adequados para a síntese de novo são bem conhecidos na técnica e podem ser adaptados para aplicações particulares. Métodos exemplares incluem, por exemplo, síntese química usando grupos de proteção adequados como, por exemplo, CEM (Masuda e cols.,(2007) Nucleic Acids Symposium Series 57:3-4), o método de β-cianoetil fosforamidita (Beaucage S.L. e cols.(1981) Tetrahedron Lett. 22:1.859); o método de H-fosfonato nucleosídeo (Garegg P. e cols.(1986) Tetrahedron Lett. 27:4.051-4; Froehler B.C. e cols.(1986) Nucl Acid Res. 14:5.399-407; Garegg P. e cols.(1986) Tetrahedron Lett. 27:4.055-8; Gaffney B.L. e cols.(1988) Tetrahedron Lett. 29:2.619 22). Essas químicas podem realizadas ou adaptadas para uso com sintetizadores automatizados de ácido nucléico que estão disponíveis comercialmente. Métodos sintéticos adequados adicionais são revelados em Uhlmann e cols. (1990) Chem. Rev. 90:544-84, e Goodchild J. (1990) Bioconjugate Chem. 1:165. A síntese de ácido nucléico também pode ser realizada usando métodos recombinantes que são bem conhecidos e convencionais na técnica, incluindo clonagem, processamento e/ou expressão de polinucleotídeos e produtos gênicos codificados por esses polinucleotídeos. O embaralhamento DNA por fragmentação aleatória e remontagemporPCRdosfragmentosgênicose polinucleotídeos sintéticos são exemplos de técnicas conhecidas que podem ser usadas para o design e planejamento de seqüências de polinucleotídeos. A mutagênese sítio-dirigida pode ser usada para alterar ácidos nucléicos e as proteínas codificadas, por exemplo, para inserir novos sítios de restrição, alterar padrões de glicosilação, alterar preferência de códons, produzir variantes de splice, introduzir mutações e semelhantes. Métodos adequados para transcrição, tradução e expressão de seqüências de ácidos nucléicos são conhecidos e convencionais na técnica (veja geralmente, “Current Protocols in Molecular Biology”, Vol. 2, Ed. Ausubel, e cols., Greene Publish. Assoc. e Wiley Interscience, Capítulo 13,1988; Glover, “DNA Cloning”, Vol. II, IRL Press, Wash., D.C., Capítulo 3,1986; Bitter, e cols., em Methods in Enzymology 153:516-544(1987);“The Molecular Biology of the Yeast Saccharomyces”, Eds. Strathern e cols., Cold Spring Harbor Press, Vols. I e II, 1982; e Sambrook e cols.,“Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Press, 1989).
[0099]A presença e/ou quantidade de um ou mais nucleotídeos modificados em uma molécula de RNA auto- replicante pode ser determinada usando qualquer método adequado. Por exemplo, um RNA auto-replicante pode ser digerido em monofosfatos (por exemplo, usando nuclease P1) e desfosforilado (por exemplo, usando uma fosfatase adequada, por exemplo, CIAP), e os nucleosídeos resultantes analisados por HPLC de fase reversa (por exemplo, usando uma coluna YMC Pack ODS-AQ (5 mícrons, 4,6 X 250 mm) e eluídos usando um gradiente de 30% de B (0-5 min) até 100% de B (5-13 min) e a 100% de B (13-40) min, taxa de fluxo (0,7 ml/min), detecção UV (comprimento de onda: 260 nm), temperatura da coluna (30°C). Tampão A (20 mM de ácido acético - acetato de amônio pH 3,5), tampão B (20 mM ácido acético - acetato de amônio pH 3,5/ metanol [90/10])).
[0100]O RNA auto-replicante pode estar associado a um sistema de liberação. O RNA auto-replicante pode ser administrado com ou sem um adjuvante.
Sistemas de liberação de RNA
[0101]Os RNAs auto-replicantes aqui descritos são adequados à liberação em diversas modalidades, por exemplo, liberação de RNA naked ou em combinação com lipídeos, polímeros ou outros compostos que facilitam a entrada nas células. As moléculas de RNA auto-replicantes podem ser introduzidas em células-alvo ou indivíduos usando qualquer técnica adequada, por exemplo, por injeção direta, microinjeção, eletroporação, lipofecção, biolística, e semelhantes. A molécula de RNA auto-replicante também pode ser introduzida nas células por meio de endocitose mediada por receptor. Veja, por exemplo, a Patente U.S. N°: 6.090.619; Wu e Wu, J. Biol. Chem.,263:14.621(1988); e Curiel e cols., Proc. Natl. Acad. Sci. U.S.A.,88:8.850 (1991). Por exemplo, a Patente U.S. N°:6.083.741 revela a introdução de um ácido nucléico exógeno em células mamíferas por associação do ácido nucléico a uma porção de policátion (por exemplo, poli-L-lisina que possui 3-100 resíduos de lisina), a qual, ela própria, é acoplada a uma porção de ligação doreceptorde integrina (por exemplo,um peptídeo cíclicoquepossui aseqüência Arg-Gly-Asp (ID.DE SEQ. N°:5)) .
[0102]As moléculas de RNA auto-replicantes podem ser liberadas nas células por meio de anfifílicos. Veja, por exemplo, a Patente U.S. N°:6.071.890. Tipicamente, uma molécula de ácido nucléico pode formar um complexo com o anfifílico catiônico. Células mamíferas colocadas em contato com o complexo podem facilmente recolhê-lo.
[0103]O RNA auto-replicante pode ser liberado como RNA naked (por exemplo, simplesmente como uma solução aquosa de RNA), mas, para aumentar a entrada nas células e também os efeitos intercelulares subseqüentes, o RNA auto-replicante é administrado preferivelmente em combinação com um sistema de liberação, por exemplo, um sistema de liberação particulado oude emulsão.Um grandenúmero de sistemas de liberação é bem conhecido por aqueles habilitados na técnica. Essessistemas deliberaçãoincluem,por exemplo, a liberação baseada em lipossomo (WO 93/24640 para Debs e Zhu (1993); Mannino e Gould-Fogerite (1988) BioTechniques 6(7):682-691; Patente U.S. N°:5.279.833 para Rose; WO 91/06309 para Brigham (1991); e Feigner e cols.(1987) Proc. Natl. Acad. Sci. U.S.A. 84:7.413-7.414), além do uso de vetores virais (por exemplo, adenovirais (veja, por exemplo, Berns e cols.(1995) Ann. NY Acad. Sci.772:95104; Ali e cols.(1994) Gene Ther.1:367-384; e Haddada e cols.(1995) Curr. Top. Microbiol. Immunol. 199 (Parte 3): 297-306 para uma revisão), de papilomavírus, retrovirais (veja, por exemplo, Buchscher e cols.(1992) J. Virol. 66(5)2.731-2.739; Johann e cols.(1992) J. Virol. 66(5): 1.635-1.640(1992); Sommerfelt e cols.,(1990) Virol.176: 58-59; Wilson e cols.(1989) J. Virol. 63:2.374-2.378; Miller e cols., J. Virol. 65:2.220-2.224(1991); Wong- Staal e cols.,PCT/US94/05700 e Rosenburg e Fauci (1993) em “Fundamental Immunology”, Terceira Edição Paul (ed.) Raven Press, Ltd., Nova York e as referências nele citadas, e Yu e cols., Gene Therapy (1994) supra), e vetores de vírus adeno-associados (veja, West e cols.(1987) Virology 160: 38-47; Patente U.S. N°:4.797.368 para Carter e cols. (1989); WO 93/24641 para Carter e cols.(1993); Kotin (1994) Human Gene Therapy 5:793-801; Muzyczka (1994) J. Clin. Invest. 94:1.351 e Samulski (supra) para uma visao geral de vetores de AAV; veja também, Patente U.S. N°: 5.173.414 para Lebkowski; Tratschin e cols. (1985) Mol. Cell. Biol. 5(11): 3.251-3.260; Tratschin, e cols. (1984) Mol. Cell. Biol., 4: 2.072-2.081; Hermonat e Muzyczka (1984) Proc. Natl. Acad. Sci. U.S.A., 81: 6.466-6.470; McLaughlin e cols. (1988) e Samulski e cols. (1989) J. Virol., 63: 3.822-3.828), e semelhantes.
[0104] Três sistemas de liberação particularmente úteis são (i) lipossomos, (ii) micropartículas de polímero atóxico e biodegradável, e (iii) emulsões óleo-em-água catiônicas submícron.
Lipossomos
[0105] Vários lipídeos anfifílicos podem formar bicamadas em um ambiente aquoso para encapsular um núcleo aquoso que contém RNA como um lipossomo. Esses lipídeos podem ter um grupo principal hidrofílico aniônico, catiônico ou zwitteriônico. A formação de lipossomos a parir de fosfolipídeos aniônicos remonta aos anos de 1960, e lipídeos catiônicos formadores de lipossomos vêm sendo estudados desde os anos de 1990. Alguns fosfolipídeos são aniônicos, enquanto outros são zwitteriônicos. Classes de fosfolipídeo adequadas incluem, sem limitação, fosfatidiletanolaminas, fosfatidilcolinas, fosfatidilserinas e fosfatidilgliceróis, e alguns fosfolipídeos úteis estão listados na Tabela 2. Lipídeos catiônicos úteis incluem, sem limitação, dioleoil trimetilamônio propano (DOTAP), 1,2-diesteariloxi-N,N- dimetil-3-aminopropano (DSDMA), 1,2-dioleiloxi-N,N-dimetil- 3-aminopropano (DODMA), 1,2-dilinoleiloxi-N,N-dimetil-3- aminopropano (DLinDMA), 1,2-dilinoleniloxi-N,N-dimetil-3- aminopropano (DLenDMA). Lipídeos zwitteriônicos incluem, sem limitação, lipídeos zwitteriônicos de acil e lipídeos zwitteriônicos de éter. Exemplos de lipídeos zwitteriônicos úteis são DPPC, DOPC e dodecilfosfocolina. Os lipídeos podem ser saturados ou insaturados.
[0106]Lipossomos podem ser formados a partir de um único lipídeo ou a partir de uma mistura de lipídeos. Uma mistura pode compreender:(i) uma mistura de lipídeos aniônicos (ii) uma mistura de lipídeos catiônicos (iii) uma mistura de lipídeos zwitteriônicos (iv) uma mistura de lipídeos aniônicos e lipídeos catiônicos (v) uma mistura de lipídeos aniônicos e lipídeos zwitteriônicos (vi) uma mistura de lipídeos zwitteriônicos e lipídeos catiônicos ou (vii) uma mistura de lipídeos aniônicos, lipídeos catiônicos e lipídeos zwitteriônicos. Similarmente,uma mistura pode compreender lipídeos tanto saturados quanto insaturados. Por exemplo, uma mistura pode compreender DSPC (zwitteriônicos,saturados),DlinDMA(catiônicos, insaturados) e/ou DMPG (aniônicos, saturados). Quando é usada uma mistura de lipídeos, nem todos os lipídeos componentes na mistura precisam ser anfifílicos, por exemplo, um ou mais lipídeos anfifílicos podem ser misturados com colesterol.
[0107]A porção hidrofílica de um lipídeo pode ser PEGuilada (ou seja, modificada por covalente adesão de um polietileno glicol). Essa modificação pode aumentar a estabilidade e evitar a adsorção não específica dos lipossomos. Por exemplo, os lipídeos podem ser conjugados ao PEG usando técnicas tais como aquelas reveladas em Heyes e cols.(2005) J. Controlled Release 107:276-87.
[0108]Uma mistura de DSPC, DlinDMA, PEG-DMPG e colesterol pode ser usada para formar lipossomos. Um aspecto separado da invenção é um lipossomo que compreende DSPC, DlinDMA, PEG-DMG e colesterol. Esse lipossomo preferivelmente encapsula RNA, por exemplo, um RNA auto- replicante, por exemplo, que codifica um imunógeno.
[0109]Lipossomos são normalmente divididos em três grupos:vesículas multilamelares (MLV); pequenas vesículas unilamelares (SUV); e grandes vesículas unilamelares (LUV). MLVs possuem múltiplas bicamadas em cada vesícula, formando vários compartimentos aquosos separados. SUVs e LUVs possuem uma bicamada única que encapsula um núcleo aquoso; SUVs tipicamente possuem um diâmetro < 50nm, e LUVs possuem um diâmetro >50nm. Lipossomos úteis com a invenção são idealmente LUVs com um diâmetro na faixa de 50-220 nm. Para uma composição que compreende uma população de LUVs com diâmetros diferentes:(i) pelo menos 80% por número devem ter diâmetros na faixa de 20-220 nm, (ii) o diâmetro médio (Zav, por intensidade) da população está idealmente na faixa de 40-200 nm, e/ou (iii) os diâmetros devem ter um índice de poli dispersibilidade <0,2.
[0110]Metodologias para preparação de lipossomos adequados são bem conhecidas na técnica; por exemplo, veja “Liposomes:MethodsandProtocols”,Volume1: “Pharmaceutical Nanocarriers: Methods and Protocols”, (ed. Weissig), Humana Press, 2009. ISBN 160327359X; Liposome Technology, volumes I, II e III. (ed. Gregoriadis). Informa Healthcare, 2006; e “Functional Polymer Colloids and Microparticles”, Volume 4(“Microspheres, Microcapsules and Liposomes”), (eds. Arshady e Guyot). Citus Books, 2002. Um método útil envolve a misturação de:(i) uma solução etanólica dos lipídeos (ii) uma solução aquosa do ácido nucléico e (iii) tampão, seguida por misturação, equilíbrio, diluição e purificação (Heyes e cols. (2005) J. Controlled Release 107: 276-87).
[0111] O RNA é preferivelmente encapsulado dentro dos lipossomos e, portanto, o lipossomo forma uma camada externa em torno de um núcleo aquoso que contém o RNA. Constatou-se que essa encapsulação protege o RNA de digestão por RNase. Os lipossomos podem incluir algum RNA externo (por exemplo, na superfície dos lipossomos), mas, preferivelmente, pelo menos metade do RNA (e idealmente substancialmente todo ele) é encapsulada.
Micropartículas poliméricas
[0112] Vários polímeros podem formar micropartículas para encapsular ou adsorver RNA. O uso de um polímero substancialmente atóxico significa que um receptor pode receber com segurança as partículas, e o uso de um polímero biodegradável significa que as partículas podem ser metabolizadas após liberação para evitar a persistência de longo prazo. Polímeros úteis também são passíveis de esterilização, para ajudar na preparação de formulações de grau farmacêutico.
[0113] Polímeros atóxicos e biodegradáveis adequados incluem, sem limitação, poli(ácidos a-hidróxi), ácidos polihidroxibutíricos, polilactonas (incluindo policaprolactonas), polidioxanonas, polivalerolactona, poliortoésteres, polianidridos, policianoacrilatos, policarbonatos derivados de tirosina, polivinil- pirrolidinonas ou poliéster-amidas, e combinações destes.
[0114] Em algumas modalidades, as micropartículas são formadas por poli(ácidos a-hidróxi), por exemplo, uma poli(lactidas) (“PLA”), copolímeros de lactida e glicolida, por exemplo, uma poli(D,L-lactida-co-glicolida) (“PLG”), e copolímeros de D,L-lactida e caprolactona. Polímeros de PLG úteis incluem aqueles que possuem uma proporção molar de lactida/glicolida que varia, por exemplo, de 20:80 a 80:20 por exemplo, 25:75,40:60,45:55,55:45,60:40,75:25.Polímeros de PLG úteis incluem aqueles que possuem um peso molecular entre, por exemplo, 5.000-200.000 Da, por exemplo, entre 10.000-100.000,20.000-70.000,40.00050.000 Da.
[0115]As micropartículas idealmente possuem um diâmetro na faixa de 0,02 μm a 8 μm. Para uma composição que compreende uma população de micropartículas com diâmetros diferentes, pelo menos 80% por número devem ter diâmetros na faixa de 0,03-7 μm.
[0116]Metodologias para preparação de micropartículas adequadas são bem conhecidas na técnica; por exemplo, veja “Functional Polymer Colloids and Microparticles”, Volume 4 (“Microspheres, microcapsules and Liposomes”),(eds. Arshady e Guyot). Citus Books, 2002;“Polymers in Drug Delivery” (eds. Uchegbu e Schatzlein). CRC Press, 2006. (em particular o capítulo 7) e “Microparticulate Systems for the Delivery of Proteins and Vaccines” (eds. Cohen e Bernstein). CRC Press, 1996. Para facilitar a adsorção de RNA, uma micropartícula pode incluir um tensoativo e/ou lipídeo catiônico, por exemplo, como revelado em O’Hagan e cols.(2001) J Virology,15:9.037-9.043; e Singh e cols. (2003) Pharmaceutical Research 20:247-251. Uma forma alternativa de produção de micropartículas poliméricas é por moldagem e cura, por exemplo, como revelado em WO 2009/132206.
[0117] As micropartículas da invenção podem ter um potencial zeta entre 40-100 mV. O RNA pode ser adsorvido às micropartículas, e a adsorção é facilitada por inclusão de materiais catiônicos (por exemplo, lipídeos catiônicos) na micropartícula.
Emulsões catiônicas óleo-em-água
[0118] Emulsões óleo-em-água são conhecidas por atuarem como adjuvantes em vacinas para influenza como, por exemplo, o adjuvante MF59™ no produto FLUAD™, e o adjuvante AS03 no produto PREPANDRIX™. A liberação de RNA pode ser obtida pelo uso de uma emulsão óleo-em-água, desde que a emulsão inclua uma ou mais moléculas catiônicas. Por exemplo, um lipídeo catiônico pode ser incluído na emulsão para fornecer uma superfície de gotícula carregada positivamente à qual o RNA carregado negativamente pode se anexar.
[0119] A emulsão compreende um ou mais óleos. Óleos adequados incluem aqueles de, por exemplo, um animal (por exemplo, peixe) ou uma fonte vegetal. O óleo é idealmente biodegradável (metabolizável) e biocompatível. Fontes para óleos vegetais incluem nozes, sementes e grãos. Óleo de amendoim, óleo de soja, óleo de coco e azeite de oliva, os mais comumente disponíveis, exemplificam os óleos de nozes. Pode ser usado o óleo de jojoba, por exemplo, obtido do feijão de jojoba. Óleos de sementes incluem óleo de açafroa, óleo de semente de algodão, óleo de semente de girassol, óleo de semente de gergelim e semelhantes. No grupo dos grãos, o óleo de milho é o mais facilmente disponível, mas o óleo de outros grãos de cereais como, por exemplo, trigo, aveia, centeio, arroz, teff, triticale e semelhantes, também pode ser usado. Ésteres de glicerol ácido graxo 6-10 carbonos e 1,2-propanodiol, embora não ocorram naturalmente em óleos de sementes, podem ser preparados por hidrólise, separação e esterificação os materiais apropriados partindo dos óleos de nozes e sementes. Gorduras e óleos do leite de mamíferos são metabolizáveis e, portanto, podem ser usados. Os procedimentos para separação, purificação, saponificação e outros meios necessários para a obtenção de óleos puros a partir de fontes animais são bem conhecidos na técnica.
[0120] A maioria dos peixes contém óleos metabolizáveis que podem ser prontamente recuperados. Por exemplo, o óleo de fígado de bacalhau, óleos de fígado de tubarão e o óleo de baleia, por exemplo, espermacete, exemplificam vários dos óleos de peixes que podem ser aqui usados. Diversos óleos de cadeia ramificada são sintetizados bioquimicamente em unidades de isopreno de 5 carbonos e são geralmente denominados terpenóides. Esqualano, o análogo saturado para esqualeno, também pode ser usado. Óleos de peixes, incluindo esqualeno e esqualano, são facilmente disponíveis por fontes comerciais ou podem ser obtidos por métodos conhecidos na técnica.
[0121] Outros óleos úteis são os tocoferóis, particularmente em combinação com esqualeno. Quando a fase oleosa de uma emulsão inclui um tocoferol, qualquer um dos α, β, Y, δ, ε ou £ tocoferóis pode ser usado, mas α- tocoferóis são preferidos. D-α-tocoferol e DL-α-tocoferol podem, ambos, serem usados. Um α-tocoferol preferido é DL- α-tocoferol. Uma combinação de óleos que compreende esqualeno e um tocoferol (por exemplo, DL-α-tocoferol) pode ser usada.
[0122] Emulsões preferidas compreendem esqualeno, um óleo de fígado de tubarão que é um terpenóide insaturado ramificado (C30H50; [(CH3)2C[=CHCH2CH2C(CH3)]2=CHCH2-]2; 2,6,10,15,19,23-hexametil-2,6,10,14,18,22-tetracosahexaeno; CAS R 7683-64-9).
[0123] O óleo na emulsão pode compreender uma combinação de óleos, por exemplo, esqualeno e pelo menos um óleo adicional.
[0124] O componente aquoso da emulsão pode ser água simples (por exemplo, água para injeção) ou pode incluir componentes adicionais, por exemplo, solutos. Por exemplo, ele pode incluir sais para formar um tampão, por exemplo, sais de citrato ou fosfato, por exemplo, sais de sódio. Tampões típicos incluem: um tampão de fosfato; um tampão Tris; um tampão de borato; um tampão de succinato; um tampão de histidina; ou um tampão de citrato. A fase aquosa tamponada é preferida, e tampões tipicamente serão incluídos na faixa de 5-20 mM.
[0125] A emulsão também inclui um lipídeo catiônico. De preferência esse lipídeo é um tensoativo que possa facilitar a formação e estabilização da emulsão. Lipídeos catiônicos úteis geralmente contém um átomo de nitrogênio que é carregado positivamente sob condições fisiológicas, por exemplo, como uma amina terciária ou quaternária. Esse nitrogênio pode estar no grupo hidrofílico principal do tensoativo anfifílico. Lipídeos catiônicos úteis incluem, sem limitação: 1,2-dioleoiloxi-3-(trimetilamônio)propano (DOTAP),3’-[N-(N’,N’-dimetilaminoetano)- carbamoil]colesterol (DC colesterol), dimetildioctadecil- amônio (DDA por exemplo, o brometo), 1,2-dimiristoil-3- trimetil-amônio-propano (DMTAP), dipalmitoil(C16:0)trimetil amônio propano (DPTAP), diestearoiltrimetilamônio propano (DSTAP). Outros lipídeos catiônicos úteis são: cloreto de benzalcônio (BAK), cloreto de benzetônio, cetramida (que contém brometo de tetradeciltrimetilamônio e possivelmente pequenas quantidades de brometo de dodeciltrimetilamônio e brometo de hexadeciltrimetil amônio),cloreto de cetilpiridínio (CPC), cloreto de cetil trimetilamônio (CTAC),N,N’,N‘-polioxietileno(10)-N-tallow-1,3- diaminopropano, brometo de dodeciltrimetilamônio, brometo de hexadeciltrimetil-amônio, brometo misto de alquil- trimetil-amônio, cloreto de benzildimetildodecilamônio, cloretodebenzildimetilhexadecil-amônio, benziltrimetilamôniometóxido,brometode cetildimetiletilamônio, brometo de dimetildioctadecil amônio (DDAB), cloreto de metilbenzetônio, cloreto de decametônio, cloreto de metil trialquil amônio misto, cloreto de metil trioctilamônio), cloreto de N,N-dimetil-N- [2(2-metil-4-(1,1,3,3etrametilbutil)-fenóxi]-etóxi)etil]- benzenometamônio (DEBDA), sais de dialquildimetilamônio, cloretode[1-(2,3-dioleiloxi)-propil]-N,N,N- trimetilamônio, 1,2-diacil-3-(trimetilamônio)propano (grupo acil = dimiristoil, dipalmitoil, diestearoil, dioleoil), 1,2-diacil-3(dimetilamônio)propano(acilgrupo= dimiristoil, dipalmitoil, diestearoil, dioleoil), 1,2- dioleoil-3-(4’-trimetil-amônio)butanoil-sn-glicerol, éster de 1,2-dioleoil 3-succinil-sn-glicerol colina, colesteril (4’-trimetilamônio)butanoato), sais de N-alquil piridínio (por exemplo, brometo de cetilpiridínio e cloreto de cetilpiridínio), sais de N-alquilpiperidínio, eletrólitos dicatiônicosdotipobola(C12Me6;C12BU6), dialquilglicetilfosforilcolina,lisolecitina,L-α dioleoilfosfatidiletanolamina,colinaésterde hemissuccinato de colesterol, lipopoliaminas, incluindo, sem limitação, dioctadecilamidoglicilespermina (DOGS), dipalmitoilfosfatidiletanol-amidoespermina(DPPES), lipopoli-L (ou D)-lisina (LPLL, LPDL), poli (L (ou D)- lisina conjugada a N-glutarilfosfatidiletanolamina, éster de didodecil glutamato com grupo amino pendente (CAGluPhCnN), éster de ditetradecil glutamato com grupo amino pendente (C14GluCnN+), derivados catiônicos de colesterol, incluindo, sem limitação, sal de colesteril-3 β-oxissuccinamidoetilenotrimetilam0nio, colesteril-3 β- oxissuccinamidoetileno-dimetilamina, sal de colesteril-3 β- carboxiamidoetilenotrimetilamônioecolesteril-3-β- carboxiamidoetilenodimetilamina. Outroslipídeos catiônicos úteis são descritos em US 2008/0085870 e US 2008/0057080, que são aqui incorporados por referência. O lipídeo catiônico é preferivelmente biodegradável (metabolizável) e biocompatível.
[0126]Além do óleo e lipídeo catiônico, uma emulsão pode incluir um tensoativo não iônico e/ou um tensoativo zwitteriônico. Esses tensoativos incluem, sem limitação: os tensoativos de ésteres de polioxietileno sorbitano (comumente denominados Tweens), especialmente polissorbato 20 e polissorbato 80; copolímeros de óxido de etileno (EO), óxido de propileno (PO) e/ou óxido de butileno (BO), vendidos sob o nome comercial DOWFAX™, por exemplo, copolímeros em bloco lineares de EO/PO; octoxinóis, que podem variar no número de repetição de grupos etóxi (oxi- 1,2-etanodiil), com octoxinol-9 (Triton X-100 ou t- octilfenoxipolietoxietanol) sendo de interesse particular; (octilfenoxi)polietoxietanol(IGEPALCA-630/NP-40); fosfolipídeoscomo,porexemplo,fosfatidilcolina (lecitina); graxos de polioxietileno derivados de lauril, cetil, estearil e oleil álcoois (conhecidos como tensoativos Brij), por exemplo, trietilenoglicol monolauril éter (Brij 30); polioxietileno-9-lauryl éter; e ésteres de sorbitano (comumente conhecidos como Spans), por exemplo, trioleato de sorbitano (Span 85) e monolaurato de sorbitano. Tensoativos preferidos para inclusão na emulsão são polissorbato 80 (Tween 80; monooleato de polioxietileno sorbitano), Span 85 (trioleato de sorbitano), lecitina e Triton X-100.
[0127]Misturas desses tensoativos podem ser incluídas na emulsão; por exemplo, misturas de Tween 80/Span 85 ou misturas de Tween 80/Triton-X100. Uma combinação de um éster de polioxietileno sorbitano como, por exemplo, monooleato de polioxietileno sorbitano (Tween 80) e um octoxinol como, por exemplo, t-octilfenoxi-polietoxietanol (Triton X-100), também é adequada. Outra combinação útil compreende laureth 9 mais um éster de polioxietileno sorbitano e/ou um octoxinol. Misturas úteis podem compreender um tensoativo com um valor de HLB na faixa de 10-20 (por exemplo, polissorbato 80, com um HLB de 15,0) e um tensoativo com um valor de HLB na faixa de 1-10 (por exemplo, trioleato de sorbitano, com um HLB de 1,8).
[0128] As quantidades preferidas de óleo (% por volume) na emulsão final estão entre 2-20%, por exemplo, 5-15%, 614%, 7-13%, 8-12%. Um teor de esqualeno de cerca de 4-6% ou cerca de 9-11% é particularmente útil.
[0129] Quantidades preferidas de tensoativos (% por peso) na emulsão final estão entre 0,001% e 8%. Por exemplo: ésteres de polioxietileno sorbitano (por exemplo, polissorbato 80) 0,2 a 4%, em particular entre 0,4-0,6%, entre 0,45-0,55%, cerca de 0,5% ou entre 1,5-2%, entre 1,82,2%, entre 1,9-2,1%, cerca de 2% ou 0,85-0,95% ou cerca de 1%; ésteres de sorbitano (por exemplo, trioleato de sorbitano) 0,02 a 2%, em particular cerca de 0,5%, ou cerca de 1%; octil- ou nonilfenoxi polioxietanóis (por exemplo, Triton X-100) 0,001 a 0,1%), em particular 0,005 a 0,02%; éteres de polioxietileno (por exemplo, laureth 9) 0,1 a 8%, preferivelmente 0,1 a 10% e, em particular, 0,1 a 1% ou cerca de 0,5%.
[0130] As quantidades absolutas de óleo e tensoativo, e sua proporção, podem ser variadas dentro de limites amplos enquanto ainda formam uma emulsão. Aqueles habilitados na técnica podem facilmente variar as proporções relativas dos componentes para obter uma emulsão desejada, mas uma proporção de peso entre 4:1 e 5:1 para óleo e tensoativo é típica (óleo em excesso).
[0131] Um importante parâmetro para assegurar atividade imunoestimulante de uma emulsão, particularmente em animais grandes, é o tamanho da gotícula de óleo (diâmetro). As emulsões mais eficazes possuem um tamanho de gotícula na faixa submícron. Adequadamente, os tamanhos de gotícula estarão na faixa de 50-750 nm. Mais adequadamente ainda, o tamanho de gotícula é menos de 250 nm, por exemplo, menos de 200 nm, menos de 150 nm. O tamanho de gotícula médio está adequadamente na faixa de 80-180 nm. Idealmente, pelo menos 80% (por número) das gotículas de óleo da emulsão possuem menos de 250 nm de diâmetro e, preferivelmente, pelo menos 90%. Aparelhos para determinação do tamanho de gotícula médio em uma emulsão, e a distribuição de tamanho, estão disponíveis comercialmente. Esses tipicamente usam as técnicas de dispersão luminosa dinâmica e/ou detecção óptica de partícula única, por exemplo, as séries de instrumentos Accusizer™ e Nicomp™ disponíveis por Particle Sizing Systems (Santa Barbara, USA), ou os instrumentos Zetasizer™ de Malvern Instruments (UK), ou os instrumentos “Particle Size Distribution Analyzer” de Horiba (Kyoto, Japão).
[0132] Idealmente, a distribuição de tamanhos de gotícula (por número) possui apenas um máximo, ou seja, há uma população de gotículas únicas distribuídas em torno de uma média (modo), ao invés de terem dois máximos. Emulsões preferidas possuem uma polidispersibilidade < 0,4, por exemplo, 0,3, 0,2, ou menos.
[0133] Emulsões adequadas com gotículas submícron e uma distribuição de tamanho estreita podem ser obtidas pelo uso de microfluidificação. Essa técnica reduz o tamanho médio da gotícula de óleo propelindo jatos de componentes de entrada através de canais geometricamente fixados sob pressão elevada e velocidade elevada. Esses jatos entram em contado com as paredes do canal, paredes da câmara e entre eles. Como resultado, forças de cisalhamento, impacto e cavitação causam uma redução do tamanho de gotícula. Etapas repetidas de microfluidificação podem ser realizadas até que seja obtida uma emulsão com uma média e distribuição de tamanho de gotícula desejadas.
[0134]Como alternativa à microfluidificação, podem ser usados métodos térmicos para causar inversão de fase. Esses métodos também podem fornecer uma emulsão submícron com uma distribuição de tamanho de partícula estreita.
[0135]Emulsões preferidas podem ser esterilizadas por filtração, ou seja, suas gotículas podem passar através de um filtro de 220 nm. Além de fornecer uma esterilização, esse procedimento também remove quaisquer gotículas grandes na emulsão.
[0136]Em certas modalidades, o lipídeo catiônico na emulsão é DOTAP. A emulsão óleo-em-água catiônica pode compreender de cerca de 0,5 mg/ml até cerca de 25 mg/ml de DOTAP. Por exemplo, a emulsão óleo-em-água catiônica pode compreender DOTAP de cerca de 0,5 mg/ml até cerca de 25 mg/ml, de cerca de 0,6 mg/ml até cerca de 25 mg/ml, de cerca de 0,7 mg/ml até cerca de 25 mg/ml, de cerca de 0,8 mg/ml até cerca de 25 mg/ml, de cerca de 0,9 mg/ml até cerca de 25 mg/ml, de cerca de 1,0 mg/ml até cerca de 25 mg/ml, de cerca de 1,1 mg/ml até cerca de 25 mg/ml, de cerca de 1,2 mg/ml até cerca de 25 mg/ml, de cerca de 1,3 mg/ml até cerca de 25 mg/ml, de cerca de 1,4 mg/ml até cerca de 25 mg/ml, de cerca de 1,5 mg/ml até cerca de 25 mg/ml, de cerca de 1,6 mg/ml até cerca de 25 mg/ml, de cerca de 1,7 mg/ml até cerca de 25 mg/ml, de cerca de 0,5 mg/ml até cerca de 24 mg/ml, de cerca de 0,5 mg/ml até cerca de 22 mg/ml, de cerca de 0,5 mg/ml até cerca de 20 mg/ml, de cerca de 0,5 mg/ml até cerca de 18 mg/ml, de cerca de 0,5 mg/ml até cerca de 15 mg/ml, de cerca de 0,5 mg/ml até cerca de 12 mg/ml, de cerca de 0,5 mg/ml até cerca de 10 mg/ml, de cerca de 0,5 mg/ml até cerca de 5 mg/ml, de cerca de 0,5 mg/ml até cerca de 2 mg/ml, de cerca de 0,5 mg/ml até cerca de 1,9 mg/ml, de cerca de 0,5 mg/ml até cerca de 1,8 mg/ml, de cerca de 0,5 mg/ml até cerca de 1,7 mg/ml, de cerca de 0,5 mg/ml até cerca de 1,6 mg/ml, de cerca de 0,6 mg/ml até cerca de 1,6 mg/ml, de cerca de 0,7 mg/ml até cerca de 1,6 mg/ml, de cerca de 0,8 mg/ml até cerca de 1,6 mg/ml, cerca de 0,5 mg/ml, cerca de 0,6 mg/ml, cerca de 0,7 mg/ml, cerca de 0,8 mg/ml, cerca de 0,9 mg/ml, cerca de 1,0 mg/ml, cerca de 1,1 mg/ml, cerca de 1,2 mg/ml, cerca de 1,3 mg/ml, cerca de 1,4 mg/ml, cerca de 1,5 mg/ml, cerca de 1,6 mg/ml, cerca de 12 mg/ml, cerca de 18 mg/ml, cerca de 20 mg/ml, cerca de 21,8 mg/ml, cerca de 24 mg/ml etc. Em uma modalidade exemplar, a emulsão óleo-em-água catiônica compreende de cerca de 0,8 mg/ml até cerca de 1,6 mg/ml de DOTAP, por exemplo, 0,8 mg/ml, 1,2 mg/ml, 1,4 mg/ml ou 1,6 mg/ml.
[0137] Em certas modalidades, o lipídeo catiônico é DC colesterol. A emulsão óleo-em-água catiônica pode compreender DC colesterol de cerca de 0,1 mg/ml até cerca de 5 mg/ml de DC colesterol. Por exemplo, a emulsão óleo- em-água catiônica pode compreender DC colesterol de cerca de 0,1 mg/ml até cerca de 5 mg/ml, de cerca de 0,2 mg/ml até cerca de 5 mg/ml, de cerca de 0,3 mg/ml até cerca de 5 mg/ml, de cerca de 0,4 mg/ml até cerca de 5 mg/ml, de cerca de 0,5 mg/ml até cerca de 5 mg/ml, de cerca de 0,62 mg/ml até cerca de 5 mg/ml, de cerca de 1 mg/ml até cerca de 5 mg/ml, de cerca de 1,5 mg/ml até cerca de 5 mg/ml, de cerca de 2 mg/ml até cerca de 5 mg/ml, de cerca de 2,46 mg/ml até cerca de 5 mg/ml, de cerca de 3 mg/ml até cerca de 5 mg/ml, de cerca de 3,5 mg/ml até cerca de 5 mg/ml, de cerca de 4 mg/ml até cerca de 5 mg/ml, de cerca de 4,5 mg/ml até cerca de 5 mg/ml, de cerca de 0,1 mg/ml até cerca de 4,92 mg/ml, de cerca de 0,1 mg/ml até cerca de 4,5 mg/ml, de cerca de 0,1 mg/ml até cerca de 4 mg/ml, de cerca de 0,1 mg/ml até cerca de 3,5 mg/ml, de cerca de 0,1 mg/ml até cerca de 3 mg/ml, de cerca de 0,1 mg/ml até cerca de 2,46 mg/ml, de cerca de 0,1 mg/ml até cerca de 2 mg/ml, de cerca de 0,1 mg/ml até cerca de 1,5 mg/ml, de cerca de 0,1 mg/ml até cerca de 1 mg/ml, de cerca de 0,1 mg/ml até cerca de 0,62 mg/ml, cerca de 0,15 mg/ml, cerca de 0,3 mg/ml, cerca de 0,6 mg/ml, cerca de 0,62 mg/ml, cerca de 0,9 mg/ml, cerca de 1,2 mg/ml, cerca de 2,46 mg/ml, cerca de 4,92 mg/ml etc. Em uma modalidade exemplar, a emulsão óleo-em-água catiônica compreende de cerca de 0,62 mg/ml até cerca de 4,92 mg/ml de DC colesterol, por exemplo, 2,46 mg/ml.
[0138] Em certas modalidades, o lipídeo catiônico é DDA. A emulsão óleo-em-água catiônica pode compreender de cerca de 0,1 mg/ml até cerca de 5 mg/ml de DDA. Por exemplo, a emulsão óleo-em-água catiônica pode compreender DDA de cerca de 0,1 mg/ml até cerca de 5 mg/ml, de cerca de 0,1 mg/ml até cerca de 4,5 mg/ml, de cerca de 0,1 mg/ml até cerca de 4 mg/ml, de cerca de 0,1 mg/ml até cerca de 3,5 mg/ml, de cerca de 0,1 mg/ml até cerca de 3 mg/ml, de cerca de 0,1 mg/ml até cerca de 2,5 mg/ml, de cerca de 0,1 mg/ml até cerca de 2 mg/ml, de cerca de 0,1 mg/ml até cerca de 1,5 mg/ml, de cerca de 0,1 mg/ml até cerca de 1,45 mg/ml, de cerca de 0,2 mg/ml até cerca de 5 mg/ml, de cerca de 0,3 mg/ml até cerca de 5 mg/ml, de cerca de 0,4 mg/ml até cerca de 5 mg/ml, de cerca de 0,5 mg/ml até cerca de 5 mg/ml, de cerca de 0,6 mg/ml até cerca de 5 mg/ml, de cerca de 0,73 mg/ml até cerca de 5 mg/ml, de cerca de 0,8 mg/ml até cerca de 5 mg/ml, de cerca de 0,9 mg/ml até cerca de 5 mg/ml, de cerca de 1,0 mg/ml até cerca de 5 mg/ml, de cerca de 1,2 mg/ml até cerca de 5 mg/ml, de cerca de 1,45 mg/ml até cerca de 5 mg/ml, de cerca de 2 mg/ml até cerca de 5 mg/ml, de cerca de 2,5 mg/ml até cerca de 5 mg/ml, de cerca de 3 mg/ml até cerca de 5 mg/ml, de cerca de 3,5 mg/ml até cerca de 5 mg/ml, de cerca de 4 mg/ml até cerca de 5 mg/ml, de cerca de 4,5 mg/ml até cerca de 5 mg/ml, cerca de 1,2 mg/ml, cerca de 1,45 mg/ml etc. Alternativamente, a emulsão óleo-em-água catiônica pode compreender DDA a cerca de 20 mg/ml, cerca de 21 mg/ml, cerca de 21,5 mg/ml, cerca de 21,6 mg/ml, cerca de 25 mg/ml. Em uma modalidade exemplar, a emulsão óleo-em-água catiônica compreende de cerca de 0,73 mg/ml até cerca de 1,45 mg/ml de DDA, por exemplo, 1,45 mg/ml.
[0139] Cateteres ou dispositivos semelhantes podem ser usados para liberar as moléculas de RNA auto-replicantes da invenção, como RNA naked ou em combinação com um sistema de liberação, em um órgão ou tecido-alvo. Cateteres adequados são revelados, por exemplo, as Patentes U.S. Nos 4.186.745, 5.397.307, 5.547.472, 5.674.192 e 6.129.705, todas aqui incorporadas por referência.
[0140] A presente invenção inclui o uso de sistemas de liberação adequados, por exemplo, lipossomos, micropartículas de polímero ou micropartículas de emulsão submícron com RNA auto-replicante encapsulado ou adsorvido, para liberar uma molécula de RNA auto-replicante que codifica duas ou mais proteínas de CMV, por exemplo, para despertar uma resposta imune isoladamente, ou em combinação com outra macromolécula. A invenção inclui lipossomos, micropartículas e emulsões submícron com moléculas de RNA auto-replicantesadsorvidase/ouencapsuladas,ecombinações destes.
[0141]As moléculas de RNA auto-replicantes associadas aos lipossomos e micropartículas de emulsão submícron podem ser eficazmente liberadas a uma célula hospedeira, e podem induzir uma resposta imune à proteína codificada pelo RNA auto-replicante.
[0142]Moléculas de RNA policistrônico auto-replicantes que codificam proteínas de CMV, e VRPs produzidas usando replicons de alfavírus policistrônico, podem ser usadas para formar complexos de proteínas de CMV em uma célula. Complexos incluem, sem limitação, gB/gH/gL; gH/gL; gH/gL/gO;gM/gN;gH/gL/UL128/UL130/UL131;e UL128/UL130/UL131.
[0143]Em algumas modalidades, combinações de VRPs são liberadas aumacélula.Combinações incluem, sem limitação: 1.UmaVRPdegH/gLe outra VRP; 2.UmaVRPdegH/gLe uma VRP degB; 3.UmaVRPdegH/gL/gO e uma VRPdegB; 4.UmaVRPdegBeumaVRPdegH/gL/UL128/UL130/UL131; 5.UmaVRPdegBeumaVRPdeUL128/UL130/UL131; 6.UmaVRPdegBeumaVRPdegM/gN; 7.Uma VRP de gB, uma VRP de gH/gL e uma VRP de UL128/UL130/UL131; 8.Uma VRP de gB, uma VRP de gH/gLgO e uma VRP de UL128/UL130/UL131; 9.Uma VRP de gB, uma VRP de gM/gN, uma VRP de gH/gL e uma VRP de UL128/UL130/UL131; 10.Uma VRP de gB, uma VRP de gM/gN, uma VRP de gH/gL/O e uma VRP de UL128/UL130/UL131; 11.Uma VRP de gH/gL e uma VRP de UL128/UL130/UL131; e
[0144] Em algumas modalidades, combinações de moléculas de RNA auto-replicantes são liberadas a uma célula. Combinações incluem, sem limitação: 1.Uma molécula de RNA auto-replicante que codifica gH/gL e uma molécula de RNA auto-replicante que codifica outra proteína; 2.Uma molécula de RNA auto-replicante que codifica gH e gL e uma molécula de RNA auto-replicante que codifica gB; 3.Uma molécula de RNA auto-replicante que codifica gH, gL e gO e uma molécula de RNA auto-replicante que codifica gB; 4.Uma molécula de RNA auto-replicante que codifica gB e uma molécula de RNA auto-replicante que codifica gH, gL, UL128, UL130 e UL131; 5.Uma molécula de RNA auto-replicante que codifica gB e uma molécula de RNA auto-replicante que codifica UL128, UL130 e UL131; 6.Uma molécula de RNA auto-replicante que codifica gB e uma molécula de RNA auto-replicante que codifica gM e gN; 7.Uma molécula de RNA auto-replicante que codifica gB, uma molécula de RNA auto-replicante que codifica gH e gL e uma molécula de RNA auto-replicante que codifica UL128, UL130 e UL131; 8.Uma molécula de RNA auto-replicante que codifica gB, uma molécula de RNA auto-replicante que codifica gH, gL e gO e uma molécula de RNA auto-replicante que codifica UL128, UL130 e UL131; 9.Uma molécula de RNA auto-replicante que codifica gB, uma molécula de RNA auto-replicante que codifica gM e gN, uma molécula de RNA auto-replicante que codifica gH e gL e uma molécula de RNA auto-replicante que codifica UL128, UL130 e UL131; 10.Uma molécula de RNA auto-replicante que codifica gB, uma molécula de RNA auto-replicante que codifica gM e gN, uma molécula de RNA auto-replicante que codifica gH, gL e gO e uma molécula de RNA auto-replicante que codifica UL128, UL130 e UL131; 11.Uma molécula de RNA auto-replicante que codifica gH e gL e uma molécula de RNA auto-replicante que codifica UL128, UL130 e UL131; e
Proteínas de CMV
[0145] Proteínas de CMV adequadas incluem gB, gH, gL, gO e podem ser de qualquer cepa de CMV. Outras proteínas de CMV adequadas incluem UL128, UL130 e UL131, e podem ser de qualquer cepa de CMV. Por exemplo, proteínas de CMV podem ser de cepas de CMV Merlin, AD169, VR1814, Towne, Toledo, TR, PH, TB40 ou Fix. Proteínas de CMV exemplares e fragmentos são aqui descritos. Essas proteínas e fragmentos podem ser codificados por qualquer seqüência de nucleotídeos adequada, incluindo seqüências que são códon- otimizadas ou não para expressão em um hospedeiro desejado, por exemplo, uma célula humana. Seqüências exemplares de proteínas de CMV e ácidos nucléicos que codificam as proteínas são fornecidos na Tabela 2. Tabela 2.
Figure img0002
Figure img0003
Proteínas gB de CMV
[0146]Uma proteína gB pode ser de comprimento total ou pode omitir uma ou mais regiões da proteína. Alternativamente, podem ser usados fragmentos de uma proteína gB. Osaminoácidos degBsão numerados deacordo com a seqüênciade aminoácidosdegBde comprimento total (gB FL de CMV) mostrada no ID. DE SEQ. N°:26, que possui 907 aminoácidos de comprimento. Regiões adequadas de uma proteína gB, que podem ser excluídas da proteína de comprimento total ou incluídas como fragmentos incluem: a seqüência sinalizadora (aminoácidos 1-24), um domínio desintegrina-like de gB-DLD (aminoácidos 57-146), um sítio de clivagem de furina (aminoácidos 459-460), uma região de repetição heptad (679-693), um domínio de expansão de membrana (aminoácidos 751-771) e um domínio citoplasmático dos aminoácidos 771-906. Em algumas modalidades, uma proteína gB inclui aminoácidos 67-86 (epitopo neutralizante AD2) e/ou aminoácidos 532-635 (epitopo imunodominante AD1). Exemplos específicos de fragmentos de gB, incluem “gB sol 692”, que inclui os primeiros 692 aminoácidos de gB, e “gB sol 750”, que inclui os primeiros 750 aminoácidos de gB. A seqüência sinalizadora, aminoácidos 1-24, pode estar presente ou ausente de gB sol 692 e gB sol 750, como desejado. Opcionalmente, a proteína gB pode ser um fragmento de gB com 10 aminoácidos ou mais de comprimento. Por exemplo, o número de aminoácidos no fragmento pode compreender 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850 ou 875 aminoácidos. Um fragmento de gB pode começar em qualquer um dos números de resduo1,2,3,4,5,6,7,8,9,10,11,12,13,14,15, 16,17,18,19,20,21,22,23,24,25,26,27,28,29,30, 31,32,33,34,35,36,37,38,39,40,41,42,43,44,45, 46,47,48,49,50,51,52,53,54,55,56,57,58,59,60, 61,62,63,64,65,66,67,68,69,70,71,72,73,74,75, 76,77,78,79,80,81,82,83,84,85,86,87,88,89,90, 91,92,93,94,95,96,97,98,99,100,101,102,103, 104,105,106,107,108,109,110,111,112,113,114,115, 116,117, 128,129, 140,141, 152,153, 164,165, 176,177, 188,189, 200,201, 212,213, 224,225, 236,237, 248,249, 260,261, 272,273, 284,285, 296,297, 308,309, 320,321, 332,333, 344,345, 356,357, 368,369, 380,381, 392,393, 404,405, 416,417, 428,429, 440,441, 452,453, 464,465, 118,119, 130,131, 142,143, 154,155, 166,167, 178,179, 190,191, 202,203, 214,215, 226,227, 238,239, 250,251, 262,263, 274,275, 286,287, 298,299, 310,311, 322,323, 334,335, 346,347, 358,359, 370,371, 382,383, 394,395, 406,407, 418,419, 430,431, 442,443, 454,455, 466,467, 120,121, 132,133, 144,145, 156,157, 168,169, 180,181, 192,193, 204,205, 216,217, 228,229, 240,241, 252,253, 264,265, 276,277, 288,289, 300,301, 312,313, 324,325, 336,337, 348,349, 360,361, 372,373, 384,385, 396,397, 408,409, 420,421, 432,433, 444,445, 456,457, 468,469, 122,123, 134,135, 146,147, 158,159, 170,171, 182,183, 194,195, 206,207, 218,219, 230,231, 242,243, 254,255, 266,267, 278,279, 290,291, 302,303, 314,315, 326,327, 338,339, 350,351, 362,363, 374,375, 386,387, 398,399, 410,411, 422,423, 434,435, 446,447, 458,459, 470,471, 124,125, 136,137, 148,149, 160,161, 172,173, 184,185, 196,197, 208,209, 220,221, 232,233, 244,245, 256,257, 268,269, 280,281, 292,293, 304,305, 316,317, 328,329, 340,341, 352,353, 364,365, 376,377, 388,389, 400,401, 412,413, 424,425, 436,437, 448,449, 460,461, 472,473, 126,127, 138,139, 150,151, 162,163, 174,175, 186,187, 198,199, 210,211, 222,223, 234,235, 246,247, 258,259, 270,271, 282,283, 294,295, 306,307, 318,319, 330,331, 342,343, 354,355, 366,367, 378,379, 390,391, 402,403, 414,415, 426,427, 438,439, 450,451, 462,463, 474,475, 477,478, 489,490, 501,502, 513,514, 525,526, 537,538, 549,550, 561,562, 573,574, 585,586, 597,598, 609,610, 621,622, 633,634, 645,646, 657,658, 669,670, 681,682, 693,694, 705,706, 717,718, 729,730, 741,742, 753,754, 765,766, 777,778, 789,790, 801,802, 813,814, 825,826, 479,480, 491,492, 503,504, 515,516, 527,528, 539,540, 551,552, 563,564, 575,576, 587,588, 599,600, 611,612, 623,624, 635,636, 647,648, 659,660, 671,672, 683,684, 695,696, 707,708, 719,720, 731,732, 743,744, 755,756, 767,768, 779,780, 791,792, 803,804, 815,816, 827,828, 481,482, 493,494, 505,506, 517,518, 529,530, 541,542, 553,554, 565,566, 577,578, 589,590, 601,602, 613,614, 625,626, 637,638, 649,650, 661,662, 673,674, 685,686, 697,698, 709,710, 721,722, 733,734, 745,746, 757,758, 769,770, 781,782, 793,794, 805,806, 817,818, 829,830, 836,837,838,839,840,841,842,843,844,845,846,847 848,849,850,851,852,853,854,855,856,857,858,859 860,861,862,863,864,865,866,867,868,869,870,871 872,873,874,875,876,877,878,879,880,881,882,883 884,885,886,887,888,889,890,891,892,893,894,895 896 ou 897. Opcionalmente, um fragmento de gB pode adicionalmente se estender no terminal N por 5,10,20 ou 30 aminoácidos a partir do resíduo de partida do fragmento. Opcionalmente, um fragmento de gB pode adicionalmente se estender no terminal C por 5,10,20 ou 30 aminoácidos a partir do último resíduo do fragmento.
Proteínas gH de CMV
[0147]Em algumas modalidades, uma proteína gH é uma proteína gH de comprimento total (gH FL de CMV, ID. DE SEQ. N°:32, por exemplo, que é uma proteína de 743 aminoácidos). gH possui um domínio de expansão de membrana e um domíniocitoplasmático que começa na posição 716 até a posição 743.A remoção de aminoácidosde717 a 743 fornece uma gH solúvel (por exemplo, gH sol de CMV, ID. DE SEQ. N°: 34). Em algumas modalidades, a proteína gH pode ser um fragmento de gH com 10 aminoácidos ou mais de comprimento. Por exemplo, o número de aminoácidos no fragmento pode compreender 10,15,20,30,40,50,60,70,80,90,100, 125,150,175,200,225,250,275,300,325,350,375,400, 425,450,475,500,525,550,575,600,625,650,675,700, ou 725 aminoácidos. Opcionalmente, a proteína gH pode ser um fragmento de gH com 10 aminoácidos ou mais de comprimento. Por exemplo, o número de aminoácidos no fragmento pode compreender 10,15,20,30,40,50,60,70, 80,90,100,125,150,175,200,225,250,275,300,325, 350,375,400,425,450,475,500,525,550,575,600,625, 650,675,700 ou 725 aminoácidos. Um fragmento de gHpode começar em qualquer um dos números de resíduo: 1,2,3,4, 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20, 21,22,23,24,25,26,27,28,29,30,31,32,33,34,35, 36,37,38,39,40,41,42,43,44,45,46,47,48,49,50, 51,52,53,54,55,56,57,58,59,60,61,62,63,64,65, 66,67,68,69,70,71,72,73,74,75,76,77,78,79,80, 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95, 96,97,98,99,100,101,102,103,104,105,106,107, 108,109,110,111,112,113,114,115,116,117,118,119, 120,121,122,123,124,125,126,127,128,129,130,131, 132,133,134,135,136,137,138,139,140,141,142,143, 144,145,146,147,148,149,150,151,152,153,154,155, 156,157,158,159,160,161,162,163,164,165,166,167, 168,169,170,171,172,173,174,175,176,177,178,179, 180,181,182,183,184,185,186,187,188,189,190,191, 192,193,194,195,196,197,198,199,200,201,202,203, 204,205,206,207,208,209,210,211,212,213,214,215, 216,217,218,219,220,221,222,223,224,225,226,227, 228,229,230,231,232,233,234,235,236,237,238,239, 240,241,242,243,244,245,246,247,248,249,250,251, 252,253,254,255,256,257,258,259,260,261,262,263, 264,265,266,267,268,269,270,271,272,273,274,275, 276,277,278,279,280,281,282,283,284,285,286,287, 288,289,290,291,292,293,294,295,296,297,298,299, 300,301,302,303,304,305,306,307,308,309,310,311, 312,313,314,315,316,317,318,319,320,321,322,323, 324,325,326,327,328,329,330,331,332,333,334,335, 336,337,338,339,340,341,342,343,344,345,346, 348,349,350,351,352,353,354,355,356,357,358, 360,361,362,363,364,365,366,367,368,369,370, 372,373,374,375,376,377,378,379,380,381,382, 384,385,386,387,388,389,390,391,392,393,394, 396,397,398,399,400,401,402,403,404,405,406, 408,409,410,411,412,413,414,415,416,417,418, 420,421,422,423,424,425,426,427,428,429,430, 432,433,434,435,436,437,438,439,440,441,442, 444,445,446,447,448,449,450,451,452,453,454, 456,457,458,459,460,461,462,463,464,465,466, 468,469,470,471,472,473,474,475,476,477,478, 480,481,482,483,484,485,486,487,488,489,490, 492,493,494,495,496,497,498,499,500,501,502, 504,505,506,507,508,509,510,511,512,513,514, 516,517,518,519,520,521,522,523,524,525,526, 528,529,530,531,532,533,534,535,536,537,538, 540,541,542,543,544,545,546,547,548,549,550, 552,553,554,555,556,557,558,559,560,561,562, 564,565,566,567,568,569,570,571,572,573,574, 576,577,578,579,580,581,582,583,584,585,586, 588,589,590,591,592,593,594,595,596,597,598, 600,601,602,603,604,605,606,607,608,609,610, 612,613,614,615,616,617,618,619,620,621,622, 624,625,626,627,628,629,630,631,632,633,634, 636,637,638,639,640,641,642,643,644,645,646, 648,649,650,651,652,653,654,655,656,657,658, 660,661,662,663,664,665,666,667,668,669,670, 672,673,674,675,676,677,678,679,680,681,682, 684,685,686,687,688,689,690,691,692,693,694, 347, 359, 371, 383, 395, 407, 419, 431, 443, 455, 467, 479, 491, 503, 515, 527, 539, 551, 563, 575, 587, 599, 611, 623, 635, 647, 659, 671, 683, 695,696,697,698,699,700,701,702,703,704,705,706,707, 708,709,710,711,712,713,714,715,716,717,718,719, 720,721,722,723,724,725,726,727,728,729,730,731ou 732.
[0148] Os resíduos de gH são numerados de acordo com a seqüência de aminoácidos de gH de comprimento total (gH FL de CMV) mostrada no ID. DE SEQ. N°: 32. Opcionalmente, um fragmento de gH pode adicionalmente se estender no terminal N por 5, 10, 20 ou 30 aminoácidos a partir do resíduo de partida do fragmento. Opcionalmente, um fragmento de gH pode adicionalmente se estender no terminal C por 5, 10, 20 ou 30 aminoácidos a partir do último resíduo do fragmento.
Proteínas gL de CMV
[0149] Em algumas modalidades, uma proteína gL é uma proteína gL de comprimento total (gL FL de CMV, ID. DE SEQ. N°: 36, por exemplo, que é uma proteína de 278 aminoácidos). Em algumas modalidades, um fragmento de gL pode ser usado. Por exemplo, o número de aminoácidos no fragmento pode compreender 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225 ou 250 aminoácidos. Um fragmento de gL pode começar em qualquer um dos números de resduo1,2,3,4,5,6,7,8,9,10,11,12,13,14,15, 16,17,18,19,20,21,22,23,24,25,26,27,28,29,30, 31,32,33,34,35,36,37,38,39,40,41,42,43,44,45, 46,47,48,49,50,51,52,53,54,55,56,57,58,59,60, 61,62,63,64,65,66,67,68,69,70,71,72,73,74,75, 76,77,78,79,80,81,82,83,84,85,86,87,88,89,90, 91,92,93,94,95,96,97,98,99,100,101,102,103, 104,105,106,107,108,109,110,111,112,113,114,115, 116,117,118,119,120,121,122,123,124,125,126,127, 128,129,130,131,132,133,134,135,136,137,138,139 140,141,142,143,144,145,146,147,148,149,150,151 152,153,154,155,156,157,158,159,160,161,162,163 164,165,166,167,168,169,170,171,172,173,174,175 176,177,178,179,180,181,182,183,184,185,186,187 188,189,190,191,192,193,194,195,196,197,198,199 200,201,202,203,204,205,206,207,208,209,210,211 212,213,214,215,216,217,218,219,220,221,222,223 224,225,226,227,228,229,230,231,232,233,234,235 236,237,238,239,240,241,242,243,244,245,246,247 248,249,250,251,252,253,254,255,256,257,258,259 260,261,262,263,264,265,266,267ou 268.
[0150] Os resíduos de gL são numerados de acordo com a seqüência de aminoácidos de gL de comprimento total (gL FL de CMV) mostrada no ID. DE SEQ. N°: 36. Opcionalmente, um fragmento de gL pode adicionalmente se estender no terminal N por 5, 10, 20 ou 30 aminoácidos a partir do resíduo de partida do fragmento. Opcionalmente, um fragmento de gL pode adicionalmente se estender no terminal C por 5, 10, 20 ou 30 aminoácidos a partir do último resíduo do fragmento.
Proteínas gO de CMV
[0151] Em algumas modalidades, uma proteína gO é uma proteína gO de comprimento total (gO FL de CMV, ID. DE SEQ. N°: 42, por exemplo, que é uma proteína de 472 aminoácidos). Em algumas modalidades, a proteína gO pode ser um fragmento de gO com 10 aminoácidos ou mais de comprimento. Por exemplo, o número de aminoácidos no fragmento pode compreender 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425 ou 450 aminoácidos. Um fragmento de gO pode começar em qualquer um dos números de resíduo: 1,23, 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19, 20,21,22,23,24,25,26,2728,29,30,31,32,33,34, 35,36,37,38,39,40,41,42,43,44,45,46,47,48,49, 5051,52,53,54,55,56,57,58,59,60,61,62,63,64, 65,66,67,68,69,70,71,72,7374,75,76,77,78,79, 80,81,82,83,84,85,86,87,88,89,90,91,92,93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 133, 150, 167, 184, 201, 218, 235, 252, 269, 286, 303, 320, 337, 354, 371, 388, 405, 422, 439, 456.
[0152] Os resíduos de gO são numerados de acordo com a seqüência de aminoácidos de gO de comprimento total (gO FL de CMV) mostrada no ID. DE SEQ. N°: 42. Opcionalmente, um fragmento de gO pode adicionalmente se estender no terminal N por 5, 10, 20 ou 30 aminoácidos a partir do resíduo de partida do fragmento. Opcionalmente, um fragmento de gO pode adicionalmente se estender no terminal C por 5, 10, 20 ou 30 aminoácidos a partir do último resíduo do fragmento.
Proteínas gM de CMV
[0153] Em algumas modalidades, uma proteína gM é uma proteína gM de comprimento total (gM FL de CMV, ID. DE SEQ. N°: 38, por exemplo, que é uma proteína de 371 aminoácidos). Em algumas modalidades, a proteína gM pode ser um fragmento de gM com 10 aminoácidos ou mais de comprimento. Por exemplo, o número de aminoácidos no fragmento pode compreender 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325 ou 350 aminoácidos. Um fragmento de gM pode começar em qualquer um dos números de resíduo: 1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17,18,19,20,21,22,23, 24,25,26,27,28,29,30,31,32,33,34,35,36,37,38, 39,40,41,42,43,44,45,46,47,48,49,50,51,52,53, 54,55,56,57,58,59,60,61,62,63,64,65,66,67,68, 69,70,71,72,73,74,75,76,77,78,79,80,81,82,83, 84,85,86,87,88,89,90,91,92,93,94,95,96,97,98, 99,100,101,102,103,104,105,106,107,108,109,110, 111,112,113,114,115,116,117,118,119,120,121,122, 123,124,125,126,127,128,129,130,131,132,133,134, 135,136,137,138,139,140,141,142,143,144,145,146, 147,148,149,150,151,152,153,154,155,156,157,158, 159,160,161,162,163,164,165,166,167,168,169,170, 171,172,173,174,175,176,177,178,179,180,181,182, 183,184,185,186,187,188,189,190,191,192,193,194, 195,196,197,198,199,200,201,202,203,204,205,206, 207,208,209,210,211,212,213,214,215,216,217,218, 219,220,221,222,223,224,225,226,227,228,229,230, 231,232,233,234,235,236,237,238,239,240,241,242, 243,244,245,246,247,248,249,250,251,252,253,254, 255,256,257,258,259,260,261,262,263,264,265,266, 267,268,269,270,271,272,273,274,275,276,277,278, 279,280,281,282,283,284,285,286,287,288,289,290, 291,292,293,294,295,296,297,298,299,300,301,302, 303,304,305,306,307,308,309,310,311,312,313,314, 315,316,317,318,319,320,321,322,323,324,325,326, 327,328,329,330,331,332,333,334,335,336,337,338, 339,340,341,342,343,344,345,346,347,348,349,350, 351,352,353,354,355,356,357,358,359,360 ou 361.
[0154]Os resíduos de gM são numerados de acordo com a seqüência de aminoácidos de gM de comprimento total (gM FL de CMV) mostrada no ID. DE SEQ. N°: 38. Opcionalmente, um fragmento de gM pode adicionalmente se estender no terminal N por 5, 10, 20 ou 30 aminoácidos a partir do resíduo de partida do fragmento. Opcionalmente, um fragmento de gM pode adicionalmente se estender no terminal C por 5, 10, 20 ou 30 aminoácidos a partir do último resíduo do fragmento.
Proteínas gN de CMV
[0155] Em algumas modalidades, uma proteína gN é uma proteína gN de comprimento total (gN FL de CMV, ID. DE SEQ. N°: 40, por exemplo, que é uma proteína de 135 aminoácidos). Em algumas modalidades, a proteína gN pode ser um fragmento de gN com 10 aminoácidos ou mais de comprimento. Por exemplo, o número de aminoácidos no fragmento pode compreender 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 ou 125 aminoácidos. Um fragmento de gN pode começar em qualquer um dos números de resíduo: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 ou 125.
[0156] Os resíduos de gN são numerados de acordo com a seqüência de aminoácidos de gN de comprimento total (gN FL de CMV) mostrada no ID. DE SEQ. N°: 40. Opcionalmente, um fragmento de gN pode adicionalmente se estender no terminal N por 5, 10, 20 ou 30 aminoácidos a partir do resíduo de partida do fragmento. Opcionalmente, um fragmento de gN pode adicionalmente se estender no terminal C por 5, 10, 20 ou 30 aminoácidos a partir do último resíduo do fragmento.
Proteínas UL128 de CMV
[0157] Em algumas modalidades, uma proteína UL128 é uma proteína UL128 de comprimento total (UL128 FL de CMV, ID. DE SEQ. N°: 44, por exemplo, que é uma proteína de 171 aminoácidos). Em algumas modalidades, a proteína UL128 pode ser um fragmento de UL128 com 10 aminoácidos ou mais de comprimento. Por exemplo, o número de aminoácidos no fragmento pode compreender 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125 ou 150 aminoácidos. Um fragmento de UL128 pode começar em qualquer um dos números de resíduo: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20,21,22,23,24,25,26,27,28,29,30,31,32,33, 34,35,36,37,38,39,40,41,42,43,44,45,46,47,48, 49,50,51,52,53,54,55,56,57,58,59,60,61,62,63, 64,65,66,67,68,69,70,71,72,73,74,75,76,77,78, 79,80,81,82,83,84,85,86,87,88,89,90,91,92,93, 94,95,96,97,98,99,100,101,102,103,104,105,106, 107,108,109,110,111,112,113,114,115,116,117,118, 119,120,121,122,123,124,125,126,127,128,129,130, 131,132,133,134,135,136,137,138,139,140,141,142, 143,144,145,146,147,148,149,150,151,152,153,154, 155,156,157,158,159,160 ou 161.
[0158]Os resíduos de UL128 são numerados de acordo com a seqüência de aminoácidos de UL128 de comprimento total (UL128 FL de CMV) mostrada no ID. DE SEQ. N°:44. Opcionalmente, um fragmento de UL128 pode adicionalmente se estender no terminal N por 5,10,20 ou 30 aminoácidos a partir do resíduo de partida do fragmento. Opcionalmente, um fragmento de UL128 pode adicionalmente se estender no terminal C por 5,10,20 ou 30 aminoácidos a partir doúltimo resíduo do fragmento.
Proteínas UL130 de CMV
[0159]Em algumas modalidades, uma proteína UL130 é uma proteína UL130 de comprimento total (UL130 FL de CMV, ID. DE SEQ. N°:46, por exemplo, que é uma proteína de 214 aminoácidos). Em algumas modalidades, a proteína UL130 pode ser um fragmento de UL130 com 10 aminoácidos ou mais de comprimento. Por exemplo, o número de aminoácidos no fragmento pode compreender 10,15,20,30,40,50,60,70, 80,90,100,125,150,175 ou 200 aminoácidos. Um fragmento deUL130podecomeçaremqualquerum dosnúmerosde resíduo: 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15, 16,17,18,19,20,21,22,23,24,25,26,27,28,29,30, 31,32,33,34,35,36,37,38,39,40,41,42,43,44,45, 46,47,48,49,50,51,52,53,54,55,56,57,58,59,60, 61,62,63,64,65,66,67,68,69,70,71,72,73,74,75, 76,77,78,79,80,81,82,83,84,85,86,87,88,89,90, 91,92,93,94,95,96,97,98,99,100,101,102,103, 104,105,106,107,108,109,110,111,112,113,114,115, 116,117,118,119,120,121,122,123,124,125,126,127, 128,129,130,131,132,133,134,135,136,137,138,139, 140,141,142,143,144,145,146,147,148,149,150,151, 152,153,154,155,156,157,158,159,160,161,162,163, 164,165,166,167,168,169,170,171,172,173,174,175, 176,177,178,179,180,181,182,183,184,185,186,187, 188,189,190,191,192,193,194,195,196,197,198,199, 200,201,202,203 ou 204.
[0160]Os resíduos de UL130 são numerados de acordo com a seqüência de aminoácidos de UL130 de comprimento total (UL130 FL de CMV) mostrada no ID. DE SEQ. N°:46. Opcionalmente, um fragmento de UL130 pode adicionalmente se estender no terminal N por 5,10,20 ou 30 aminoácidos a partir do resíduo de partida do fragmento. Opcionalmente, um fragmento de UL130 pode adicionalmente se estender no terminal C por 5,10,20 ou 30 aminoácidos a partir do último resíduo do fragmento.
Proteínas UL131 de CMV
[0161]Em algumas modalidades, uma proteína UL131 é uma proteína UL131 de comprimento total (UL131 de CMV, ID. DE SEQ. N°:48, por exemplo, que é uma proteína de 129 aminoácidos). Em algumas modalidades, a proteína UL131 pode ser um fragmento de UL131 com 10 aminoácidos ou mais de comprimento. Por exemplo, o número de aminoácidos no fragmento pode compreender 10,15,20,30,40,50,60,70, 80,90,100,125,150,175 ou 200 aminoácidos. Um fragmento de UL131 pode começar em qualquer um dos números de resduo1,2,3,4,5,6,7,8,9,10,11,12,13,14,15, 16,17,18,19,20,21,22,23,24,25,26,27,28,29,30, 31,32,33,34,35,36,37,38,39,40,41,42,43,44,45, 46,47,48,49,50,51,52,53,54,55,56,57,58,59,60, 61,62,63,64,65,66,67,68,69,70,71,72,73,74,75, 76,77,78,79,80,81,82,83,84,85,86,87,88,89,90, 91,92,93,94,95,96,97,98,99,100,101,102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119.
[0162]Os resíduos de UL131 são numerados de acordo com a seqüência de aminoácidos de UL131 de comprimento total (UL131 FL de CMV) mostrada no ID. DE SEQ. N°:48. Opcionalmente, um fragmento de UL131 pode adicionalmente se estender no terminal N por 5,10,20 ou 30 aminoácidos a partir do resíduo de partida do fragmento. Opcionalmente, um fragmento de UL131 pode adicionalmente se estender no terminal C por 5,10,20 ou 30 aminoácidos a partir do último resíduo do fragmento.
[0163]Como estabelecido acima, a invenção está relacionada às moléculas de ácido nucléico policistrônico recombinante que contêm uma primeira seqüência que codifica uma primeira proteína do herpesvírus, ou fragmento desta, e uma segunda seqüência que codifica uma segunda proteína do herpesvírus, ou fragmento desta. Conseqüentemente, a descrição apresentada anteriormente de certas modalidades preferidas, por exemplo, VRPs de alfavírus e RNAs auto- replicantes que contêm seqüências que codificam duas ou mais proteínas de CMV ou fragmentos destas, é ilustrativa da invenção, mas não limita o escopo da invenção. Será observado que as seqüências que codificam proteínas de CMV nessas modalidades preferidas podem ser substituídas com seqüências que codificam proteínas, por exemplo, gH e gL, ou destas, que possuem 10 aminoácidos de comprimento ou mais, de outros herpesvírus como, por exemplo, HHV-1, HHV- 2, HHV-3, HHV-4, HHV-6, HHV-7 e HHV-8. Por exemplo, proteínas VZV (HHV-3) adequadas incluem gB, gE, gH, gI e gL, e fragmentos destas, que possuem 10 aminoácidos de comprimento ou mais, e podem ser de qualquer cepa de VZV. Por exemplo, proteínas de VZV ou fragmentos destas podem ser das cepas de VZV pOka, Dumas, HJO, CA123 ou DR. Essas proteínas de VZV exemplares, e fragmentos destas, podem ser codificadas por qualquer seqüência de nucleotídeos adequada, incluindo seqüências que são códon-otimizadas ou não para expressão em um hospedeiro desejado, por exemplo, uma célula humana. São aqui fornecidas seqüências exemplares de proteínas de VZV.
[0164] Por exemplo, em uma modalidade, a molécula de ácido nucléico policistrônico contém uma primeira seqüência que codifica a proteína gH de VZV ou fragmento desta, e uma segunda seqüência que codifica uma proteína gL de VZV ou fragmento desta.
[0165] Em algumas modalidades, cada uma das seqüências que codificam uma proteína do herpesvírus ou fragmento que estão presentes na molécula de ácido nucléico policistrônico está ligada operacionalmente aos seus próprios elementos de controle. Por exemplo, cada uma das seqüências que codificam uma proteína do herpesvírus ou fragmento está ligada operacionalmente ao seu próprio promotor subgenômico. Dessa forma, a molécula de ácido nucléico policistrônico, por exemplo, um replicon de alfavírus, pode conter dois, três, quatro ou cinco promotores subgenômicos, cada um dos quais controla a expressão de uma proteína do herpesvírus ou fragmento. Quando esse tipo de molécula de ácido nucléico policistrônico é um RNA auto-replicante, por exemplo, um replicon de alfavírus, ele pode ser empacotado como uma VRP, ou se associar ou ser formulada com um sistema de liberação de RNA.
MÉTODOS E USOS
[0166] Em algumas modalidades, as moléculas de RNA auto- replicantes ou VRPs são administradas a um indivíduo para estimular uma resposta imune. Nessas modalidades, as moléculas de RNA auto-replicantes ou VRPs tipicamente estão presentes em uma composição que pode compreender um veículo farmaceuticamente aceitável e, opcionalmente, um adjuvante. Veja, por exemplo, U.S. 6.299.884, U.S. 7.641.911, U.S. 7.306.805 e US 2007/0207090.
[0167] A resposta imune pode compreender uma resposta imune humoral, uma resposta imune mediada por células, ou ambas. Em algumas modalidades, uma resposta imune é induzida contra cada proteína de CMV liberada. Uma resposta imune mediada por células pode compreender uma resposta de célula T helper (T), uma resposta de célula T citotóxica CD8+ (CTL), ou ambas. Em algumas modalidades, a resposta imune compreende uma resposta imune humoral, e os anticorpos são anticorpos neutralizantes. Anticorpos neutralizantes bloqueiam a infecção viral das células. CMV infecta células epiteliais e também células de fibroblastos. Em algumas modalidades, a resposta imune reduz ou evita a infecção de ambos os tipos de células. As respostas de anticorpos neutralizantes podem ser complemento-dependentes ou complemento-independentes. Em algumas modalidades, a resposta de anticorpo neutralizante é complemento-independente. Em algumas modalidades, a resposta de anticorpo neutralizante efetua neutralização cruzada, ou seja, um anticorpo gerado contra uma composição administrada neutraliza um vírus CMV de uma cepa diferente da cepa usada na composição.
[0168] Uma medida útil da potência do anticorpo na técnica é a “titulação de 50% de neutralização”. Para determinar a titulação 50% neutralizante, soro de animais imunizados é diluído para avaliar o quanto diluído o soro pode ser e ainda reter a habilidade para bloquear a entrada de 50% dos vírus nas células. Por exemplo, uma titulação de 700 significa que soro reteve a habilidade para neutralizar 50% dos vírus após ser diluído 700 vezes. Dessa forma, titulações maiores indicam respostas de anticorpos neutralizantes mais potentes. Em algumas modalidades, essa titulação está em uma faixa que possui um limite inferior de cerca de 200, cerca de 400, cerca de 600, cerca de 800, cerca de 1.000, cerca de 1.500, cerca de 2.000, cerca de 2.500, cerca de 3.000, cerca de 3.500, cerca de 4.000, cerca de 4.500, cerca de 5.000, cerca de 5.500, cerca de 6.000, cerca de 6.500, ou cerca de 7.000. A faixa de titulação de 50% de neutralização pode ter um limite superior de cerca de 400, cerca de 600, cerca de 800, cerca de 1.000, cerca de 1.500, cerca de 2.000, cerca de 2.500, cerca de 3.000, cerca de 3.500, cerca de 4.000, cerca de 4.500, cerca de 5.000, cerca de 5.500, cerca de 6.000, cerca de 6.500, cerca de 7.000, cerca de 8.000, cerca de 9.000, cerca de 10.000, cerca de 11.000, cerca de 12.000, cerca de 13.000, cerca de 14.000, cerca de 15.000, cerca de 16.000, cerca de 17.000, cerca de 18.000, cerca de 19.000, cerca de 20.000, cerca de 21.000, cerca de 22.000, cerca de 23.000, cerca de 24.000, cerca de 25.000, cerca de 26.000, cerca de 27.000, cerca de 28.000, cerca de 29.000 ou cerca de 30.000. Por exemplo, a titulação de 50% de neutralização pode ser cerca de 3.000 até cerca de 6.500. “Cerca de” significa mais ou menos 10% do valor citado. A titulação de neutralização pode ser medida como descrito nos exemplos específicos, abaixo.
[0169]Uma resposta imune pode ser estimulada por administração de VRPs ou RNA auto-replicante a um indivíduo, tipicamente um mamífero, incluindo um ser humano. Emalgumas modalidades, a respostaimune induzida é uma resposta imune protetora, ou seja, a resposta reduz o risco ou agravidade da infecção pelo CMV.A estimulação de uma resposta imune protetora é particularmente desejável em algumas populações particularmente em risco para infecção e doença pelo CMV. Por exemplo, populações em risco incluem pacientes submetidos a transplantes de órgãos sólidos (SOT), pacientes submetidos ao transplante de medula óssea e pacientes submetidos ao transplante de células-tronco hematopoiéticas (HSCT). VRPs podem ser administradas a um doador de transplante pré-transplante, ou a um receptor de transplante pré- e/ou pós-transplante. Como a transmissão vertical de mãepara filhoéuma fonte comumde infecçãoem bebês, a administração de VRPs ou RNA auto-replicante a uma mulher que podeengravidaréparticularmenteútil.
[0170]Qualquer via de administração adequada pode ser usada. Por exemplo, uma composição pode ser administrada por via intramuscular, intraperitoneal, subcutânea ou transdérmica. Algumas modalidades serão administradas por uma via intramucosa como, por exemplo, por via intra-oral, intranasal, intravaginal e intra-retal. As composições podem ser administradas de acordo com qualquer posologia adequada.
[0171]Todas as patentes, pedidos de patentes e referências citadas nessa revelação, incluindo seqüências de nucleotídeos e de aminoácidos referidas pelo número de acesso, são expressamente aqui incorporados por referência. A revelação acima é uma descrição geral. Uma compreensão mais complete pode ser obtida por referência aos exemplos específicos seguintes, que são fornecidos apenas com fins ilustrativos.
EXEMPLO 1 Liberação de antígenos de CMV individuais usando uma plataforma de VRP
[0172] Cada uma das glicoproteínas de CMV gB e gH induz respostas neutralizantes, e gB é o antígeno dominante entre anticorpos em soros humanos que neutralizam infecção de fibroblastos (Britt e cols. (1990) J. Virol. 64(3): 1.07985). Os experimentos seguintes demonstram em camundongos uma resposta neutralizante contra esses antígenos liberados usando uma plataforma de VRP.
[0173] Cada antígeno de CMV foi clonado em um vetor pcDNA-6His (Invitrogen) e testado quanto à expressão de proteína antes da clonagem em um vetor de replicon de alfavírus, pVCR 2.1 SalI/XbaI derivado do plasmídeo descrito por Perri e cols. (J. Virol. 77(19): 10.394-10.403 (2003)) produzindo as construções mostradas na Figura 2. pVCR 2.1 SalI/XbaI é um vetor de RNA auto-replicante que, quando eletroporado com RNA helper defeituoso de capsídeo e glicoproteína, forma uma partícula infecciosa de alfavírus.
[0174] Vetores pVCR foram usados para fazer RNA que foi eletroporado em células de rim de filhote de hamster (BHKV) na presença de RNAs helper defeituosos de capsídeo e glicoproteína derivados do vírus da encefalite eqüina venezuelana (VEE). Após eletroporação, o sobrenadante contendo partículas do vetor de alfavírus (VRPs) secretadas foi coletado, purificado, titulado e usado for estudos de imunização de camundongos. Os camundongos foram imunizados com 1 x 106 unidades infecciosas (IU)/camundongo em uma série de duas imunizações, com intervalo de três semanas. A sangria terminal foi feita três semanas após a segunda imunização.
VRPs de gB, gH e gL monocistrônicas
[0175]Duas versões diferentes de gB solúvel foram construídas: “gB sol 750” desprovida do domínio de expansão transmembrana e do domínio citoplasmático; e “gB sol 692” também desprovida de uma região hidrofóbica (FIG. 2A) e é similar à construção de Reap e cols. Uma gH solúvel desprovida do domínio de expansão transmembrana e do domínio citoplasmático (“gH sol 716”) também foi construída (FIG. 2C). Soros de camundongos imunizados foram analisados em vários ensaios. Imunoblot (dados não mostrados) e ensaios de imunofluorescência foram usados para confirmar respostas de anticorpo específicas para os antígenos. Ensaios de neutralização foram usados para demonstrar que as respostas de anticorpo despertadas eram capazes de neutralizar a infecção pelo CMV.
[0176]Soros de camundongos imunizados foram examinados por imunofluorescênciaparareconhecimentode gB em células 293T transfectadas com construções que expressam gB-6His. As células foram sondadas com anticorpos anti-His (“anti- 6His”), um anticorpo monoclonal para gB (“anti-gB 27-156”) ou soros de camundongo coletados reunidos em pool. O soro pré-imune foi negativo em todos os casos. Em células transfectadas com construções que expressam gB FL-6His, fixadas e permeabilizadas, a coloração anti-6His revelou um padrão de expressão de expressão de superfície com um padrão citoplasmático punctiforme que corresponde, mais provavelmente, à via de tráfego endocítico/exocítico. Tanto anti-gB 27-156 quanto os soros de camundongo reunidos em pool mostraram um padrão de expressão similar. Soros de camundongos imunizados com cada um de VRPs de gB FL, VRPs de gB sol 750 e VRPs de gB sol 692 mostrou o mesmo padrão de expressão.
[0177] Camundongos imunizados com VRPs de gH FL e VRPs de gH sol 716 produziram anticorpos específicos para gH. A análise por imunofluorescência de células 293T transfectadas com construções que expressam gH FL-6His detectou forte reconhecimento de gH por anti-6His, anti-gH e soros de camundongo reunidos em pool. Soros coletados de camundongos imunizados com VRPs de gL produziram uma resposta de anticorpo específica como determinado por análise imunoblot e imunofluorescência. VRPs de gL não despertaram uma resposta neutralizante.
[0178] Soros de camundongos imunizados com VRPs de gB ou VRPs de gH foram analisados quanto á presença de anticorpos neutralizantes usando um ensaio de neutralização de CMV. Soros em várias diluições foram pré-incubados com vírus CMV TB40UL32EGFP (“TB40-GFP”, um isolado clínico modificado geneticamente para expressar GFP) e depois adicionadas às células epiteliais ARPE-19 e incubados por 5 dias. Com 5 dias pós-infecção, as células GFP-positivas foram contadas. Nesse ensaio, células incubadas com soro contendo anticorpos neutralizantes tinham menos células GFP- positivas comparadas com células incubadas com vírus isoladamente ou com vírus incubado com soros pré-imunes. Soros de camundongos imunizados com VRPs de gB, VRPs de gB FL, VRPs de gB sol 750 ou VRPs de gB sol 692 tinham forte atividade neutralizante na presença de complemento de porquinho-da-índia (titulação de 50% de neutralização em uma diluição de soro de 1:1.280-1:2.560; FIG. 3). Soros de camundongos imunizados com VRPs de gH FL ou VRPs de gH sol tinham alguma atividade neutralizante que era independente de complemento de porquinho-da-índia (FIG. 3).
EXEMPLO 2 Construção de vetores de alfavírus policistrônicos
[0179]CMV produz vários complexos multiproteínas durante infecção. Para determinar se um único replicon que expressa todos os componentes de um complexo desejado pode ser usado para produzir o complexo de CMV em um indivíduo, ou se os componentes do complexo poderiam ser co-liberados por múltiplos vetores de replicon, projetamosuma plataforma que permite a expressão controlada de múltiplas proteínas de CMV.
[0180]Um vetor de alfavírus (pVCR 2.1 SalI XbaI) foi modificado para permitir a montagem de múltiplos promotores subgenômicos (SGP) e genes de interesse (GOI). O sítio ApaI de pVCR 2.1 SalI/XbaI em 11026-31bp foi alterado de GGGCCC (ID. DE SEQ. N°:7) paraGGCGCC(ID. DE SEQ. N°:8). Os sítios de restrição ClaI e PmlI adicionados na região imediatamente abaixo do primeiro promotor subgenômico e sítios de inserção de SalI-XbaI. A seqüência em 7727-7754 bp foi alterada de ctcgatgtacttccgaggaactgatgtg (ID. DE SEQ. N°:9) para ATCGATGTACTTCCGAGGAACTCACGTG (ID. DE SEQ. N°:10).
[0181]Um sistema de vetor-ponte (shuttling) foi projetado para permitir a inserção de um GOI diretamente abaixo de um SGP usando os sítios SalI-XbaI. pcDNA 3.1(-) C foi modificado como mostrado a seguir. Três sítios SalI foram deletados:posições 1046-1051 bp, 3332-3337 bp e 5519-21,1-3 bp deGTCGAC(ID. DE SEQ. N°:11) para GTCTAC (ID. DE SEQ. N°:12). pcDNA 3.1(-) C foi modificado para fazer a mutação de um sítio XbaI na posição 916-921 bp de TCTAGA (ID. DE SEQ. N°:13) paraTCAAGA(ID. DE SEQ. N°: 14). pcDNA 3.1(-) C foi modificado para adicionar um sítio a ClaI e um sítio SacII nas posições 942-947 (ClaI) e 950955 (SacII) bp de ctggatatctgcag (ID. DE SEQ. N°:15) para ATCGATATCCGCGG(ID. DE SEQ. N°:16).
[0182]Após os sítios de restrição terem sido adicionados e a seqüência resultante ser verificada, a regiãode7611-7689bp (ctataactctctacggctaacctgaatggactacgacatagtctagtcgac caagcctctagacggcgcgcccaccca) (ID. DE SEQ. N°:17) foi amplificada pelo vetor de alfavírus pVCR 2.1 modificado usando os seguintes iniciadores: SGP S-X Not F direto: 5’-ATAAGAATGCGGCCGCCTATAACTCTCTACGGCTAACC-3’ (ID. DE SEQ. N°:18) SGPS-XClaRreverso: 5’-CCATCGATTGGGTGGGCGCGCCGTCTAG-3’ (ID. DE SEQ. N°:19) ou SGPS-XClaFdireto: 5’-CCATCGATCTATAACTCTCTACGGCTAACC3’(ID.DESEQ.N°:20)e SGPS-XSacRreverso: 5’-TCCCCGCGGTGGGTGGGCGCGCCGTCTAG-3’(ID.DESEQ.N°:21).
[0183]As regiões amplificadas foram adicionadas no vetor pcDNA 3.1(-) C modificado para fazer vetores-ponte (pcDNA SV) entre sítios apropriados (NotI-ClaI ou ClaI- SacII). A inserção do NotI-SGP Sal-Xba-ClaI forma o cassete de pcDNA SV 2, a inserção do ClaI-SGP Sal-Xba-SacII forma o cassete de pcDNA SV 3. Esses cassetes de SV foram seqüenciados. O cassete de pcDNA SV 2 contém 12 bp adicionais entre o sítio XbaI e o sítio ClaI (CCACTGTGATCG) (ID. DE SEQ. N°:22), porque o sítio Clal não foi cortado no cassete de vetor pcDNA SV 2. Um sítio PmlI foi, portanto, adicionado. Para o cassete de pcDNASV 2, o sítio PmlI foi inserido em 1012 bp (CACGTG) (ID. DESEQ. N°:23). Para o cassete 3, o sítio PmlI foi adicionadoem 935-940 bp (ACTGTG (ID. DE SEQ. N°:24) foi alterado para CACGTG (ID. DE SEQ. N°:23).
[0184]Para cada vetor policistrônico, o primeiro gene era inserido diretamente no vetor pVCR 2.1 modificado usando os sítios SalI-XbaI. O segundo gene foi ligado no cassete de pcDNA SV 2 usando SalI-XbaI e excisado usando NotI-PmlI, NotI-SacII ou PCRed usando iniciadores para NotI-ClaI e digerido usando NotI e ClaI. O inserto resultante, SGP- SalI-GOI-Xba, foi ligado no vetor pVCR 2.1 modificado usando os sítios NotI-PmlI, NotI-SacII ou NotI- ClaI. O inserto NotI-ClaI foi usado apenas quando um gene desejado na construção continha um sítio PmlI.
[0185]Em alguns casos, um terceiro gene foi ligado no cassete de pcDNA SV 3 usando SalI-XbaI e excisado usando PmlI-SacII ou PCRed usando iniciadores para ClaI-SacII e digerido usando ClaI e SacII. O inserto resultante, SGP- SalI-GOI-XbaI, foi ligado no vetor pVCR 2.1 modificado usando PmlI-SacII ou ClaI-SacII.
[0186]A digestão com SalI-XbaI foi usada para validar a construção do DNA de vetor policistrônico. Após digestão com SalI-XbaI, a eletroforese em gel de agarose foi realizada para confirmar a presença dos GOIs. O DNA de vetor policistrônico foi então linearizado com PmeI de um dia para o outro, purificado usando o kit de purificação por PCR de Qiagen, e usado como modelo para fazer o RNA usando o kit Ambion mMessage mMachine. A qualidade do RNA foi verificada por processamento de uma alíquota da amostra em um gel de agarose de RNA.
Expressão por um vetor policistrônico
[0187]ProteínasfluorescentesGFP(proteína fluorescente verde) e mCherry (proteína fluorescente vermelha) foram usadas como os GOIs para avaliar a habilidade do vetor policistrônico para expressar as duas proteínas. Preparamos um vetor bicistrônico no qual GFP poderia ser expressa usando um primeiro promotor subgenômico e mCherry seria expressa por um segundo promotor subgenômico (FIG. 4A). Polinucleotídeos contendo seqüências codificadoras para essas proteínas foram inseridosusandosítiosSalI-XbaI.Oprimeiro polinucleotídeo (GFP) foi inserido diretamente no vetor de replicon de alfavírus modificado. O segundo polinucleotídeo (mCherry) foi inserido primeiro em um vetor-ponte que contém um promotor subgenômico diretamente acima da seqüência codificadora. Um fragmento contendo tanto o segundopromotorsubgenômicoquantoosegundo polinucleotídeo foi isolado e ligado no vetor de replicon dealfavírusmodificadocontendooprimeiro polinucleotídeo, fornecendo um replicon de alfavírus com múltiplos promotores subgenômicos.
[0188]VRPs foram produzidas em células BHKV por eletroporação de RNAs de replicon com RNAs helper defeituosos para Cap e Gly. As VRPs foram coletadas 24 horas após eletroporação e usadas para infectar células BHKV em uma multiplicidade de infecção (MOI) de 20 unidades infecciosas (IU) por célula.
[0189] O experimento testou quatro conjuntos de VRPs: uma VRP que expressa somente GFP; uma VRP que expressa mCherry; uma VRP que expressa somente GFP e uma VRP que expressa somente mCherry, ambas em uma MOI de 20 IU/célula; e uma VRP que contém o vetor bicistrônico GFP(1)- SGPmCherry(2). As células BHKV infectadas por VRP foram examinadas 24 horas pós-infecção para determinar o percentual de colocalização. Quase todas as células foram positivas para GFP ou mCherry quando infectadas isoladamente. As células infectadas com duas VRPs separadas apareceram verdes ou vermelhas. Muito poucas células eram amarelas, indicando que poucas células expressavam GFP e mCherry em níveis iguais e que havia um nível baixo de coinfecção. Esses dados foram confirmados usando análise por FACS (FIG. 4B).
[0190] Em contraste, as células infectadas com alfavírus contendo o vetor bicistrônico GFP(1)-SGPmCherry(2) eram todas amarelas, o que indica expressão aproximadamente igual de GFP e mCherry. Esse estudo demonstra que múltiplas proteínas podem ser expressas com sucesso por um único vetor de replicon de alfavírus policistrônico.
EXEMPLO 3 Produção de complexos de CMV
[0191] Esse exemplo demonstra que complexos de proteínas de CMV podem ser formados em uma célula após liberação dos componentes do complexo por um vetor de replicon de alfavírus policistrônico.
Complexos de gH/gL e de gH/gL/gO
[0192] Replicons de gH/gL e gH/gL/gO de alfavírus policistrônicos foram construídos como descrito acima (mostrado esquematicamente na FIG. 5A). VRPs contendo gH, gL, gO, gH/gL e gH/gL/gO que codificam replicons foram produzidas em células BHKV como descrito acima e usadas para infectar células BHKV para demonstrar a formação de complexo in vitro. Células ARPE-19 infectadas com VRP produziram complexos de gH/gL ligados por dissulfeto. gO não alterou de forma detectável a associação de gH/gL (FIG. 5B).
[0193] Foram realizados estudos de imunofluorescência para avaliar a localização de gH e gL liberadas isoladamente e quando liberadas usando um alfavírus policistrônico para verificar a relocalização das proteínas quando co-expressas. A localização de gH não pareceu se alterar na presença ou ausência de gL ou gL/gO. A localização de gL se alterou quando na presença de gH e gH/gO.
[0194] Finalmente, a associação de gH/gL foi examinada por meio de imunoprecipitação. Um anticorpo de gH comercial (Genway) foi usado para investigar a associação de gH e gL. Em todos os casos, o anticorpo de gH imunoprecipitou eficientemente gH (FIG. 5C). Quando no gH estava presente, gL não foi imunoprecipitada. Quando gL era expressa na presença de gH ou gH/gO, houve uma associação de gL com gH (FIG. 5C).
[0195] A relocalização de gL na presença de gH e a associação de gH/gL (com ou sem gO) indica que todos os componentes dos replicons de alfavírus policistrônico foram expressos e associados para formar um complexo.
EXEMPLO 4 VRPs que efetuam a formação de complexo de gH/gL in vitro induzem resposta imune potente para CMV que é qualitativamente e quantitativamente superior à resposta imune despertada às VRPs de gB.
[0196]Esse exemplo demonstra a indução de respostas imunes robustas aos complexos formados por liberação de VRPs policistrônicas de gH/gL ou VRPs de gH/gL/gO comparadas com as respostas imunes obtidas usando VRPs que liberam componentes únicos ou VRPs de componente único administradas em combinação ou com respostas despertadas por VRPs de gB.
[0197]Os camundongos foram infectados três vezes com VRPs administradas com intervalo de 3 semanas (106 IU por camundongo; 5 camundongos BalbC/grupo). Soros coletados de imunizações com VRPs únicas e policistrônicas foram avaliados quanto aos anticorpos neutralizantes usando um ensaio de neutralização de CMV como descrito acima. A titulação de neutralização foi medida da forma seguinte. Várias diluições de soros foram pré-incubadas com TB40- UL32-EGFP na presença ou ausência de complemento de porquinho-da-índia e depois adicionadas às células epiteliais ARPE-19 ou células de fibroblastos MRC-5 e incubadas por 5 dias. Após infecção por 5 dias com o vírus, as células GFP-positivas foram contadas. Os resultados para as células ARPE-19 são mostrados na FIG. 6A, na FIG. 6B e na FIG. 6C. Os resultados para as células MRC-5 são mostradosna FIG.7A e na FIG.7B.
[0198]Soros de camundongos imunizados com VRPs de gH FL tiveram baixa atividade neutralizante complemento- independente (FIG. 6A e FIG. 6B). Nenhuma atividade neutralizante foi observada usando soros de camundongos imunizadossomentecomgL ou gOna presença ou ausência de complemento de porquinho-da-índia (FIG. 6C). Soros de imunizaçãoreunidos empoolcomvárias proteínas gB deCMV (gB FL, gB sol 750 e gB sol 692) demonstraram forte atividade neutralizante na presença de complemento de porquinho-da-índia, comumatitulação de 50% de neutralização em uma diluição dos soros de 1:1.280. No entanto, não houve atividade neutralizante na ausência de complemento de porquinho-da-índia em células ARPE-19 para os soros de gB reunidos em pool. VRPs que expressam proteínas de CMV únicas (VRPs de gH ou de gL ou a co- administração de VRPs de gH, de gL e de gO- a 106 IU/camundongo/VRP) não aumentam a atividade neutralizante além daquela de gH isoladamente.
[0199]Em contraste, soros de camundongos imunizados com VRPs bicistrônicas gH/gL ou tricistrônicas gH/gL/gO (1 x 106 IU/camundongo) demonstraram respostas neutralizantes robustas. Além disso, as respostas foram similares na presença e ausência de complemento de porquinho-da-índia, mostrando que VRPs policistrônicas induziram com sucesso uma resposta imune complemento-independente (FIG. 6C). A titulação de 50% de neutralização foi em uma diluição de soros de 1:3500-6400+ em células ARPE-19 com vírus CMV TB40-GFP. Essa titulação é aproximadamente uma titulação 34 vezes maior do que a titulação de 50% de neutralização complemento-dependente para soros de gB reunidos em pool.
[0200]Os resultados nas células de fibroblastos MRC-5 foram similares àqueles em células ARPE-19 (FIGS. 7A e 7B). Soros de camundongos imunizados com VRPs bicistrônicas gH/gL ou tricistrônicas gH/gL/gO demonstraram forte atividade neutralizante comparadoscomsoros decamundongos imunizados com VRPs que codificam gH isoladamente, gL isoladamente ou gO isoladamente, ecomsoros decamundongos imunizados por co-administração deVRPsde gH eVRPs de gL, ou co-administração de VRPs de gH,VRPsde gL eVRPs de gO. Esses resultados demonstram que a administração das VRPs policistrônicas induziu uma resposta imune que forneceboa neutralização complemento-independente de infecção peloCMV de células de fibroblastos. Para avaliar a amplitude e a potência dos soros imunes de gH/gL contra diferentes cepas de CMV, comparamos a habilidade dos soros para bloquear a infecção de fibroblastos e células epiteliais com seis cepasde CMV diferentes. A Figura 8 mostra que ossoros de gH/gLneutralizam potentemente a infecção de ambosos tipos de células com uma ampla gama de cepas.
[0201]Esses dados também demonstram forte atividade neutralizante para soros de camundongos imunizados com as VRPs policistrônicas, mas não com os pools mistos de VRPs que expressam apenas uma proteína. Isso mostra que replicons policistrônicos que codificam os componentes de um complexo de proteínas em um único replicon resultam na produção eficiente do complexo in situ. Além disso, como as proteínas de CMV da cepa Merlin foram usadas para estimular essas respostas, os dados in vitro obtidos usando a cepa TB40 do vírus CMV demonstram que os anticorpos neutralizantesinduzidosporliberaçãodasVRPs policistrônicas são anticorpos de neutralização cruzada.
EXEMPLO 5 Síntese de RNA
[0202]DNA de plasmídeo que codifica replicons de alfavírus (veja FIGS. 14-16) serviram como um modelo para a síntese de RNA in vitro. Replicons de alfavírus contêm os elementos genéticos necessários para a replicação de RNA, mas não possuem aqueles que codificam produtos gênicos necessários à montagem de partícula; Os genes estruturais do genoma do alfavírus são substituídos por seqüências que codificam uma proteína heterólogo. Após liberação dos replicons às células eucarióticas, o RNA de fita positiva é traduzido para produzir quatro proteínas não estruturais, que juntas replicam o RNA genômico e transcrevem mRNAs subgenômicos abundantes que codificam o produto gênico heterólogo ou o gene de interesse (GOI). Em função da ausência de expressão das proteínas estruturais de alfavírus, os replicons são incapazes de induzir a geração de partículas infecciosas. Um promotor de bacteriófago (T7 ou SP6) acima do cDNA de alfavírus facilita a síntese do RNA do replicon in vitro e a ribozima do vírus da hepatite delta (HDV) imediatamente abaixo da cauda de poli(A) gera a extremidade 3’ correta por meio de sua atividade de autoclivagem.
[0203]A fim de permitir a formação de um complexo antigênico de proteínas, a expressão dos componentes individuais do referido complexo na mesma célula é de importância fundamental. Em teoria, isso pode ser obtido por co-transfecção de células com os genes que codificam os componentes individuais. No entanto, no caso de RNAs de replicon de alfavírus liberados de forma não viral ou por VRP, essa estratégia é impedida por co-liberação ineficiente de múltiplos RNAs à mesma célula ou, alternativamente, por lançamento ineficiente de múltiplos RNAs auto-replicantes em uma célula individual. Uma forma potencialmente mais eficiente para facilitar a co-expressão de componentes de um complexo de proteínas é liberar os respectivos genes como parte da mesma molécula de RNA auto- replicante. Para essa finalidade, projetamos construções de replicon de alfavírus que codificam múltiplos genes de interesse. Cada GOI é precedido por seu próprio promotor subgenômico que é reconhecido pelo maquinário de transcrição do alfavírus. Dessa forma, múltiplas espécies de RNA mensageiro subgenômico são sintetizadas em uma célula individual, o que permite a montagem de complexos de proteínas multicomponentes.
[0204] Após linearização do DNA de plasmídeo abaixo da ribozima de HDV com uma endonuclease de restrição adequada, os transcritos foram sintetizados in vitro usando RNA polimerase DNA-dependente derivada de bacteriófago T7. As transcrições foram realizadas por 2 horas a 37°C na presença de 7,5 mM de cada um dos trifosfatos de nucleosídeo (ATP, CTP, GTP e UTP) seguindo as instruções fornecidas pelo fabricante (Ambion, Austin, TX). Após a transcrição, o DNA-modelo foi digerido com TURBO DNase (Ambion, Austin, TX). O RNA do replicon foi precipitado com LiCl e reconstituído em água livre de nuclease. RNA sem cobertura foi coberto pós-transcrição com enzima de cobertura do vírus de vacínia (VCE) usando o sistema de cobertura ScriptCap m7G (Epicentre Biotechnologies, Madison, WI) como definido no manual do usuário. RNA oberto pós-transcrição foi precipitado com LiCl e reconstituído em água livre de nuclease. A concentração das amostras de RNA foi determinada por medição da densidade óptica a 260 nm. A integridade dos transcritos in vitro foi confirmada por eletroforese em gel de agarose desnaturante.
Formulação de nanopartícula de lipídeo (LNP)
[0205]1,2-dilinoleiloxi-N,N-dimetil-3-aminopropano (DlinDMA) foi sintetizado usando um procedimento publicado previamente [Heyes, J., Palmer, L., Bremner, K., MacLachlan, I. “Cationic Lipide Saturation Influences Intracellular Delivery of Encapsulated Nucleic Acids”, Journal of Controlled Release,107:276-287(2005)].1,2- Diaestearoil-sn-glicero-3-fosfocolina (DSPC) foi adquirida de Genzyme. Colesterol foi obtido de Sigma-Aldrich (St. Lois, MO). 1,2-Dimiristoil-sn-glicero-3-fosfoetanolamina-N- [metóxi(polietileno glicol)-2000] (sal de amônio) (PEG DMG 2000) foi obtido de Avanti Polar Lipids.
[0206]LNPs (RV01(14)) foram formuladas usando o seguinte método: batelada de 150 μg, (fibras ocas PES e sem mustang):soluções de estoque frescas de lipídeo em etanol foram preparadas; 37 mg de DlinDMA, 11,8 mg de DSPC, 27,8 mg de colesterol e 8,07 mg de PEG DMG 2000 foram pesados e dissolvidos em 7,55 ml de etanol. A solução de estoque de lipídeo recém preparada foi gentilmente agitada a 37°C por cerca de 15 minutos para formar uma mistura homogênea. A seguir, 453μl do estoque foramadicionadosa 1,547 mlde etanolparaproduzir umasolução de estoquede lipídeode trabalho de 2 ml. Essa quantidade de lipídeos foi usada para formar LNPs com 150 μg de RNA em uma proporção de N:P (nitrogênio para fosfato) de 8:1. O nitrogênio ionizável em DlinDMA (o lipídeo catiônico) e fosfatos no RNA são usados para esse cálculo. Foipresumido quecada μg de moléculade RNA auto-replicante contém 3 nanomoles de fosfato aniônico, foi presumido que cada μg de DlinDMAcontém 1,6nanomolede nitrogênio catiônico. Uma solução de RNA de trabalho de 2 mltambém foi preparada a parir de uma soluçãodeestoque deaproximadamente 1 μg/μl, em 100 mM de tampãodecitrato (pH 6) (Teknova). Três frascos de vidro de 20 ml (com barras de agitação) foram enxaguados com solução de RNase Away (Molecular BioProducts) e lavados com bastante água MilliQ antes do uso para descontaminar os frascos de RNAses. Um dos frascos foi usado para a solução de trabalho de RNA e os outros para coleta do lipídeo e misturas de RNA (como descrito mais tarde). As soluções de trabalho de lipídeo e de RNA foram aquecidas a 37°C por 10 min antes de serem carregadasem seringas luer-lok de 3cc(BD Medical). Dois ml de tampão de citrato (pH 6) foram carregados em outra seringa de 3 cc. As seringas que contêm RNA e os lipídeos foram conectadas a misturador em “T” (junção PEEK™ com 500 μm de diâmetro interno) usando tubulação de FEP ([etileno-propileno fluorinado] 2 mm de diâmetro interno x 3 mm de diâmetro externo, Idex Health Science, Oak Harbor, WA). A saída do misturador em “T” também era uma tubulação de FEP (2 mm de diâmetro interno x 3 mm). A terceira seringa contendo o tampão de citrato foi conectada a um pedaço separado de tubulação (2 mm de diâmetro interno x 3 mm de diâmetro externo). Todas as seringas foram então conduzidas em uma taxa de fluxo de 7 ml/min usando uma bomba de seringas (de kdScientific, modelo N° KDS-220). As saídas do tubo foram posicionadas para coletar as misturas emum frasco devidro de20 ml(durante agitação). A barra deagitaçãofoiretiradaefoipermitido que a solução de etanol/aquosa se equilibrasse até a temperatura ambiente por 1 h. A seguir, a mistura foi carregada em uma seringa de 5 cc (BD Medical), que foi ajustada a um pedaço de tubulação de FEP (2 mm de diâmetro interno x 3 mm de diâmetro externo) e, em outra seringa de 5 cc com comprimento igual de tubulação de FEP, um volume igual de tampão de citrato 100 mM (pH 6) foi carregado. As duas seringas foram conduzidas emumataxa defluxode 7 ml/min usando uma bomba de seringase amisturafinalcoletada em um frasco de vidro de 20 ml (durante agitação). A seguir, as LNPs foram concentradas até 2 ml e dialisadas contra 1015 volumes de PBS IX (de Teknova) usando o sistema de filtração de fluxo tangencial (TFF) antes da recuperação do produto final. O sistema de TFF e as membranas de filtração em fibra oca foram adquiridos de Spectrum Labs e foram usados de acordo com as instruções do fabricante. Foram usadas membranas de filtração em fibra oca de poli(éter sulfona) (PES) (número da peça P-C1-100E-100-01N) com um valor de corte de poro de 100 kD e 20 cm2 de área de superfície. Para experimentos in vitro e in vivo, as formulações foram diluídas até a concentração de RNA necessária com PBS IX (de Teknova).
Tamanho de partícula
[0207]O tamanho de partícula foi medido usando um Zetasizer Nano ZS (Malvern Instruments, Worcestershire, GB) de acordo com as instruções do fabricante. Os tamanhos de partículas são registrados como a média Z com o índice de polidispersibilidade (pdi). Lipossomos foram diluídos em PBS IX antes da medição.
Eficiência da encapsulação e concentração de RNA
[0208] A percentagem de RNA encapsulado e a concentração de RNA foram determinadas pelo kit de reagente de RNA Quant-iT RiboGreen (Invitrogen). As instruções do fabricante foram seguidas no ensaio. O padrão de RNA ribossômico fornecido no kit foi usado para gerar uma curva-padrão. LNPs obtidas pelo método 1 ou pelos métodos 2-5 foram diluídas dez vezes ou cem vezes, respectivamente, em tampão IX TE (do kit), antes da adição do corante. Separadamente, LNPs foram diluídas dez ou 100 vezes em tampão IX TE contendo Triton X 0,5% (Sigma-Aldrich), antes da adição do corante. A seguir, uma quantidade igual de corante foi adicionada a cada solução e depois aproximadamente 180 μl de cada solução após adição do corante foram carregados em duplicata em uma placa de cultura de tecido de 96 poços (obtida de VWR, N° de Catálogo 353072). A fluorescência (excitação: 485 nm; emissão: 528 nm) foi lida em uma leitora de microplacas (de BioTek Instruments, Inc.).
[0209] Triton X foi usado para romper as LNPs, fornecendo uma leitura de fluorescência que corresponde à quantidade de RNA total e a amostra sem Triton X forneceu a fluorescência que corresponde ao RNA não encapsulado. O % de encapsulação de RNA foi determinado da seguinte forma: encapsulação de RNA de LNP (%) = [(Ft-Fi)/Ft] X 100, em que Ft é a intensidade de fluorescência de LNPs com adição de Triton X e Fi é a intensidade de fluorescência da solução de LNP sem adição de detergente. Esses valores (Ft e Fi) foram obtidos após subtração da intensidade de fluorescência de um branco (tampão IX TE). A concentração de RNA encapsulado foi obtida por comparação de Ft-Fi com a curva-padrão gerada. Todas as formulações de LNP foram dosadas in vivo com base na dose encapsulada.
Partículas de replicon viral (VRP)
[0210]Para comparar vacinas de RNA as abordagens tradicionais de RNA-com vetor para obtenção da expressão in vivo de genes repórteres ou antígenos, utilizamos partículas de replicon viral (VRPs), produzidas em células BHK pelos métodos descritos por Perri e cols.(J. Virol. 77(19):10.394-10.403(2003)), que codificam a expressão dos mesmos antígenos que as construções de RNA correspondentes. Nesse sistema, o antígeno consistiu em replicons quiméricos de alfavírus (VCR) derivados do genoma do vírus da encefalite eqüina venezuelana (VEEV) modificado geneticamente para conter as seqüências do terminal 3’ (UTR 3’) do vírus Sindbis e um sinal de empacotamento do vírus Sindbis (PS) (veja a FIG. 2 de Perri e cols.). Os replicons foram empacotados em VRPs por sua co-eletroporação em células de rim de filhote de hamster (BHK) juntamente com RNAs helper defeituosos que codificam o capsídeo do vírus Sindbis e genes de glicoproteína (veja a FIG. 2 de Perri e cols.). As VRPs foram então coletadas e parcialmente purificadas por ultracentrifugação em uma almofada de sacarose, e concentradas em um concentrador Amicon. O estoque de VRP resultante foi titulado por métodos padronizados e inoculado em animais em fluido de cultura ou outros tampões isotônicos. Uma quimera de partícula de replicon de alfavírus derivada do vírus da encefalite eqüina venezuelana e do vírus Sindbis é um vetor potente de liberação de vacina à base de gene (J. Virol. 77,10.394 10.403).
Estudos murídeos de imunogenicidade
[0211]Grupos de 10 machos de camundongos BALB/c com idade de 8-10 semanas e pesando cerca de 20 g foram imunizados com 1 x 106 IU (VRP) ou 1,0 μg (RNA) no dia 0, 21 e 42 com sangrias realizadas 3 semanas após a 2a vacinação e 3 semanas após a 3a vacinação. Todos os animais foram injetados no quadríceps nas duas patas traseiras, cada uma recebendo um volume equivalente (50 μl por local).
Ensaio de microneutralização
[0212]Amostras de soro foram testadas quanto à presença de anticorpos neutralizantes por um teste de neutralização de redução de infecção. Diluições seriais de duas vezes de HI-soro (em DMEM com FBS HI 10%) foram adicionadas a um volume igual de CMV (cepa TB40 ou isolado clínico 8819) previamente titulado para gerar aproximadamente 200IU/50 μl. As cepas VR1814, Towne, AD169 e o isolado clínico 8822 também foram usados. Misturas de soro/vírus foram incubadas por 2 horas a 37°C e CO25%, para permitir que ocorra a neutralização do vírus, e depois 50 μl dessa mistura (contendo aproximadamente 200 IU) foram inoculados em poços em duplicata de células ARPE-19 em placas de 96 poços pela metade. As placas foram incubadas por 40-44 horas. A menos que observado de forma diferente, o número de focos infectados positivos foi determinado por imunocoloração com um anticorpo monoclonal IE1 de CMV conjugado a AlexaFluor 488, seguida por contagem automatizada. A titulação de neutralização é definida como a reciproca da diluição de soro que produz uma redução de 50% no número de focos vírus-positivos por poço, em relação aos controles (sem soro).
Imunogenicidade de RNA formulado em VRPs de gH/gL e LNP
[0213]O replicon A323 que expressa a glicoproteína de superfície B (gB) de CMV, o replicon A160 que expressa o complexo da membrana da glicoproteína H e L de comprimento total (gH/gL) e o replicon A322 que expressa o complexo da membrana da forma solúvel de glicoproteína H e L (gH sol/gL) foram usados para esse experimento. Camundongos BALB/c, 10 animais por grupo, receberam vacinações intramusculares bilaterais (50 por pata) nos dias 0,21 e 42 com VRPs que expressam gB (1 x 106 IU), VRPs que expressam gH/gL (1 x 106 IU), VRPs que expressam gH sol/gL (1 x 106 IU) e PBS como os controles. Os três grupos de teste receberam RNA auto-replicante (A160,A322 ou A323) formulado em LNP (RV01(14)). Soro foi coletado para análise imunológica nos dias 39(3wp2) e 63(3wp3).
Resultados
[0214]O tamanho e a percentagem de RNA encapsulado nas formulações RV01(14) feitas para o experimento são mostrados na Tabela 3.Tabela 3.
Figure img0004
Figure img0005
[0215] As titulações 50% neutralizantes para os soros terminais (dia 63, três semanas após a vacinação final) são mostradas na Tabela 4.Tabela 4.
Figure img0006
[0216]RNA que expressa uma forma de comprimento total ou uma forma solúvel presumida do complexo de gH/gL de HCMV desperta titulações elevadas de anticorpo neutralizante, como avaliado em células epiteliais usando duas cepas de HCMV diferentes. As titulações médias despertadas pelos RNAs de gH/gL são pelo menos tão elevadas quanto à titulação média para as VRPs de gH/gL correspondentes (veja a FIG. 17).
EXEMPLO 6 Ácidos nucléicos bicistrônicos e pentacistrônicos que codificam proteínas de CMV
[0217] Replicons bicistrônicos e pentacistrônicos de alfavírus adicionais que expressam complexos de glicoproteínas de citomegalovírus humano (HCMV) foram preparados, e são mostrados esquematicamente nas FIGS. 18 e 20. Os replicons de alfavírus eram baseados no vírus da encefalite eqüina venezuelana (VEE). Os replicons foram empacotados em partículas de replicon viral (VRPs), encapsulados em nanopartículas lipídicas (LNP) ou formulados com uma nanoemulsão catiônica (CNE). A expressão das proteínas de HCMV codificadas e de complexos de proteínas de cada um dos replicons foi confirmada por imunoblot, coimunoprecipitação e citometria de fluxo. A citometria de fluxo foi usada para verificar a expressão do complexo pentamérico gH/gL/UL128/UL130/UL131 pelos replicons pentaméricos que codificam os componentes protéicos do complexo, usando anticorpos humanos monoclonais específicos para epitopos conformacionais presentes no complexo pentamérico (Macagno e cols. (2010), J. Virol. 84(2): 1.005-13). A FIG. 19 mostra que esses anticorpos se ligam às células BHKV transfectadas com RNA do replicon que expressa o complexo pentamérico Hg/gL/UL128/UL130/UL131 de HCMV (A527). Resultados similares foram obtidos quando as células foram infectadas com VRPs feitas pela mesma construção de replicon. Isso mostra que replicons projetados para expressar o complexo pentamérico na verdade expressam o antígeno desejado, e não o subproduto potencial gH/gL.
[0218] As VRPs, RNA encapsulado em LNPs e RNA formulado com CNE foram usados para imunizar camundongos Balb/c por injeções intramusculares no quadríceps traseiro. Os camundongos foram imunizados três vezes, com intervalo de três semanas, e amostras de soro foram coletadas entes de cada imunização, bem como três semanas após a terceira imunização e a imunização final. Os soros foram avaliados em ensaios de microneutralização para medir a potência da resposta de anticorpo neutralizante que foi despertada pelas vacinações. As titulações são expressas como titulação 50% neutralizante.
[0219] A imunogenicidade de diversas configurações diferentes de um cassete de expressão bicistrônico para um complexo de gH/gL de HCMV solúvel em VRPs foi avaliada. A FIG. 20 mostra que VRPs que expressam o complexo de gH/gL de comprimento total ancorado à membrana despertaram anticorpos neutralizantes potentes em titulações ligeiramente maiores que o complexo solúvel (gH sol/gL) expresso por um cassete de expressão bicistrônico similar. A alteração da ordem dos genes que codificam gH sol e gL ou a substituição de um dos promotores subgenômicos com um IRES ou um sítio FMDV 2A não aumentam substancialmente a imunogenicidade.
[0220] A amplitude e potência da atividade neutralizante de HCMV em soros de camundongos imunizados com VRPs de VEE/SIN que expressam gH/gL foram avaliadas pela utilização dos soros para bloquear a infecção de fibroblastos e células epiteliais com diferentes cepas de HCMV. A Tabela 5 mostra que soros imunes de gH/gL foram ampla e potentemente neutralizantes contra seis cepas de HCMV diferentes em ambos os tipos de células na ausência de complemento. A adição de complemento teve um ligeiro efeito negativo sobre a potência neutralizante dos soros. Tabela 5. Titulações de anticorpo neutralizante em soros de camundongos imunizados com VRPs derivadas de pVCR que expressam gH/gL.
Figure img0007
[0221]A imunogenicidade de LNP-RNAs encapsulados que codificam o complexo pentamérico (A526 e A527) comparados com LNP-RNA encapsulado (A160) e VRPs (gH-SGPgL modificado por pVCR) que expressam gH/gL foi avaliada. A Tabela 6 mostra que replicons que expressam o complexo pentamérico despertaram mais potentemente anticorpos neutralizantes do que replicons que expressam gH/gL.Tabela 6. Titulações de anticorpo neutralizante.
Figure img0008
[0222] O replicon de RNA pentacistrônico à base de VEE que despertou as maiores titulações de anticorpos neutralizantes (A527) foi empacotado como VRPs, e a imunogenicidade das VRPs foi comparada com VRPs que expressam gH/gL e replicons encapsulados em LNP que expressam gH/gL e complexo pentamérico. A Tabela 7 mostra que as VRPs que expressam o complexo pentamérico despertaram titulações de anticorpos neutralizantes maiores do que as VRPs que expressam gH/gL. Além disso, 106 unidades infecciosas de VRPs são pelo menos tão potentes quanto 1 μg de LNP-RNA encapsulado quando as VRPs e o RNA codificavam os mesmos complexos de proteínas.Tabela 7. Titulações de anticorpo neutralizante. Soros foram coletados três semanas após a segunda imunização.
Figure img0009
Figure img0010
[0223]Aamplitudee potênciada atividadeneutralizante de HCMVemsoros decamundongosimunizados com RNA à base de VEEquecodificao complexopentamérico(A527)foram avaliadas por utilização dos soros para bloquear a infecção de fibroblastos e células epiteliais com diferentes cepas de HCMV. A Tabela 8 mostra que soros imunes anti- gH/gL/UL128/UL130/UL131 neutralizam ampla e potentemente a infecção de células epiteliais. Esse efeito era complemento-independente. Em contraste, Os soros tiveram um efeito reduzido ou não detectável sobre a infecção de fibroblastos. Esses resultados são os esperados para soros imunes que contêm principalmente anticorpos específicos para o complexo pentamérico de gH/gL/UL128/UL130/UL131,, pois o complexo pentamérico não é necessário para a infecção de fibroblastos e, conseqüentemente, anticorpos para UL128, UL130 e UL131 não bloqueiam a infecção de fibroblastos (Adler e cols. (2006), J. Gen. Virol. 87 (Parte9): 2.451-60; Wang e Shenk (2005), Proc. Natl. Acad. Sci. U.S.A. 102(50): 18.153-8). Dessa forma, esses dados demonstram que os replicons pentaméricos que codificam o complexo pentamérico gH/gL/UL128/UL130/UL131 despertam especificamente anticorpos para o complexo in vivo. Tabela 8. Titulações de anticorpo neutralizante em soros de camundongos imunizados com o replicon de RNA A527 encapsulado em LNPs. O replicon expressa o complexo pentamérico de HCMV usando promotores subgenômicos e IRESes.
Figure img0011
[0224] Para verificar se replicons bicistrônicos e pentacistrônicos que expressam as gH/gL e os complexos pentaméricos despertariam anticorpos neutralizantes em formulações diferentes, ratos algodão foram imunizados com replicons bicistrônicos ou pentacistrônicos misturados com uma nanoemulsão catiônica (CNE). A Tabela 9 mostra que replicons em CNE despertaram titulações de anticorpo neutralizante comparáveis com as dos mesmos replicons encapsulados em LNPs.Tabela 9. Titulações de anticorpo neutralizante. Os soros foram coletados três semanas após a segunda imunização.
Figure img0012
Figure img0013
EXEMPLO 7 Replicons que codificam proteínas de VZV
[0225] Ácidos nucléicos que codificam proteínas de VZV foram clonados em um vetor de replicon de VEE para produzir replicons monocistrônicos que codificam gB, gH, gL, gE e gI, e para produzir replicons bicistrônicos que codificam gH/gL ou gE/gI. Nos replicons bicistrônicos, a expressão de cada quadro de leitura aberta de VZV foi dirigida por um promotor subgenômico separado.
[0226] Para preparar RNA do replicon, plasmídeo que codifica o replicon foi linearizado por digestão com PmeI, e o plasmídeo linearizado foi extraído com fenol/clorofórmio/álcool isoamílico, precipitado em acetato de sódio/etanol e ressuspenso em 20 μl de água livre de RNase.
[0227] RNA foi preparado por transcrição in vitro de 1 μg de DNA linearizado usando o kit MEGAscript T7 (AMBION# AM 1333). Uma reação de 20 μl foi preparada de acordo com as instruções do fabricante sem análogo de cap, e incubada por 2 horas a 32°C. TURBO DNase (1 μl) foi adicionada, e a mistura foi incubada por 30 minutos a 32°C. Água livre de RNase (30 μl) e solução de acetato de amônio (30 μl) foram adicionadas. A solução foi misturada e resfriada por pelo menos 30 minutos a -20°C. A seguir, a solução foi centrifugada em velocidade máxima por 25 minutos a 4°C. O sobrenadante foi descartado, e o pélete foi enxaguado com etanol 70%, e novamente centrifugado em velocidade máxima por 10 minutos a 4°C. O pélete foi seco ao ar e ressuspenso em 50 μl de água livre de RNase. A concentração de RNA foi medida e a qualidade verificada em um gel desnaturante.
[0228] O RNA foi coberto usando o Sistema ScriptCap m7G Capping (Epicentre #SCCE0625). A reação foi escalonada por combinação do RNA e água livre de RNase. O RNA foi então desnaturado por 5-10 minutos a 65°C. O RNA desnaturado foi transferido rapidamente para o gelo e os seguintes reagentes foram adicionados na seguinte ordem: Tampão de Capping ScriptCap, 10 mM de GTP, 2 mM de SAM recém preparado, inibidor de RNase ScriptGuard, e enzima de Capping ScriptCap. A mistura foi incubada por 60 minutos a 37°C. A reação foi interrompida por adição de água livre de RNase e 7,5 M de LiCl, bem misturada e a mistura foi armazenada por pelo menos 30 minutos a -20°C. A seguir, a mistura foi centrifugada em velocidade máxima por 25 minutos a 4°C, o pélete foi enxaguado com etanol 70%, novamente centrifugado em velocidade máxima por 10 minutos a 4°C e o pélete foi seco ao ar. O pélete foi ressuspenso em água livre de RNase. A concentração de RNA foi medida e a qualidade verificada em um gel desnaturante.
Transfecção de RNA
[0229] Células (células BHK-V) foram semeadas em placas de 6 poços e levadas até 90-95% de confluência no momento da transfecção. Para cada transfecção, 3 g de RNA foram diluídos em 50 ml de meio OPTIMEM em um primeiro tubo. Lipofectamina 2000 foi adicionada a um segundo tubo que continha 50 ml de meio OPTIMEM. O primeiro e o segundo tubos foram combinados e guardados por 20 minutos em temperatura ambiente. Os meios de cultura nas placas de 6 poços foram substituídos com meio fresco, e o complexo de RNA-Lipofectamina foi colocado sobre as células e misturado por agitação suave da placa. As placas foram incubadas por 24 horas a 37°C em uma incubadora de CO2.
[0230] A expressão das proteínas de VZV em células transfectadas foi avaliada por western blot e imunofluorescência. Para western blots, lisados de células transfectadas foram separados por eletroforese (5 μg de proteínas totais/raia) e submetidos ao blot. Uma suspensão viral depurada (7 μg de proteína total/raia) derivada da cepa da vacina OKA/Merck foi usada como um controle positivo. Os blots foram sondados usando anticorpos disponíveis comercialmente (diluição de 1:1.000) que se ligam às proteínas de VZV.
[0231] Para imunofluorescência, as células transfectadas foram coletadas e semeadas em uma placa de 96 poços, e a coloração intracelular foi feita usando mAbs de camundongo disponíveis comercialmente (faixa de diluição de 1:1001:400). Os péletes de células foram fixados e permeabilizados com soluções Citofix-Citoperm. Um reagente secundário, F(ab’)2 de cabra anti-camundongo marcado com Alexa488 (diluição de final 1:400), foi usado.
[0232] A expressão de proteínas de VZV gE e gI foi detectada em células transfectadas com construções monocistrônicas (gE ou gI), e a expressão tanto de gE quanto de gI foi detectada em células transfectadas com uma construção bicistrônica de gE/gI em western blots usando anticorpos de camundongo disponíveis comercialmente, 13B1 para gE e 8C4 para gI. A expressão de proteína gB de VZV foi detectada em células transfectadas com uma construção monocistrônica que codifica gB por imunofluorescência usando anticorpo 10G6 disponível comercialmente. A expressão do complexo de proteínas de VZV gH/gL foi detectada por imunofluorescência em células transfectadas com construção monocistrônica de gH e monocistrônica de gL, ou com uma construção bicistrônica de gH/gL. O complexo de gH/gL foi detectado usando anticorpo SG3 disponível comercialmente.
Estudos murídeos de imunogenicidade
[0233]Grupos de 8 machos de camundongos BALB/c com idade de 6-8 semanas e pesando cerca de 20 g foram imunizados por via intramuscular com 7,0 ou 1,0 μg de RNA de replicon formulado com uma CNE ou LNP (RV01) nos dias 0, 21 e 42. Amostras de sangue foram coletadas dos animais imunizados 3 semanas após a 2a imunização e 3 semanas após a 3a imunização. Os grupos são mostrados na Tabela 10. Tabela 10
Figure img0014
Figure img0015
Resposta imune a antígenos de VZV
[0234]Amostras de soro foram testadas quanto à presença de anticorpos para gB, por coloração intracelular de células MRC-5 transfectadas com replicon de VZV. As células MRC-5 foram mantidas em meio de Eagle modificado por Dulbecco com soro bovino fetal 10%. Um inóculo da cepa de VZV Oka (obtido de ATCC) foi usado para infectar uma cultura de células MRC-5, e células inteiras infectadas foram usadas para subpassagem do vírus. A proporção entre células infectadas e não infectadas foi de 1:10. Trinta horas após a infecção, as células foram dispersas por tripsina para semeadura em uma placa de 96 poços para realizar uma coloração intracelular com pools de soros de camundongos (faixa de diluição de 1:200 até 1:800) obtidos após imunização. mAbs comerciais foram usados como controles para quantificar o nível de infecção. Os péletes de células foram fixados e permeabilizados com soluções de Citofix-Citoperm. Um reagente secundário, F(ab’)2 anti- camundongo de cabra marcado com Alexa488, foi usado (diluição final de 1:400).
[0235] Anticorpos comerciais para gB (10G6), gH (SG3) e gE (13B1 (SBA) e 8612 (Millipore)) foram usados como controles positivos, e cada um corou intracelularmente células MRC-5 infectadas. Soros imunes obtidos 3 semanas após a terceira imunização com 1 ou 7 μg de RNA formulado com CNE ou LNP foram diluídos a 1/200, 1/400 e 1/800 e usados para corar intracelularmente células MRC-5 infectadas. Os resultados são mostrados na FIG. 21 (Estudo 1, grupos 1, 5, 7, 9, 11, 13 e 15, formulação de CNE) e FIG. 22 (Estudo 2, grupos 1-7, formulação de LNP).
Ensaio de neutralização
[0236] Cada soro de camundongo imunizado foi diluído serialmente por incrementos de duas vezes começando em 1:20 em um meio de cultura padronizado, e adicionado até o volume igual de suspensão de VZV na presença de complemento de porquinho-da-índia. Após incubação por 1 hora a 37°C, a linhagem de células epiteliais humanas A549 foi adicionada. As células infectadas podem ser medidas após uma semana de cultura por contagem das placas formadas na cultura sob microscópio. A partir do número de placas, o % de inibição de cada diluição de soro foi calculado. Uma tabela para cada amostra de soro foi deita por tabulação do valor do % de inibição contra a escala logarítmica do fator de diluição. Subseqüentemente, uma linha aproximada de relacionamento entre o fator de diluição e o % de inibição foi desenhada. A seguir, a titulação de 50% de neutralização foi determinada como o fator de diluição onde a linha cruzada no valor de 50% de inibição.
[0237] A Tabela 11 mostra que soros obtidos de camundongos imunizados com gE monocistrônica, gE/gI bicistrônica e gH/gL bicistrônica continham titulações robustas de anticorpo neutralizante. Tabela 11. Titulações de neutralização de soros decamundongo reunidos em pools imunizados com 7 μg de RNA.
Figure img0016
* soros pré-imunes reunidos em pool
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Varnum S.M., Streblow D.N., Monroe M.E., Smith P., Auberry K.J., Pasa-Tolic L., Wang D., Camp II D.G., Rodland K., Wiley, Britt W., Shenk T., Smith R.D. e Nelson J.A. 2004. “Identification of proteins in human cytomegalovirus (HCMV) particles:the HCMVproteome”.J.Virol. 78:10.96066. (Errata, 78:13395). Ljungman P.,Griffiths P. e PayaC.2002.“Definitions of cytomegalovirus infection and disease in transplant recipients”. Clin. Infect. Dis. 34:1.094-97. Rubin R. 2002. “Clinical approach to infection in the compromised host”, páginas 573-679. Em: R. Rubin e L.S. Young (ed.), “Infection in the organ transplant recipient”. Kluwer Academic Press, Nova York, NY. Stagno S. e Britt W.J. 2005.“Cytomegalovirus”, páginas 389-424. Em: J.S. Remington e J.O. Klein (ed.), “Infectious diseases of the fetus and newborn infant”,6a Edição. WB Saunders, Philadelphia, PA. Britt W.J., Vugler L., Butfiloski E.J. e Stephens E.B. 1990. “Cell surface expression of human cytomegalovirus (HCMV) gp55-116(gB):use of HCMV-vaccinia recombinant virus infected cells in analysis of the human neutralizing antibody response”. J. Virol. 64:1.079-85. Reap E.A., Dryga S.A., Morris J., Rivers B., Norberg P.K., Olmsted R.A. e Chulay J.D. 2007.“Cellular and Humoral Immune Responses to Alphavirus Replicon Vaccines Expressing Cytomegalovirus pp65, IL1 and gB proteins”. Clin. Vacc. Immunol. 14:748-55. Balasuriya U.B.R., Heidner H.W., Hedges J.F., Williams J.C., Davis N.L., Johnston R.E. e MacLachlan N.J. 2000. “Expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan equine encephalitis virus replicon particles”. J. Virol. 74: 10.623-30. Dunn W., Chou C., Li H., Hai R., Patterson D., Stoic V., Zhu H. e Liu F. 2003. “Functional profiling of a human cytomegalovirus genome”. Proc. Natl. Acad. Sci. U.S.A. 100: 14.223-28. Hobom U., Brune W., Messerle M., Hahn G. e Kosinowski U.H. 2000. “Fast screening procedures for random transposon llibraries of cloned herpesvirus genomes: mutational analysis of human cytomegalovirus envelope glycoprotein genes”. J. Virol. 74: 7.720-29. Ryckman B.J., Chase M.C. e Johnson D.C. 2009. “HCMV TR strain glycoprotein O acts as a chaperone promoting gH/gL incorporation into virions, but is not present in virions”. J. Virol. Wille P.T., Knoche A.J., Nelson J.A., Jarvis M.A. e Johnson J.C. 2009. “An HCMV gO-null mutant fails to incorporate gH/gL into the virion envelope and is unable to enter fibroblasts, epithelial, and endothelial cells”. J. Virol. Shimamura M., Mach M. e Britt W.J. 2006. “Human Cytomegalovirus infection elicits a glycoprotein M (gM)/gN- specific virus-neutralizing antibody response”. J. Virol. 80: 4.591-4.600. Cha T.A., Tom E., Kemble G.W., Duke G.M., Mocarski E.S. e Spaete R.R. 1996. “Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains”.J.Virol.70:78-83. WangD.e Shenk T.2005.“Human cytomegalovirus virion protein complex required for epithelial and endothelial cell tropism”. Proc. Natl. Acad. Sci. U.S.A. 102:18.153 58. Adler B., Scrivano L., Ruzcics Z., Rupp B., Sinzger C. e Kosinowski U. 2006. “Role of human cytomegalovirus UL131A in cell type-specific virus entry and release”. J. Gen. Virol. 87:2.451-60. Ryckman B.J., Rainish B.L., Chase M.C., Borton J.A., Nelson J.A., Jarvis J.A. e Johnson D.C. 2008. “Characterization of the human cytomegalovirus gH/gL/UL128- UL131 complex that mediates entry into epithelial and endothelial cells”. J. Virol. 82:60-70.
SEQUÊNCIAS
CMV gB FL :1- atggaaagccggatctggtgcctggtcgtgtgcgtgaacctgtgcatcgtgtgcctgggagccg ccgtgagcagcagcagcaccagaggcaccagcgccacacacagccaccacagcagccacaccac ctctgccgcccacagcagatccggcagcgtgtcccagagagtgaccagcagccagaccgtgtcc cacggcgtgaacgagacaatctacaacaccaccctgaagtacggcgacgtcgtgggcgtgaata ccaccaagtacccctacagagtgtgcagcatggcccagggcaccgacctgatcagattcgagcg gaacatcgtgtgcaccagcatgaagcccatcaacgaggacctggacgagggcatcatggtggtg tacaagagaaacatcgtggcccacaccttcaaagtgcgggtgtaccagaaggtgctgaccttcc ggcggagctacgcctacatccacaccacatacctgctgggcagcaacaccgagtacgtggcccc tcccatgtgggagatccaccacatcaacagccacagccagtgctacagcagctacagccgcgtg atcgccggcacagtgttcgtggcctaccaccgggacagctacgagaacaagaccatgcagctga tgcccgacgactacagcaacacccacagcaccagatacgtgaccgtgaaggaccagtggcacag cagaggcagcacctggctgtaccgggagacatgcaacctgaactgcatggtcaccatcaccacc gccagaagcaagtacccttaccacttcttcgccacctccaccggcgacgtggtggacatcagcc ccttctacaacggcaccaaccggaacgccagctacttcggcgagaacgccgacaagttcttcat cttccccaactacaccatcgtgtccgacttcggcagacccaacagcgctctggaaacccacaga ctggtggcctttctggaacgggccgacagcgtgatcagctgggacatccaggacgagaagaacg tgacctgccagctgaccttctgggaggcctctgagagaaccatcagaagcgaggccgaggacag ctaccacttcagcagcgccaagatgaccgccaccttcctgagcaagaaacaggaagtgaacatg agcgactccgccctggactgcgtgagggacgaggccatcaacaagctgcagcagatcttcaaca ccagctacaaccagacctacgagaagtatggcaatgtgtccgtgttcgagacaacaggcggcct ggtggtgttctggcagggcatcaagcagaaaagcctggtggagctggaacggctcgccaaccgg tccagcctgaacctgacccacaaccggaccaagcggagcaccgacggcaacaacgcaacccacc tgtccaacatggaaagcgtgcacaacctggtgtacgcacagctgcagttcacctacgacaccct gcggggctacatcaacagagccctggcccagatcgccgaggcttggtgcgtggaccagcggcgg accctggaagtgttcaaagagctgtccaagatcaaccccagcgccatcctgagcgccatctaca acaagcctatcgccgccagattcatgggcgacgtgctgggcctggccagctgcgtgaccatcaa ccagaccagcgtgaaggtgctgcgggacatgaacgtgaaagagagcccaggccgctgctactcc agacccgtggtcatcttcaacttcgccaacagctcctacgtgcagtacggccagctgggcgagg acaacgagatcctgctggggaaccaccggaccgaggaatgccagctgcccagcctgaagatctt tatcgccggcaacagcgcctacgagtatgtggactacctgttcaagcggatgatcgacctgagc agcatctccaccgtggacagcatgatcgccctggacatcgaccccctggaaaacaccgacttcc gggtgctggaactgtacagccagaaagagctgcggagcagcaacgtgttcgacctggaagagat catgcgggagttcaacagctacaagcagcgcgtgaaatacgtggaggacaaggtggtggacccc ctgcctccttacctgaagggcctggacgacctgatgagcggactgggcgctgccggaaaagccg tgggagtggccattggagctgtgggcggagctgtggcctctgtcgtggaaggcgtcgccacctt tctgaagaaccccttcggcgccttcaccatcatcctggtggccattgccgtcgtgatcatcacc tacctgatctacacccggcagcggagactgtgtacccagcccctgcagaacctgttcccctacc tggtgtccgccgatggcaccacagtgaccagcggctccaccaaggataccagcctgcaggcccc acccagctacgaagagagcgtgtacaacagcggcagaaagggccctggccctcccagctctgat gccagcacagccgcccctccctacaccaacgagcaggcctaccagatgctgctggccctggcta gactggatgccgagcagagggcccagcagaacggcaccgacagcctggatggcagaaccggcac ccaggacaagggccagaagcccaacctgctggaccggctgcggcaccggaagaacggctaccgg cacctgaaggacagcgacgaggaagagaacgtctgataa - 2727 CMV gB FL MESRIWCLVVCVNLCIVCLGAAVSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVS HGVNETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFERNIVCTSMKPINEDLDEGIMVV YKRNIVAHTFKVRVYQKVLTFRRSYAYIHTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRV IAGTVFVAYHRDSYENKTMQLMPDDYSNTHSTRYVTVKDQWHSRGSTWLYRETCNLNCMVTITT ARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSALETHR LVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNM SDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANR SSLNLTHNRTKRSTDGNNATHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQIAEAWCVDQRR TLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVKVLRDMNVKESPGRCYS RPVVIFNFANSSYVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLS SISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKVVDP LPPYLKGLDDLMSGLGAAGKAVGVAIGAVGGAVASVVEGVATFLKNPFGAFTIILVAIAVVIIT YLIYTRQRRLCTQPLQNLFPYLVSADGTTVTSGSTKDTSLQAPPSYEESVYNSGRKGPGPPSSD ASTAAPPYTNEQAYQMLLALARLDAEQRAQQNGTDSLDGRTGTQDKGQKPNLLDRLRHRKNGYR HLKDSDEEENV-- CMV gB sol 750 : 1-atggaaagccggatctggtgcctggtcgtgtgcgtgaacctgtgcatcgtgtgcctgggagccg ccgtgagcagcagcagcaccagaggcaccagcgccacacacagccaccacagcagccacaccac ctctgccgcccacagcagatccggcagcgtgtcccagagagtgaccagcagccagaccgtgtcc cacggcgtgaacgagacaatctacaacaccaccctgaagtacggcgacgtcgtgggcgtgaata ccaccaagtacccctacagagtgtgcagcatggcccagggcaccgacctgatcagattcgagcg gaacatcgtgtgcaccagcatgaagcccatcaacgaggacctggacgagggcatcatggtggtg tacaagagaaacatcgtggcccacaccttcaaagtgcgggtgtaccagaaggtgctgaccttcc ggcggagctacgcctacatccacaccacatacctgctgggcagcaacaccgagtacgtggcccc tcccatgtgggagatccaccacatcaacagccacagccagtgctacagcagctacagccgcgtg atcgccggcacagtgttcgtggcctaccaccgggacagctacgagaacaagaccatgcagctga tgcccgacgactacagcaacacccacagcaccagatacgtgaccgtgaaggaccagtggcacag cagaggcagcacctggctgtaccgggagacatgcaacctgaactgcatggtcaccatcaccacc gccagaagcaagtacccttaccacttcttcgccacctccaccggcgacgtggtggacatcagcc ccttctacaacggcaccaaccggaacgccagctacttcggcgagaacgccgacaagttcttcat cttccccaactacaccatcgtgtccgacttcggcagacccaacagcgctctggaaacccacaga ctggtggcctttctggaacgggccgacagcgtgatcagctgggacatccaggacgagaagaacg tgacctgccagctgaccttctgggaggcctctgagagaaccatcagaagcgaggccgaggacag ctaccacttcagcagcgccaagatgaccgccaccttcctgagcaagaaacaggaagtgaacatg agcgactccgccctggactgcgtgagggacgaggccatcaacaagctgcagcagatcttcaaca ccagctacaaccagacctacgagaagtatggcaatgtgtccgtgttcgagacaacaggcggcct ggtggtgttctggcagggcatcaagcagaaaagcctggtggagctggaacggctcgccaaccgg tccagcctgaacctgacccacaaccggaccaagcggagcaccgacggcaacaacgcaacccacc tgtccaacatggaaagcgtgcacaacctggtgtacgcacagctgcagttcacctacgacaccct gcggggctacatcaacagagccctggcccagatcgccgaggcttggtgcgtggaccagcggcgg accctggaagtgttcaaagagctgtccaagatcaaccccagcgccatcctgagcgccatctaca acaagcctatcgccgccagattcatgggcgacgtgctgggcctggccagctgcgtgaccatcaa ccagaccagcgtgaaggtgctgcgggacatgaacgtgaaagagagcccaggccgctgctactcc agacccgtggtcatcttcaacttcgccaacagctcctacgtgcagtacggccagctgggcgagg acaacgagatcctgctggggaaccaccggaccgaggaatgccagctgcccagcctgaagatctt tatcgccggcaacagcgcctacgagtatgtggactacctgttcaagcggatgatcgacctgagc agcatctccaccgtggacagcatgatcgccctggacatcgaccccctggaaaacaccgacttcc gggtgctggaactgtacagccagaaagagctgcggagcagcaacgtgttcgacctggaagagat catgcgggagttcaacagctacaagcagcgcgtgaaatacgtggaggacaaggtggtggacccc ctgcctccttacctgaagggcctggacgacctgatgagcggactgggcgctgccggaaaagccg tgggagtggccattggagctgtgggcggagctgtggcctctgtcgtggaaggcgtcgccacctt tctgaagaactgataa - 2256 Cmv gB sol 750 MESRIWCLVVCVNLCIVCLGAAVSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTS SQTVSHGVNETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFERNIVCTSMKPIN EDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRRSYAYIHTTYLLGSNTEYVAPPMWEIH HINSHSQCYSSYSRVIAGTVFVAYHRDSYENKTMQLMPDDYSNTHSTRYVTVKDQWHSR GSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENAD KFFIFPNYTIVSDFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASER TIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYE KYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNRTKRSTDGNNATHLSN MESVHNLVYAQLQFTYDTLRGYINRALAQIAEAWCVDQRRTLEVFKELSKINPSAILSA IYNKPIAARFMGDVLGLASCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSYV QYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMI ALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKVVDPLPPY LKGLDDLMSGLGAAGKAVGVAIGAVGGAVASVVEGVATFLKN-- CMV gB sol 692 : 1-atggaaagccggatctggtgcctggtcgtgtgcgtgaacctgtgcatcgtgtgcctgggagccg ccgtgagcagcagcagcaccagaggcaccagcgccacacacagccaccacagcagccacaccac ctctgccgcccacagcagatccggcagcgtgtcccagagagtgaccagcagccagaccgtgtcc cacggcgtgaacgagacaatctacaacaccaccctgaagtacggcgacgtcgtgggcgtgaata ccaccaagtacccctacagagtgtgcagcatggcccagggcaccgacctgatcagattcgagcg gaacatcgtgtgcaccagcatgaagcccatcaacgaggacctggacgagggcatcatggtggtg tacaagagaaacatcgtggcccacaccttcaaagtgcgggtgtaccagaaggtgctgaccttcc ggcggagctacgcctacatccacaccacatacctgctgggcagcaacaccgagtacgtggcccc tcccatgtgggagatccaccacatcaacagccacagccagtgctacagcagctacagccgcgtg atcgccggcacagtgttcgtggcctaccaccgggacagctacgagaacaagaccatgcagctga tgcccgacgactacagcaacacccacagcaccagatacgtgaccgtgaaggaccagtggcacag cagaggcagcacctggctgtaccgggagacatgcaacctgaactgcatggtcaccatcaccacc gccagaagcaagtacccttaccacttcttcgccacctccaccggcgacgtggtggacatcagcc ccttctacaacggcaccaaccggaacgccagctacttcggcgagaacgccgacaagttcttcat cttccccaactacaccatcgtgtccgacttcggcagacccaacagcgctctggaaacccacaga ctggtggcctttctggaacgggccgacagcgtgatcagctgggacatccaggacgagaagaacg tgacctgccagctgaccttctgggaggcctctgagagaaccatcagaagcgaggccgaggacag ctaccacttcagcagcgccaagatgaccgccaccttcctgagcaagaaacaggaagtgaacatg agcgactccgccctggactgcgtgagggacgaggccatcaacaagctgcagcagatcttcaaca ccagctacaaccagacctacgagaagtatggcaatgtgtccgtgttcgagacaacaggcggcct ggtggtgttctggcagggcatcaagcagaaaagcctggtggagctggaacggctcgccaaccgg tccagcctgaacctgacccacaaccggaccaagcggagcaccgacggcaacaacgcaacccacc tgtccaacatggaaagcgtgcacaacctggtgtacgcacagctgcagttcacctacgacaccct gcggggctacatcaacagagccctggcccagatcgccgaggcttggtgcgtggaccagcggcgg accctggaagtgttcaaagagctgtccaagatcaaccccagcgccatcctgagcgccatctaca acaagcctatcgccgccagattcatgggcgacgtgctgggcctggccagctgcgtgaccatcaa ccagaccagcgtgaaggtgctgcgggacatgaacgtgaaagagagcccaggccgctgctactcc agacccgtggtcatcttcaacttcgccaacagctcctacgtgcagtacggccagctgggcgagg acaacgagatcctgctggggaaccaccggaccgaggaatgccagctgcccagcctgaagatctt tatcgccggcaacagcgcctacgagtatgtggactacctgttcaagcggatgatcgacctgagc agcatctccaccgtggacagcatgatcgccctggacatcgaccccctggaaaacaccgacttcc gggtgctggaactgtacagccagaaagagctgcggagcagcaacgtgttcgacctggaagagat catgcgggagttcaacagctacaagcagtgataa -2082 Cmv gB sol 692; MESRIWCLVVCVNLCIVCLGAAVSSSSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSHGVNET IYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFERNIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKV RVYQKVLTFRRSYAYIHTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIAGTVFVAYHRDSYENKT MQLMPDDYSNTHSTRYVTVKDQWHSRGSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFY NGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWE ASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSV FETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNRTKRSTDGNNATHLSNMESVHNLVYAQLQFTYDT LRGYINRALAQIAEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVK VLRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVD YLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQ— CMV gH FL : 1-atgaggcctggcctgccctcctacctgatcatcctggccgtgtgcctgttcagccacctgctgtccagca gatacggcgccgaggccgtgagcgagcccctggacaaggctttccacctgctgctgaacacctacggcag acccatccggtttctgcgggagaacaccacccagtgcacctacaacagcagcctgcggaacagcaccgtc gtgagagagaacgccatcagcttcaactttttccagagctacaaccagtactacgtgttccacatgccca gatgcctgtttgccggccctctggccgagcagttcctgaaccaggtggacctgaccgagacactggaaag ataccagcagcggctgaatacctacgccctggtgtccaaggacctggccagctaccggtcctttagccag cagctcaaggctcaggatagcctcggcgagcagcctaccaccgtgccccctcccatcgacctgagcatcc cccacgtgtggatgcctccccagaccacccctcacggctggaccgagagccacaccacctccggcctgca cagaccccacttcaaccagacctgcatcctgttcgacggccacgacctgctgtttagcaccgtgaccccc tgcctgcaccagggcttctacctgatcgacgagctgagatacgtgaagatcaccctgaccgaggatttct tcgtggtcaccgtgtccatcgacgacgacacccccatgctgctgatcttcggccacctgcccagagtgct gttcaaggccccctaccagcgggacaacttcatcctgcggcagaccgagaagcacgagctgctggtgctg gtcaagaaggaccagctgaaccggcactcctacctgaaggaccccgacttcctggacgccgccctggact tcaactacctggacctgagcgccctgctgagaaacagcttccacagatacgccgtggacgtgctgaagtc cggacggtgccagatgctcgatcggcggaccgtggagatggccttcgcctatgccctcgccctgttcgcc gctgccagacaggaagaggctggcgcccaggtgtcagtgcccagagccctggatagacaggccgccctgc tgcagatccaggaattcatgatcacctgcctgagccagaccccccctagaaccaccctgctgctgtaccc cacagccgtggatctggccaagagggccctgtggacccccaaccagatcaccgacatcacaagcctcgtg cggctcgtgtacatcctgagcaagcagaaccagcagcacctgatcccccagtgggccctgagacagatcg ccgacttcgccctgaagctgcacaagacccatctggccagctttctgagcgccttcgccaggcaggaact gtacctgatgggcagcctggtccacagcatgctggtgcataccaccgagcggcgggagatcttcatcgtg gagacaggcctgtgtagcctggccgagctgtcccactttacccagctgctggcccaccctcaccacgagt acctgagcgacctgtacaccccctgcagcagcagcggcagacgggaccacagcctggaacggctgaccag actgttccccgatgccaccgtgcctgctacagtgcctgccgccctgtccatcctgtccaccatgcagccc agcaccctggaaaccttccccgacctgttctgcctgcccctgggcgagagctttagcgccctgaccgtgt ccgagcacgtgtcctacatcgtgaccaatcagtacctgatcaagggcatcagctaccccgtgtccaccac agtcgtgggccagagcctgatcatcacccagaccgacagccagaccaagtgcgagctgacccggaacatg cacaccacacacagcatcaccgtggccctgaacatcagcctggaaaactgcgctttctgtcagtctgccc tgctggaatacgacgatacccagggcgtgatcaacatcatgtacatgcacgacagcgacgacgtgctgtt cgccctggacccctacaacgaggtggtggtgtccagcccccggacccactacctgatgctgctgaagaac ggcaccgtgctggaagtgaccgacgtggtggtggacgccaccgacagcagactgctgatgatgagcgtgt acgccctgagcgccatcatcggcatctacctgctgtaccggatgctgaaaacctgctgataa -2232 Cmv gH FL; MRPGLPSYLIILAVCLFSHLLSSRYGAEAVSEPLDKAFHLLLNTYGRPIRFLRENTTQC TYNSSLRNSTVVRENAISFNFFQSYNQYYVFHMPRCLFAGPLAEQFLNQVDLTETLERY QQRLNTYALVSKDLASYRSFSQQLKAQDSLGEQPTTVPPPIDLSIPHVWMPPQTTPHGW TESHTTSGLHRPHFNQTCILFDGHDLLFSTVTPCLHQGFYLIDELRYVKITLTEDFFVV TVSIDDDTPMLLIFGHLPRVLFKAPYQRDNFILRQTEKHELLVLVKKDQLNRHSYLKDP DFLDAALDFNYLDLSALLRNSFHRYAVDVLKSGRCQMLDRRTVEMAFAYALALFAAARQ EEAGAQVSVPRALDRQAALLQIQEFMITCLSQTPPRTTLLLYPTAVDLAKRALWTPNQI TDITSLVRLVYILSKQNQQHLIPQWALRQIADFALKLHKTHLASFLSAFARQELYLMGS LVHSMLVHTTERREIFIVETGLCSLAELSHFTQLLAHPHHEYLSDLYTPCSSSGRRDHS LERLTRLFPDATVPATVPAALSILSTMQPSTLETFPDLFCLPLGESFSALTVSEHVSYI VTNQYLIKGISYPVSTTVVGQSLIITQTDSQTKCELTRNMHTTHSITVALNISLENCAF CQSALLEYDDTQGVINIMYMHDSDDVLFALDPYNEVVVSSPRTHYLMLLKNGTVLEVTD VVVDATDSRLLMMSVYALSAIIGIYLLYRMLKTC-- CMV gH sol : 1-atgaggcctggcctgccctcctacctgatcatcctggccgtgtgcctgttcagccacctgctgt ccagcagatacggcgccgaggccgtgagcgagcccctggacaaggctttccacctgctgctgaa cacctacggcagacccatccggtttctgcgggagaacaccacccagtgcacctacaacagcagc ctgcggaacagcaccgtcgtgagagagaacgccatcagcttcaactttttccagagctacaacc agtactacgtgttccacatgcccagatgcctgtttgccggccctctggccgagcagttcctgaa ccaggtggacctgaccgagacactggaaagataccagcagcggctgaatacctacgccctggtg tccaaggacctggccagctaccggtcctttagccagcagctcaaggctcaggatagcctcggcg agcagcctaccaccgtgccccctcccatcgacctgagcatcccccacgtgtggatgcctcccca gaccacccctcacggctggaccgagagccacaccacctccggcctgcacagaccccacttcaac cagacctgcatcctgttcgacggccacgacctgctgtttagcaccgtgaccccctgcctgcacc agggcttctacctgatcgacgagctgagatacgtgaagatcaccctgaccgaggatttcttcgt ggtcaccgtgtccatcgacgacgacacccccatgctgctgatcttcggccacctgcccagagtg ctgttcaaggccccctaccagcgggacaacttcatcctgcggcagaccgagaagcacgagctgc tggtgctggtcaagaaggaccagctgaaccggcactcctacctgaaggaccccgacttcctgga cgccgccctggacttcaactacctggacctgagcgccctgctgagaaacagcttccacagatac gccgtggacgtgctgaagtccggacggtgccagatgctcgatcggcggaccgtggagatggcct tcgcctatgccctcgccctgttcgccgctgccagacaggaagaggctggcgcccaggtgtcagt gcccagagccctggatagacaggccgccctgctgcagatccaggaattcatgatcacctgcctg agccagaccccccctagaaccaccctgctgctgtaccccacagccgtggatctggccaagaggg ccctgtggacccccaaccagatcaccgacatcacaagcctcgtgcggctcgtgtacatcctgag caagcagaaccagcagcacctgatcccccagtgggccctgagacagatcgccgacttcgccctg aagctgcacaagacccatctggccagctttctgagcgccttcgccaggcaggaactgtacctga tgggcagcctggtccacagcatgctggtgcataccaccgagcggcgggagatcttcatcgtgga gacaggcctgtgtagcctggccgagctgtcccactttacccagctgctggcccaccctcaccac gagtacctgagcgacctgtacaccccctgcagcagcagcggcagacgggaccacagcctggaac ggctgaccagactgttccccgatgccaccgtgcctgctacagtgcctgccgccctgtccatcct gtccaccatgcagcccagcaccctggaaaccttccccgacctgttctgcctgcccctgggcgag agctttagcgccctgaccgtgtccgagcacgtgtcctacatcgtgaccaatcagtacctgatca agggcatcagctaccccgtgtccaccacagtcgtgggccagagcctgatcatcacccagaccga cagccagaccaagtgcgagctgacccggaacatgcacaccacacacagcatcaccgtggccctg aacatcagcctggaaaactgcgctttctgtcagtctgccctgctggaatacgacgatacccagg gcgtgatcaacatcatgtacatgcacgacagcgacgacgtgctgttcgccctggacccctacaa cgaggtggtggtgtccagcccccggacccactacctgatgctgctgaagaacggcaccgtgctg gaagtgaccgacgtggtggtggacgccaccgactgataa -2151 CMV gH sol; MRPGLPSYLIILAVCLFSHLLSSRYGAEAVSEPLDKAFHLLLNTYGRPIRFLRENTTQC TYNSSLRNSTVVRENAISFNFFQSYNQYYVFHMPRCLFAGPLAEQFLNQVDLTETLERY QQRLNTYALVSKDLASYRSFSQQLKAQDSLGEQPTTVPPPIDLSIPHVWMPPQTTPHGW TESHTTSGLHRPHFNQTCILFDGHDLLFSTVTPCLHQGFYLIDELRYVKITLTEDFFVV TVSIDDDTPMLLIFGHLPRVLFKAPYQRDNFILRQTEKHELLVLVKKDQLNRHSYLKDP DFLDAALDFNYLDLSALLRNSFHRYAVDVLKSGRCQMLDRRTVEMAFAYALALFAAARQ EEAGAQVSVPRALDRQAALLQIQEFMITCLSQTPPRTTLLLYPTAVDLAKRALWTPNQI TDITSLVRLVYILSKQNQQHLIPQWALRQIADFALKLHKTHLASFLSAFARQELYLMGS LVHSMLVHTTERREIFIVETGLCSLAELSHFTQLLAHPHHEYLSDLYTPCSSSGRRDHS LERLTRLFPDATVPATVPAALSILSTMQPSTLETFPDLFCLPLGESFSALTVSEHVSYI VTNQYLIKGISYPVSTTVVGQSLIITQTDSQTKCELTRNMHTTHSITVALNISLENCAF CQSALLEYDDTQGVINIMYMHDSDDVLFALDPYNEVVVSSPRTHYLMLLKNGTVLEVTD VVVDATD-- CMV gL fl: 1-atgtgcagaaggcccgactgcggcttcagcttcagccctggacccgtgatcctgctgtggtgct gcctgctgctgcctatcgtgtcctctgccgccgtgtctgtggcccctacagccgccgagaaggt gccagccgagtgccccgagctgaccagaagatgcctgctgggcgaggtgttcgagggcgacaag tacgagagctggctgcggcccctggtcaacgtgaccggcagagatggccccctgagccagctga tccggtacagacccgtgacccccgaggccgccaatagcgtgctgctggacgaggccttcctgga taccctggccctgctgtacaacaaccccgaccagctgagagccctgctgaccctgctgtccagc gacaccgcccccagatggatgaccgtgatgcggggctacagcgagtgtggagatggcagccctg ccgtgtacacctgcgtggacgacctgtgcagaggctacgacctgaccagactgagctacggccg gtccatcttcacagagcacgtgctgggcttcgagctggtgccccccagcctgttcaacgtggtg gtggccatccggaacgaggccaccagaaccaacagagccgtgcggctgcctgtgtctacagccg ctgcacctgagggcatcacactgttctacggcctgtacaacgccgtgaaagagttctgcctccg gcaccagctggatccccccctgctgagacacctggacaagtactacgccggcctgcccccagag ctgaagcagaccagagtgaacctgcccgcccacagcagatatggccctcaggccgtggacgcca gatgataa - 840 CMV gL FL; MCRRPDCGFSFSPGPVILLWCCLLLPIVSSAAVSVAPTAAEKVPAECPELTRRCLLGEV FEGDKYESWLRPLVNVTGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQ LRALLTLLSSDTAPRWMTVMRGYSECGDGSPAVYTCVDDLCRGYDLTRLSYGRSIFTEH VLGFELVPPSLFNVVVAIRNEATRTNRAVRLPVSTAAAPEGITLFYGLYNAVKEFCLRH QLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDAR-- CMV gM FL: 1-atggcccccagccacgtggacaaagtgaacacccggacttggagcgccagcatcgtgttcatgg tgctgaccttcgtgaacgtgtccgtgcacctggtgctgtccaacttcccccacctgggctaccc ctgcgtgtactaccacgtggtggacttcgagcggctgaacatgagcgcctacaacgtgatgcac ctgcacacccccatgctgtttctggacagcgtgcagctcgtgtgctacgccgtgttcatgcagc tggtgtttctggccgtgaccatctactacctcgtgtgctggatcaagatcagcatgcggaagga caagggcatgagcctgaaccagagcacccgggacatcagctacatgggcgacagcctgaccgcc ttcctgttcatcctgagcatggacaccttccagctgttcaccctgaccatgagcttccggctgc ccagcatgatcgccttcatggccgccgtgcactttttctgtctgaccatcttcaacgtgtccat ggtcacccagtaccggtcctacaagcggagcctgttcttcttctcccggctgcaccccaagctg aagggcaccgtgcagttccggaccctgatcgtgaacctggtggaggtggccctgggcttcaata ccaccgtggtggctatggccctgtgctacggcttcggcaacaacttcttcgtgcggaccggcca tatggtgctggccgtgttcgtggtgtacgccatcatcagcatcatctactttctgctgatcgag gccgtgttcttccagtacgtgaaggtgcagttcggctaccatctgggcgcctttttcggcctgt gcggcctgatctaccccatcgtgcagtacgacaccttcctgagcaacgagtaccggaccggcat cagctggtccttcggaatgctgttcttcatctgggccatgttcaccacctgcagagccgtgcgg tacttcagaggcagaggcagcggctccgtgaagtaccaggccctggccacagcctctggcgaag aggtggccgccctgagccaccacgacagcctggaaagcagacggctgcgggaggaagaggacga cgacgacgaggacttcgaggacgcctgataa - 1119 CMV gM FL; MAPSHVDKVNTRTWSASIVFMVLTFVNVSVHLVLSNFPHLGYPCVYYHVVDFERLNMSA YNVMHLHTPMLFLDSVQLVCYAVFMQLVFLAVTIYYLVCWIKISMRKDKGMSLNQSTRD ISYMGDSLTAFLFILSMDTFQLFTLTMSFRLPSMIAFMAAVHFFCLTIFNVSMVTQYRS YKRSLFFFSRLHPKLKGTVQFRTLIVNLVEVALGFNTTVVAMALCYGFGNNFFVRTGHM VLAVFVVYAIISIIYFLLIEAVFFQYVKVQFGYHLGAFFGLCGLIYPIVQYDTFLSNEY RTGISWSFGMLFFIWAMFTTCRAVRYFRGRGSGSVKYQALATASGEEVAALSHHDSLES RRLREEEDDDDEDFEDA-- CMV gN FL: 1-atggaatggaacaccctggtcctgggcctgctggtgctgtctgtcgtggccagcagcaacaaca catccacagccagcacccctagacctagcagcagcacccacgccagcactaccgtgaaggctac caccgtggccaccacaagcaccaccactgctaccagcaccagctccaccacctctgccaagcct ggctctaccacacacgaccccaacgtgatgaggccccacgcccacaacgacttctacaacgctc actgcaccagccacatgtacgagctgtccctgagcagctttgccgcctggtggaccatgctgaa cgccctgatcctgatgggcgccttctgcatcgtgctgcggcactgctgcttccagaacttcacc gccaccaccaccaagggctactgataa -411 CMV gN FL; MEWNTLVLGLLVLSVVASSNNTSTASTPRPSSSTHASTTVKATTVATTSTTTATSTSST TSAKPGSTTHDPNVMRPHAHNDFYNAHCTSHMYELSLSSFAAWWTMLNALILMGAFCIV LRHCCFQNFTATTTKGY-- CMV gO FL: 1- atgggcaagaaagaaatgatcatggtcaagggcatccccaagatcatgctgctgattagcatca cctttctgctgctgtccctgatcaactgcaacgtgctggtcaacagccggggcaccagaagatc ctggccctacaccgtgctgtcctaccggggcaaagagatcctgaagaagcagaaagaggacatc ctgaagcggctgatgagcaccagcagcgacggctaccggttcctgatgtaccccagccagcaga aattccacgccatcgtgatcagcatggacaagttcccccaggactacatcctggccggacccat ccggaacgacagcatcacccacatgtggttcgacttctacagcacccagctgcggaagcccgcc aaatacgtgtacagcgagtacaaccacaccgcccacaagatcaccctgaggcctcccccttgtg gcaccgtgcccagcatgaactgcctgagcgagatgctgaacgtgtccaagcggaacgacaccgg cgagaagggctgcggcaacttcaccaccttcaaccccatgttcttcaacgtgccccggtggaac accaagctgtacatcggcagcaacaaagtgaacgtggacagccagaccatctactttctgggcc tgaccgccctgctgctgagatacgcccagcggaactgcacccggtccttctacctggtcaacgc catgagccggaacctgttccgggtgcccaagtacatcaacggcaccaagctgaagaacaccatg cggaagctgaagcggaagcaggccctggtcaaagagcagccccagaagaagaacaagaagtccc agagcaccaccaccccctacctgagctacaccacctccaccgccttcaacgtgaccaccaacgt gacctacagcgccacagccgccgtgaccagagtggccacaagcaccaccggctaccggcccgac agcaactttatgaagtccatcatggccacccagctgagagatctggccacctgggtgtacacca ccctgcggtacagaaacgagcccttctgcaagcccgaccggaacagaaccgccgtgagcgagtt catgaagaatacccacgtgctgatcagaaacgagacaccctacaccatctacggcaccctggac atgagcagcctgtactacaacgagacaatgagcgtggagaacgagacagccagcgacaacaacg aaaccacccccacctcccccagcacccggttccagcggaccttcatcgaccccctgtgggacta cctggacagcctgctgttcctggacaagatccggaacttcagcctgcagctgcccgcctacggc aatctgaccccccctgagcacagaagggccgccaacctgagcaccctgaacagcctgtggtggt ggagccagtgataa -1422 CMV gO FL; MGKKEMIMVKGIPKIMLLISITFLLLSLINCNVLVNSRGTRRSWPYTVLSYRGKEILKK QKEDILKRLMSTSSDGYRFLMYPSQQKFHAIVISMDKFPQDYILAGPIRNDSITHMWFD FYSTQLRKPAKYVYSEYNHTAHKITLRPPPCGTVPSMNCLSEMLNVSKRNDTGEKGCGN FTTFNPMFFNVPRWNTKLYIGSNKVNVDSQTIYFLGLTALLLRYAQRNCTRSFYLVNAM SRNLFRVPKYINGTKLKNTMRKLKRKQALVKEQPQKKNKKSQSTTTPYLSYTTSTAFNV TTNVTYSATAAVTRVATSTTGYRPDSNFMKSIMATQLRDLATWVYTTLRYRNEPFCKPD RNRTAVSEFMKNTHVLIRNETPYTIYGTLDMSSLYYNETMSVENETASDNNETTPTSPS TRFQRTFIDPLWDYLDSLLFLDKIRNFSLQLPAYGNLTPPEHRRAANLSTLNSLWWWSQ- CMV UL128 FL : 1-atgagccccaaggacctgacccccttcctgacaaccctgtggctgctcctgggccatagcagag tgcctagagtgcgggccgaggaatgctgcgagttcatcaacgtgaaccacccccccgagcggtg ctacgacttcaagatgtgcaaccggttcaccgtggccctgagatgccccgacggcgaagtgtgc tacagccccgagaaaaccgccgagatccggggcatcgtgaccaccatgacccacagcctgaccc ggcaggtggtgcacaacaagctgaccagctgcaactacaaccccctgtacctggaagccgacgg ccggatcagatgcggcaaagtgaacgacaaggcccagtacctgctgggagccgccggaagcgtg ccctaccggtggatcaacctggaatacgacaagatcacccggatcgtgggcctggaccagtacc tggaaagcgtgaagaagcacaagcggctggacgtgtgcagagccaagatgggctacatgctgca gtgataa -519 CMV UL128 FL; MSPKDLTPFLTTLWLLLGHSRVPRVRAEECCEFINVNHPPERCYDFKMCNRFTVALRCP DGEVCYSPEKTAEIRGIVTTMTHSLTRQVVHNKLTSCNYNPLYLEADGRIRCGKVNDKA QYLLGAAGSVPYRWINLEYDKITRIVGLDQYLESVKKHKRLDVCRAKMGYMLQ-- CMV UL130 FL: 1-atgctgcggctgctgctgagacaccacttccactgcctgctgctgtgtgccgtgtgggccaccc cttgtctggccagcccttggagcaccctgaccgccaaccagaaccctagccccccttggtccaa gctgacctacagcaagccccacgacgccgccaccttctactgcccctttctgtaccccagccct cccagaagccccctgcagttcagcggcttccagagagtgtccaccggccctgagtgccggaacg agacactgtacctgctgtacaaccgggagggccagacactggtggagcggagcagcacctgggt gaaaaaagtgatctggtatctgagcggccggaaccagaccatcctgcagcggatgcccagaacc gccagcaagcccagcgacggcaacgtgcagatcagcgtggaggacgccaaaatcttcggcgccc acatggtgcccaagcagaccaagctgctgagattcgtggtcaacgacggcaccagatatcagat gtgcgtgatgaagctggaaagctgggcccacgtgttccgggactactccgtgagcttccaggtc cggctgaccttcaccgaggccaacaaccagacctacaccttctgcacccaccccaacctgatcg tgtgataa - 648 CMV UL130FL; MLRLLLRHHFHCLLLCAVWATPCLASPWSTLTANQNPSPPWSKLTYSKPHDAATFYCPF LYPSPPRSPLQFSGFQRVSTGPECRNETLYLLYNREGQTLVERSSTWVKKVIWYLSGRN QTILQRMPRTASKPSDGNVQISVEDAKIFGAHMVPKQTKLLRFVVNDGTRYQMCVMKLE SWAHVFRDYSVSFQVRLTFTEANNQTYTFCTHPNLIV-- CMV UL131 FL: 1-atgcggctgtgcagagtgtggctgtccgtgtgcctgtgtgccgtggtgctgggccagtgccaga gagagacagccgagaagaacgactactaccgggtgccccactactgggatgcctgcagcagagc cctgcccgaccagacccggtacaaatacgtggagcagctcgtggacctgaccctgaactaccac tacgacgccagccacggcctggacaacttcgacgtgctgaagcggatcaacgtgaccgaggtgt ccctgctgatcagcgacttccggcggcagaacagaagaggcggcaccaacaagcggaccacctt caacgccgctggctctctggcccctcacgccagatccctggaattcagcgtgcggctgttcgcc aactgataa - 393 CMV UL131 FL; MRLCRVWLSVCLCAVVLGQCQRETAEKNDYYRVPHYWDACSRALPDQTRYKYVEQLVDL TLNYHYDASHGLDNFDVLKRINVTEVSLLISDFRRQNRRGGTNKRTTFNAAGSLAPHAR SLEFSVRLFAN-- Sequência de nucleotídeo EMCV IRES; aacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttcca ccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcat tcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagca gttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaacc ccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggc ggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctca agcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctgg ggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaa ccacggggacgtggttttcctttgaaaaacacgataat Sequência de nucleotídeo EV71 IRES; gtacctttgtacgcctgttttataccccctccctgatttgcaacttagaagcaa cgcaaaccagatcaatagtaggtgtgacataccagtcgcatcttgatcaagcacttctg tatccccggaccgagtatcaatagactgtgcacacggttgaaggagaaaacgtccgtta cccggctaactacttcgagaagcctagtaacgccattgaagttgcagagtgtttcgctc agcactccccccgtgtagatcaggtcgatgagtcaccgcattccccacgggcgaccgtg gcggtggctgcgttggcggcctgcctatggggtaacccataggacgctctaatacggac atggcgtgaagagtctattgagctagttagtagtcctccggcccctgaatgcggctaat cctaactgcggagcacatacccttaatccaaagggcagtgtgtcgtaacgggcaactct gcagcggaaccgactactttgggtgtccgtgtttctttttattcttgtattggctgctt atggtgacaattaaagaattgttaccatatagctattggattggccatccagtgtcaaa cagagctattgtatatctctttgttggattcacacctctcactcttgaaacgttacaca ccctcaattacattatactgctgaacacgaagcg Promotor subgenômico VEE5’-CTCTCTACGGCTAACCTGAATGGA-3’ Vetor modificado pVCR de gH sol-SGP gL cgcgtcggctacaattaatacataaccttatgtatcatacacatacgatttaggtgacactata gatgggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttg acatcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcagtttgaggtaga agccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaa ctgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgca gaatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacag attgtataagtatgcaactaagctgaagaaaaactgtaaggaaataactgataaggaattggac aagaaaatgaaggagctcgccgccgtcatgagcgaccctgacctggaaactgagactatgtgcc tccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggt tgacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggc tttgacaccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaact gggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgttatggagcg gtcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaacaatgttctattc tctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctg tatttcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacgg gtacgtcgttaaaagaatagctatcagtccaggcctgtatgggaagccttcaggctatgctgct acgatgcaccgcgagggattcttgtgctgcaaagtgacagacacattgaacggggagagggtct cttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatactggcaac agatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaac ggtcgcacccagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcat ttgctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacgaga tagacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatctatttataag cgcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcccagga taggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaa ggagccgtcacctctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggct aaggaggtgcgtgaagccgaggagttgcgcgcagctctaccacctttggcagctgatgttgagg agcccactctggaagccgatgtagacttgatgttacaagaggctggggccggctcagtggagac acctcgtggcttgataaaggttaccagctacgctggcgaggacaagatcggctcttacgctgtg ctttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaag tcatagtgataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagt agtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtgccacc attgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccacacatggaggag cgctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacct gtacgacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggc gagctggtggatcctcccttccatgaattcgcctacgagagtctgagaacacgaccagccgctc cttaccaagtaccaaccataggggtgtatggcgtgccaggatcaggcaagtctggcatcattaa aagcgcagtcaccaaaaaagatctagtggtgagcgccaagaaagaaaactgtgcagaaattata agggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttga atggatgcaaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtac tctcagagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaacag tgcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcacacaagtct tccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgtttta cgacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagt accaaacctaagcaggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaa tagattacaaaggcaacgaaataatgacggcagctgcctctcaagggctgacccgtaaaggtgt gtatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacccacctcagaacatgtgaac gtcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggataa aaacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagca tgatgccatcatgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggca aacgtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgaccactg aacaatggaacactgtggattattttgaaacggacaaagctcactcagcagagatagtattgaa ccaactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgtt ccgttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaata aagaagtggtccgtcagctctctcgcaggtacccacaactgcctcgggcagttgccactggaag agtctatgacatgaacactggtacactgcgcaattatgatccgcgcataaacctagtacctgta aacagaagactgcctcatgctttagtcctccaccataatgaacacccacagagtgacttttctt cattcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccagg caaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggc atcccaggtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaatacc atcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagcttgtct gcatctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagcgaa agcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcac ttgaagagacggaagttctgtttgtattcattgggtacgatcgcaaggcccgtacgcacaatcc ttacaagctttcatcaaccttgaccaacatttatacaggttccagactccacgaagccggatgt gcaccctcatatcatgtggtgcgaggggatattgccacggccaccgaaggagtgattataaatg ctgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccgga aagcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacat atcattcatgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttgg cagaggcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattcc actgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaaccatttg ctgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaa tgactctcaaggaagcagtggctaggagagaagcagtggaggagatatgcatatccgacgactc ttcagtgacagaacctgatgcagagctggtgagggtgcatccgaagagttctttggctggaagg aagggctacagcacaagcgatggcaaaactttctcatatttggaagggaccaagtttcaccagg cggccaaggatatagcagaaattaatgccatgtggcccgttgcaacggaggccaatgagcaggt atgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcg gaagcctcctcaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagag tacagcgcctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaa gtatagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaagtg cctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccggagc catcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatga gaccaggactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttg ctgtcagatggcccgacccaccaggtgctgcaagtcgaggcagacattcacgggccgccctctg tatctagctcatcctggtccattcctcatgcatccgactttgatgtggacagtttatccatact tgacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactcttacttc gcaaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctc cacatcccgctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagagggat cacgggagaaaccgtgggatacgcggttacacacaatagcgagggcttcttgctatgcaaagtt actgacacagtaaaaggagaacgggtatcgttccctgtgtgcacgtacatcccggccaccataa actcgagaaccagcctggtctccaacccgccaggcgtaaatagggtgattacaagagaggagtt tgaggcgttcgtagcacaacaacaatgacggtttgatgcgggtgcatacatcttttcctccgac accggtcaagggcatttacaacaaaaatcagtaaggcaaacggtgctatccgaagtggtgttgg agaggaccgaattggagatttcgtatgccccgcgcctcgaccaagaaaaagaagaattactacg caagaaattacagttaaatcccacacctgctaacagaagcagataccagtccaggaaggtggag aacatgaaagccataacagctagacgtattctgcaaggcctagggcattatttgaaggcagaag gaaaagtggagtgctaccgaaccctgcatcctgttcctttgtattcatctagtgtgaaccgtgc cttttcaagccccaaggtcgcagtggaagcctgtaacgccatgttgaaagagaactttccgact gtggcttcttactgtattattccagagtacgatgcctatttggacatggttgacggagcttcat gctgcttagacactgccagtttttgccctgcaaagctgcgcagctttccaaagaaacactccta tttggaacccacaatacgatcggcagtgccttcagcgatccagaacacgctccagaacgtcctg gcagctgccacaaaaagaaattgcaatgtcacgcaaatgagagaattgcccgtattggattcgg cggcctttaatgtggaatgcttcaagaaatatgcgtgtaataatgaatattgggaaacgtttaa agaaaaccccatcaggcttactgaagaaaacgtggtaaattacattaccaaattaaaaggacca aaagctgctgctctttttgcgaagacacataatttgaatatgttgcaggacataccaatggaca ggtttgtaatggacttaaagagagacgtgaaagtgactccaggaacaaaacatactgaagaacg gcccaaggtacaggtgatccaggctgccgatccgctagcaacagcgtatctgtgcggaatccac cgagagctggttaggagattaaatgcggtcctgcttccgaacattcatacactgtttgatatgt cggctgaagactttgacgctattatagccgagcacttccagcctggggattgtgttctggaaac tgacatcgcgtcgtttgataaaagtgaggacgacgccatggctctgaccgcgttaatgattctg gaagacttaggtgtggacgcagagctgttgacgctgattgaggcggctttcggcgaaatttcat caatacatttgcccactaaaactaaatttaaattcggagccatgatgaaatctggaatgttcct cacactgtttgtgaacacagtcattaacattgtaatcgcaagcagagtgttgagagaacggcta accggatcaccatgtgcagcattcattggagatgacaatatcgtgaaaggagtcaaatcggaca aattaatggcagacaggtgcgccacctggttgaatatggaagtcaagattatagatgctgtggt gggcgagaaagcgccttatttctgtggagggtttattttgtgtgactccgtgaccggcacagcg tgccgtgtggcagaccccctaaaaaggctgtttaagcttggcaaacctctggcagcagacgatg aacatgatgatgacaggagaagggcattgcatgaagagtcaacacgctggaaccgagtgggtat tctttcagagctgtgcaaggcagtagaatcaaggtatgaaaccgtaggaacttccatcatagtt atggccatgactactctagctagcagtgttaaatcattcagctacctgagaggggcccctataa ctctctacggctaacctgaatggactacgacatagtctagtcgacgccaccatgaggcctggcc tgccctcctacctgatcatcctggccgtgtgcctgttcagccacctgctgtccagcagatacgg cgccgaggccgtgagcgagcccctggacaaggctttccacctgctgctgaacacctacggcaga cccatccggtttctgcgggagaacaccacccagtgcacctacaacagcagcctgcggaacagca ccgtcgtgagagagaacgccatcagcttcaactttttccagagctacaaccagtactacgtgtt ccacatgcccagatgcctgtttgccggccctctggccgagcagttcctgaaccaggtggacctg accgagacactggaaagataccagcagcggctgaatacctacgccctggtgtccaaggacctgg ccagctaccggtcctttagccagcagctcaaggctcaggatagcctcggcgagcagcctaccac cgtgccccctcccatcgacctgagcatcccccacgtgtggatgcctccccagaccacccctcac ggctggaccgagagccacaccacctccggcctgcacagaccccacttcaaccagacctgcatcc tgttcgacggccacgacctgctgtttagcaccgtgaccccctgcctgcaccagggcttctacct gatcgacgagctgagatacgtgaagatcaccctgaccgaggatttcttcgtggtcaccgtgtcc atcgacgacgacacccccatgctgctgatcttcggccacctgcccagagtgctgttcaaggccc cctaccagcgggacaacttcatcctgcggcagaccgagaagcacgagctgctggtgctggtcaa gaaggaccagctgaaccggcactcctacctgaaggaccccgacttcctggacgccgccctggac ttcaactacctggacctgagcgccctgctgagaaacagcttccacagatacgccgtggacgtgc tgaagtccggacggtgccagatgctcgatcggcggaccgtggagatggccttcgcctatgccct cgccctgttcgccgctgccagacaggaagaggctggcgcccaggtgtcagtgcccagagccctg gatagacaggccgccctgctgcagatccaggaattcatgatcacctgcctgagccagacccccc ctagaaccaccctgctgctgtaccccacagccgtggatctggccaagagggccctgtggacccc caaccagatcaccgacatcacaagcctcgtgcggctcgtgtacatcctgagcaagcagaaccag cagcacctgatcccccagtgggccctgagacagatcgccgacttcgccctgaagctgcacaaga cccatctggccagctttctgagcgccttcgccaggcaggaactgtacctgatgggcagcctggt ccacagcatgctggtgcataccaccgagcggcgggagatcttcatcgtggagacaggcctgtgt agcctggccgagctgtcccactttacccagctgctggcccaccctcaccacgagtacctgagcg acctgtacaccccctgcagcagcagcggcagacgggaccacagcctggaacggctgaccagact gttccccgatgccaccgtgcctgctacagtgcctgccgccctgtccatcctgtccaccatgcag cccagcaccctggaaaccttccccgacctgttctgcctgcccctgggcgagagctttagcgccc tgaccgtgtccgagcacgtgtcctacatcgtgaccaatcagtacctgatcaagggcatcagcta ccccgtgtccaccacagtcgtgggccagagcctgatcatcacccagaccgacagccagaccaag tgcgagctgacccggaacatgcacaccacacacagcatcaccgtggccctgaacatcagcctgg aaaactgcgctttctgtcagtctgccctgctggaatacgacgatacccagggcgtgatcaacat catgtacatgcacgacagcgacgacgtgctgttcgccctggacccctacaacgaggtggtggtg tccagcccccggacccactacctgatgctgctgaagaacggcaccgtgctggaagtgaccgacg tggtggtggacgccaccgactgataatctagacggcgcgcccacccagcggccgcctataactc tctacggctaacctgaatggactacgacatagtctagtcgacgccaccatgtgcagaaggcccg actgcggcttcagcttcagccctggacccgtgatcctgctgtggtgctgcctgctgctgcctat cgtgtcctctgccgccgtgtctgtggcccctacagccgccgagaaggtgccagccgagtgcccc gagctgaccagaagatgcctgctgggcgaggtgttcgagggcgacaagtacgagagctggctgc ggcccctggtcaacgtgaccggcagagatggccccctgagccagctgatccggtacagacccgt gacccccgaggccgccaatagcgtgctgctggacgaggccttcctggataccctggccctgctg tacaacaaccccgaccagctgagagccctgctgaccctgctgtccagcgacaccgcccccagat ggatgaccgtgatgcggggctacagcgagtgtggagatggcagccctgccgtgtacacctgcgt ggacgacctgtgcagaggctacgacctgaccagactgagctacggccggtccatcttcacagag cacgtgctgggcttcgagctggtgccccccagcctgttcaacgtggtggtggccatccggaacg aggccaccagaaccaacagagccgtgcggctgcctgtgtctacagccgctgcacctgagggcat cacactgttctacggcctgtacaacgccgtgaaagagttctgcctccggcaccagctggatccc cccctgctgagacacctggacaagtactacgccggcctgcccccagagctgaagcagaccagag tgaacctgcccgcccacagcagatatggccctcaggccgtggacgccagatgataatctagacg gcgcgcccacccaatcgatgtacttccgaggaactcacgtgcataatgcatcaggctggtacat tagatccccgcttaccgcgggcaatatagcaacactaaaaactcgatgtacttccgaggaagcg cagtgcataatgctgcgcagtgttgccacataaccactatattaaccatttatctagcggacgc caaaaactcaatgtatttctgaggaagcgtggtgcataatgccacgcagcgtctgcataacttt tattatttcttttattaatcaacaaaattttgtttttaacatttcaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaagggtcggcatggcatctccacctcctcgcggtccgacctgggc atccgaaggaggacgcacgtccactcggatggctaagggagagccacgagctcctgtttaaacc agctccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtc gtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccag ctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggc gaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtga ccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccac gttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgct ttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccct gatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttcca aactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatt tcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatat taacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttattt ttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataat attgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggc attttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcag ttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttc gccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatc ccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggtt gagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtg ctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaa ggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccg gagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaa cgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactg gatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttatt gctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatg gtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaa tagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttac tcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcc tttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccc cgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaa acaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttc cgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagtt aggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttacca gtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccgg ataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgac ctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggaga aaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccag ggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatt tttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacgg ttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtgg ataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcag cgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacg caattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctcccggctcg tatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattac gccaagcgcgcaattaaccctcactaaagggaacaaaagctgggtaccggcgcca vetor modificado pVCR de gH FL-SGP gL cgcgtcggctacaattaatacataaccttatgtatcatacacatacgatttaggtgacactata gatgggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttg acatcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcagtttgaggtaga agccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaa ctgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgca gaatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacag attgtataagtatgcaactaagctgaagaaaaactgtaaggaaataactgataaggaattggac aagaaaatgaaggagctcgccgccgtcatgagcgaccctgacctggaaactgagactatgtgcc tccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggt tgacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggc tttgacaccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaact gggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgttatggagcg gtcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaacaatgttctattc tctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctg tatttcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacgg gtacgtcgttaaaagaatagctatcagtccaggcctgtatgggaagccttcaggctatgctgct acgatgcaccgcgagggattcttgtgctgcaaagtgacagacacattgaacggggagagggtct cttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatactggcaac agatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaac ggtcgcacccagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcat ttgctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacgaga tagacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatctatttataag cgcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcccagga taggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaa ggagccgtcacctctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggct aaggaggtgcgtgaagccgaggagttgcgcgcagctctaccacctttggcagctgatgttgagg agcccactctggaagccgatgtagacttgatgttacaagaggctggggccggctcagtggagac acctcgtggcttgataaaggttaccagctacgctggcgaggacaagatcggctcttacgctgtg ctttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaag tcatagtgataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagt agtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtgccacc attgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccacacatggaggag cgctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacct gtacgacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggc gagctggtggatcctcccttccatgaattcgcctacgagagtctgagaacacgaccagccgctc cttaccaagtaccaaccataggggtgtatggcgtgccaggatcaggcaagtctggcatcattaa aagcgcagtcaccaaaaaagatctagtggtgagcgccaagaaagaaaactgtgcagaaattata agggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttga atggatgcaaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtac tctcagagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaacag tgcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcacacaagtct tccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgtttta cgacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagt accaaacctaagcaggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaa tagattacaaaggcaacgaaataatgacggcagctgcctctcaagggctgacccgtaaaggtgt gtatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacccacctcagaacatgtgaac gtcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggataa aaacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagca tgatgccatcatgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggca aacgtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgaccactg aacaatggaacactgtggattattttgaaacggacaaagctcactcagcagagatagtattgaa ccaactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgtt ccgttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaata aagaagtggtccgtcagctctctcgcaggtacccacaactgcctcgggcagttgccactggaag agtctatgacatgaacactggtacactgcgcaattatgatccgcgcataaacctagtacctgta aacagaagactgcctcatgctttagtcctccaccataatgaacacccacagagtgacttttctt cattcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccagg caaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggc atcccaggtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaatacc atcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagcttgtct gcatctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagcgaa agcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcac ttgaagagacggaagttctgtttgtattcattgggtacgatcgcaaggcccgtacgcacaatcc ttacaagctttcatcaaccttgaccaacatttatacaggttccagactccacgaagccggatgt gcaccctcatatcatgtggtgcgaggggatattgccacggccaccgaaggagtgattataaatg ctgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccgga aagcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacat atcattcatgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttgg cagaggcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattcc actgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaaccatttg ctgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaa tgactctcaaggaagcagtggctaggagagaagcagtggaggagatatgcatatccgacgactc ttcagtgacagaacctgatgcagagctggtgagggtgcatccgaagagttctttggctggaagg aagggctacagcacaagcgatggcaaaactttctcatatttggaagggaccaagtttcaccagg cggccaaggatatagcagaaattaatgccatgtggcccgttgcaacggaggccaatgagcaggt atgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcg gaagcctcctcaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagag tacagcgcctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaa gtatagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaagtg cctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccggagc catcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatga gaccaggactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttg ctgtcagatggcccgacccaccaggtgctgcaagtcgaggcagacattcacgggccgccctctg tatctagctcatcctggtccattcctcatgcatccgactttgatgtggacagtttatccatact tgacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactcttacttc gcaaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctc cacatcccgctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagagggat cacgggagaaaccgtgggatacgcggttacacacaatagcgagggcttcttgctatgcaaagtt actgacacagtaaaaggagaacgggtatcgttccctgtgtgcacgtacatcccggccaccataa actcgagaaccagcctggtctccaacccgccaggcgtaaatagggtgattacaagagaggagtt tgaggcgttcgtagcacaacaacaatgacggtttgatgcgggtgcatacatcttttcctccgac accggtcaagggcatttacaacaaaaatcagtaaggcaaacggtgctatccgaagtggtgttgg agaggaccgaattggagatttcgtatgccccgcgcctcgaccaagaaaaagaagaattactacg caagaaattacagttaaatcccacacctgctaacagaagcagataccagtccaggaaggtggag aacatgaaagccataacagctagacgtattctgcaaggcctagggcattatttgaaggcagaag gaaaagtggagtgctaccgaaccctgcatcctgttcctttgtattcatctagtgtgaaccgtgc cttttcaagccccaaggtcgcagtggaagcctgtaacgccatgttgaaagagaactttccgact gtggcttcttactgtattattccagagtacgatgcctatttggacatggttgacggagcttcat gctgcttagacactgccagtttttgccctgcaaagctgcgcagctttccaaagaaacactccta tttggaacccacaatacgatcggcagtgccttcagcgatccagaacacgctccagaacgtcctg gcagctgccacaaaaagaaattgcaatgtcacgcaaatgagagaattgcccgtattggattcgg cggcctttaatgtggaatgcttcaagaaatatgcgtgtaataatgaatattgggaaacgtttaa agaaaaccccatcaggcttactgaagaaaacgtggtaaattacattaccaaattaaaaggacca aaagctgctgctctttttgcgaagacacataatttgaatatgttgcaggacataccaatggaca ggtttgtaatggacttaaagagagacgtgaaagtgactccaggaacaaaacatactgaagaacg gcccaaggtacaggtgatccaggctgccgatccgctagcaacagcgtatctgtgcggaatccac cgagagctggttaggagattaaatgcggtcctgcttccgaacattcatacactgtttgatatgt cggctgaagactttgacgctattatagccgagcacttccagcctggggattgtgttctggaaac tgacatcgcgtcgtttgataaaagtgaggacgacgccatggctctgaccgcgttaatgattctg gaagacttaggtgtggacgcagagctgttgacgctgattgaggcggctttcggcgaaatttcat caatacatttgcccactaaaactaaatttaaattcggagccatgatgaaatctggaatgttcct cacactgtttgtgaacacagtcattaacattgtaatcgcaagcagagtgttgagagaacggcta accggatcaccatgtgcagcattcattggagatgacaatatcgtgaaaggagtcaaatcggaca aattaatggcagacaggtgcgccacctggttgaatatggaagtcaagattatagatgctgtggt gggcgagaaagcgccttatttctgtggagggtttattttgtgtgactccgtgaccggcacagcg tgccgtgtggcagaccccctaaaaaggctgtttaagcttggcaaacctctggcagcagacgatg aacatgatgatgacaggagaagggcattgcatgaagagtcaacacgctggaaccgagtgggtat tctttcagagctgtgcaaggcagtagaatcaaggtatgaaaccgtaggaacttccatcatagtt atggccatgactactctagctagcagtgttaaatcattcagctacctgagaggggcccctataa ctctctacggctaacctgaatggactacgacatagtctagtcgacgccaccatgaggcctggcc tgccctcctacctgatcatcctggccgtgtgcctgttcagccacctgctgtccagcagatacgg cgccgaggccgtgagcgagcccctggacaaggctttccacctgctgctgaacacctacggcaga cccatccggtttctgcgggagaacaccacccagtgcacctacaacagcagcctgcggaacagca ccgtcgtgagagagaacgccatcagcttcaactttttccagagctacaaccagtactacgtgtt ccacatgcccagatgcctgtttgccggccctctggccgagcagttcctgaaccaggtggacctg accgagacactggaaagataccagcagcggctgaatacctacgccctggtgtccaaggacctgg ccagctaccggtcctttagccagcagctcaaggctcaggatagcctcggcgagcagcctaccac cgtgccccctcccatcgacctgagcatcccccacgtgtggatgcctccccagaccacccctcac ggctggaccgagagccacaccacctccggcctgcacagaccccacttcaaccagacctgcatcc tgttcgacggccacgacctgctgtttagcaccgtgaccccctgcctgcaccagggcttctacct gatcgacgagctgagatacgtgaagatcaccctgaccgaggatttcttcgtggtcaccgtgtcc atcgacgacgacacccccatgctgctgatcttcggccacctgcccagagtgctgttcaaggccc cctaccagcgggacaacttcatcctgcggcagaccgagaagcacgagctgctggtgctggtcaa gaaggaccagctgaaccggcactcctacctgaaggaccccgacttcctggacgccgccctggac ttcaactacctggacctgagcgccctgctgagaaacagcttccacagatacgccgtggacgtgc tgaagtccggacggtgccagatgctcgatcggcggaccgtggagatggccttcgcctatgccct cgccctgttcgccgctgccagacaggaagaggctggcgcccaggtgtcagtgcccagagccctg gatagacaggccgccctgctgcagatccaggaattcatgatcacctgcctgagccagacccccc ctagaaccaccctgctgctgtaccccacagccgtggatctggccaagagggccctgtggacccc caaccagatcaccgacatcacaagcctcgtgcggctcgtgtacatcctgagcaagcagaaccag cagcacctgatcccccagtgggccctgagacagatcgccgacttcgccctgaagctgcacaaga cccatctggccagctttctgagcgccttcgccaggcaggaactgtacctgatgggcagcctggt ccacagcatgctggtgcataccaccgagcggcgggagatcttcatcgtggagacaggcctgtgt agcctggccgagctgtcccactttacccagctgctggcccaccctcaccacgagtacctgagcg acctgtacaccccctgcagcagcagcggcagacgggaccacagcctggaacggctgaccagact gttccccgatgccaccgtgcctgctacagtgcctgccgccctgtccatcctgtccaccatgcag cccagcaccctggaaaccttccccgacctgttctgcctgcccctgggcgagagctttagcgccc tgaccgtgtccgagcacgtgtcctacatcgtgaccaatcagtacctgatcaagggcatcagcta ccccgtgtccaccacagtcgtgggccagagcctgatcatcacccagaccgacagccagaccaag tgcgagctgacccggaacatgcacaccacacacagcatcaccgtggccctgaacatcagcctgg aaaactgcgctttctgtcagtctgccctgctggaatacgacgatacccagggcgtgatcaacat catgtacatgcacgacagcgacgacgtgctgttcgccctggacccctacaacgaggtggtggtg tccagcccccggacccactacctgatgctgctgaagaacggcaccgtgctggaagtgaccgacg tggtggtggacgccaccgacagcagactgctgatgatgagcgtgtacgccctgagcgccatcat cggcatctacctgctgtaccggatgctgaaaacctgctgataatctagacggcgcgcccaccca gcggccgcctataactctctacggctaacctgaatggactacgacatagtctagtcgacgccac catgtgcagaaggcccgactgcggcttcagcttcagccctggacccgtgatcctgctgtggtgc tgcctgctgctgcctatcgtgtcctctgccgccgtgtctgtggcccctacagccgccgagaagg tgccagccgagtgccccgagctgaccagaagatgcctgctgggcgaggtgttcgagggcgacaa gtacgagagctggctgcggcccctggtcaacgtgaccggcagagatggccccctgagccagctg atccggtacagacccgtgacccccgaggccgccaatagcgtgctgctggacgaggccttcctgg ataccctggccctgctgtacaacaaccccgaccagctgagagccctgctgaccctgctgtccag cgacaccgcccccagatggatgaccgtgatgcggggctacagcgagtgtggagatggcagccct gccgtgtacacctgcgtggacgacctgtgcagaggctacgacctgaccagactgagctacggcc ggtccatcttcacagagcacgtgctgggcttcgagctggtgccccccagcctgttcaacgtggt ggtggccatccggaacgaggccaccagaaccaacagagccgtgcggctgcctgtgtctacagcc gctgcacctgagggcatcacactgttctacggcctgtacaacgccgtgaaagagttctgcctcc ggcaccagctggatccccccctgctgagacacctggacaagtactacgccggcctgcccccaga gctgaagcagaccagagtgaacctgcccgcccacagcagatatggccctcaggccgtggacgcc agatgataatctagacggcgcgcccacccaatcgatgtacttccgaggaactcacgtgcataat gcatcaggctggtacattagatccccgcttaccgcgggcaatatagcaacactaaaaactcgat gtacttccgaggaagcgcagtgcataatgctgcgcagtgttgccacataaccactatattaacc atttatctagcggacgccaaaaactcaatgtatttctgaggaagcgtggtgcataatgccacgc agcgtctgcataacttttattatttcttttattaatcaacaaaattttgtttttaacatttcaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagggtcggcatggcatctccacctcct cgcggtccgacctgggcatccgaaggaggacgcacgtccactcggatggctaagggagagccac gagctcctgtttaaaccagctccaattcgccctatagtgagtcgtattacgcgcgctcactggc cgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagca catccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagt tgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggt ggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttc ccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttag ggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacg tagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaat agtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttat aagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgc gaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaa cccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctg ataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgccctt attcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaa aagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaa gatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgcta tgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactatt ctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagt aagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgaca acgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgcc ttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcc tgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccgg caacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttc cggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgc agcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaac tgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaag gatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttc cactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcg taatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaaga gctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttctt ctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctc tgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactc aagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagccc agcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgcca cgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcg cacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctc tgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagca acgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgtt atcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagc cgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgc ctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagc gggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacac tttatgctcccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaaca gctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctgggtac cggcgcca vetor modificado pVCR de gH sol-SGP gL-SGP gO cgcgtcggctacaattaatacataaccttatgtatcatacacatacgatttaggtgacactata gatgggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttg acatcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcagtttgaggtaga agccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaa ctgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgca gaatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacag attgtataagtatgcaactaagctgaagaaaaactgtaaggaaataactgataaggaattggac aagaaaatgaaggagctcgccgccgtcatgagcgaccctgacctggaaactgagactatgtgcc tccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggt tgacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggc tttgacaccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaact gggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgttatggagcg gtcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaacaatgttctattc tctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctg tatttcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacgg gtacgtcgttaaaagaatagctatcagtccaggcctgtatgggaagccttcaggctatgctgct acgatgcaccgcgagggattcttgtgctgcaaagtgacagacacattgaacggggagagggtct cttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatactggcaac agatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaac ggtcgcacccagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcat ttgctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacgaga tagacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatctatttataag cgcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcccagga taggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaa ggagccgtcacctctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggct aaggaggtgcgtgaagccgaggagttgcgcgcagctctaccacctttggcagctgatgttgagg agcccactctggaagccgatgtagacttgatgttacaagaggctggggccggctcagtggagac acctcgtggcttgataaaggttaccagctacgctggcgaggacaagatcggctcttacgctgtg ctttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaag tcatagtgataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagt agtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtgccacc attgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccacacatggaggag cgctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacct gtacgacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggc gagctggtggatcctcccttccatgaattcgcctacgagagtctgagaacacgaccagccgctc cttaccaagtaccaaccataggggtgtatggcgtgccaggatcaggcaagtctggcatcattaa aagcgcagtcaccaaaaaagatctagtggtgagcgccaagaaagaaaactgtgcagaaattata agggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttga atggatgcaaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtac tctcagagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaacag tgcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcacacaagtct tccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgtttta cgacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagt accaaacctaagcaggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaa tagattacaaaggcaacgaaataatgacggcagctgcctctcaagggctgacccgtaaaggtgt gtatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacccacctcagaacatgtgaac gtcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggataa aaacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagca tgatgccatcatgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggca aacgtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgaccactg aacaatggaacactgtggattattttgaaacggacaaagctcactcagcagagatagtattgaa ccaactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgtt ccgttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaata aagaagtggtccgtcagctctctcgcaggtacccacaactgcctcgggcagttgccactggaag agtctatgacatgaacactggtacactgcgcaattatgatccgcgcataaacctagtacctgta aacagaagactgcctcatgctttagtcctccaccataatgaacacccacagagtgacttttctt cattcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccagg caaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggc atcccaggtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaatacc atcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagcttgtct gcatctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagcgaa agcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcac ttgaagagacggaagttctgtttgtattcattgggtacgatcgcaaggcccgtacgcacaatcc ttacaagctttcatcaaccttgaccaacatttatacaggttccagactccacgaagccggatgt gcaccctcatatcatgtggtgcgaggggatattgccacggccaccgaaggagtgattataaatg ctgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccgga aagcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacat atcattcatgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttgg cagaggcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattcc actgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaaccatttg ctgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaa tgactctcaaggaagcagtggctaggagagaagcagtggaggagatatgcatatccgacgactc ttcagtgacagaacctgatgcagagctggtgagggtgcatccgaagagttctttggctggaagg aagggctacagcacaagcgatggcaaaactttctcatatttggaagggaccaagtttcaccagg cggccaaggatatagcagaaattaatgccatgtggcccgttgcaacggaggccaatgagcaggt atgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcg gaagcctcctcaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagag tacagcgcctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaa gtatagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaagtg cctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccggagc catcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatga gaccaggactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttg ctgtcagatggcccgacccaccaggtgctgcaagtcgaggcagacattcacgggccgccctctg tatctagctcatcctggtccattcctcatgcatccgactttgatgtggacagtttatccatact tgacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactcttacttc gcaaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctc cacatcccgctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagagggat cacgggagaaaccgtgggatacgcggttacacacaatagcgagggcttcttgctatgcaaagtt actgacacagtaaaaggagaacgggtatcgttccctgtgtgcacgtacatcccggccaccataa actcgagaaccagcctggtctccaacccgccaggcgtaaatagggtgattacaagagaggagtt tgaggcgttcgtagcacaacaacaatgacggtttgatgcgggtgcatacatcttttcctccgac accggtcaagggcatttacaacaaaaatcagtaaggcaaacggtgctatccgaagtggtgttgg agaggaccgaattggagatttcgtatgccccgcgcctcgaccaagaaaaagaagaattactacg caagaaattacagttaaatcccacacctgctaacagaagcagataccagtccaggaaggtggag aacatgaaagccataacagctagacgtattctgcaaggcctagggcattatttgaaggcagaag gaaaagtggagtgctaccgaaccctgcatcctgttcctttgtattcatctagtgtgaaccgtgc cttttcaagccccaaggtcgcagtggaagcctgtaacgccatgttgaaagagaactttccgact gtggcttcttactgtattattccagagtacgatgcctatttggacatggttgacggagcttcat gctgcttagacactgccagtttttgccctgcaaagctgcgcagctttccaaagaaacactccta tttggaacccacaatacgatcggcagtgccttcagcgatccagaacacgctccagaacgtcctg gcagctgccacaaaaagaaattgcaatgtcacgcaaatgagagaattgcccgtattggattcgg cggcctttaatgtggaatgcttcaagaaatatgcgtgtaataatgaatattgggaaacgtttaa agaaaaccccatcaggcttactgaagaaaacgtggtaaattacattaccaaattaaaaggacca aaagctgctgctctttttgcgaagacacataatttgaatatgttgcaggacataccaatggaca ggtttgtaatggacttaaagagagacgtgaaagtgactccaggaacaaaacatactgaagaacg gcccaaggtacaggtgatccaggctgccgatccgctagcaacagcgtatctgtgcggaatccac cgagagctggttaggagattaaatgcggtcctgcttccgaacattcatacactgtttgatatgt cggctgaagactttgacgctattatagccgagcacttccagcctggggattgtgttctggaaac tgacatcgcgtcgtttgataaaagtgaggacgacgccatggctctgaccgcgttaatgattctg gaagacttaggtgtggacgcagagctgttgacgctgattgaggcggctttcggcgaaatttcat caatacatttgcccactaaaactaaatttaaattcggagccatgatgaaatctggaatgttcct cacactgtttgtgaacacagtcattaacattgtaatcgcaagcagagtgttgagagaacggcta accggatcaccatgtgcagcattcattggagatgacaatatcgtgaaaggagtcaaatcggaca aattaatggcagacaggtgcgccacctggttgaatatggaagtcaagattatagatgctgtggt gggcgagaaagcgccttatttctgtggagggtttattttgtgtgactccgtgaccggcacagcg tgccgtgtggcagaccccctaaaaaggctgtttaagcttggcaaacctctggcagcagacgatg aacatgatgatgacaggagaagggcattgcatgaagagtcaacacgctggaaccgagtgggtat tctttcagagctgtgcaaggcagtagaatcaaggtatgaaaccgtaggaacttccatcatagtt atggccatgactactctagctagcagtgttaaatcattcagctacctgagaggggcccctataa ctctctacggctaacctgaatggactacgacatagtctagtcgacgccaccatgaggcctggcc tgccctcctacctgatcatcctggccgtgtgcctgttcagccacctgctgtccagcagatacgg cgccgaggccgtgagcgagcccctggacaaggctttccacctgctgctgaacacctacggcaga cccatccggtttctgcgggagaacaccacccagtgcacctacaacagcagcctgcggaacagca ccgtcgtgagagagaacgccatcagcttcaactttttccagagctacaaccagtactacgtgtt ccacatgcccagatgcctgtttgccggccctctggccgagcagttcctgaaccaggtggacctg accgagacactggaaagataccagcagcggctgaatacctacgccctggtgtccaaggacctgg ccagctaccggtcctttagccagcagctcaaggctcaggatagcctcggcgagcagcctaccac cgtgccccctcccatcgacctgagcatcccccacgtgtggatgcctccccagaccacccctcac ggctggaccgagagccacaccacctccggcctgcacagaccccacttcaaccagacctgcatcc tgttcgacggccacgacctgctgtttagcaccgtgaccccctgcctgcaccagggcttctacct gatcgacgagctgagatacgtgaagatcaccctgaccgaggatttcttcgtggtcaccgtgtcc atcgacgacgacacccccatgctgctgatcttcggccacctgcccagagtgctgttcaaggccc cctaccagcgggacaacttcatcctgcggcagaccgagaagcacgagctgctggtgctggtcaa gaaggaccagctgaaccggcactcctacctgaaggaccccgacttcctggacgccgccctggac ttcaactacctggacctgagcgccctgctgagaaacagcttccacagatacgccgtggacgtgc tgaagtccggacggtgccagatgctcgatcggcggaccgtggagatggccttcgcctatgccct cgccctgttcgccgctgccagacaggaagaggctggcgcccaggtgtcagtgcccagagccctg gatagacaggccgccctgctgcagatccaggaattcatgatcacctgcctgagccagacccccc ctagaaccaccctgctgctgtaccccacagccgtggatctggccaagagggccctgtggacccc caaccagatcaccgacatcacaagcctcgtgcggctcgtgtacatcctgagcaagcagaaccag cagcacctgatcccccagtgggccctgagacagatcgccgacttcgccctgaagctgcacaaga cccatctggccagctttctgagcgccttcgccaggcaggaactgtacctgatgggcagcctggt ccacagcatgctggtgcataccaccgagcggcgggagatcttcatcgtggagacaggcctgtgt agcctggccgagctgtcccactttacccagctgctggcccaccctcaccacgagtacctgagcg acctgtacaccccctgcagcagcagcggcagacgggaccacagcctggaacggctgaccagact gttccccgatgccaccgtgcctgctacagtgcctgccgccctgtccatcctgtccaccatgcag cccagcaccctggaaaccttccccgacctgttctgcctgcccctgggcgagagctttagcgccc tgaccgtgtccgagcacgtgtcctacatcgtgaccaatcagtacctgatcaagggcatcagcta ccccgtgtccaccacagtcgtgggccagagcctgatcatcacccagaccgacagccagaccaag tgcgagctgacccggaacatgcacaccacacacagcatcaccgtggccctgaacatcagcctgg aaaactgcgctttctgtcagtctgccctgctggaatacgacgatacccagggcgtgatcaacat catgtacatgcacgacagcgacgacgtgctgttcgccctggacccctacaacgaggtggtggtg tccagcccccggacccactacctgatgctgctgaagaacggcaccgtgctggaagtgaccgacg tggtggtggacgccaccgactgataatctagacggcgcgcccacccagcggccgcctataactc tctacggctaacctgaatggactacgacatagtctagtcgacgccaccatgtgcagaaggcccg actgcggcttcagcttcagccctggacccgtgatcctgctgtggtgctgcctgctgctgcctat cgtgtcctctgccgccgtgtctgtggcccctacagccgccgagaaggtgccagccgagtgcccc gagctgaccagaagatgcctgctgggcgaggtgttcgagggcgacaagtacgagagctggctgc ggcccctggtcaacgtgaccggcagagatggccccctgagccagctgatccggtacagacccgt gacccccgaggccgccaatagcgtgctgctggacgaggccttcctggataccctggccctgctg tacaacaaccccgaccagctgagagccctgctgaccctgctgtccagcgacaccgcccccagat ggatgaccgtgatgcggggctacagcgagtgtggagatggcagccctgccgtgtacacctgcgt ggacgacctgtgcagaggctacgacctgaccagactgagctacggccggtccatcttcacagag cacgtgctgggcttcgagctggtgccccccagcctgttcaacgtggtggtggccatccggaacg aggccaccagaaccaacagagccgtgcggctgcctgtgtctacagccgctgcacctgagggcat cacactgttctacggcctgtacaacgccgtgaaagagttctgcctccggcaccagctggatccc cccctgctgagacacctggacaagtactacgccggcctgcccccagagctgaagcagaccagag tgaacctgcccgcccacagcagatatggccctcaggccgtggacgccagatgataatctagacg gcgcgcccacccaatcgatctataactctctacggctaacctgaatggactacgacatagtcta gtcgacgccaccatgggcaagaaagaaatgatcatggtcaagggcatccccaagatcatgctgc tgattagcatcacctttctgctgctgtccctgatcaactgcaacgtgctggtcaacagccgggg caccagaagatcctggccctacaccgtgctgtcctaccggggcaaagagatcctgaagaagcag aaagaggacatcctgaagcggctgatgagcaccagcagcgacggctaccggttcctgatgtacc ccagccagcagaaattccacgccatcgtgatcagcatggacaagttcccccaggactacatcct ggccggacccatccggaacgacagcatcacccacatgtggttcgacttctacagcacccagctg cggaagcccgccaaatacgtgtacagcgagtacaaccacaccgcccacaagatcaccctgaggc ctcccccttgtggcaccgtgcccagcatgaactgcctgagcgagatgctgaacgtgtccaagcg gaacgacaccggcgagaagggctgcggcaacttcaccaccttcaaccccatgttcttcaacgtg ccccggtggaacaccaagctgtacatcggcagcaacaaagtgaacgtggacagccagaccatct actttctgggcctgaccgccctgctgctgagatacgcccagcggaactgcacccggtccttcta cctggtcaacgccatgagccggaacctgttccgggtgcccaagtacatcaacggcaccaagctg aagaacaccatgcggaagctgaagcggaagcaggccctggtcaaagagcagccccagaagaaga acaagaagtcccagagcaccaccaccccctacctgagctacaccacctccaccgccttcaacgt gaccaccaacgtgacctacagcgccacagccgccgtgaccagagtggccacaagcaccaccggc taccggcccgacagcaactttatgaagtccatcatggccacccagctgagagatctggccacct gggtgtacaccaccctgcggtacagaaacgagcccttctgcaagcccgaccggaacagaaccgc cgtgagcgagttcatgaagaatacccacgtgctgatcagaaacgagacaccctacaccatctac ggcaccctggacatgagcagcctgtactacaacgagacaatgagcgtggagaacgagacagcca gcgacaacaacgaaaccacccccacctcccccagcacccggttccagcggaccttcatcgaccc cctgtgggactacctggacagcctgctgttcctggacaagatccggaacttcagcctgcagctg cccgcctacggcaatctgaccccccctgagcacagaagggccgccaacctgagcaccctgaaca gcctgtggtggtggagccagtgataatctagacggcgcgcccacccaccgcgggcaatatagca acactaaaaactcgatgtacttccgaggaagcgcagtgcataatgctgcgcagtgttgccacat aaccactatattaaccatttatctagcggacgccaaaaactcaatgtatttctgaggaagcgtg gtgcataatgccacgcagcgtctgcataacttttattatttcttttattaatcaacaaaatttt gtttttaacatttcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagggtcggcat ggcatctccacctcctcgcggtccgacctgggcatccgaaggaggacgcacgtccactcggatg gctaagggagagccacgagctcctgtttaaaccagctccaattcgccctatagtgagtcgtatt acgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaact taatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgat cgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaa gcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgc tcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaat cgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatt agggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttgga gtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtc tattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgattt aacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcgg ggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctca tgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaaca tttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaa acgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactgg atctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcac ttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggt cgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatctta cggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggc caacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggg gatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagc gtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactact tactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccactt ctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggt ctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacac gacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactg attaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttc atttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatccctta acgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagat cctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggttt gtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagat accaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccg cctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtc ttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacgggggg ttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgag ctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcaggg tcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgt cgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagccta tggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcaca tgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctga taccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgc ccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggt ttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggc accccaggctttacactttatgctcccggctcgtatgttgtgtggaattgtgagcggataacaa tttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctgggtaccggcgcca vetor modificado pVCR de gH FL-SGP gL-SGP gO cgcgtcggctacaattaatacataaccttatgtatcatacacatacgatttaggtgacactata gatgggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttg acatcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcagtttgaggtaga agccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaa ctgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgca gaatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacag attgtataagtatgcaactaagctgaagaaaaactgtaaggaaataactgataaggaattggac aagaaaatgaaggagctcgccgccgtcatgagcgaccctgacctggaaactgagactatgtgcc tccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggt tgacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggc tttgacaccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaact gggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgttatggagcg gtcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaacaatgttctattc tctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctg tatttcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacgg gtacgtcgttaaaagaatagctatcagtccaggcctgtatgggaagccttcaggctatgctgct acgatgcaccgcgagggattcttgtgctgcaaagtgacagacacattgaacggggagagggtct cttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatactggcaac agatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaac ggtcgcacccagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcat ttgctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacgaga tagacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatctatttataag cgcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcccagga taggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaa ggagccgtcacctctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggct aaggaggtgcgtgaagccgaggagttgcgcgcagctctaccacctttggcagctgatgttgagg agcccactctggaagccgatgtagacttgatgttacaagaggctggggccggctcagtggagac acctcgtggcttgataaaggttaccagctacgctggcgaggacaagatcggctcttacgctgtg ctttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaag tcatagtgataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagt agtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtgccacc attgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccacacatggaggag cgctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacct gtacgacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggc gagctggtggatcctcccttccatgaattcgcctacgagagtctgagaacacgaccagccgctc cttaccaagtaccaaccataggggtgtatggcgtgccaggatcaggcaagtctggcatcattaa aagcgcagtcaccaaaaaagatctagtggtgagcgccaagaaagaaaactgtgcagaaattata agggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttga atggatgcaaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtac tctcagagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaacag tgcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcacacaagtct tccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgtttta cgacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagt accaaacctaagcaggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaa tagattacaaaggcaacgaaataatgacggcagctgcctctcaagggctgacccgtaaaggtgt gtatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacccacctcagaacatgtgaac gtcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggataa aaacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagca tgatgccatcatgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggca aacgtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgaccactg aacaatggaacactgtggattattttgaaacggacaaagctcactcagcagagatagtattgaa ccaactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgtt ccgttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaata aagaagtggtccgtcagctctctcgcaggtacccacaactgcctcgggcagttgccactggaag agtctatgacatgaacactggtacactgcgcaattatgatccgcgcataaacctagtacctgta aacagaagactgcctcatgctttagtcctccaccataatgaacacccacagagtgacttttctt cattcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccagg caaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggc atcccaggtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaatacc atcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagcttgtct gcatctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagcgaa agcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcac ttgaagagacggaagttctgtttgtattcattgggtacgatcgcaaggcccgtacgcacaatcc ttacaagctttcatcaaccttgaccaacatttatacaggttccagactccacgaagccggatgt gcaccctcatatcatgtggtgcgaggggatattgccacggccaccgaaggagtgattataaatg ctgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccgga aagcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacat atcattcatgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttgg cagaggcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattcc actgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaaccatttg ctgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaa tgactctcaaggaagcagtggctaggagagaagcagtggaggagatatgcatatccgacgactc ttcagtgacagaacctgatgcagagctggtgagggtgcatccgaagagttctttggctggaagg aagggctacagcacaagcgatggcaaaactttctcatatttggaagggaccaagtttcaccagg cggccaaggatatagcagaaattaatgccatgtggcccgttgcaacggaggccaatgagcaggt atgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcg gaagcctcctcaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagag tacagcgcctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaa gtatagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaagtg cctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccggagc catcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatga gaccaggactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttg ctgtcagatggcccgacccaccaggtgctgcaagtcgaggcagacattcacgggccgccctctg tatctagctcatcctggtccattcctcatgcatccgactttgatgtggacagtttatccatact tgacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactcttacttc gcaaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctc cacatcccgctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagagggat cacgggagaaaccgtgggatacgcggttacacacaatagcgagggcttcttgctatgcaaagtt actgacacagtaaaaggagaacgggtatcgttccctgtgtgcacgtacatcccggccaccataa actcgagaaccagcctggtctccaacccgccaggcgtaaatagggtgattacaagagaggagtt tgaggcgttcgtagcacaacaacaatgacggtttgatgcgggtgcatacatcttttcctccgac accggtcaagggcatttacaacaaaaatcagtaaggcaaacggtgctatccgaagtggtgttgg agaggaccgaattggagatttcgtatgccccgcgcctcgaccaagaaaaagaagaattactacg caagaaattacagttaaatcccacacctgctaacagaagcagataccagtccaggaaggtggag aacatgaaagccataacagctagacgtattctgcaaggcctagggcattatttgaaggcagaag gaaaagtggagtgctaccgaaccctgcatcctgttcctttgtattcatctagtgtgaaccgtgc cttttcaagccccaaggtcgcagtggaagcctgtaacgccatgttgaaagagaactttccgact gtggcttcttactgtattattccagagtacgatgcctatttggacatggttgacggagcttcat gctgcttagacactgccagtttttgccctgcaaagctgcgcagctttccaaagaaacactccta tttggaacccacaatacgatcggcagtgccttcagcgatccagaacacgctccagaacgtcctg gcagctgccacaaaaagaaattgcaatgtcacgcaaatgagagaattgcccgtattggattcgg cggcctttaatgtggaatgcttcaagaaatatgcgtgtaataatgaatattgggaaacgtttaa agaaaaccccatcaggcttactgaagaaaacgtggtaaattacattaccaaattaaaaggacca aaagctgctgctctttttgcgaagacacataatttgaatatgttgcaggacataccaatggaca ggtttgtaatggacttaaagagagacgtgaaagtgactccaggaacaaaacatactgaagaacg gcccaaggtacaggtgatccaggctgccgatccgctagcaacagcgtatctgtgcggaatccac cgagagctggttaggagattaaatgcggtcctgcttccgaacattcatacactgtttgatatgt cggctgaagactttgacgctattatagccgagcacttccagcctggggattgtgttctggaaac tgacatcgcgtcgtttgataaaagtgaggacgacgccatggctctgaccgcgttaatgattctg gaagacttaggtgtggacgcagagctgttgacgctgattgaggcggctttcggcgaaatttcat caatacatttgcccactaaaactaaatttaaattcggagccatgatgaaatctggaatgttcct cacactgtttgtgaacacagtcattaacattgtaatcgcaagcagagtgttgagagaacggcta accggatcaccatgtgcagcattcattggagatgacaatatcgtgaaaggagtcaaatcggaca aattaatggcagacaggtgcgccacctggttgaatatggaagtcaagattatagatgctgtggt gggcgagaaagcgccttatttctgtggagggtttattttgtgtgactccgtgaccggcacagcg tgccgtgtggcagaccccctaaaaaggctgtttaagcttggcaaacctctggcagcagacgatg aacatgatgatgacaggagaagggcattgcatgaagagtcaacacgctggaaccgagtgggtat tctttcagagctgtgcaaggcagtagaatcaaggtatgaaaccgtaggaacttccatcatagtt atggccatgactactctagctagcagtgttaaatcattcagctacctgagaggggcccctataa ctctctacggctaacctgaatggactacgacatagtctagtcgacgccaccatgaggcctggcc tgccctcctacctgatcatcctggccgtgtgcctgttcagccacctgctgtccagcagatacgg cgccgaggccgtgagcgagcccctggacaaggctttccacctgctgctgaacacctacggcaga cccatccggtttctgcgggagaacaccacccagtgcacctacaacagcagcctgcggaacagca ccgtcgtgagagagaacgccatcagcttcaactttttccagagctacaaccagtactacgtgtt ccacatgcccagatgcctgtttgccggccctctggccgagcagttcctgaaccaggtggacctg accgagacactggaaagataccagcagcggctgaatacctacgccctggtgtccaaggacctgg ccagctaccggtcctttagccagcagctcaaggctcaggatagcctcggcgagcagcctaccac cgtgccccctcccatcgacctgagcatcccccacgtgtggatgcctccccagaccacccctcac ggctggaccgagagccacaccacctccggcctgcacagaccccacttcaaccagacctgcatcc tgttcgacggccacgacctgctgtttagcaccgtgaccccctgcctgcaccagggcttctacct gatcgacgagctgagatacgtgaagatcaccctgaccgaggatttcttcgtggtcaccgtgtcc atcgacgacgacacccccatgctgctgatcttcggccacctgcccagagtgctgttcaaggccc cctaccagcgggacaacttcatcctgcggcagaccgagaagcacgagctgctggtgctggtcaa gaaggaccagctgaaccggcactcctacctgaaggaccccgacttcctggacgccgccctggac ttcaactacctggacctgagcgccctgctgagaaacagcttccacagatacgccgtggacgtgc tgaagtccggacggtgccagatgctcgatcggcggaccgtggagatggccttcgcctatgccct cgccctgttcgccgctgccagacaggaagaggctggcgcccaggtgtcagtgcccagagccctg gatagacaggccgccctgctgcagatccaggaattcatgatcacctgcctgagccagacccccc ctagaaccaccctgctgctgtaccccacagccgtggatctggccaagagggccctgtggacccc caaccagatcaccgacatcacaagcctcgtgcggctcgtgtacatcctgagcaagcagaaccag cagcacctgatcccccagtgggccctgagacagatcgccgacttcgccctgaagctgcacaaga cccatctggccagctttctgagcgccttcgccaggcaggaactgtacctgatgggcagcctggt ccacagcatgctggtgcataccaccgagcggcgggagatcttcatcgtggagacaggcctgtgt agcctggccgagctgtcccactttacccagctgctggcccaccctcaccacgagtacctgagcg acctgtacaccccctgcagcagcagcggcagacgggaccacagcctggaacggctgaccagact gttccccgatgccaccgtgcctgctacagtgcctgccgccctgtccatcctgtccaccatgcag cccagcaccctggaaaccttccccgacctgttctgcctgcccctgggcgagagctttagcgccc tgaccgtgtccgagcacgtgtcctacatcgtgaccaatcagtacctgatcaagggcatcagcta ccccgtgtccaccacagtcgtgggccagagcctgatcatcacccagaccgacagccagaccaag tgcgagctgacccggaacatgcacaccacacacagcatcaccgtggccctgaacatcagcctgg aaaactgcgctttctgtcagtctgccctgctggaatacgacgatacccagggcgtgatcaacat catgtacatgcacgacagcgacgacgtgctgttcgccctggacccctacaacgaggtggtggtg tccagcccccggacccactacctgatgctgctgaagaacggcaccgtgctggaagtgaccgacg tggtggtggacgccaccgacagcagactgctgatgatgagcgtgtacgccctgagcgccatcat cggcatctacctgctgtaccggatgctgaaaacctgctgataatctagacggcgcgcccaccca gcggccgcctataactctctacggctaacctgaatggactacgacatagtctagtcgacgccac catgtgcagaaggcccgactgcggcttcagcttcagccctggacccgtgatcctgctgtggtgc tgcctgctgctgcctatcgtgtcctctgccgccgtgtctgtggcccctacagccgccgagaagg tgccagccgagtgccccgagctgaccagaagatgcctgctgggcgaggtgttcgagggcgacaa gtacgagagctggctgcggcccctggtcaacgtgaccggcagagatggccccctgagccagctg atccggtacagacccgtgacccccgaggccgccaatagcgtgctgctggacgaggccttcctgg ataccctggccctgctgtacaacaaccccgaccagctgagagccctgctgaccctgctgtccag cgacaccgcccccagatggatgaccgtgatgcggggctacagcgagtgtggagatggcagccct gccgtgtacacctgcgtggacgacctgtgcagaggctacgacctgaccagactgagctacggcc ggtccatcttcacagagcacgtgctgggcttcgagctggtgccccccagcctgttcaacgtggt ggtggccatccggaacgaggccaccagaaccaacagagccgtgcggctgcctgtgtctacagcc gctgcacctgagggcatcacactgttctacggcctgtacaacgccgtgaaagagttctgcctcc ggcaccagctggatccccccctgctgagacacctggacaagtactacgccggcctgcccccaga gctgaagcagaccagagtgaacctgcccgcccacagcagatatggccctcaggccgtggacgcc agatgataatctagacggcgcgcccacccaatcgatctataactctctacggctaacctgaatg gactacgacatagtctagtcgacgccaccatgggcaagaaagaaatgatcatggtcaagggcat ccccaagatcatgctgctgattagcatcacctttctgctgctgtccctgatcaactgcaacgtg ctggtcaacagccggggcaccagaagatcctggccctacaccgtgctgtcctaccggggcaaag agatcctgaagaagcagaaagaggacatcctgaagcggctgatgagcaccagcagcgacggcta ccggttcctgatgtaccccagccagcagaaattccacgccatcgtgatcagcatggacaagttc ccccaggactacatcctggccggacccatccggaacgacagcatcacccacatgtggttcgact tctacagcacccagctgcggaagcccgccaaatacgtgtacagcgagtacaaccacaccgccca caagatcaccctgaggcctcccccttgtggcaccgtgcccagcatgaactgcctgagcgagatg ctgaacgtgtccaagcggaacgacaccggcgagaagggctgcggcaacttcaccaccttcaacc ccatgttcttcaacgtgccccggtggaacaccaagctgtacatcggcagcaacaaagtgaacgt ggacagccagaccatctactttctgggcctgaccgccctgctgctgagatacgcccagcggaac tgcacccggtccttctacctggtcaacgccatgagccggaacctgttccgggtgcccaagtaca tcaacggcaccaagctgaagaacaccatgcggaagctgaagcggaagcaggccctggtcaaaga gcagccccagaagaagaacaagaagtcccagagcaccaccaccccctacctgagctacaccacc tccaccgccttcaacgtgaccaccaacgtgacctacagcgccacagccgccgtgaccagagtgg ccacaagcaccaccggctaccggcccgacagcaactttatgaagtccatcatggccacccagct gagagatctggccacctgggtgtacaccaccctgcggtacagaaacgagcccttctgcaagccc gaccggaacagaaccgccgtgagcgagttcatgaagaatacccacgtgctgatcagaaacgaga caccctacaccatctacggcaccctggacatgagcagcctgtactacaacgagacaatgagcgt ggagaacgagacagccagcgacaacaacgaaaccacccccacctcccccagcacccggttccag cggaccttcatcgaccccctgtgggactacctggacagcctgctgttcctggacaagatccgga acttcagcctgcagctgcccgcctacggcaatctgaccccccctgagcacagaagggccgccaa cctgagcaccctgaacagcctgtggtggtggagccagtgataatctagacggcgcgcccaccca ccgcgggcaatatagcaacactaaaaactcgatgtacttccgaggaagcgcagtgcataatgct gcgcagtgttgccacataaccactatattaaccatttatctagcggacgccaaaaactcaatgt atttctgaggaagcgtggtgcataatgccacgcagcgtctgcataacttttattatttctttta ttaatcaacaaaattttgtttttaacatttcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaagggtcggcatggcatctccacctcctcgcggtccgacctgggcatccgaaggaggac gcacgtccactcggatggctaagggagagccacgagctcctgtttaaaccagctccaattcgcc ctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaac cctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcg aagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgcc ctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgcc agcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttc cccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcga ccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggttttt cgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacac tcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggtt aaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatt taggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattc aaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaag agtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctg tttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagt gggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgt tttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccg ggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagt cacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatg agtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctt ttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagc cataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaacta ttaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggata aagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctgg agccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgt atcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctg agataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatacttta gattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctc atgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatca aaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccacc gctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggc ttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttca agaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccag tggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcgg tcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactga gatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggta tccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctgg tatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgt caggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttg ctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattacc gcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcg aggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatg cagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagt tagctcactcattaggcaccccaggctttacactttatgctcccggctcgtatgttgtgtggaa ttgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaat taaccctcactaaagggaacaaaagctgggtaccggcgcca Vetor A526: SGP-gH-SGP-gL-SGP-UL128-2A-UL130-2Amod-UL131 ATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGA GGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTC ACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGG ACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCA TTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAA AACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACC CTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGT TTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTC GCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACT CTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGA GCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCT GTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACT TACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAG AATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTC TTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAG CTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCT GGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTAC CTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAA GGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAAC ATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTG CCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACA AGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGA GGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTG GAAGCCGATGTAGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAA AGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAA GAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAA GGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGG ACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCA CCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAG CACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAG GGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGC CGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAA AGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACG TCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACA CCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCC ATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCC TGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAA ATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACT AAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAG GGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCT GACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAA CATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGA TAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGA TGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGT TGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTG TGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTT TGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGG GATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACC CACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGA TCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACAC CCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGT TGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGA TTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATAC CATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATC TGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGG TGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTT CTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGA CCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGA TATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTG TGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGAC TGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGA AGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCA GTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACC ATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAAT GACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTG ACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCA CAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGA AATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGC ATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTT GCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTAC TGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCT ATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAG ACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAAC CGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGT TTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTAT CTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCT GGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAG TTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAA GAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAA TAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGA ACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAG CACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACA ACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTAT GCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTA ACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGG CCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTAT TCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAG AGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGG AGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCC TATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAG CTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAA TGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGG CTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGA AGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGT GAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCG CTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGA ACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGG GGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCG TTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAA TTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCT CACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGA TCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAG ACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTA TTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAA AGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGC ATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTA TGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGC TACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAA GATGAGGCCTGGCCTGCCCTCCTACCTGATCATCCTGGCCGTGTGCCTGTTCAGCCACCTGCTGTCCAGC AGATACGGCGCCGAGGCCGTGAGCGAGCCCCTGGACAAGGCTTTCCACCTGCTGCTGAACACCTACGGCA GACCCATCCGGTTTCTGCGGGAGAACACCACCCAGTGCACCTACAACAGCAGCCTGCGGAACAGCACCGT CGTGAGAGAGAACGCCATCAGCTTCAACTTTTTCCAGAGCTACAACCAGTACTACGTGTTCCACATGCCC AGATGCCTGTTTGCCGGCCCTCTGGCCGAGCAGTTCCTGAACCAGGTGGACCTGACCGAGACACTGGAAA GATACCAGCAGCGGCTGAATACCTACGCCCTGGTGTCCAAGGACCTGGCCAGCTACCGGTCCTTTAGCCA GCAGCTCAAGGCTCAGGATAGCCTCGGCGAGCAGCCTACCACCGTGCCCCCTCCCATCGACCTGAGCATC CCCCACGTGTGGATGCCTCCCCAGACCACCCCTCACGGCTGGACCGAGAGCCACACCACCTCCGGCCTGC ACAGACCCCACTTCAACCAGACCTGCATCCTGTTCGACGGCCACGACCTGCTGTTTAGCACCGTGACCCC CTGCCTGCACCAGGGCTTCTACCTGATCGACGAGCTGAGATACGTGAAGATCACCCTGACCGAGGATTTC TTCGTGGTCACCGTGTCCATCGACGACGACACCCCCATGCTGCTGATCTTCGGCCACCTGCCCAGAGTGC TGTTCAAGGCCCCCTACCAGCGGGACAACTTCATCCTGCGGCAGACCGAGAAGCACGAGCTGCTGGTGCT GGTCAAGAAGGACCAGCTGAACCGGCACTCCTACCTGAAGGACCCCGACTTCCTGGACGCCGCCCTGGAC TTCAACTACCTGGACCTGAGCGCCCTGCTGAGAAACAGCTTCCACAGATACGCCGTGGACGTGCTGAAGT CCGGACGGTGCCAGATGCTCGATCGGCGGACCGTGGAGATGGCCTTCGCCTATGCCCTCGCCCTGTTCGC CGCTGCCAGACAGGAAGAGGCTGGCGCCCAGGTGTCAGTGCCCAGAGCCCTGGATAGACAGGCCGCCCTG CTGCAGATCCAGGAATTCATGATCACCTGCCTGAGCCAGACCCCCCCTAGAACCACCCTGCTGCTGTACC CCACAGCCGTGGATCTGGCCAAGAGGGCCCTGTGGACCCCCAACCAGATCACCGACATCACAAGCCTCGT GCGGCTCGTGTACATCCTGAGCAAGCAGAACCAGCAGCACCTGATCCCCCAGTGGGCCCTGAGACAGATC GCCGACTTCGCCCTGAAGCTGCACAAGACCCATCTGGCCAGCTTTCTGAGCGCCTTCGCCAGGCAGGAAC TGTACCTGATGGGCAGCCTGGTCCACAGCATGCTGGTGCATACCACCGAGCGGCGGGAGATCTTCATCGT GGAGACAGGCCTGTGTAGCCTGGCCGAGCTGTCCCACTTTACCCAGCTGCTGGCCCACCCTCACCACGAG TACCTGAGCGACCTGTACACCCCCTGCAGCAGCAGCGGCAGACGGGACCACAGCCTGGAACGGCTGACCA GACTGTTCCCCGATGCCACCGTGCCTGCTACAGTGCCTGCCGCCCTGTCCATCCTGTCCACCATGCAGCC CAGCACCCTGGAAACCTTCCCCGACCTGTTCTGCCTGCCCCTGGGCGAGAGCTTTAGCGCCCTGACCGTG TCCGAGCACGTGTCCTACATCGTGACCAATCAGTACCTGATCAAGGGCATCAGCTACCCCGTGTCCACCA CAGTCGTGGGCCAGAGCCTGATCATCACCCAGACCGACAGCCAGACCAAGTGCGAGCTGACCCGGAACAT GCACACCACACACAGCATCACCGTGGCCCTGAACATCAGCCTGGAAAACTGCGCTTTCTGTCAGTCTGCC CTGCTGGAATACGACGATACCCAGGGCGTGATCAACATCATGTACATGCACGACAGCGACGACGTGCTGT TCGCCCTGGACCCCTACAACGAGGTGGTGGTGTCCAGCCCCCGGACCCACTACCTGATGCTGCTGAAGAA CGGCACCGTGCTGGAAGTGACCGACGTGGTGGTGGACGCCACCGACAGCAGACTGCTGATGATGAGCGTG TACGCCCTGAGCGCCATCATCGGCATCTACCTGCTGTACCGGATGCTGAAAACCTGCTGATAATCTAGAG GCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGATGTGCAGAAG GCCCGACTGCGGCTTCAGCTTCAGCCCTGGACCCGTGATCCTGCTGTGGTGCTGCCTGCTGCTGCCTATC GTGTCCTCTGCCGCCGTGTCTGTGGCCCCTACAGCCGCCGAGAAGGTGCCAGCCGAGTGCCCCGAGCTGA CCAGAAGATGCCTGCTGGGCGAGGTGTTCGAGGGCGACAAGTACGAGAGCTGGCTGCGGCCCCTGGTCAA CGTGACCGGCAGAGATGGCCCCCTGAGCCAGCTGATCCGGTACAGACCCGTGACCCCCGAGGCCGCCAAT AGCGTGCTGCTGGACGAGGCCTTCCTGGATACCCTGGCCCTGCTGTACAACAACCCCGACCAGCTGAGAG CCCTGCTGACCCTGCTGTCCAGCGACACCGCCCCCAGATGGATGACCGTGATGCGGGGCTACAGCGAGTG TGGAGATGGCAGCCCTGCCGTGTACACCTGCGTGGACGACCTGTGCAGAGGCTACGACCTGACCAGACTG AGCTACGGCCGGTCCATCTTCACAGAGCACGTGCTGGGCTTCGAGCTGGTGCCCCCCAGCCTGTTCAACG TGGTGGTGGCCATCCGGAACGAGGCCACCAGAACCAACAGAGCCGTGCGGCTGCCTGTGTCTACAGCCGC TGCACCTGAGGGCATCACACTGTTCTACGGCCTGTACAACGCCGTGAAAGAGTTCTGCCTCCGGCACCAG CTGGATCCCCCCCTGCTGAGACACCTGGACAAGTACTACGCCGGCCTGCCCCCAGAGCTGAAGCAGACCA GAGTGAACCTGCCCGCCCACAGCAGATATGGCCCTCAGGCCGTGGACGCCAGATGATAACGCCGGCGGCC CCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGATGAGCCCCAAGGA CCTGACCCCCTTCCTGACAACCCTGTGGCTGCTCCTGGGCCATAGCAGAGTGCCTAGAGTGCGGGCCGAG GAATGCTGCGAGTTCATCAACGTGAACCACCCCCCCGAGCGGTGCTACGACTTCAAGATGTGCAACCGGT TCACCGTGGCCCTGAGATGCCCCGACGGCGAAGTGTGCTACAGCCCCGAGAAAACCGCCGAGATCCGGGG CATCGTGACCACCATGACCCACAGCCTGACCCGGCAGGTGGTGCACAACAAGCTGACCAGCTGCAACTAC AACCCCCTGTACCTGGAAGCCGACGGCCGGATCAGATGCGGCAAAGTGAACGACAAGGCCCAGTACCTGC TGGGAGCCGCCGGAAGCGTGCCCTACCGGTGGATCAACCTGGAATACGACAAGATCACCCGGATCGTGGG CCTGGACCAGTACCTGGAAAGCGTGAAGAAGCACAAGCGGCTGGACGTGTGCAGAGCCAAGATGGGCTAC ATGCTGCAGCTGTTGAATTTTGACCTTCTTAAGCTTGCGGGAGACGTCGAGTCCAACCCCGGGCCCATGC TGCGGCTGCTGCTGAGACACCACTTCCACTGCCTGCTGCTGTGTGCCGTGTGGGCCACCCCTTGTCTGGC CAGCCCTTGGAGCACCCTGACCGCCAACCAGAACCCTAGCCCCCCTTGGTCCAAGCTGACCTACAGCAAG CCCCACGACGCCGCCACCTTCTACTGCCCCTTTCTGTACCCCAGCCCTCCCAGAAGCCCCCTGCAGTTCA GCGGCTTCCAGAGAGTGTCCACCGGCCCTGAGTGCCGGAACGAGACACTGTACCTGCTGTACAACCGGGA GGGCCAGACACTGGTGGAGCGGAGCAGCACCTGGGTGAAAAAAGTGATCTGGTATCTGAGCGGCCGGAAC CAGACCATCCTGCAGCGGATGCCCAGAACCGCCAGCAAGCCCAGCGACGGCAACGTGCAGATCAGCGTGG AGGACGCCAAAATCTTCGGCGCCCACATGGTGCCCAAGCAGACCAAGCTGCTGAGATTCGTGGTCAACGA CGGCACCAGATATCAGATGTGCGTGATGAAGCTGGAAAGCTGGGCCCACGTGTTCCGGGACTACTCCGTG AGCTTCCAGGTCCGGCTGACCTTCACCGAGGCCAACAACCAGACCTACACCTTCTGCACCCACCCCAACC TGATCGTGCTGCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCCATAT GCGGCTGTGCAGAGTGTGGCTGTCCGTGTGCCTGTGTGCCGTGGTGCTGGGCCAGTGCCAGAGAGAGACA GCCGAGAAGAACGACTACTACCGGGTGCCCCACTACTGGGATGCCTGCAGCAGAGCCCTGCCCGACCAGA CCCGGTACAAATACGTGGAGCAGCTCGTGGACCTGACCCTGAACTACCACTACGACGCCAGCCACGGCCT GGACAACTTCGACGTGCTGAAGCGGATCAACGTGACCGAGGTGTCCCTGCTGATCAGCGACTTCCGGCGG CAGAACAGAAGAGGCGGCACCAACAAGCGGACCACCTTCAACGCCGCTGGCTCTCTGGCCCCTCACGCCA GATCCCTGGAATTCAGCGTGCGGCTGTTCGCCAACTGATAACGTTGCATCCTGCAGGATACAGCAGCAAT TGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTCTTT TCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAG GGTCGGCATGGCATCTCCACCTCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGACGCACGTCCACTCGG ATGGCTAAGGGAGAGCCACGTTTAAACGCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCC CTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGT AACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCA GATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCC ACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCT GGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGCTAGCGGAGTGTATACTGGCTTACTAT GTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCA GCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTG CGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGG AAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGA CGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGGCGGCTCCCT CGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCAT TCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTT CAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCAC CACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAA CTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCA GAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCA AAACGATCTCAAGAAGATCATCTTATTAAGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGA TTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATC AATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTATTAGAAAAATTCATCCAGCAGACGATAAAAC GCAATACGCTGGCTATCCGGTGCCGCAATGCCATACAGCACCAGAAAACGATCCGCCCATTCGCCGCCCA GTTCTTCCGCAATATCACGGGTGGCCAGCGCAATATCCTGATAACGATCCGCCACGCCCAGACGGCCGCA ATCAATAAAGCCGCTAAAACGGCCATTTTCCACCATAATGTTCGGCAGGCACGCATCACCATGGGTCACC ACCAGATCTTCGCCATCCGGCATGCTCGCTTTCAGACGCGCAAACAGCTCTGCCGGTGCCAGGCCCTGAT GTTCTTCATCCAGATCATCCTGATCCACCAGGCCCGCTTCCATACGGGTACGCGCACGTTCAATACGATG TTTCGCCTGATGATCAAACGGACAGGTCGCCGGGTCCAGGGTATGCAGACGACGCATGGCATCCGCCATA ATGCTCACTTTTTCTGCCGGCGCCAGATGGCTAGACAGCAGATCCTGACCCGGCACTTCGCCCAGCAGCA GCCAATCACGGCCCGCTTCGGTCACCACATCCAGCACCGCCGCACACGGAACACCGGTGGTGGCCAGCCA GCTCAGACGCGCCGCTTCATCCTGCAGCTCGTTCAGCGCACCGCTCAGATCGGTTTTCACAAACAGCACC GGACGACCCTGCGCGCTCAGACGAAACACCGCCGCATCAGAGCAGCCAATGGTCTGCTGCGCCCAATCAT AGCCAAACAGACGTTCCACCCACGCTGCCGGGCTACCCGCATGCAGGCCATCCTGTTCAATCATACTCTT CCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATT TAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAA TATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGC AAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGCGCTCCCATTCG CCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCG AAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACACGCGTAATACG ACTCACTATAG Vetor A527: SGP-gH-SGP-gL-SGP-UL128-EMCV-UL130-EV71-UL131 ATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGA GGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTC ACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGG ACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCA TTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAA AACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACC CTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGT TTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTC GCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACT CTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGA GCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCT GTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACT TACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAG AATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTC TTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAG CTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCT GGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTAC CTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAA GGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAAC ATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTG CCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACA AGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGA GGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTG GAAGCCGATGTAGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAA AGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAA GAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAA GGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGG ACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCA CCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAG CACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAG GGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGC CGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAA AGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACG TCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACA CCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCC ATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCC TGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAA ATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACT AAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAG GGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCT GACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAA CATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGA TAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGA TGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGT TGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTG TGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTT TGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGG GATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACC CACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGA TCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACAC CCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGT TGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGA TTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATAC CATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATC TGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGG TGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTT CTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGA CCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGA TATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTG TGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGAC TGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGA AGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCA GTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACC ATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAAT GACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTG ACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCA CAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGA AATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGC ATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTT GCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTAC TGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCT ATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAG ACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAAC CGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGT TTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTAT CTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCT GGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAG TTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAA GAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAA TAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGA ACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAG CACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACA ACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTAT GCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTA ACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGG CCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTAT TCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAG AGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGG AGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCC TATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAG CTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAA TGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGG CTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGA AGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGT GAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCG CTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGA ACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGG GGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCG TTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAA TTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCT CACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGA TCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAG ACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTA TTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAA AGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGC ATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTA TGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGC TACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAA GATGAGGCCTGGCCTGCCCTCCTACCTGATCATCCTGGCCGTGTGCCTGTTCAGCCACCTGCTGTCCAGC AGATACGGCGCCGAGGCCGTGAGCGAGCCCCTGGACAAGGCTTTCCACCTGCTGCTGAACACCTACGGCA GACCCATCCGGTTTCTGCGGGAGAACACCACCCAGTGCACCTACAACAGCAGCCTGCGGAACAGCACCGT CGTGAGAGAGAACGCCATCAGCTTCAACTTTTTCCAGAGCTACAACCAGTACTACGTGTTCCACATGCCC AGATGCCTGTTTGCCGGCCCTCTGGCCGAGCAGTTCCTGAACCAGGTGGACCTGACCGAGACACTGGAAA GATACCAGCAGCGGCTGAATACCTACGCCCTGGTGTCCAAGGACCTGGCCAGCTACCGGTCCTTTAGCCA GCAGCTCAAGGCTCAGGATAGCCTCGGCGAGCAGCCTACCACCGTGCCCCCTCCCATCGACCTGAGCATC CCCCACGTGTGGATGCCTCCCCAGACCACCCCTCACGGCTGGACCGAGAGCCACACCACCTCCGGCCTGC ACAGACCCCACTTCAACCAGACCTGCATCCTGTTCGACGGCCACGACCTGCTGTTTAGCACCGTGACCCC CTGCCTGCACCAGGGCTTCTACCTGATCGACGAGCTGAGATACGTGAAGATCACCCTGACCGAGGATTTC TTCGTGGTCACCGTGTCCATCGACGACGACACCCCCATGCTGCTGATCTTCGGCCACCTGCCCAGAGTGC TGTTCAAGGCCCCCTACCAGCGGGACAACTTCATCCTGCGGCAGACCGAGAAGCACGAGCTGCTGGTGCT GGTCAAGAAGGACCAGCTGAACCGGCACTCCTACCTGAAGGACCCCGACTTCCTGGACGCCGCCCTGGAC TTCAACTACCTGGACCTGAGCGCCCTGCTGAGAAACAGCTTCCACAGATACGCCGTGGACGTGCTGAAGT CCGGACGGTGCCAGATGCTCGATCGGCGGACCGTGGAGATGGCCTTCGCCTATGCCCTCGCCCTGTTCGC CGCTGCCAGACAGGAAGAGGCTGGCGCCCAGGTGTCAGTGCCCAGAGCCCTGGATAGACAGGCCGCCCTG CTGCAGATCCAGGAATTCATGATCACCTGCCTGAGCCAGACCCCCCCTAGAACCACCCTGCTGCTGTACC CCACAGCCGTGGATCTGGCCAAGAGGGCCCTGTGGACCCCCAACCAGATCACCGACATCACAAGCCTCGT GCGGCTCGTGTACATCCTGAGCAAGCAGAACCAGCAGCACCTGATCCCCCAGTGGGCCCTGAGACAGATC GCCGACTTCGCCCTGAAGCTGCACAAGACCCATCTGGCCAGCTTTCTGAGCGCCTTCGCCAGGCAGGAAC TGTACCTGATGGGCAGCCTGGTCCACAGCATGCTGGTGCATACCACCGAGCGGCGGGAGATCTTCATCGT GGAGACAGGCCTGTGTAGCCTGGCCGAGCTGTCCCACTTTACCCAGCTGCTGGCCCACCCTCACCACGAG TACCTGAGCGACCTGTACACCCCCTGCAGCAGCAGCGGCAGACGGGACCACAGCCTGGAACGGCTGACCA GACTGTTCCCCGATGCCACCGTGCCTGCTACAGTGCCTGCCGCCCTGTCCATCCTGTCCACCATGCAGCC CAGCACCCTGGAAACCTTCCCCGACCTGTTCTGCCTGCCCCTGGGCGAGAGCTTTAGCGCCCTGACCGTG TCCGAGCACGTGTCCTACATCGTGACCAATCAGTACCTGATCAAGGGCATCAGCTACCCCGTGTCCACCA CAGTCGTGGGCCAGAGCCTGATCATCACCCAGACCGACAGCCAGACCAAGTGCGAGCTGACCCGGAACAT GCACACCACACACAGCATCACCGTGGCCCTGAACATCAGCCTGGAAAACTGCGCTTTCTGTCAGTCTGCC CTGCTGGAATACGACGATACCCAGGGCGTGATCAACATCATGTACATGCACGACAGCGACGACGTGCTGT TCGCCCTGGACCCCTACAACGAGGTGGTGGTGTCCAGCCCCCGGACCCACTACCTGATGCTGCTGAAGAA CGGCACCGTGCTGGAAGTGACCGACGTGGTGGTGGACGCCACCGACAGCAGACTGCTGATGATGAGCGTG TACGCCCTGAGCGCCATCATCGGCATCTACCTGCTGTACCGGATGCTGAAAACCTGCTGATAATCTAGAG GCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGATGTGCAGAAG GCCCGACTGCGGCTTCAGCTTCAGCCCTGGACCCGTGATCCTGCTGTGGTGCTGCCTGCTGCTGCCTATC GTGTCCTCTGCCGCCGTGTCTGTGGCCCCTACAGCCGCCGAGAAGGTGCCAGCCGAGTGCCCCGAGCTGA CCAGAAGATGCCTGCTGGGCGAGGTGTTCGAGGGCGACAAGTACGAGAGCTGGCTGCGGCCCCTGGTCAA CGTGACCGGCAGAGATGGCCCCCTGAGCCAGCTGATCCGGTACAGACCCGTGACCCCCGAGGCCGCCAAT AGCGTGCTGCTGGACGAGGCCTTCCTGGATACCCTGGCCCTGCTGTACAACAACCCCGACCAGCTGAGAG CCCTGCTGACCCTGCTGTCCAGCGACACCGCCCCCAGATGGATGACCGTGATGCGGGGCTACAGCGAGTG TGGAGATGGCAGCCCTGCCGTGTACACCTGCGTGGACGACCTGTGCAGAGGCTACGACCTGACCAGACTG AGCTACGGCCGGTCCATCTTCACAGAGCACGTGCTGGGCTTCGAGCTGGTGCCCCCCAGCCTGTTCAACG TGGTGGTGGCCATCCGGAACGAGGCCACCAGAACCAACAGAGCCGTGCGGCTGCCTGTGTCTACAGCCGC TGCACCTGAGGGCATCACACTGTTCTACGGCCTGTACAACGCCGTGAAAGAGTTCTGCCTCCGGCACCAG CTGGATCCCCCCCTGCTGAGACACCTGGACAAGTACTACGCCGGCCTGCCCCCAGAGCTGAAGCAGACCA GAGTGAACCTGCCCGCCCACAGCAGATATGGCCCTCAGGCCGTGGACGCCAGATGATAACGCCGGCGGCC CCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGATGAGCCCCAAGGA CCTGACCCCCTTCCTGACAACCCTGTGGCTGCTCCTGGGCCATAGCAGAGTGCCTAGAGTGCGGGCCGAG GAATGCTGCGAGTTCATCAACGTGAACCACCCCCCCGAGCGGTGCTACGACTTCAAGATGTGCAACCGGT TCACCGTGGCCCTGAGATGCCCCGACGGCGAAGTGTGCTACAGCCCCGAGAAAACCGCCGAGATCCGGGG CATCGTGACCACCATGACCCACAGCCTGACCCGGCAGGTGGTGCACAACAAGCTGACCAGCTGCAACTAC AACCCCCTGTACCTGGAAGCCGACGGCCGGATCAGATGCGGCAAAGTGAACGACAAGGCCCAGTACCTGC TGGGAGCCGCCGGAAGCGTGCCCTACCGGTGGATCAACCTGGAATACGACAAGATCACCCGGATCGTGGG CCTGGACCAGTACCTGGAAAGCGTGAAGAAGCACAAGCGGCTGGACGTGTGCAGAGCCAAGATGGGCTAC ATGCTGCAGTGATAAGGCGCGCCAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGT CTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTC TTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGG AAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCC CCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAA CCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACA AGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTT TACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGA AAAACACGATAATATGCTGCGGCTGCTGCTGAGACACCACTTCCACTGCCTGCTGCTGTGTGCCGTGTGG GCCACCCCTTGTCTGGCCAGCCCTTGGAGCACCCTGACCGCCAACCAGAACCCTAGCCCCCCTTGGTCCA AGCTGACCTACAGCAAGCCCCACGACGCCGCCACCTTCTACTGCCCCTTTCTGTACCCCAGCCCTCCCAG AAGCCCCCTGCAGTTCAGCGGCTTCCAGAGAGTGTCCACCGGCCCTGAGTGCCGGAACGAGACACTGTAC CTGCTGTACAACCGGGAGGGCCAGACACTGGTGGAGCGGAGCAGCACCTGGGTGAAAAAAGTGATCTGGT ATCTGAGCGGCCGGAACCAGACCATCCTGCAGCGGATGCCCAGAACCGCCAGCAAGCCCAGCGACGGCAA CGTGCAGATCAGCGTGGAGGACGCCAAAATCTTCGGAGCCCACATGGTGCCCAAGCAGACCAAGCTGCTG AGATTCGTGGTCAACGACGGCACCAGATATCAGATGTGCGTGATGAAGCTGGAAAGCTGGGCCCACGTGT TCCGGGACTACTCCGTGAGCTTCCAGGTCCGGCTGACCTTCACCGAGGCCAACAACCAGACCTACACCTT CTGCACCCACCCCAACCTGATCGTGTGATAAGTACCTTTGTACGCCTGTTTTATACCCCCTCCCTGATTT GCAACTTAGAAGCAACGCAAACCAGATCAATAGTAGGTGTGACATACCAGTCGCATCTTGATCAAGCACT TCTGTATCCCCGGACCGAGTATCAATAGACTGTGCACACGGTTGAAGGAGAAAACGTCCGTTACCCGGCT AACTACTTCGAGAAGCCTAGTAACGCCATTGAAGTTGCAGAGTGTTTCGCTCAGCACTCCCCCCGTGTAG ATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCTATG GGGTAACCCATAGGACGCTCTAATACGGACATGGCGTGAAGAGTCTATTGAGCTAGTTAGTAGTCCTCCG GCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACATACCCTTAATCCAAAGGGCAGTGTGTCGTAACGG GCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCTTTTTATTCTTGTATTGGCTGCTTATG GTGACAATTAAAGAATTGTTACCATATAGCTATTGGATTGGCCATCCAGTGTCAAACAGAGCTATTGTAT ATCTCTTTGTTGGATTCACACCTCTCACTCTTGAAACGTTACACACCCTCAATTACATTATACTGCTGAA CACGAAGCGCATATGCGGCTGTGCAGAGTGTGGCTGTCCGTGTGCCTGTGTGCCGTGGTGCTGGGCCAGT GCCAGAGAGAGACAGCCGAGAAGAACGACTACTACCGGGTGCCCCACTACTGGGATGCCTGCAGCAGAGC CCTGCCCGACCAGACCCGGTACAAATACGTGGAGCAGCTCGTGGACCTGACCCTGAACTACCACTACGAC GCCAGCCACGGCCTGGACAACTTCGACGTGCTGAAGCGGATCAACGTGACCGAGGTGTCCCTGCTGATCA GCGACTTCCGGCGGCAGAACAGAAGAGGCGGCACCAACAAGCGGACCACCTTCAACGCCGCTGGCTCTCT GGCCCCTCACGCCAGATCCCTGGAATTCAGCGTGCGGCTGTTCGCCAACTGATAACGTTGCATCCTGCAG GATACAGCAGCAATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTA TTTTATTTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAGGGTCGGCATGGCATCTCCACCTCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGAC GCACGTCCACTCGGATGGCTAAGGGAGAGCCACGTTTAAACGCTAGAGCAAGACGTTTCCCGTTGAATAT GGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTT ATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCT GAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAA TCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGG GGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGCTAGCGGAGTGTA TACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCT GCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCT CGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAA GATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGC ATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCC CCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGC CGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGC ACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAG ACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGC CGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAA GAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGA TTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAAGGGGTCTGACGCTCAGTGGAACGAAAA CTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAA TGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTATTAGAAAAATTCATCCA GCAGACGATAAAACGCAATACGCTGGCTATCCGGTGCCGCAATGCCATACAGCACCAGAAAACGATCCGC CCATTCGCCGCCCAGTTCTTCCGCAATATCACGGGTGGCCAGCGCAATATCCTGATAACGATCCGCCACG CCCAGACGGCCGCAATCAATAAAGCCGCTAAAACGGCCATTTTCCACCATAATGTTCGGCAGGCACGCAT CACCATGGGTCACCACCAGATCTTCGCCATCCGGCATGCTCGCTTTCAGACGCGCAAACAGCTCTGCCGG TGCCAGGCCCTGATGTTCTTCATCCAGATCATCCTGATCCACCAGGCCCGCTTCCATACGGGTACGCGCA CGTTCAATACGATGTTTCGCCTGATGATCAAACGGACAGGTCGCCGGGTCCAGGGTATGCAGACGACGCA TGGCATCCGCCATAATGCTCACTTTTTCTGCCGGCGCCAGATGGCTAGACAGCAGATCCTGACCCGGCAC TTCGCCCAGCAGCAGCCAATCACGGCCCGCTTCGGTCACCACATCCAGCACCGCCGCACACGGAACACCG GTGGTGGCCAGCCAGCTCAGACGCGCCGCTTCATCCTGCAGCTCGTTCAGCGCACCGCTCAGATCGGTTT TCACAAACAGCACCGGACGACCCTGCGCGCTCAGACGAAACACCGCCGCATCAGAGCAGCCAATGGTCTG CTGCGCCCAATCATAGCCAAACAGACGTTCCACCCACGCTGCCGGGCTACCCGCATGCAGGCCATCCTGT TCAATCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACA TATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAA ATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAAT AGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAG GGCGCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGGTGCGGGCCTCTTCGCTAT TACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTC ACACGCGTAATACGACTCACTATAG Vetor A531: SGP-gHsol-SGP-gL ATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGA GGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTC ACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGG ACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCA TTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAA AACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACC CTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGT TTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTC GCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACT CTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGA GCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCT GTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACT TACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAG AATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTC TTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAG CTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCT GGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTAC CTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAA GGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAAC ATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTG CCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACA AGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGA GGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTG GAAGCCGATGTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAA AGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAA GAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAA GGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGG ACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCA CCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAG CACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAG GGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGC CGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAA AGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACG TCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACA CCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCC ATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCC TGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAA ATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACT AAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAG GGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCT GACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAA CATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGA TAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGA TGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGT TGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTG TGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTT TGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGG GATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACC CACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGA TCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACAC CCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGT TGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGA TTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATAC CATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATC TGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGG TGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTT CTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGA CCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGA TATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTG TGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGAC TGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGA AGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCA GTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACC ATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAAT GACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTG ACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCA CAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGA AATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGC ATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTT GCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTAC TGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCT ATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAG ACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAAC CGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGT TTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTAT CTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCT GGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAG TTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAA GAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAA TAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGA ACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAG CACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACA ACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTAT GCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTA ACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGG CCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTAT TCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAG AGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGG AGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCC TATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAG CTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAA TGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGG CTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGA AGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGT GAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCG CTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGA ACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGG GGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCG TTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAA TTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCT CACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGA TCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAG ACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTA TTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAA AGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGC ATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTA TGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGC TACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAA GATGAGGCCTGGCCTGCCCTCCTACCTGATCATCCTGGCCGTGTGCCTGTTCAGCCACCTGCTGTCCAGC AGATACGGCGCCGAGGCCGTGAGCGAGCCCCTGGACAAGGCTTTCCACCTGCTGCTGAACACCTACGGCA GACCCATCCGGTTTCTGCGGGAGAACACCACCCAGTGCACCTACAACAGCAGCCTGCGGAACAGCACCGT CGTGAGAGAGAACGCCATCAGCTTCAACTTTTTCCAGAGCTACAACCAGTACTACGTGTTCCACATGCCC AGATGCCTGTTTGCCGGCCCTCTGGCCGAGCAGTTCCTGAACCAGGTGGACCTGACCGAGACACTGGAAA GATACCAGCAGCGGCTGAATACCTACGCCCTGGTGTCCAAGGACCTGGCCAGCTACCGGTCCTTTAGCCA GCAGCTCAAGGCTCAGGATAGCCTCGGCGAGCAGCCTACCACCGTGCCCCCTCCCATCGACCTGAGCATC CCCCACGTGTGGATGCCTCCCCAGACCACCCCTCACGGCTGGACCGAGAGCCACACCACCTCCGGCCTGC ACAGACCCCACTTCAACCAGACCTGCATCCTGTTCGACGGCCACGACCTGCTGTTTAGCACCGTGACCCC CTGCCTGCACCAGGGCTTCTACCTGATCGACGAGCTGAGATACGTGAAGATCACCCTGACCGAGGATTTC TTCGTGGTCACCGTGTCCATCGACGACGACACCCCCATGCTGCTGATCTTCGGCCACCTGCCCAGAGTGC TGTTCAAGGCCCCCTACCAGCGGGACAACTTCATCCTGCGGCAGACCGAGAAGCACGAGCTGCTGGTGCT GGTCAAGAAGGACCAGCTGAACCGGCACTCCTACCTGAAGGACCCCGACTTCCTGGACGCCGCCCTGGAC TTCAACTACCTGGACCTGAGCGCCCTGCTGAGAAACAGCTTCCACAGATACGCCGTGGACGTGCTGAAGT CCGGACGGTGCCAGATGCTCGATCGGCGGACCGTGGAGATGGCCTTCGCCTATGCCCTCGCCCTGTTCGC CGCTGCCAGACAGGAAGAGGCTGGCGCCCAGGTGTCAGTGCCCAGAGCCCTGGATAGACAGGCCGCCCTG CTGCAGATCCAGGAATTCATGATCACCTGCCTGAGCCAGACCCCCCCTAGAACCACCCTGCTGCTGTACC CCACAGCCGTGGATCTGGCCAAGAGGGCCCTGTGGACCCCCAACCAGATCACCGACATCACAAGCCTCGT GCGGCTCGTGTACATCCTGAGCAAGCAGAACCAGCAGCACCTGATCCCCCAGTGGGCCCTGAGACAGATC GCCGACTTCGCCCTGAAGCTGCACAAGACCCATCTGGCCAGCTTTCTGAGCGCCTTCGCCAGGCAGGAAC TGTACCTGATGGGCAGCCTGGTCCACAGCATGCTGGTGCATACCACCGAGCGGCGGGAGATCTTCATCGT GGAGACAGGCCTGTGTAGCCTGGCCGAGCTGTCCCACTTTACCCAGCTGCTGGCCCACCCTCACCACGAG TACCTGAGCGACCTGTACACCCCCTGCAGCAGCAGCGGCAGACGGGACCACAGCCTGGAACGGCTGACCA GACTGTTCCCCGATGCCACCGTGCCTGCTACAGTGCCTGCCGCCCTGTCCATCCTGTCCACCATGCAGCC CAGCACCCTGGAAACCTTCCCCGACCTGTTCTGCCTGCCCCTGGGCGAGAGCTTTAGCGCCCTGACCGTG TCCGAGCACGTGTCCTACATCGTGACCAATCAGTACCTGATCAAGGGCATCAGCTACCCCGTGTCCACCA CAGTCGTGGGCCAGAGCCTGATCATCACCCAGACCGACAGCCAGACCAAGTGCGAGCTGACCCGGAACAT GCACACCACACACAGCATCACCGTGGCCCTGAACATCAGCCTGGAAAACTGCGCTTTCTGTCAGTCTGCC CTGCTGGAATACGACGATACCCAGGGCGTGATCAACATCATGTACATGCACGACAGCGACGACGTGCTGT TCGCCCTGGACCCCTACAACGAGGTGGTGGTGTCCAGCCCCCGGACCCACTACCTGATGCTGCTGAAGAA CGGCACCGTGCTGGAAGTGACCGACGTGGTGGTGGACGCCACCGACTGATAATCTAGAGGCCCCTATAAC TCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGATGTGCAGAAGGCCCGACTGCG GCTTCAGCTTCAGCCCTGGACCCGTGATCCTGCTGTGGTGCTGCCTGCTGCTGCCTATCGTGTCCTCTGC CGCCGTGTCTGTGGCCCCTACAGCCGCCGAGAAGGTGCCAGCCGAGTGCCCCGAGCTGACCAGAAGATGC CTGCTGGGCGAGGTGTTCGAGGGCGACAAGTACGAGAGCTGGCTGCGGCCCCTGGTCAACGTGACCGGCA GAGATGGCCCCCTGAGCCAGCTGATCCGGTACAGACCCGTGACCCCCGAGGCCGCCAATAGCGTGCTGCT GGACGAGGCCTTCCTGGATACCCTGGCCCTGCTGTACAACAACCCCGACCAGCTGAGAGCCCTGCTGACC CTGCTGTCCAGCGACACCGCCCCCAGATGGATGACCGTGATGCGGGGCTACAGCGAGTGTGGAGATGGCA GCCCTGCCGTGTACACCTGCGTGGACGACCTGTGCAGAGGCTACGACCTGACCAGACTGAGCTACGGCCG GTCCATCTTCACAGAGCACGTGCTGGGCTTCGAGCTGGTGCCCCCCAGCCTGTTCAACGTGGTGGTGGCC ATCCGGAACGAGGCCACCAGAACCAACAGAGCCGTGCGGCTGCCTGTGTCTACAGCCGCTGCACCTGAGG GCATCACACTGTTCTACGGCCTGTACAACGCCGTGAAAGAGTTCTGCCTCCGGCACCAGCTGGATCCCCC CCTGCTGAGACACCTGGACAAGTACTACGCCGGCCTGCCCCCAGAGCTGAAGCAGACCAGAGTGAACCTG CCCGCCCACAGCAGATATGGCCCTCAGGCCGTGGACGCCAGATGATAAGCGGCCGCATACAGCAGCAATT GGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTCTTTT CTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGG GTCGGCATGGCATCTCCACCTCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGACGCACGTCCACTCGGA TGGCTAAGGGAGAGCCACGTTTAAACACGTGATATCTGGCCTCATGGGCCTTCCTTTCACTGCCCGCTTT CCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTC TCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAA AGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCT GACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGG CGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGC CTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTC GTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACT ATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAG CAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGA ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCG GCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGG ATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAAT CAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTATTAGAAAAATTCATCCAGCAGACGATAAAA CGCAATACGCTGGCTATCCGGTGCCGCAATGCCATACAGCACCAGAAAACGATCCGCCCATTCGCCGCCC AGTTCTTCCGCAATATCACGGGTGGCCAGCGCAATATCCTGATAACGATCCGCCACGCCCAGACGGCCGC AATCAATAAAGCCGCTAAAACGGCCATTTTCCACCATAATGTTCGGCAGGCACGCATCACCATGGGTCAC CACCAGATCTTCGCCATCCGGCATGCTCGCTTTCAGACGCGCAAACAGCTCTGCCGGTGCCAGGCCCTGA TGTTCTTCATCCAGATCATCCTGATCCACCAGGCCCGCTTCCATACGGGTACGCGCACGTTCAATACGAT GTTTCGCCTGATGATCAAACGGACAGGTCGCCGGGTCCAGGGTATGCAGACGACGCATGGCATCCGCCAT AATGCTCACTTTTTCTGCCGGCGCCAGATGGCTAGACAGCAGATCCTGACCCGGCACTTCGCCCAGCAGC AGCCAATCACGGCCCGCTTCGGTCACCACATCCAGCACCGCCGCACACGGAACACCGGTGGTGGCCAGCC AGCTCAGACGCGCCGCTTCATCCTGCAGCTCGTTCAGCGCACCGCTCAGATCGGTTTTCACAAACAGCAC CGGACGACCCTGCGCGCTCAGACGAAACACCGCCGCATCAGAGCAGCCAATGGTCTGCTGCGCCCAATCA TAGCCAAACAGACGTTCCACCCACGCTGCCGGGCTACCCGCATGCAGGCCATCCTGTTCAATCATACTCT TCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTAT TTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTA ATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGG CAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGCGCTCCCATTC GCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGC GAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACACGCGTAATAC GACTCACTATAG Vetor A532: SGP-gHsol-2A-gL ATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGA GGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTC ACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGG ACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCA TTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAA AACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACC CTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGT TTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTC GCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACT CTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGA GCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCT GTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACT TACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAG AATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTC TTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAG CTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCT GGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTAC CTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAA GGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAAC ATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTG CCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACA AGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGA GGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTG GAAGCCGATGTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAA AGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAA GAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAA GGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGG ACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCA CCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAG CACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAG GGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGC CGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAA AGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACG TCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACA CCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCC ATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCC TGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAA ATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACT AAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAG GGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCT GACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAA CATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGA TAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGA TGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGT TGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTG TGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTT TGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGG GATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACC CACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGA TCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACAC CCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGT TGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGA TTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATAC CATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATC TGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGG TGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTT CTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGA CCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGA TATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTG TGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGAC TGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGA AGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCA GTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACC ATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAAT GACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTG ACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCA CAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGA AATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGC ATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTT GCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTAC TGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCT ATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAG ACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAAC CGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGT TTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTAT CTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCT GGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAG TTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAA GAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAA TAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGA ACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAG CACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACA ACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTAT GCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTA ACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGG CCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTAT TCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAG AGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGG AGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCC TATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAG CTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAA TGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGG CTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGA AGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGT GAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCG CTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGA ACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGG GGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCG TTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAA TTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCT CACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGA TCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAG ACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTA TTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAA AGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGC ATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTA TGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGC TACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAA GATGAGGCCTGGCCTGCCCTCCTACCTGATCATCCTGGCCGTGTGCCTGTTCAGCCACCTGCTGTCCAGC AGATACGGCGCCGAGGCCGTGAGCGAGCCCCTGGACAAGGCTTTCCACCTGCTGCTGAACACCTACGGCA GACCCATCCGGTTTCTGCGGGAGAACACCACCCAGTGCACCTACAACAGCAGCCTGCGGAACAGCACCGT CGTGAGAGAGAACGCCATCAGCTTCAACTTTTTCCAGAGCTACAACCAGTACTACGTGTTCCACATGCCC AGATGCCTGTTTGCCGGCCCTCTGGCCGAGCAGTTCCTGAACCAGGTGGACCTGACCGAGACACTGGAAA GATACCAGCAGCGGCTGAATACCTACGCCCTGGTGTCCAAGGACCTGGCCAGCTACCGGTCCTTTAGCCA GCAGCTCAAGGCTCAGGATAGCCTCGGCGAGCAGCCTACCACCGTGCCCCCTCCCATCGACCTGAGCATC CCCCACGTGTGGATGCCTCCCCAGACCACCCCTCACGGCTGGACCGAGAGCCACACCACCTCCGGCCTGC ACAGACCCCACTTCAACCAGACCTGCATCCTGTTCGACGGCCACGACCTGCTGTTTAGCACCGTGACCCC CTGCCTGCACCAGGGCTTCTACCTGATCGACGAGCTGAGATACGTGAAGATCACCCTGACCGAGGATTTC TTCGTGGTCACCGTGTCCATCGACGACGACACCCCCATGCTGCTGATCTTCGGCCACCTGCCCAGAGTGC TGTTCAAGGCCCCCTACCAGCGGGACAACTTCATCCTGCGGCAGACCGAGAAGCACGAGCTGCTGGTGCT GGTCAAGAAGGACCAGCTGAACCGGCACTCCTACCTGAAGGACCCCGACTTCCTGGACGCCGCCCTGGAC TTCAACTACCTGGACCTGAGCGCCCTGCTGAGAAACAGCTTCCACAGATACGCCGTGGACGTGCTGAAGT CCGGACGGTGCCAGATGCTCGATCGGCGGACCGTGGAGATGGCCTTCGCCTATGCCCTCGCCCTGTTCGC CGCTGCCAGACAGGAAGAGGCTGGCGCCCAGGTGTCAGTGCCCAGAGCCCTGGATAGACAGGCCGCCCTG CTGCAGATCCAGGAATTCATGATCACCTGCCTGAGCCAGACCCCCCCTAGAACCACCCTGCTGCTGTACC CCACAGCCGTGGATCTGGCCAAGAGGGCCCTGTGGACCCCCAACCAGATCACCGACATCACAAGCCTCGT GCGGCTCGTGTACATCCTGAGCAAGCAGAACCAGCAGCACCTGATCCCCCAGTGGGCCCTGAGACAGATC GCCGACTTCGCCCTGAAGCTGCACAAGACCCATCTGGCCAGCTTTCTGAGCGCCTTCGCCAGGCAGGAAC TGTACCTGATGGGCAGCCTGGTCCACAGCATGCTGGTGCATACCACCGAGCGGCGGGAGATCTTCATCGT GGAGACAGGCCTGTGTAGCCTGGCCGAGCTGTCCCACTTTACCCAGCTGCTGGCCCACCCTCACCACGAG TACCTGAGCGACCTGTACACCCCCTGCAGCAGCAGCGGCAGACGGGACCACAGCCTGGAACGGCTGACCA GACTGTTCCCCGATGCCACCGTGCCTGCTACAGTGCCTGCCGCCCTGTCCATCCTGTCCACCATGCAGCC CAGCACCCTGGAAACCTTCCCCGACCTGTTCTGCCTGCCCCTGGGCGAGAGCTTTAGCGCCCTGACCGTG TCCGAGCACGTGTCCTACATCGTGACCAATCAGTACCTGATCAAGGGCATCAGCTACCCCGTGTCCACCA CAGTCGTGGGCCAGAGCCTGATCATCACCCAGACCGACAGCCAGACCAAGTGCGAGCTGACCCGGAACAT GCACACCACACACAGCATCACCGTGGCCCTGAACATCAGCCTGGAAAACTGCGCTTTCTGTCAGTCTGCC CTGCTGGAATACGACGATACCCAGGGCGTGATCAACATCATGTACATGCACGACAGCGACGACGTGCTGT TCGCCCTGGACCCCTACAACGAGGTGGTGGTGTCCAGCCCCCGGACCCACTACCTGATGCTGCTGAAGAA CGGCACCGTGCTGGAAGTGACCGACGTGGTGGTGGACGCCACCGACCTGTTGAATTTTGACCTTCTTAAG CTTGCGGGAGACGTCGAGTCCAACCCCGGGCCCATGTGCAGAAGGCCCGACTGCGGCTTCAGCTTCAGCC CTGGACCCGTGATCCTGCTGTGGTGCTGCCTGCTGCTGCCTATCGTGTCCTCTGCCGCCGTGTCTGTGGC CCCTACAGCCGCCGAGAAGGTGCCAGCCGAGTGCCCCGAGCTGACCAGAAGATGCCTGCTGGGCGAGGTG TTCGAGGGCGACAAGTACGAGAGCTGGCTGCGGCCCCTGGTCAACGTGACCGGCAGAGATGGCCCCCTGA GCCAGCTGATCCGGTACAGACCCGTGACCCCCGAGGCCGCCAATAGCGTGCTGCTGGACGAGGCCTTCCT GGATACCCTGGCCCTGCTGTACAACAACCCCGACCAGCTGAGAGCCCTGCTGACCCTGCTGTCCAGCGAC ACCGCCCCCAGATGGATGACCGTGATGCGGGGCTACAGCGAGTGTGGAGATGGCAGCCCTGCCGTGTACA CCTGCGTGGACGACCTGTGCAGAGGCTACGACCTGACCAGACTGAGCTACGGCCGGTCCATCTTCACAGA GCACGTGCTGGGCTTCGAGCTGGTGCCCCCCAGCCTGTTCAACGTGGTGGTGGCCATCCGGAACGAGGCC ACCAGAACCAACAGAGCCGTGCGGCTGCCTGTGTCTACAGCCGCTGCACCTGAGGGCATCACACTGTTCT ACGGCCTGTACAACGCCGTGAAAGAGTTCTGCCTCCGGCACCAGCTGGATCCCCCCCTGCTGAGACACCT GGACAAGTACTACGCCGGCCTGCCCCCAGAGCTGAAGCAGACCAGAGTGAACCTGCCCGCCCACAGCAGA TATGGCCCTCAGGCCGTGGACGCCAGATGATAAGCGGCCGCATACAGCAGCAATTGGCAAGCTGCTTACA TAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTCTTTTCTTTTCCGAATCGGA TTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGTCGGCATGGCATCT CCACCTCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGACGCACGTCCACTCGGATGGCTAAGGGAGAGC CACGTTTAAACACGTGATATCTGGCCTCATGGGCCTTCCTTTCACTGCCCGCTTTCCAGTCGGGAAACCT GTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCCTCGCTC ACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAAAAGGC CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAA AATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAA GCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGG AAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTG GGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGT AGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATC TGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCG CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCC TTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGA TTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATAT ATGAGTAAACTTGGTCTGACAGTTATTAGAAAAATTCATCCAGCAGACGATAAAACGCAATACGCTGGCT ATCCGGTGCCGCAATGCCATACAGCACCAGAAAACGATCCGCCCATTCGCCGCCCAGTTCTTCCGCAATA TCACGGGTGGCCAGCGCAATATCCTGATAACGATCCGCCACGCCCAGACGGCCGCAATCAATAAAGCCGC TAAAACGGCCATTTTCCACCATAATGTTCGGCAGGCACGCATCACCATGGGTCACCACCAGATCTTCGCC ATCCGGCATGCTCGCTTTCAGACGCGCAAACAGCTCTGCCGGTGCCAGGCCCTGATGTTCTTCATCCAGA TCATCCTGATCCACCAGGCCCGCTTCCATACGGGTACGCGCACGTTCAATACGATGTTTCGCCTGATGAT CAAACGGACAGGTCGCCGGGTCCAGGGTATGCAGACGACGCATGGCATCCGCCATAATGCTCACTTTTTC TGCCGGCGCCAGATGGCTAGACAGCAGATCCTGACCCGGCACTTCGCCCAGCAGCAGCCAATCACGGCCC GCTTCGGTCACCACATCCAGCACCGCCGCACACGGAACACCGGTGGTGGCCAGCCAGCTCAGACGCGCCG CTTCATCCTGCAGCTCGTTCAGCGCACCGCTCAGATCGGTTTTCACAAACAGCACCGGACGACCCTGCGC GCTCAGACGAAACACCGCCGCATCAGAGCAGCCAATGGTCTGCTGCGCCCAATCATAGCCAAACAGACGT TCCACCCACGCTGCCGGGCTACCCGCATGCAGGCCATCCTGTTCAATCATACTCTTCCTTTTTCAATATT ATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACA AATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAAT TCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAA ATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGCGCTCCCATTCGCCATTCAGGCTGCG CAACTGTTGGGAAGGGCGTTTCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGC TGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACACGCGTAATACGACTCACTATAG Vetor A533: SGP-gHsol-EV71-gL ATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGA GGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTC ACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGG ACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCA TTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAA AACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACC CTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGT TTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTC GCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACT CTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGA GCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCT GTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACT TACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAG AATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTC TTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAG CTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCT GGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTAC CTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAA GGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAAC ATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTG CCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACA AGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGA GGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTG GAAGCCGATGTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAA AGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAA GAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAA GGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGG ACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCA CCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAG CACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAG GGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGC CGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAA AGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACG TCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACA CCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCC ATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCC TGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAA ATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACT AAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAG GGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCT GACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAA CATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGA TAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGA TGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGT TGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTG TGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTT TGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGG GATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACC CACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGA TCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACAC CCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGT TGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGA TTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATAC CATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATC TGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGG TGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTT CTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGA CCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGA TATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTG TGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGAC TGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGA AGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCA GTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACC ATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAAT GACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTG ACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCA CAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGA AATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGC ATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTT GCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTAC TGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCT ATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAG ACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAAC CGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGT TTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTAT CTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCT GGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAG TTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAA GAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAA TAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGA ACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAG CACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACA ACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTAT GCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTA ACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGG CCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTAT TCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAG AGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGG AGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCC TATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAG CTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAA TGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGG CTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGA AGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGT GAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCG CTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGA ACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGG GGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCG TTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAA TTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCT CACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGA TCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAG ACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTA TTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAA AGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGC ATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTA TGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGC TACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAA GATGAGGCCTGGCCTGCCCTCCTACCTGATCATCCTGGCCGTGTGCCTGTTCAGCCACCTGCTGTCCAGC AGATACGGCGCCGAGGCCGTGAGCGAGCCCCTGGACAAGGCTTTCCACCTGCTGCTGAACACCTACGGCA GACCCATCCGGTTTCTGCGGGAGAACACCACCCAGTGCACCTACAACAGCAGCCTGCGGAACAGCACCGT CGTGAGAGAGAACGCCATCAGCTTCAACTTTTTCCAGAGCTACAACCAGTACTACGTGTTCCACATGCCC AGATGCCTGTTTGCCGGCCCTCTGGCCGAGCAGTTCCTGAACCAGGTGGACCTGACCGAGACACTGGAAA GATACCAGCAGCGGCTGAATACCTACGCCCTGGTGTCCAAGGACCTGGCCAGCTACCGGTCCTTTAGCCA GCAGCTCAAGGCTCAGGATAGCCTCGGCGAGCAGCCTACCACCGTGCCCCCTCCCATCGACCTGAGCATC CCCCACGTGTGGATGCCTCCCCAGACCACCCCTCACGGCTGGACCGAGAGCCACACCACCTCCGGCCTGC ACAGACCCCACTTCAACCAGACCTGCATCCTGTTCGACGGCCACGACCTGCTGTTTAGCACCGTGACCCC CTGCCTGCACCAGGGCTTCTACCTGATCGACGAGCTGAGATACGTGAAGATCACCCTGACCGAGGATTTC TTCGTGGTCACCGTGTCCATCGACGACGACACCCCCATGCTGCTGATCTTCGGCCACCTGCCCAGAGTGC TGTTCAAGGCCCCCTACCAGCGGGACAACTTCATCCTGCGGCAGACCGAGAAGCACGAGCTGCTGGTGCT GGTCAAGAAGGACCAGCTGAACCGGCACTCCTACCTGAAGGACCCCGACTTCCTGGACGCCGCCCTGGAC TTCAACTACCTGGACCTGAGCGCCCTGCTGAGAAACAGCTTCCACAGATACGCCGTGGACGTGCTGAAGT CCGGACGGTGCCAGATGCTCGATCGGCGGACCGTGGAGATGGCCTTCGCCTATGCCCTCGCCCTGTTCGC CGCTGCCAGACAGGAAGAGGCTGGCGCCCAGGTGTCAGTGCCCAGAGCCCTGGATAGACAGGCCGCCCTG CTGCAGATCCAGGAATTCATGATCACCTGCCTGAGCCAGACCCCCCCTAGAACCACCCTGCTGCTGTACC CCACAGCCGTGGATCTGGCCAAGAGGGCCCTGTGGACCCCCAACCAGATCACCGACATCACAAGCCTCGT GCGGCTCGTGTACATCCTGAGCAAGCAGAACCAGCAGCACCTGATCCCCCAGTGGGCCCTGAGACAGATC GCCGACTTCGCCCTGAAGCTGCACAAGACCCATCTGGCCAGCTTTCTGAGCGCCTTCGCCAGGCAGGAAC TGTACCTGATGGGCAGCCTGGTCCACAGCATGCTGGTGCATACCACCGAGCGGCGGGAGATCTTCATCGT GGAGACAGGCCTGTGTAGCCTGGCCGAGCTGTCCCACTTTACCCAGCTGCTGGCCCACCCTCACCACGAG TACCTGAGCGACCTGTACACCCCCTGCAGCAGCAGCGGCAGACGGGACCACAGCCTGGAACGGCTGACCA GACTGTTCCCCGATGCCACCGTGCCTGCTACAGTGCCTGCCGCCCTGTCCATCCTGTCCACCATGCAGCC CAGCACCCTGGAAACCTTCCCCGACCTGTTCTGCCTGCCCCTGGGCGAGAGCTTTAGCGCCCTGACCGTG TCCGAGCACGTGTCCTACATCGTGACCAATCAGTACCTGATCAAGGGCATCAGCTACCCCGTGTCCACCA CAGTCGTGGGCCAGAGCCTGATCATCACCCAGACCGACAGCCAGACCAAGTGCGAGCTGACCCGGAACAT GCACACCACACACAGCATCACCGTGGCCCTGAACATCAGCCTGGAAAACTGCGCTTTCTGTCAGTCTGCC CTGCTGGAATACGACGATACCCAGGGCGTGATCAACATCATGTACATGCACGACAGCGACGACGTGCTGT TCGCCCTGGACCCCTACAACGAGGTGGTGGTGTCCAGCCCCCGGACCCACTACCTGATGCTGCTGAAGAA CGGCACCGTGCTGGAAGTGACCGACGTGGTGGTGGACGCCACCGACTGATAATCTAGATTAAAACAGCTG TGGGTTGTTCCCACCCACAGGGCCCACTGGGCGCTAGCACTCTGATTTTACGAAATCCTTGTGCGCCTGT TTTATATCCCTTCCCTAATTCGAAACGTAGAAGCAATGCGCACCACTGATCAATAGTAGGCGTAACGCGC CAGTTACGTCATGATCAAGCATATCTGTTCCCCCGGACTGAGTATCAATAGACTGCTTACGCGGTTGAAG GAGAAAACGTTCGTTATCCGGCTAACTACTTCGAGAAGCCCAGTAACACCATGGAAGCTGCAGGGTGTTT CGCTCAGCACTTCCCCCGTGTAGATCAGGTCGATGAGCCACTGCAATCCCCACAGGTGACTGTGGCAGTG GCTGCGTTGGCGGCCTGCCTATGGGGAGACCCATAGGACGCTCTAATGTGGACATGGTGCGAAGAGCCTA TTGAGCTAGTTAGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACATGCCTTCAACC CAGAGGGTAGTGTGTCGTAATGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCTTTT TATTCTTATATTGGCTGCTTATGGTGACAATTACAGAATTGTTACCATATAGCTATTGGATTGGCCATCC GGTGTGTAATAGAGCTGTTATATACCTATTTGTTGGCTTTGTACCACTAACTTTAAAATCTATAACTACC CTCAACTTTATATTAACCCTCAATACAGTTGAACATGTGCAGAAGGCCCGACTGCGGCTTCAGCTTCAGC CCTGGACCCGTGATCCTGCTGTGGTGCTGCCTGCTGCTGCCTATCGTGTCCTCTGCCGCCGTGTCTGTGG CCCCTACAGCCGCCGAGAAGGTGCCAGCCGAGTGCCCCGAGCTGACCAGAAGATGCCTGCTGGGCGAGGT GTTCGAGGGCGACAAGTACGAGAGCTGGCTGCGGCCCCTGGTCAACGTGACCGGCAGAGATGGCCCCCTG AGCCAGCTGATCCGGTACAGACCCGTGACCCCCGAGGCCGCCAATAGCGTGCTGCTGGACGAGGCCTTCC TGGATACCCTGGCCCTGCTGTACAACAACCCCGACCAGCTGAGAGCCCTGCTGACCCTGCTGTCCAGCGA CACCGCCCCCAGATGGATGACCGTGATGCGGGGCTACAGCGAGTGTGGAGATGGCAGCCCTGCCGTGTAC ACCTGCGTGGACGACCTGTGCAGAGGCTACGACCTGACCAGACTGAGCTACGGCCGGTCCATCTTCACAG AGCACGTGCTGGGCTTCGAGCTGGTGCCCCCCAGCCTGTTCAACGTGGTGGTGGCCATCCGGAACGAGGC CACCAGAACCAACAGAGCCGTGCGGCTGCCTGTGTCTACAGCCGCTGCACCTGAGGGCATCACACTGTTC TACGGCCTGTACAACGCCGTGAAAGAGTTCTGCCTCCGGCACCAGCTGGATCCCCCCCTGCTGAGACACC TGGACAAGTACTACGCCGGCCTGCCCCCAGAGCTGAAGCAGACCAGAGTGAACCTGCCCGCCCACAGCAG ATATGGCCCTCAGGCCGTGGACGCCAGATGATAAGCGGCCGCATACAGCAGCAATTGGCAAGCTGCTTAC ATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTCTTTTCTTTTCCGAATCGG ATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGTCGGCATGGCATC TCCACCTCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGACGCACGTCCACTCGGATGGCTAAGGGAGAG CCACGTTTAAACACGTGATATCTGGCCTCATGGGCCTTCCTTTCACTGCCCGCTTTCCAGTCGGGAAACC TGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCCTCGCT CACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAAAAGG CCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAA AAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGA AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGG GAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCT GGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCC AACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATG TAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTAT CTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACC GCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATC CTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATA TATGAGTAAACTTGGTCTGACAGTTATTAGAAAAATTCATCCAGCAGACGATAAAACGCAATACGCTGGC TATCCGGTGCCGCAATGCCATACAGCACCAGAAAACGATCCGCCCATTCGCCGCCCAGTTCTTCCGCAAT ATCACGGGTGGCCAGCGCAATATCCTGATAACGATCCGCCACGCCCAGACGGCCGCAATCAATAAAGCCG CTAAAACGGCCATTTTCCACCATAATGTTCGGCAGGCACGCATCACCATGGGTCACCACCAGATCTTCGC CATCCGGCATGCTCGCTTTCAGACGCGCAAACAGCTCTGCCGGTGCCAGGCCCTGATGTTCTTCATCCAG ATCATCCTGATCCACCAGGCCCGCTTCCATACGGGTACGCGCACGTTCAATACGATGTTTCGCCTGATGA TCAAACGGACAGGTCGCCGGGTCCAGGGTATGCAGACGACGCATGGCATCCGCCATAATGCTCACTTTTT CTGCCGGCGCCAGATGGCTAGACAGCAGATCCTGACCCGGCACTTCGCCCAGCAGCAGCCAATCACGGCC CGCTTCGGTCACCACATCCAGCACCGCCGCACACGGAACACCGGTGGTGGCCAGCCAGCTCAGACGCGCC GCTTCATCCTGCAGCTCGTTCAGCGCACCGCTCAGATCGGTTTTCACAAACAGCACCGGACGACCCTGCG CGCTCAGACGAAACACCGCCGCATCAGAGCAGCCAATGGTCTGCTGCGCCCAATCATAGCCAAACAGACG TTCCACCCACGCTGCCGGGCTACCCGCATGCAGGCCATCCTGTTCAATCATACTCTTCCTTTTTCAATAT TATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAAC AAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAA TTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATA AATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGCGCTCCCATTCGCCATTCAGGCTGC GCAACTGTTGGGAAGGGCGTTTCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTG CTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACACGCGTAATACGACTCACTATAG Vetor A534: SGP-gL-EV71-gH ATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGA GGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTC ACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGG ACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCA TTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAA AACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACC CTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGT TTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTC GCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACT CTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGA GCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCT GTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACT TACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAG AATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTC TTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAG CTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCT GGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTAC CTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAA GGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAAC ATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTG CCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACA AGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGA GGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTG GAAGCCGATGTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAA AGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAA GAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAA GGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGG ACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCA CCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAG CACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAG GGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGC CGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAA AGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACG TCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACA CCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCC ATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCC TGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAA ATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACT AAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAG GGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCT GACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAA CATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGA TAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGA TGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGT TGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTG TGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTT TGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGG GATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACC CACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGA TCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACAC CCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGT TGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGA TTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATAC CATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATC TGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGG TGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTT CTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGA CCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGA TATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTG TGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGAC TGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGA AGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCA GTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACC ATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAAT GACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTG ACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCA CAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGA AATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGC ATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTT GCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTAC TGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCT ATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAG ACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAAC CGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGT TTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTAT CTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCT GGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAG TTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAA GAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAA TAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGA ACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAG CACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACA ACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTAT GCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTA ACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGG CCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTAT TCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAG AGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGG AGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCC TATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAG CTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAA TGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGG CTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGA AGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGT GAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCG CTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGA ACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGG GGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCG TTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAA TTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCT CACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGA TCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAG ACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTA TTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAA AGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGC ATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTA TGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGC TACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAA GATGTGCAGAAGGCCCGACTGCGGCTTCAGCTTCAGCCCTGGACCCGTGATCCTGCTGTGGTGCTGCCTG CTGCTGCCTATCGTGTCCTCTGCCGCCGTGTCTGTGGCCCCTACAGCCGCCGAGAAGGTGCCAGCCGAGT GCCCCGAGCTGACCAGAAGATGCCTGCTGGGCGAGGTGTTCGAGGGCGACAAGTACGAGAGCTGGCTGCG GCCCCTGGTCAACGTGACCGGCAGAGATGGCCCCCTGAGCCAGCTGATCCGGTACAGACCCGTGACCCCC GAGGCCGCCAATAGCGTGCTGCTGGACGAGGCCTTCCTGGATACCCTGGCCCTGCTGTACAACAACCCCG ACCAGCTGAGAGCCCTGCTGACCCTGCTGTCCAGCGACACCGCCCCCAGATGGATGACCGTGATGCGGGG CTACAGCGAGTGTGGAGATGGCAGCCCTGCCGTGTACACCTGCGTGGACGACCTGTGCAGAGGCTACGAC CTGACCAGACTGAGCTACGGCCGGTCCATCTTCACAGAGCACGTGCTGGGCTTCGAGCTGGTGCCCCCCA GCCTGTTCAACGTGGTGGTGGCCATCCGGAACGAGGCCACCAGAACCAACAGAGCCGTGCGGCTGCCTGT GTCTACAGCCGCTGCACCTGAGGGCATCACACTGTTCTACGGCCTGTACAACGCCGTGAAAGAGTTCTGC CTCCGGCACCAGCTGGATCCCCCCCTGCTGAGACACCTGGACAAGTACTACGCCGGCCTGCCCCCAGAGC TGAAGCAGACCAGAGTGAACCTGCCCGCCCACAGCAGATATGGCCCTCAGGCCGTGGACGCCAGATGATA ATCTAGATTAAAACAGCTGTGGGTTGTTCCCACCCACAGGGCCCACTGGGCGCTAGCACTCTGATTTTAC GAAATCCTTGTGCGCCTGTTTTATATCCCTTCCCTAATTCGAAACGTAGAAGCAATGCGCACCACTGATC AATAGTAGGCGTAACGCGCCAGTTACGTCATGATCAAGCATATCTGTTCCCCCGGACTGAGTATCAATAG ACTGCTTACGCGGTTGAAGGAGAAAACGTTCGTTATCCGGCTAACTACTTCGAGAAGCCCAGTAACACCA TGGAAGCTGCAGGGTGTTTCGCTCAGCACTTCCCCCGTGTAGATCAGGTCGATGAGCCACTGCAATCCCC ACAGGTGACTGTGGCAGTGGCTGCGTTGGCGGCCTGCCTATGGGGAGACCCATAGGACGCTCTAATGTGG ACATGGTGCGAAGAGCCTATTGAGCTAGTTAGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGC GGAGCACATGCCTTCAACCCAGAGGGTAGTGTGTCGTAATGGGCAACTCTGCAGCGGAACCGACTACTTT GGGTGTCCGTGTTTCTTTTTATTCTTATATTGGCTGCTTATGGTGACAATTACAGAATTGTTACCATATA GCTATTGGATTGGCCATCCGGTGTGTAATAGAGCTGTTATATACCTATTTGTTGGCTTTGTACCACTAAC TTTAAAATCTATAACTACCCTCAACTTTATATTAACCCTCAATACAGTTGAACATGAGGCCTGGCCTGCC CTCCTACCTGATCATCCTGGCCGTGTGCCTGTTCAGCCACCTGCTGTCCAGCAGATACGGCGCCGAGGCC GTGAGCGAGCCCCTGGACAAGGCTTTCCACCTGCTGCTGAACACCTACGGCAGACCCATCCGGTTTCTGC GGGAGAACACCACCCAGTGCACCTACAACAGCAGCCTGCGGAACAGCACCGTCGTGAGAGAGAACGCCAT CAGCTTCAACTTTTTCCAGAGCTACAACCAGTACTACGTGTTCCACATGCCCAGATGCCTGTTTGCCGGC CCTCTGGCCGAGCAGTTCCTGAACCAGGTGGACCTGACCGAGACACTGGAAAGATACCAGCAGCGGCTGA ATACCTACGCCCTGGTGTCCAAGGACCTGGCCAGCTACCGGTCCTTTAGCCAGCAGCTCAAGGCTCAGGA TAGCCTCGGCGAGCAGCCTACCACCGTGCCCCCTCCCATCGACCTGAGCATCCCCCACGTGTGGATGCCT CCCCAGACCACCCCTCACGGCTGGACCGAGAGCCACACCACCTCCGGCCTGCACAGACCCCACTTCAACC AGACCTGCATCCTGTTCGACGGCCACGACCTGCTGTTTAGCACCGTGACCCCCTGCCTGCACCAGGGCTT CTACCTGATCGACGAGCTGAGATACGTGAAGATCACCCTGACCGAGGATTTCTTCGTGGTCACCGTGTCC ATCGACGACGACACCCCCATGCTGCTGATCTTCGGCCACCTGCCCAGAGTGCTGTTCAAGGCCCCCTACC AGCGGGACAACTTCATCCTGCGGCAGACCGAGAAGCACGAGCTGCTGGTGCTGGTCAAGAAGGACCAGCT GAACCGGCACTCCTACCTGAAGGACCCCGACTTCCTGGACGCCGCCCTGGACTTCAACTACCTGGACCTG AGCGCCCTGCTGAGAAACAGCTTCCACAGATACGCCGTGGACGTGCTGAAGTCCGGACGGTGCCAGATGC TCGATCGGCGGACCGTGGAGATGGCCTTCGCCTATGCCCTCGCCCTGTTCGCCGCTGCCAGACAGGAAGA GGCTGGCGCCCAGGTGTCAGTGCCCAGAGCCCTGGATAGACAGGCCGCCCTGCTGCAGATCCAGGAATTC ATGATCACCTGCCTGAGCCAGACCCCCCCTAGAACCACCCTGCTGCTGTACCCCACAGCCGTGGATCTGG CCAAGAGGGCCCTGTGGACCCCCAACCAGATCACCGACATCACAAGCCTCGTGCGGCTCGTGTACATCCT GAGCAAGCAGAACCAGCAGCACCTGATCCCCCAGTGGGCCCTGAGACAGATCGCCGACTTCGCCCTGAAG CTGCACAAGACCCATCTGGCCAGCTTTCTGAGCGCCTTCGCCAGGCAGGAACTGTACCTGATGGGCAGCC TGGTCCACAGCATGCTGGTGCATACCACCGAGCGGCGGGAGATCTTCATCGTGGAGACAGGCCTGTGTAG CCTGGCCGAGCTGTCCCACTTTACCCAGCTGCTGGCCCACCCTCACCACGAGTACCTGAGCGACCTGTAC ACCCCCTGCAGCAGCAGCGGCAGACGGGACCACAGCCTGGAACGGCTGACCAGACTGTTCCCCGATGCCA CCGTGCCTGCTACAGTGCCTGCCGCCCTGTCCATCCTGTCCACCATGCAGCCCAGCACCCTGGAAACCTT CCCCGACCTGTTCTGCCTGCCCCTGGGCGAGAGCTTTAGCGCCCTGACCGTGTCCGAGCACGTGTCCTAC ATCGTGACCAATCAGTACCTGATCAAGGGCATCAGCTACCCCGTGTCCACCACAGTCGTGGGCCAGAGCC TGATCATCACCCAGACCGACAGCCAGACCAAGTGCGAGCTGACCCGGAACATGCACACCACACACAGCAT CACCGTGGCCCTGAACATCAGCCTGGAAAACTGCGCTTTCTGTCAGTCTGCCCTGCTGGAATACGACGAT ACCCAGGGCGTGATCAACATCATGTACATGCACGACAGCGACGACGTGCTGTTCGCCCTGGACCCCTACA ACGAGGTGGTGGTGTCCAGCCCCCGGACCCACTACCTGATGCTGCTGAAGAACGGCACCGTGCTGGAAGT GACCGACGTGGTGGTGGACGCCACCGACTGATAAGCGGCCGCATACAGCAGCAATTGGCAAGCTGCTTAC ATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTCTTTTCTTTTCCGAATCGG ATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGTCGGCATGGCATC TCCACCTCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGACGCACGTCCACTCGGATGGCTAAGGGAGAG CCACGTTTAAACACGTGATATCTGGCCTCATGGGCCTTCCTTTCACTGCCCGCTTTCCAGTCGGGAAACC TGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCCTCGCT CACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAAAAGG CCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAA AAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGA AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGG GAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCT GGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCC AACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATG TAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTAT CTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACC GCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATC CTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATA TATGAGTAAACTTGGTCTGACAGTTATTAGAAAAATTCATCCAGCAGACGATAAAACGCAATACGCTGGC TATCCGGTGCCGCAATGCCATACAGCACCAGAAAACGATCCGCCCATTCGCCGCCCAGTTCTTCCGCAAT ATCACGGGTGGCCAGCGCAATATCCTGATAACGATCCGCCACGCCCAGACGGCCGCAATCAATAAAGCCG CTAAAACGGCCATTTTCCACCATAATGTTCGGCAGGCACGCATCACCATGGGTCACCACCAGATCTTCGC CATCCGGCATGCTCGCTTTCAGACGCGCAAACAGCTCTGCCGGTGCCAGGCCCTGATGTTCTTCATCCAG ATCATCCTGATCCACCAGGCCCGCTTCCATACGGGTACGCGCACGTTCAATACGATGTTTCGCCTGATGA TCAAACGGACAGGTCGCCGGGTCCAGGGTATGCAGACGACGCATGGCATCCGCCATAATGCTCACTTTTT CTGCCGGCGCCAGATGGCTAGACAGCAGATCCTGACCCGGCACTTCGCCCAGCAGCAGCCAATCACGGCC CGCTTCGGTCACCACATCCAGCACCGCCGCACACGGAACACCGGTGGTGGCCAGCCAGCTCAGACGCGCC GCTTCATCCTGCAGCTCGTTCAGCGCACCGCTCAGATCGGTTTTCACAAACAGCACCGGACGACCCTGCG CGCTCAGACGAAACACCGCCGCATCAGAGCAGCCAATGGTCTGCTGCGCCCAATCATAGCCAAACAGACG TTCCACCCACGCTGCCGGGCTACCCGCATGCAGGCCATCCTGTTCAATCATACTCTTCCTTTTTCAATAT TATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAAC AAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAA TTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATA AATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGCGCTCCCATTCGCCATTCAGGCTGC GCAACTGTTGGGAAGGGCGTTTCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTG CTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACACGCGTAATACGACTCACTATAG Vetor A535: SGP-342-EV71-gHsol-2A-gL ATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGA GGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTC ACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGG ACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCA TTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAA AACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACC CTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGT TTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTC GCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACT CTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGA GCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCT GTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACT TACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAG AATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTC TTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAG CTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCT GGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTAC CTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAA GGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAAC ATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTG CCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACA AGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGA GGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTG GAAGCCGATGTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAA AGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAA GAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAA GGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGG ACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCA CCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAG CACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAG GGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGC CGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAA AGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACG TCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACA CCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCC ATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCC TGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAA ATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACT AAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAG GGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCT GACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAA CATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGA TAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGA TGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGT TGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTG TGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTT TGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGG GATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACC CACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGA TCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACAC CCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGT TGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGA TTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATAC CATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATC TGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGG TGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTT CTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGA CCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGA TATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTG TGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGAC TGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGA AGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCA GTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACC ATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAAT GACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTG ACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCA CAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGA AATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGC ATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTT GCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTAC TGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCT ATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAG ACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAAC CGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGT TTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTAT CTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCT GGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAG TTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAA GAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAA TAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGA ACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAG CACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACA ACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTAT GCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTA ACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGG CCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTAT TCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAG AGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGG AGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCC TATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAG CTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAA TGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGG CTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGA AGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGT GAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCG CTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGA ACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGG GGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCG TTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAA TTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCT CACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGA TCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAG ACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTA TTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAA AGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGC ATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTA TGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGC TACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAA GCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTGTATATCCATTT TCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAATAAGATGGATTGCACGTAGGTTC TCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACTGACAATCGGCTGCTCTGATGCC GCCGTGATCCGGTTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGA ATGAACTGAAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGTCTAGAC TGGCGCGCCAAACCTGCAGGTTAAAACAGCTGTGGGTTGTTCCCACCCACAGGGCCCACTGGGCGCTAGC ACTCTGATTTTACGAAATCCTTGTGCGCCTGTTTTATATCCCTTCCCTAATTCGAAACGTAGAAGCAATG CGCACCACTGATCAATAGTAGGCGTAACGCGCCAGTTACGTCATGATCAAGCATATCTGTTCCCCCGGAC TGAGTATCAATAGACTGCTTACGCGGTTGAAGGAGAAAACGTTCGTTATCCGGCTAACTACTTCGAGAAG CCCAGTAACACCATGGAAGCTGCAGGGTGTTTCGCTCAGCACTTCCCCCGTGTAGATCAGGTCGATGAGC CACTGCAATCCCCACAGGTGACTGTGGCAGTGGCTGCGTTGGCGGCCTGCCTATGGGGAGACCCATAGGA CGCTCTAATGTGGACATGGTGCGAAGAGCCTATTGAGCTAGTTAGTAGTCCTCCGGCCCCTGAATGCGGC TAATCCTAACTGCGGAGCACATGCCTTCAACCCAGAGGGTAGTGTGTCGTAATGGGCAACTCTGCAGCGG AACCGACTACTTTGGGTGTCCGTGTTTCTTTTTATTCTTATATTGGCTGCTTATGGTGACAATTACAGAA TTGTTACCATATAGCTATTGGATTGGCCATCCGGTGTGTAATAGAGCTGTTATATACCTATTTGTTGGCT TTGTACCACTAACTTTAAAATCTATAACTACCCTCAACTTTATATTAACCCTCAATACAGTTGAACATGA GGCCTGGCCTGCCCTCCTACCTGATCATCCTGGCCGTGTGCCTGTTCAGCCACCTGCTGTCCAGCAGATA CGGCGCCGAGGCCGTGAGCGAGCCCCTGGACAAGGCTTTCCACCTGCTGCTGAACACCTACGGCAGACCC ATCCGGTTTCTGCGGGAGAACACCACCCAGTGCACCTACAACAGCAGCCTGCGGAACAGCACCGTCGTGA GAGAGAACGCCATCAGCTTCAACTTTTTCCAGAGCTACAACCAGTACTACGTGTTCCACATGCCCAGATG CCTGTTTGCCGGCCCTCTGGCCGAGCAGTTCCTGAACCAGGTGGACCTGACCGAGACACTGGAAAGATAC CAGCAGCGGCTGAATACCTACGCCCTGGTGTCCAAGGACCTGGCCAGCTACCGGTCCTTTAGCCAGCAGC TCAAGGCTCAGGATAGCCTCGGCGAGCAGCCTACCACCGTGCCCCCTCCCATCGACCTGAGCATCCCCCA CGTGTGGATGCCTCCCCAGACCACCCCTCACGGCTGGACCGAGAGCCACACCACCTCCGGCCTGCACAGA CCCCACTTCAACCAGACCTGCATCCTGTTCGACGGCCACGACCTGCTGTTTAGCACCGTGACCCCCTGCC TGCACCAGGGCTTCTACCTGATCGACGAGCTGAGATACGTGAAGATCACCCTGACCGAGGATTTCTTCGT GGTCACCGTGTCCATCGACGACGACACCCCCATGCTGCTGATCTTCGGCCACCTGCCCAGAGTGCTGTTC AAGGCCCCCTACCAGCGGGACAACTTCATCCTGCGGCAGACCGAGAAGCACGAGCTGCTGGTGCTGGTCA AGAAGGACCAGCTGAACCGGCACTCCTACCTGAAGGACCCCGACTTCCTGGACGCCGCCCTGGACTTCAA CTACCTGGACCTGAGCGCCCTGCTGAGAAACAGCTTCCACAGATACGCCGTGGACGTGCTGAAGTCCGGA CGGTGCCAGATGCTCGATCGGCGGACCGTGGAGATGGCCTTCGCCTATGCCCTCGCCCTGTTCGCCGCTG CCAGACAGGAAGAGGCTGGCGCCCAGGTGTCAGTGCCCAGAGCCCTGGATAGACAGGCCGCCCTGCTGCA GATCCAGGAATTCATGATCACCTGCCTGAGCCAGACCCCCCCTAGAACCACCCTGCTGCTGTACCCCACA GCCGTGGATCTGGCCAAGAGGGCCCTGTGGACCCCCAACCAGATCACCGACATCACAAGCCTCGTGCGGC TCGTGTACATCCTGAGCAAGCAGAACCAGCAGCACCTGATCCCCCAGTGGGCCCTGAGACAGATCGCCGA CTTCGCCCTGAAGCTGCACAAGACCCATCTGGCCAGCTTTCTGAGCGCCTTCGCCAGGCAGGAACTGTAC CTGATGGGCAGCCTGGTCCACAGCATGCTGGTGCATACCACCGAGCGGCGGGAGATCTTCATCGTGGAGA CAGGCCTGTGTAGCCTGGCCGAGCTGTCCCACTTTACCCAGCTGCTGGCCCACCCTCACCACGAGTACCT GAGCGACCTGTACACCCCCTGCAGCAGCAGCGGCAGACGGGACCACAGCCTGGAACGGCTGACCAGACTG TTCCCCGATGCCACCGTGCCTGCTACAGTGCCTGCCGCCCTGTCCATCCTGTCCACCATGCAGCCCAGCA CCCTGGAAACCTTCCCCGACCTGTTCTGCCTGCCCCTGGGCGAGAGCTTTAGCGCCCTGACCGTGTCCGA GCACGTGTCCTACATCGTGACCAATCAGTACCTGATCAAGGGCATCAGCTACCCCGTGTCCACCACAGTC GTGGGCCAGAGCCTGATCATCACCCAGACCGACAGCCAGACCAAGTGCGAGCTGACCCGGAACATGCACA CCACACACAGCATCACCGTGGCCCTGAACATCAGCCTGGAAAACTGCGCTTTCTGTCAGTCTGCCCTGCT GGAATACGACGATACCCAGGGCGTGATCAACATCATGTACATGCACGACAGCGACGACGTGCTGTTCGCC CTGGACCCCTACAACGAGGTGGTGGTGTCCAGCCCCCGGACCCACTACCTGATGCTGCTGAAGAACGGCA CCGTGCTGGAAGTGACCGACGTGGTGGTGGACGCCACCGACCTGTTGAATTTTGACCTTCTTAAGCTTGC GGGAGACGTCGAGTCCAACCCCGGGCCCATGTGCAGAAGGCCCGACTGCGGCTTCAGCTTCAGCCCTGGA CCCGTGATCCTGCTGTGGTGCTGCCTGCTGCTGCCTATCGTGTCCTCTGCCGCCGTGTCTGTGGCCCCTA CAGCCGCCGAGAAGGTGCCAGCCGAGTGCCCCGAGCTGACCAGAAGATGCCTGCTGGGCGAGGTGTTCGA GGGCGACAAGTACGAGAGCTGGCTGCGGCCCCTGGTCAACGTGACCGGCAGAGATGGCCCCCTGAGCCAG CTGATCCGGTACAGACCCGTGACCCCCGAGGCCGCCAATAGCGTGCTGCTGGACGAGGCCTTCCTGGATA CCCTGGCCCTGCTGTACAACAACCCCGACCAGCTGAGAGCCCTGCTGACCCTGCTGTCCAGCGACACCGC CCCCAGATGGATGACCGTGATGCGGGGCTACAGCGAGTGTGGAGATGGCAGCCCTGCCGTGTACACCTGC GTGGACGACCTGTGCAGAGGCTACGACCTGACCAGACTGAGCTACGGCCGGTCCATCTTCACAGAGCACG TGCTGGGCTTCGAGCTGGTGCCCCCCAGCCTGTTCAACGTGGTGGTGGCCATCCGGAACGAGGCCACCAG AACCAACAGAGCCGTGCGGCTGCCTGTGTCTACAGCCGCTGCACCTGAGGGCATCACACTGTTCTACGGC CTGTACAACGCCGTGAAAGAGTTCTGCCTCCGGCACCAGCTGGATCCCCCCCTGCTGAGACACCTGGACA AGTACTACGCCGGCCTGCCCCCAGAGCTGAAGCAGACCAGAGTGAACCTGCCCGCCCACAGCAGATATGG CCCTCAGGCCGTGGACGCCAGATGATAAGCGGCCGCATACAGCAGCAATTGGCAAGCTGCTTACATAGAA CTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTCTTTTCTTTTCCGAATCGGATTTTG TTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGTCGGCATGGCATCTCCACC TCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGACGCACGTCCACTCGGATGGCTAAGGGAGAGCCACGT TTAAACACGTGATATCTGGCCTCATGGGCCTTCCTTTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGT GCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCCTCGCTCACTGA CTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAAAAGGCCAGGA ACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCG ACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCC CTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCG TGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTG TGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCG GTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCG GTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGC TCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGT AGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGA TCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATC AAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG TAAACTTGGTCTGACAGTTATTAGAAAAATTCATCCAGCAGACGATAAAACGCAATACGCTGGCTATCCG GTGCCGCAATGCCATACAGCACCAGAAAACGATCCGCCCATTCGCCGCCCAGTTCTTCCGCAATATCACG GGTGGCCAGCGCAATATCCTGATAACGATCCGCCACGCCCAGACGGCCGCAATCAATAAAGCCGCTAAAA CGGCCATTTTCCACCATAATGTTCGGCAGGCACGCATCACCATGGGTCACCACCAGATCTTCGCCATCCG GCATGCTCGCTTTCAGACGCGCAAACAGCTCTGCCGGTGCCAGGCCCTGATGTTCTTCATCCAGATCATC CTGATCCACCAGGCCCGCTTCCATACGGGTACGCGCACGTTCAATACGATGTTTCGCCTGATGATCAAAC GGACAGGTCGCCGGGTCCAGGGTATGCAGACGACGCATGGCATCCGCCATAATGCTCACTTTTTCTGCCG GCGCCAGATGGCTAGACAGCAGATCCTGACCCGGCACTTCGCCCAGCAGCAGCCAATCACGGCCCGCTTC GGTCACCACATCCAGCACCGCCGCACACGGAACACCGGTGGTGGCCAGCCAGCTCAGACGCGCCGCTTCA TCCTGCAGCTCGTTCAGCGCACCGCTCAGATCGGTTTTCACAAACAGCACCGGACGACCCTGCGCGCTCA GACGAAACACCGCCGCATCAGAGCAGCCAATGGTCTGCTGCGCCCAATCATAGCCAAACAGACGTTCCAC CCACGCTGCCGGGCTACCCGCATGCAGGCCATCCTGTTCAATCATACTCTTCCTTTTTCAATATTATTGA AGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAG GGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCG TTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAA AAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGCGCTCCCATTCGCCATTCAGGCTGCGCAACT GTTGGGAAGGGCGTTTCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAA GGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACACGCGTAATACGACTCACTATAG Vetor A536: SGP-342-EV71-gHsol-EMCV-gL ATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGA GGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTC ACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGG ACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCA TTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAA AACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACC CTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGT TTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTC GCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACT CTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGA GCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCT GTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACT TACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAG AATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTC TTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAG CTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCT GGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTAC CTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAA GGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAAC ATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTG CCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACA AGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGA GGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTG GAAGCCGATGTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAA AGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAA GAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAA GGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGG ACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCA CCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAG CACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAG GGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGC CGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAA AGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACG TCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACA CCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCC ATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCC TGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAA ATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACT AAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAG GGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCT GACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAA CATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGA TAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGA TGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGT TGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTG TGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTT TGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGG GATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACC CACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGA TCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACAC CCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGT TGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGA TTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATAC CATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATC TGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGG TGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTT CTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGA CCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGA TATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTG TGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGAC TGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGA AGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCA GTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACC ATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAAT GACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTG ACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCA CAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGA AATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGC ATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTT GCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTAC TGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCT ATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAG ACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAAC CGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGT TTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTAT CTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCT GGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAG TTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAA GAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAA TAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGA ACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAG CACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACA ACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTAT GCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTA ACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGG CCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTAT TCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAG AGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGG AGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCC TATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAG CTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAA TGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGG CTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGA AGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGT GAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCG CTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGA ACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGG GGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCG TTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAA TTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCT CACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGA TCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAG ACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTA TTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAA AGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGC ATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTA TGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGC TACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAA GCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTGTATATCCATTT TCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAATAAGATGGATTGCACGTAGGTTC TCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACTGACAATCGGCTGCTCTGATGCC GCCGTGATCCGGTTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGA ATGAACTGAAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGTCTAGAC TGGCGCGCCAAACCTGCAGGTTAAAACAGCTGTGGGTTGTTCCCACCCACAGGGCCCACTGGGCGCTAGC ACTCTGATTTTACGAAATCCTTGTGCGCCTGTTTTATATCCCTTCCCTAATTCGAAACGTAGAAGCAATG CGCACCACTGATCAATAGTAGGCGTAACGCGCCAGTTACGTCATGATCAAGCATATCTGTTCCCCCGGAC TGAGTATCAATAGACTGCTTACGCGGTTGAAGGAGAAAACGTTCGTTATCCGGCTAACTACTTCGAGAAG CCCAGTAACACCATGGAAGCTGCAGGGTGTTTCGCTCAGCACTTCCCCCGTGTAGATCAGGTCGATGAGC CACTGCAATCCCCACAGGTGACTGTGGCAGTGGCTGCGTTGGCGGCCTGCCTATGGGGAGACCCATAGGA CGCTCTAATGTGGACATGGTGCGAAGAGCCTATTGAGCTAGTTAGTAGTCCTCCGGCCCCTGAATGCGGC TAATCCTAACTGCGGAGCACATGCCTTCAACCCAGAGGGTAGTGTGTCGTAATGGGCAACTCTGCAGCGG AACCGACTACTTTGGGTGTCCGTGTTTCTTTTTATTCTTATATTGGCTGCTTATGGTGACAATTACAGAA TTGTTACCATATAGCTATTGGATTGGCCATCCGGTGTGTAATAGAGCTGTTATATACCTATTTGTTGGCT TTGTACCACTAACTTTAAAATCTATAACTACCCTCAACTTTATATTAACCCTCAATACAGTTGAACATGA GGCCTGGCCTGCCCTCCTACCTGATCATCCTGGCCGTGTGCCTGTTCAGCCACCTGCTGTCCAGCAGATA CGGCGCCGAGGCCGTGAGCGAGCCCCTGGACAAGGCTTTCCACCTGCTGCTGAACACCTACGGCAGACCC ATCCGGTTTCTGCGGGAGAACACCACCCAGTGCACCTACAACAGCAGCCTGCGGAACAGCACCGTCGTGA GAGAGAACGCCATCAGCTTCAACTTTTTCCAGAGCTACAACCAGTACTACGTGTTCCACATGCCCAGATG CCTGTTTGCCGGCCCTCTGGCCGAGCAGTTCCTGAACCAGGTGGACCTGACCGAGACACTGGAAAGATAC CAGCAGCGGCTGAATACCTACGCCCTGGTGTCCAAGGACCTGGCCAGCTACCGGTCCTTTAGCCAGCAGC TCAAGGCTCAGGATAGCCTCGGCGAGCAGCCTACCACCGTGCCCCCTCCCATCGACCTGAGCATCCCCCA CGTGTGGATGCCTCCCCAGACCACCCCTCACGGCTGGACCGAGAGCCACACCACCTCCGGCCTGCACAGA CCCCACTTCAACCAGACCTGCATCCTGTTCGACGGCCACGACCTGCTGTTTAGCACCGTGACCCCCTGCC TGCACCAGGGCTTCTACCTGATCGACGAGCTGAGATACGTGAAGATCACCCTGACCGAGGATTTCTTCGT GGTCACCGTGTCCATCGACGACGACACCCCCATGCTGCTGATCTTCGGCCACCTGCCCAGAGTGCTGTTC AAGGCCCCCTACCAGCGGGACAACTTCATCCTGCGGCAGACCGAGAAGCACGAGCTGCTGGTGCTGGTCA AGAAGGACCAGCTGAACCGGCACTCCTACCTGAAGGACCCCGACTTCCTGGACGCCGCCCTGGACTTCAA CTACCTGGACCTGAGCGCCCTGCTGAGAAACAGCTTCCACAGATACGCCGTGGACGTGCTGAAGTCCGGA CGGTGCCAGATGCTCGATCGGCGGACCGTGGAGATGGCCTTCGCCTATGCCCTCGCCCTGTTCGCCGCTG CCAGACAGGAAGAGGCTGGCGCCCAGGTGTCAGTGCCCAGAGCCCTGGATAGACAGGCCGCCCTGCTGCA GATCCAGGAATTCATGATCACCTGCCTGAGCCAGACCCCCCCTAGAACCACCCTGCTGCTGTACCCCACA GCCGTGGATCTGGCCAAGAGGGCCCTGTGGACCCCCAACCAGATCACCGACATCACAAGCCTCGTGCGGC TCGTGTACATCCTGAGCAAGCAGAACCAGCAGCACCTGATCCCCCAGTGGGCCCTGAGACAGATCGCCGA CTTCGCCCTGAAGCTGCACAAGACCCATCTGGCCAGCTTTCTGAGCGCCTTCGCCAGGCAGGAACTGTAC CTGATGGGCAGCCTGGTCCACAGCATGCTGGTGCATACCACCGAGCGGCGGGAGATCTTCATCGTGGAGA CAGGCCTGTGTAGCCTGGCCGAGCTGTCCCACTTTACCCAGCTGCTGGCCCACCCTCACCACGAGTACCT GAGCGACCTGTACACCCCCTGCAGCAGCAGCGGCAGACGGGACCACAGCCTGGAACGGCTGACCAGACTG TTCCCCGATGCCACCGTGCCTGCTACAGTGCCTGCCGCCCTGTCCATCCTGTCCACCATGCAGCCCAGCA CCCTGGAAACCTTCCCCGACCTGTTCTGCCTGCCCCTGGGCGAGAGCTTTAGCGCCCTGACCGTGTCCGA GCACGTGTCCTACATCGTGACCAATCAGTACCTGATCAAGGGCATCAGCTACCCCGTGTCCACCACAGTC GTGGGCCAGAGCCTGATCATCACCCAGACCGACAGCCAGACCAAGTGCGAGCTGACCCGGAACATGCACA CCACACACAGCATCACCGTGGCCCTGAACATCAGCCTGGAAAACTGCGCTTTCTGTCAGTCTGCCCTGCT GGAATACGACGATACCCAGGGCGTGATCAACATCATGTACATGCACGACAGCGACGACGTGCTGTTCGCC CTGGACCCCTACAACGAGGTGGTGGTGTCCAGCCCCCGGACCCACTACCTGATGCTGCTGAAGAACGGCA CCGTGCTGGAAGTGACCGACGTGGTGGTGGACGCCACCGACTGATAACGCCGGCGCCCCCCCCTAACGTT ACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGT CTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCC TCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGA CAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCC AAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGT TGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCC CATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAAC GTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATAATAATATGTGCAGAAGGC CCGACTGCGGCTTCAGCTTCAGCCCTGGACCCGTGATCCTGCTGTGGTGCTGCCTGCTGCTGCCTATCGT GTCCTCTGCCGCCGTGTCTGTGGCCCCTACAGCCGCCGAGAAGGTGCCAGCCGAGTGCCCCGAGCTGACC AGAAGATGCCTGCTGGGCGAGGTGTTCGAGGGCGACAAGTACGAGAGCTGGCTGCGGCCCCTGGTCAACG TGACCGGCAGAGATGGCCCCCTGAGCCAGCTGATCCGGTACAGACCCGTGACCCCCGAGGCCGCCAATAG CGTGCTGCTGGACGAGGCCTTCCTGGATACCCTGGCCCTGCTGTACAACAACCCCGACCAGCTGAGAGCC CTGCTGACCCTGCTGTCCAGCGACACCGCCCCCAGATGGATGACCGTGATGCGGGGCTACAGCGAGTGTG GAGATGGCAGCCCTGCCGTGTACACCTGCGTGGACGACCTGTGCAGAGGCTACGACCTGACCAGACTGAG CTACGGCCGGTCCATCTTCACAGAGCACGTGCTGGGCTTCGAGCTGGTGCCCCCCAGCCTGTTCAACGTG GTGGTGGCCATCCGGAACGAGGCCACCAGAACCAACAGAGCCGTGCGGCTGCCTGTGTCTACAGCCGCTG CACCTGAGGGCATCACACTGTTCTACGGCCTGTACAACGCCGTGAAAGAGTTCTGCCTCCGGCACCAGCT GGATCCCCCCCTGCTGAGACACCTGGACAAGTACTACGCCGGCCTGCCCCCAGAGCTGAAGCAGACCAGA GTGAACCTGCCCGCCCACAGCAGATATGGCCCTCAGGCCGTGGACGCCAGATGATAAGCGGCCGCATACA GCAGCAATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTAT TTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAGGGTCGGCATGGCATCTCCACCTCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGACGCACGT CCACTCGGATGGCTAAGGGAGAGCCACGTTTAAACACGTGATATCTGGCCTCATGGGCCTTCCTTTCACT GCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTAT TGGGCGCTCTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTA ATGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTC CGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATA CCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCG GTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTAT CCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAA CAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTAC ACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCT CTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG AAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCA CGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAA GTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTATTAGAAAAATTCATCCAGCAG ACGATAAAACGCAATACGCTGGCTATCCGGTGCCGCAATGCCATACAGCACCAGAAAACGATCCGCCCAT TCGCCGCCCAGTTCTTCCGCAATATCACGGGTGGCCAGCGCAATATCCTGATAACGATCCGCCACGCCCA GACGGCCGCAATCAATAAAGCCGCTAAAACGGCCATTTTCCACCATAATGTTCGGCAGGCACGCATCACC ATGGGTCACCACCAGATCTTCGCCATCCGGCATGCTCGCTTTCAGACGCGCAAACAGCTCTGCCGGTGCC AGGCCCTGATGTTCTTCATCCAGATCATCCTGATCCACCAGGCCCGCTTCCATACGGGTACGCGCACGTT CAATACGATGTTTCGCCTGATGATCAAACGGACAGGTCGCCGGGTCCAGGGTATGCAGACGACGCATGGC ATCCGCCATAATGCTCACTTTTTCTGCCGGCGCCAGATGGCTAGACAGCAGATCCTGACCCGGCACTTCG CCCAGCAGCAGCCAATCACGGCCCGCTTCGGTCACCACATCCAGCACCGCCGCACACGGAACACCGGTGG TGGCCAGCCAGCTCAGACGCGCCGCTTCATCCTGCAGCTCGTTCAGCGCACCGCTCAGATCGGTTTTCAC AAACAGCACCGGACGACCCTGCGCGCTCAGACGAAACACCGCCGCATCAGAGCAGCCAATGGTCTGCTGC GCCCAATCATAGCCAAACAGACGTTCCACCCACGCTGCCGGGCTACCCGCATGCAGGCCATCCTGTTCAA TCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATT TGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTG TAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGC CGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGCG CTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGGTGCGGGCCTCTTCGCTATTACG CCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACAC GCGTAATACGACTCACTATAG Vetor A537: SGP-342-EV71-gL-EMCV-gHsol ATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGA GGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTC ACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGG ACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCA TTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAA AACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACC CTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGT TTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTC GCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACT CTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGA GCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCT GTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACT TACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAG AATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTC TTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAG CTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCT GGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTAC CTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAA GGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAAC ATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTG CCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACA AGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGA GGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTG GAAGCCGATGTCGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAA AGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAA GAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAA GGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGG ACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCA CCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAG CACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAG GGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGC CGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAA AGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACG TCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACA CCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCC ATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCC TGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAA ATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACT AAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAG GGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCT GACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAA CATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGA TAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGA TGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGT TGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTG TGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTT TGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGG GATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACC CACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGA TCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACAC CCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGT TGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGA TTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATAC CATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATC TGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGG TGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTT CTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGA CCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGA TATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTG TGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGAC TGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGA AGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCA GTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACC ATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAAT GACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTG ACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCA CAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGA AATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGC ATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTT GCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTAC TGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCT ATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAG ACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAAC CGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGT TTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTAT CTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCT GGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAG TTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAA GAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAA TAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGA ACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAG CACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACA ACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTAT GCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTA ACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGG CCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTAT TCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAG AGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGG AGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCC TATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAG CTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAA TGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGG CTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGA AGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGT GAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCG CTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGA ACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGG GGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCG TTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAA TTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCT CACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGA TCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAG ACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTA TTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAA AGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGC ATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTA TGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGC TACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAA GCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTGTATATCCATTT TCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAATAAGATGGATTGCACGTAGGTTC TCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACTGACAATCGGCTGCTCTGATGCC GCCGTGATCCGGTTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGA ATGAACTGAAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGTCTAGAC TGGCGCGCCAAACCTGCAGGTTAAAACAGCTGTGGGTTGTTCCCACCCACAGGGCCCACTGGGCGCTAGC ACTCTGATTTTACGAAATCCTTGTGCGCCTGTTTTATATCCCTTCCCTAATTCGAAACGTAGAAGCAATG CGCACCACTGATCAATAGTAGGCGTAACGCGCCAGTTACGTCATGATCAAGCATATCTGTTCCCCCGGAC TGAGTATCAATAGACTGCTTACGCGGTTGAAGGAGAAAACGTTCGTTATCCGGCTAACTACTTCGAGAAG CCCAGTAACACCATGGAAGCTGCAGGGTGTTTCGCTCAGCACTTCCCCCGTGTAGATCAGGTCGATGAGC CACTGCAATCCCCACAGGTGACTGTGGCAGTGGCTGCGTTGGCGGCCTGCCTATGGGGAGACCCATAGGA CGCTCTAATGTGGACATGGTGCGAAGAGCCTATTGAGCTAGTTAGTAGTCCTCCGGCCCCTGAATGCGGC TAATCCTAACTGCGGAGCACATGCCTTCAACCCAGAGGGTAGTGTGTCGTAATGGGCAACTCTGCAGCGG AACCGACTACTTTGGGTGTCCGTGTTTCTTTTTATTCTTATATTGGCTGCTTATGGTGACAATTACAGAA TTGTTACCATATAGCTATTGGATTGGCCATCCGGTGTGTAATAGAGCTGTTATATACCTATTTGTTGGCT TTGTACCACTAACTTTAAAATCTATAACTACCCTCAACTTTATATTAACCCTCAATACAGTTGAACATGT GCAGAAGGCCCGACTGCGGCTTCAGCTTCAGCCCTGGACCCGTGATCCTGCTGTGGTGCTGCCTGCTGCT GCCTATCGTGTCCTCTGCCGCCGTGTCTGTGGCCCCTACAGCCGCCGAGAAGGTGCCAGCCGAGTGCCCC GAGCTGACCAGAAGATGCCTGCTGGGCGAGGTGTTCGAGGGCGACAAGTACGAGAGCTGGCTGCGGCCCC TGGTCAACGTGACCGGCAGAGATGGCCCCCTGAGCCAGCTGATCCGGTACAGACCCGTGACCCCCGAGGC CGCCAATAGCGTGCTGCTGGACGAGGCCTTCCTGGATACCCTGGCCCTGCTGTACAACAACCCCGACCAG CTGAGAGCCCTGCTGACCCTGCTGTCCAGCGACACCGCCCCCAGATGGATGACCGTGATGCGGGGCTACA GCGAGTGTGGAGATGGCAGCCCTGCCGTGTACACCTGCGTGGACGACCTGTGCAGAGGCTACGACCTGAC CAGACTGAGCTACGGCCGGTCCATCTTCACAGAGCACGTGCTGGGCTTCGAGCTGGTGCCCCCCAGCCTG TTCAACGTGGTGGTGGCCATCCGGAACGAGGCCACCAGAACCAACAGAGCCGTGCGGCTGCCTGTGTCTA CAGCCGCTGCACCTGAGGGCATCACACTGTTCTACGGCCTGTACAACGCCGTGAAAGAGTTCTGCCTCCG GCACCAGCTGGATCCCCCCCTGCTGAGACACCTGGACAAGTACTACGCCGGCCTGCCCCCAGAGCTGAAG CAGACCAGAGTGAACCTGCCCGCCCACAGCAGATATGGCCCTCAGGCCGTGGACGCCAGATGATAACGCC GGCGCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTA TTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCA TTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCC TCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGC GACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCC ACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAA GGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTT TAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGAT AATAATATGAGGCCTGGCCTGCCCTCCTACCTGATCATCCTGGCCGTGTGCCTGTTCAGCCACCTGCTGT CCAGCAGATACGGCGCCGAGGCCGTGAGCGAGCCCCTGGACAAGGCTTTCCACCTGCTGCTGAACACCTA CGGCAGACCCATCCGGTTTCTGCGGGAGAACACCACCCAGTGCACCTACAACAGCAGCCTGCGGAACAGC ACCGTCGTGAGAGAGAACGCCATCAGCTTCAACTTTTTCCAGAGCTACAACCAGTACTACGTGTTCCACA TGCCCAGATGCCTGTTTGCCGGCCCTCTGGCCGAGCAGTTCCTGAACCAGGTGGACCTGACCGAGACACT GGAAAGATACCAGCAGCGGCTGAATACCTACGCCCTGGTGTCCAAGGACCTGGCCAGCTACCGGTCCTTT AGCCAGCAGCTCAAGGCTCAGGATAGCCTCGGCGAGCAGCCTACCACCGTGCCCCCTCCCATCGACCTGA GCATCCCCCACGTGTGGATGCCTCCCCAGACCACCCCTCACGGCTGGACCGAGAGCCACACCACCTCCGG CCTGCACAGACCCCACTTCAACCAGACCTGCATCCTGTTCGACGGCCACGACCTGCTGTTTAGCACCGTG ACCCCCTGCCTGCACCAGGGCTTCTACCTGATCGACGAGCTGAGATACGTGAAGATCACCCTGACCGAGG ATTTCTTCGTGGTCACCGTGTCCATCGACGACGACACCCCCATGCTGCTGATCTTCGGCCACCTGCCCAG AGTGCTGTTCAAGGCCCCCTACCAGCGGGACAACTTCATCCTGCGGCAGACCGAGAAGCACGAGCTGCTG GTGCTGGTCAAGAAGGACCAGCTGAACCGGCACTCCTACCTGAAGGACCCCGACTTCCTGGACGCCGCCC TGGACTTCAACTACCTGGACCTGAGCGCCCTGCTGAGAAACAGCTTCCACAGATACGCCGTGGACGTGCT GAAGTCCGGACGGTGCCAGATGCTCGATCGGCGGACCGTGGAGATGGCCTTCGCCTATGCCCTCGCCCTG TTCGCCGCTGCCAGACAGGAAGAGGCTGGCGCCCAGGTGTCAGTGCCCAGAGCCCTGGATAGACAGGCCG CCCTGCTGCAGATCCAGGAATTCATGATCACCTGCCTGAGCCAGACCCCCCCTAGAACCACCCTGCTGCT GTACCCCACAGCCGTGGATCTGGCCAAGAGGGCCCTGTGGACCCCCAACCAGATCACCGACATCACAAGC CTCGTGCGGCTCGTGTACATCCTGAGCAAGCAGAACCAGCAGCACCTGATCCCCCAGTGGGCCCTGAGAC AGATCGCCGACTTCGCCCTGAAGCTGCACAAGACCCATCTGGCCAGCTTTCTGAGCGCCTTCGCCAGGCA GGAACTGTACCTGATGGGCAGCCTGGTCCACAGCATGCTGGTGCATACCACCGAGCGGCGGGAGATCTTC ATCGTGGAGACAGGCCTGTGTAGCCTGGCCGAGCTGTCCCACTTTACCCAGCTGCTGGCCCACCCTCACC ACGAGTACCTGAGCGACCTGTACACCCCCTGCAGCAGCAGCGGCAGACGGGACCACAGCCTGGAACGGCT GACCAGACTGTTCCCCGATGCCACCGTGCCTGCTACAGTGCCTGCCGCCCTGTCCATCCTGTCCACCATG CAGCCCAGCACCCTGGAAACCTTCCCCGACCTGTTCTGCCTGCCCCTGGGCGAGAGCTTTAGCGCCCTGA CCGTGTCCGAGCACGTGTCCTACATCGTGACCAATCAGTACCTGATCAAGGGCATCAGCTACCCCGTGTC CACCACAGTCGTGGGCCAGAGCCTGATCATCACCCAGACCGACAGCCAGACCAAGTGCGAGCTGACCCGG AACATGCACACCACACACAGCATCACCGTGGCCCTGAACATCAGCCTGGAAAACTGCGCTTTCTGTCAGT CTGCCCTGCTGGAATACGACGATACCCAGGGCGTGATCAACATCATGTACATGCACGACAGCGACGACGT GCTGTTCGCCCTGGACCCCTACAACGAGGTGGTGGTGTCCAGCCCCCGGACCCACTACCTGATGCTGCTG AAGAACGGCACCGTGCTGGAAGTGACCGACGTGGTGGTGGACGCCACCGACTGATAAGCGGCCGCATACA GCAGCAATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTAT TTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAGGGTCGGCATGGCATCTCCACCTCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGACGCACGT CCACTCGGATGGCTAAGGGAGAGCCACGTTTAAACACGTGATATCTGGCCTCATGGGCCTTCCTTTCACT GCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTAT TGGGCGCTCTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTA ATGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTC CGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATA CCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCG GTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTAT CCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAA CAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTAC ACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCT CTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG AAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCA CGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAA GTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTATTAGAAAAATTCATCCAGCAG ACGATAAAACGCAATACGCTGGCTATCCGGTGCCGCAATGCCATACAGCACCAGAAAACGATCCGCCCAT TCGCCGCCCAGTTCTTCCGCAATATCACGGGTGGCCAGCGCAATATCCTGATAACGATCCGCCACGCCCA GACGGCCGCAATCAATAAAGCCGCTAAAACGGCCATTTTCCACCATAATGTTCGGCAGGCACGCATCACC ATGGGTCACCACCAGATCTTCGCCATCCGGCATGCTCGCTTTCAGACGCGCAAACAGCTCTGCCGGTGCC AGGCCCTGATGTTCTTCATCCAGATCATCCTGATCCACCAGGCCCGCTTCCATACGGGTACGCGCACGTT CAATACGATGTTTCGCCTGATGATCAAACGGACAGGTCGCCGGGTCCAGGGTATGCAGACGACGCATGGC ATCCGCCATAATGCTCACTTTTTCTGCCGGCGCCAGATGGCTAGACAGCAGATCCTGACCCGGCACTTCG CCCAGCAGCAGCCAATCACGGCCCGCTTCGGTCACCACATCCAGCACCGCCGCACACGGAACACCGGTGG TGGCCAGCCAGCTCAGACGCGCCGCTTCATCCTGCAGCTCGTTCAGCGCACCGCTCAGATCGGTTTTCAC AAACAGCACCGGACGACCCTGCGCGCTCAGACGAAACACCGCCGCATCAGAGCAGCCAATGGTCTGCTGC GCCCAATCATAGCCAAACAGACGTTCCACCCACGCTGCCGGGCTACCCGCATGCAGGCCATCCTGTTCAA TCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATT TGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTG TAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGC CGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGCG CTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGGTGCGGGCCTCTTCGCTATTACG CCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACAC GCGTAATACGACTCACTATAG Vetor A554: SGP-gH-SGP-gL-SGP-UL128-SGP-UL130-SGP-UL131 ATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGA GGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTC ACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGG ACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCA TTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAA AACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACC CTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGT TTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTC GCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACT CTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGA GCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCT GTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACT TACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAG AATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTC TTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAG CTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCT GGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTAC CTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAA GGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAAC ATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTG CCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACA AGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGA GGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTG GAAGCCGATGTAGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAA AGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAA GAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAA GGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGG ACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCA CCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAG CACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAG GGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGC CGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAA AGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACG TCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACA CCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCC ATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCC TGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAA ATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACT AAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAG GGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCT GACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAA CATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGA TAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGA TGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGT TGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTG TGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTT TGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGG GATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACC CACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGA TCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACAC CCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGT TGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGA TTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATAC CATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATC TGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGG TGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTT CTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGA CCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGA TATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTG TGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGAC TGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGA AGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCA GTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACC ATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAAT GACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTG ACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCA CAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGA AATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGC ATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTT GCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTAC TGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCT ATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAG ACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAAC CGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGT TTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTAT CTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCT GGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAG TTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAA GAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAA TAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGA ACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAG CACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACA ACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTAT GCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTA ACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGG CCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTAT TCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAG AGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGG AGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCC TATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAG CTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAA TGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGG CTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGA AGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGT GAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCG CTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGA ACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGG GGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCG TTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAA TTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCT CACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGA TCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAG ACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTA TTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAA AGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGC ATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTA TGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGC TACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAA GATGAGGCCTGGCCTGCCCTCCTACCTGATCATCCTGGCCGTGTGCCTGTTCAGCCACCTGCTGTCCAGC AGATACGGCGCCGAGGCCGTGAGCGAGCCCCTGGACAAGGCTTTCCACCTGCTGCTGAACACCTACGGCA GACCCATCCGGTTTCTGCGGGAGAACACCACCCAGTGCACCTACAACAGCAGCCTGCGGAACAGCACCGT CGTGAGAGAGAACGCCATCAGCTTCAACTTTTTCCAGAGCTACAACCAGTACTACGTGTTCCACATGCCC AGATGCCTGTTTGCCGGCCCTCTGGCCGAGCAGTTCCTGAACCAGGTGGACCTGACCGAGACACTGGAAA GATACCAGCAGCGGCTGAATACCTACGCCCTGGTGTCCAAGGACCTGGCCAGCTACCGGTCCTTTAGCCA GCAGCTCAAGGCTCAGGATAGCCTCGGCGAGCAGCCTACCACCGTGCCCCCTCCCATCGACCTGAGCATC CCCCACGTGTGGATGCCTCCCCAGACCACCCCTCACGGCTGGACCGAGAGCCACACCACCTCCGGCCTGC ACAGACCCCACTTCAACCAGACCTGCATCCTGTTCGACGGCCACGACCTGCTGTTTAGCACCGTGACCCC CTGCCTGCACCAGGGCTTCTACCTGATCGACGAGCTGAGATACGTGAAGATCACCCTGACCGAGGATTTC TTCGTGGTCACCGTGTCCATCGACGACGACACCCCCATGCTGCTGATCTTCGGCCACCTGCCCAGAGTGC TGTTCAAGGCCCCCTACCAGCGGGACAACTTCATCCTGCGGCAGACCGAGAAGCACGAGCTGCTGGTGCT GGTCAAGAAGGACCAGCTGAACCGGCACTCCTACCTGAAGGACCCCGACTTCCTGGACGCCGCCCTGGAC TTCAACTACCTGGACCTGAGCGCCCTGCTGAGAAACAGCTTCCACAGATACGCCGTGGACGTGCTGAAGT CCGGACGGTGCCAGATGCTCGATCGGCGGACCGTGGAGATGGCCTTCGCCTATGCCCTCGCCCTGTTCGC CGCTGCCAGACAGGAAGAGGCTGGCGCCCAGGTGTCAGTGCCCAGAGCCCTGGATAGACAGGCCGCCCTG CTGCAGATCCAGGAATTCATGATCACCTGCCTGAGCCAGACCCCCCCTAGAACCACCCTGCTGCTGTACC CCACAGCCGTGGATCTGGCCAAGAGGGCCCTGTGGACCCCCAACCAGATCACCGACATCACAAGCCTCGT GCGGCTCGTGTACATCCTGAGCAAGCAGAACCAGCAGCACCTGATCCCCCAGTGGGCCCTGAGACAGATC GCCGACTTCGCCCTGAAGCTGCACAAGACCCATCTGGCCAGCTTTCTGAGCGCCTTCGCCAGGCAGGAAC TGTACCTGATGGGCAGCCTGGTCCACAGCATGCTGGTGCATACCACCGAGCGGCGGGAGATCTTCATCGT GGAGACAGGCCTGTGTAGCCTGGCCGAGCTGTCCCACTTTACCCAGCTGCTGGCCCACCCTCACCACGAG TACCTGAGCGACCTGTACACCCCCTGCAGCAGCAGCGGCAGACGGGACCACAGCCTGGAACGGCTGACCA GACTGTTCCCCGATGCCACCGTGCCTGCTACAGTGCCTGCCGCCCTGTCCATCCTGTCCACCATGCAGCC CAGCACCCTGGAAACCTTCCCCGACCTGTTCTGCCTGCCCCTGGGCGAGAGCTTTAGCGCCCTGACCGTG TCCGAGCACGTGTCCTACATCGTGACCAATCAGTACCTGATCAAGGGCATCAGCTACCCCGTGTCCACCA CAGTCGTGGGCCAGAGCCTGATCATCACCCAGACCGACAGCCAGACCAAGTGCGAGCTGACCCGGAACAT GCACACCACACACAGCATCACCGTGGCCCTGAACATCAGCCTGGAAAACTGCGCTTTCTGTCAGTCTGCC CTGCTGGAATACGACGATACCCAGGGCGTGATCAACATCATGTACATGCACGACAGCGACGACGTGCTGT TCGCCCTGGACCCCTACAACGAGGTGGTGGTGTCCAGCCCCCGGACCCACTACCTGATGCTGCTGAAGAA CGGCACCGTGCTGGAAGTGACCGACGTGGTGGTGGACGCCACCGACAGCAGACTGCTGATGATGAGCGTG TACGCCCTGAGCGCCATCATCGGCATCTACCTGCTGTACCGGATGCTGAAAACCTGCTGATAATCTAGAG GCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGATGTGCAGAAG GCCCGACTGCGGCTTCAGCTTCAGCCCTGGACCCGTGATCCTGCTGTGGTGCTGCCTGCTGCTGCCTATC GTGTCCTCTGCCGCCGTGTCTGTGGCCCCTACAGCCGCCGAGAAGGTGCCAGCCGAGTGCCCCGAGCTGA CCAGAAGATGCCTGCTGGGCGAGGTGTTCGAGGGCGACAAGTACGAGAGCTGGCTGCGGCCCCTGGTCAA CGTGACCGGCAGAGATGGCCCCCTGAGCCAGCTGATCCGGTACAGACCCGTGACCCCCGAGGCCGCCAAT AGCGTGCTGCTGGACGAGGCCTTCCTGGATACCCTGGCCCTGCTGTACAACAACCCCGACCAGCTGAGAG CCCTGCTGACCCTGCTGTCCAGCGACACCGCCCCCAGATGGATGACCGTGATGCGGGGCTACAGCGAGTG TGGAGATGGCAGCCCTGCCGTGTACACCTGCGTGGACGACCTGTGCAGAGGCTACGACCTGACCAGACTG AGCTACGGCCGGTCCATCTTCACAGAGCACGTGCTGGGCTTCGAGCTGGTGCCCCCCAGCCTGTTCAACG TGGTGGTGGCCATCCGGAACGAGGCCACCAGAACCAACAGAGCCGTGCGGCTGCCTGTGTCTACAGCCGC TGCACCTGAGGGCATCACACTGTTCTACGGCCTGTACAACGCCGTGAAAGAGTTCTGCCTCCGGCACCAG CTGGATCCCCCCCTGCTGAGACACCTGGACAAGTACTACGCCGGCCTGCCCCCAGAGCTGAAGCAGACCA GAGTGAACCTGCCCGCCCACAGCAGATATGGCCCTCAGGCCGTGGACGCCAGATGATAACGCCGGCGGCC CCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGATGAGCCCCAAGGA CCTGACCCCCTTCCTGACAACCCTGTGGCTGCTCCTGGGCCATAGCAGAGTGCCTAGAGTGCGGGCCGAG GAATGCTGCGAGTTCATCAACGTGAACCACCCCCCCGAGCGGTGCTACGACTTCAAGATGTGCAACCGGT TCACCGTGGCCCTGAGATGCCCCGACGGCGAAGTGTGCTACAGCCCCGAGAAAACCGCCGAGATCCGGGG CATCGTGACCACCATGACCCACAGCCTGACCCGGCAGGTGGTGCACAACAAGCTGACCAGCTGCAACTAC AACCCCCTGTACCTGGAAGCCGACGGCCGGATCAGATGCGGCAAAGTGAACGACAAGGCCCAGTACCTGC TGGGAGCCGCCGGAAGCGTGCCCTACCGGTGGATCAACCTGGAATACGACAAGATCACCCGGATCGTGGG CCTGGACCAGTACCTGGAAAGCGTGAAGAAGCACAAGCGGCTGGACGTGTGCAGAGCCAAGATGGGCTAC ATGCTGCAGTGATAAGGCGCGCCGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTC TAGTCCGCCAAGATGCTGCGGCTGCTGCTGAGACACCACTTCCACTGCCTGCTGCTGTGTGCCGTGTGGG CCACCCCTTGTCTGGCCAGCCCTTGGAGCACCCTGACCGCCAACCAGAACCCTAGCCCCCCTTGGTCCAA GCTGACCTACAGCAAGCCCCACGACGCCGCCACCTTCTACTGCCCCTTTCTGTACCCCAGCCCTCCCAGA AGCCCCCTGCAGTTCAGCGGCTTCCAGAGAGTGTCCACCGGCCCTGAGTGCCGGAACGAGACACTGTACC TGCTGTACAACCGGGAGGGCCAGACACTGGTGGAGCGGAGCAGCACCTGGGTGAAAAAAGTGATCTGGTA TCTGAGCGGCCGGAACCAGACCATCCTGCAGCGGATGCCCAGAACCGCCAGCAAGCCCAGCGACGGCAAC GTGCAGATCAGCGTGGAGGACGCCAAAATCTTCGGAGCCCACATGGTGCCCAAGCAGACCAAGCTGCTGA GATTCGTGGTCAACGACGGCACCAGATATCAGATGTGCGTGATGAAGCTGGAAAGCTGGGCCCACGTGTT CCGGGACTACTCCGTGAGCTTCCAGGTCCGGCTGACCTTCACCGAGGCCAACAACCAGACCTACACCTTC TGCACCCACCCCAACCTGATCGTGTGATAAGCGGCCGCGCCCCTATAACTCTCTACGGCTAACCTGAATG GACTACGACATAGTCTAGTCCGCCAAGATGCGGCTGTGCAGAGTGTGGCTGTCCGTGTGCCTGTGTGCCG TGGTGCTGGGCCAGTGCCAGAGAGAGACAGCCGAGAAGAACGACTACTACCGGGTGCCCCACTACTGGGA TGCCTGCAGCAGAGCCCTGCCCGACCAGACCCGGTACAAATACGTGGAGCAGCTCGTGGACCTGACCCTG AACTACCACTACGACGCCAGCCACGGCCTGGACAACTTCGACGTGCTGAAGCGGATCAACGTGACCGAGG TGTCCCTGCTGATCAGCGACTTCCGGCGGCAGAACAGAAGAGGCGGCACCAACAAGCGGACCACCTTCAA CGCCGCTGGCTCTCTGGCCCCTCACGCCAGATCCCTGGAATTCAGCGTGCGGCTGTTCGCCAACTGATAA CGTTGCATCCTGCAGGATACAGCAGCAATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCC GCCTTAAAATTTTTATTTTATTTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGTCGGCATGGCATCTCCACCTCCTCGCGGTCCGACCTGGG CATCCGAAGGAGGACGCACGTCCACTCGGATGGCTAAGGGAGAGCCACGTTTAAACGCTAGAGCAAGACG TTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCA TGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATA AATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAA AGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTT CTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAG CGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGC AGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCA CTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCT GGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCC GCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAG ATACCAGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCA TTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCA AGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGA GTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGT CTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAG TTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGT TTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAAGGGGTCTGACGC TCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATC CTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTATT AGAAAAATTCATCCAGCAGACGATAAAACGCAATACGCTGGCTATCCGGTGCCGCAATGCCATACAGCAC CAGAAAACGATCCGCCCATTCGCCGCCCAGTTCTTCCGCAATATCACGGGTGGCCAGCGCAATATCCTGA TAACGATCCGCCACGCCCAGACGGCCGCAATCAATAAAGCCGCTAAAACGGCCATTTTCCACCATAATGT TCGGCAGGCACGCATCACCATGGGTCACCACCAGATCTTCGCCATCCGGCATGCTCGCTTTCAGACGCGC AAACAGCTCTGCCGGTGCCAGGCCCTGATGTTCTTCATCCAGATCATCCTGATCCACCAGGCCCGCTTCC ATACGGGTACGCGCACGTTCAATACGATGTTTCGCCTGATGATCAAACGGACAGGTCGCCGGGTCCAGGG TATGCAGACGACGCATGGCATCCGCCATAATGCTCACTTTTTCTGCCGGCGCCAGATGGCTAGACAGCAG ATCCTGACCCGGCACTTCGCCCAGCAGCAGCCAATCACGGCCCGCTTCGGTCACCACATCCAGCACCGCC GCACACGGAACACCGGTGGTGGCCAGCCAGCTCAGACGCGCCGCTTCATCCTGCAGCTCGTTCAGCGCAC CGCTCAGATCGGTTTTCACAAACAGCACCGGACGACCCTGCGCGCTCAGACGAAACACCGCCGCATCAGA GCAGCCAATGGTCTGCTGCGCCCAATCATAGCCAAACAGACGTTCCACCCACGCTGCCGGGCTACCCGCA TGCAGGCCATCCTGTTCAATCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTC TCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCG AAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCT CATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTT GAGTGGCCGCTACAGGGCGCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGGTGC GGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCC AGGGTTTTCCCAGTCACACGCGTAATACGACTCACTATAG Vetor A555: SGP-gHsol-SGP-gL-SGP-UL128-SGP-UL130-SGP-UL131 ATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGA GGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTC ACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGG ACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCA TTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAA AACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACC CTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGT TTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTC GCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACT CTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGA GCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCT GTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACT TACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAG AATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTC TTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAG CTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCT GGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTAC CTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAA GGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAAC ATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTG CCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACA AGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGA GGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTG GAAGCCGATGTAGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAA AGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAA GAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAA GGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGG ACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCA CCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAG CACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAG GGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGC CGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAA AGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACG TCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACA CCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCC ATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCC TGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAA ATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACT AAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAG GGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCT GACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAA CATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGA TAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGA TGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGT TGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTG TGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTT TGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGG GATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACC CACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGA TCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACAC CCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGT TGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGA TTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATAC CATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATC TGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGG TGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTT CTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGA CCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGA TATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTG TGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGAC TGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGA AGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCA GTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACC ATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAAT GACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTG ACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCA CAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGA AATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGC ATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTT GCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTAC TGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCT ATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAG ACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAAC CGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGT TTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTAT CTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCT GGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAG TTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAA GAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAA TAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGA ACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAG CACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACA ACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTAT GCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTA ACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGG CCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTAT TCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAG AGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGG AGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCC TATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAG CTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAA TGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGG CTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGA AGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGT GAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCG CTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGA ACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGG GGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCG TTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAA TTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCT CACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGA TCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAG ACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTA TTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAA AGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGC ATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTA TGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGC TACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAA GATGAGGCCTGGCCTGCCCTCCTACCTGATCATCCTGGCCGTGTGCCTGTTCAGCCACCTGCTGTCCAGC AGATACGGCGCCGAGGCCGTGAGCGAGCCCCTGGACAAGGCTTTCCACCTGCTGCTGAACACCTACGGCA GACCCATCCGGTTTCTGCGGGAGAACACCACCCAGTGCACCTACAACAGCAGCCTGCGGAACAGCACCGT CGTGAGAGAGAACGCCATCAGCTTCAACTTTTTCCAGAGCTACAACCAGTACTACGTGTTCCACATGCCC AGATGCCTGTTTGCCGGCCCTCTGGCCGAGCAGTTCCTGAACCAGGTGGACCTGACCGAGACACTGGAAA GATACCAGCAGCGGCTGAATACCTACGCCCTGGTGTCCAAGGACCTGGCCAGCTACCGGTCCTTTAGCCA GCAGCTCAAGGCTCAGGATAGCCTCGGCGAGCAGCCTACCACCGTGCCCCCTCCCATCGACCTGAGCATC CCCCACGTGTGGATGCCTCCCCAGACCACCCCTCACGGCTGGACCGAGAGCCACACCACCTCCGGCCTGC ACAGACCCCACTTCAACCAGACCTGCATCCTGTTCGACGGCCACGACCTGCTGTTTAGCACCGTGACCCC CTGCCTGCACCAGGGCTTCTACCTGATCGACGAGCTGAGATACGTGAAGATCACCCTGACCGAGGATTTC TTCGTGGTCACCGTGTCCATCGACGACGACACCCCCATGCTGCTGATCTTCGGCCACCTGCCCAGAGTGC TGTTCAAGGCCCCCTACCAGCGGGACAACTTCATCCTGCGGCAGACCGAGAAGCACGAGCTGCTGGTGCT GGTCAAGAAGGACCAGCTGAACCGGCACTCCTACCTGAAGGACCCCGACTTCCTGGACGCCGCCCTGGAC TTCAACTACCTGGACCTGAGCGCCCTGCTGAGAAACAGCTTCCACAGATACGCCGTGGACGTGCTGAAGT CCGGACGGTGCCAGATGCTCGATCGGCGGACCGTGGAGATGGCCTTCGCCTATGCCCTCGCCCTGTTCGC CGCTGCCAGACAGGAAGAGGCTGGCGCCCAGGTGTCAGTGCCCAGAGCCCTGGATAGACAGGCCGCCCTG CTGCAGATCCAGGAATTCATGATCACCTGCCTGAGCCAGACCCCCCCTAGAACCACCCTGCTGCTGTACC CCACAGCCGTGGATCTGGCCAAGAGGGCCCTGTGGACCCCCAACCAGATCACCGACATCACAAGCCTCGT GCGGCTCGTGTACATCCTGAGCAAGCAGAACCAGCAGCACCTGATCCCCCAGTGGGCCCTGAGACAGATC GCCGACTTCGCCCTGAAGCTGCACAAGACCCATCTGGCCAGCTTTCTGAGCGCCTTCGCCAGGCAGGAAC TGTACCTGATGGGCAGCCTGGTCCACAGCATGCTGGTGCATACCACCGAGCGGCGGGAGATCTTCATCGT GGAGACAGGCCTGTGTAGCCTGGCCGAGCTGTCCCACTTTACCCAGCTGCTGGCCCACCCTCACCACGAG TACCTGAGCGACCTGTACACCCCCTGCAGCAGCAGCGGCAGACGGGACCACAGCCTGGAACGGCTGACCA GACTGTTCCCCGATGCCACCGTGCCTGCTACAGTGCCTGCCGCCCTGTCCATCCTGTCCACCATGCAGCC CAGCACCCTGGAAACCTTCCCCGACCTGTTCTGCCTGCCCCTGGGCGAGAGCTTTAGCGCCCTGACCGTG TCCGAGCACGTGTCCTACATCGTGACCAATCAGTACCTGATCAAGGGCATCAGCTACCCCGTGTCCACCA CAGTCGTGGGCCAGAGCCTGATCATCACCCAGACCGACAGCCAGACCAAGTGCGAGCTGACCCGGAACAT GCACACCACACACAGCATCACCGTGGCCCTGAACATCAGCCTGGAAAACTGCGCTTTCTGTCAGTCTGCC CTGCTGGAATACGACGATACCCAGGGCGTGATCAACATCATGTACATGCACGACAGCGACGACGTGCTGT TCGCCCTGGACCCCTACAACGAGGTGGTGGTGTCCAGCCCCCGGACCCACTACCTGATGCTGCTGAAGAA CGGCACCGTGCTGGAAGTGACCGACGTGGTGGTGGACGCCACCGACTGATAATCTAGAGGCCCCTATAAC TCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGATGTGCAGAAGGCCCGACTGCG GCTTCAGCTTCAGCCCTGGACCCGTGATCCTGCTGTGGTGCTGCCTGCTGCTGCCTATCGTGTCCTCTGC CGCCGTGTCTGTGGCCCCTACAGCCGCCGAGAAGGTGCCAGCCGAGTGCCCCGAGCTGACCAGAAGATGC CTGCTGGGCGAGGTGTTCGAGGGCGACAAGTACGAGAGCTGGCTGCGGCCCCTGGTCAACGTGACCGGCA GAGATGGCCCCCTGAGCCAGCTGATCCGGTACAGACCCGTGACCCCCGAGGCCGCCAATAGCGTGCTGCT GGACGAGGCCTTCCTGGATACCCTGGCCCTGCTGTACAACAACCCCGACCAGCTGAGAGCCCTGCTGACC CTGCTGTCCAGCGACACCGCCCCCAGATGGATGACCGTGATGCGGGGCTACAGCGAGTGTGGAGATGGCA GCCCTGCCGTGTACACCTGCGTGGACGACCTGTGCAGAGGCTACGACCTGACCAGACTGAGCTACGGCCG GTCCATCTTCACAGAGCACGTGCTGGGCTTCGAGCTGGTGCCCCCCAGCCTGTTCAACGTGGTGGTGGCC ATCCGGAACGAGGCCACCAGAACCAACAGAGCCGTGCGGCTGCCTGTGTCTACAGCCGCTGCACCTGAGG GCATCACACTGTTCTACGGCCTGTACAACGCCGTGAAAGAGTTCTGCCTCCGGCACCAGCTGGATCCCCC CCTGCTGAGACACCTGGACAAGTACTACGCCGGCCTGCCCCCAGAGCTGAAGCAGACCAGAGTGAACCTG CCCGCCCACAGCAGATATGGCCCTCAGGCCGTGGACGCCAGATGATAACGCCGGCGGCCCCTATAACTCT CTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGATGAGCCCCAAGGACCTGACCCCCT TCCTGACAACCCTGTGGCTGCTCCTGGGCCATAGCAGAGTGCCTAGAGTGCGGGCCGAGGAATGCTGCGA GTTCATCAACGTGAACCACCCCCCCGAGCGGTGCTACGACTTCAAGATGTGCAACCGGTTCACCGTGGCC CTGAGATGCCCCGACGGCGAAGTGTGCTACAGCCCCGAGAAAACCGCCGAGATCCGGGGCATCGTGACCA CCATGACCCACAGCCTGACCCGGCAGGTGGTGCACAACAAGCTGACCAGCTGCAACTACAACCCCCTGTA CCTGGAAGCCGACGGCCGGATCAGATGCGGCAAAGTGAACGACAAGGCCCAGTACCTGCTGGGAGCCGCC GGAAGCGTGCCCTACCGGTGGATCAACCTGGAATACGACAAGATCACCCGGATCGTGGGCCTGGACCAGT ACCTGGAAAGCGTGAAGAAGCACAAGCGGCTGGACGTGTGCAGAGCCAAGATGGGCTACATGCTGCAGTG ATAAGGCGCGCCAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTAT TTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCAT TCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCT CTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCG ACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCA CGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAG GATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTT AGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATA ATATGCTGCGGCTGCTGCTGAGACACCACTTCCACTGCCTGCTGCTGTGTGCCGTGTGGGCCACCCCTTG TCTGGCCAGCCCTTGGAGCACCCTGACCGCCAACCAGAACCCTAGCCCCCCTTGGTCCAAGCTGACCTAC AGCAAGCCCCACGACGCCGCCACCTTCTACTGCCCCTTTCTGTACCCCAGCCCTCCCAGAAGCCCCCTGC AGTTCAGCGGCTTCCAGAGAGTGTCCACCGGCCCTGAGTGCCGGAACGAGACACTGTACCTGCTGTACAA CCGGGAGGGCCAGACACTGGTGGAGCGGAGCAGCACCTGGGTGAAAAAAGTGATCTGGTATCTGAGCGGC CGGAACCAGACCATCCTGCAGCGGATGCCCAGAACCGCCAGCAAGCCCAGCGACGGCAACGTGCAGATCA GCGTGGAGGACGCCAAAATCTTCGGAGCCCACATGGTGCCCAAGCAGACCAAGCTGCTGAGATTCGTGGT CAACGACGGCACCAGATATCAGATGTGCGTGATGAAGCTGGAAAGCTGGGCCCACGTGTTCCGGGACTAC TCCGTGAGCTTCCAGGTCCGGCTGACCTTCACCGAGGCCAACAACCAGACCTACACCTTCTGCACCCACC CCAACCTGATCGTGTGATAAGTACCTTTGTACGCCTGTTTTATACCCCCTCCCTGATTTGCAACTTAGAA GCAACGCAAACCAGATCAATAGTAGGTGTGACATACCAGTCGCATCTTGATCAAGCACTTCTGTATCCCC GGACCGAGTATCAATAGACTGTGCACACGGTTGAAGGAGAAAACGTCCGTTACCCGGCTAACTACTTCGA GAAGCCTAGTAACGCCATTGAAGTTGCAGAGTGTTTCGCTCAGCACTCCCCCCGTGTAGATCAGGTCGAT GAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCTATGGGGTAACCCAT AGGACGCTCTAATACGGACATGGCGTGAAGAGTCTATTGAGCTAGTTAGTAGTCCTCCGGCCCCTGAATG CGGCTAATCCTAACTGCGGAGCACATACCCTTAATCCAAAGGGCAGTGTGTCGTAACGGGCAACTCTGCA GCGGAACCGACTACTTTGGGTGTCCGTGTTTCTTTTTATTCTTGTATTGGCTGCTTATGGTGACAATTAA AGAATTGTTACCATATAGCTATTGGATTGGCCATCCAGTGTCAAACAGAGCTATTGTATATCTCTTTGTT GGATTCACACCTCTCACTCTTGAAACGTTACACACCCTCAATTACATTATACTGCTGAACACGAAGCGCA TATGCGGCTGTGCAGAGTGTGGCTGTCCGTGTGCCTGTGTGCCGTGGTGCTGGGCCAGTGCCAGAGAGAG ACAGCCGAGAAGAACGACTACTACCGGGTGCCCCACTACTGGGATGCCTGCAGCAGAGCCCTGCCCGACC AGACCCGGTACAAATACGTGGAGCAGCTCGTGGACCTGACCCTGAACTACCACTACGACGCCAGCCACGG CCTGGACAACTTCGACGTGCTGAAGCGGATCAACGTGACCGAGGTGTCCCTGCTGATCAGCGACTTCCGG CGGCAGAACAGAAGAGGCGGCACCAACAAGCGGACCACCTTCAACGCCGCTGGCTCTCTGGCCCCTCACG CCAGATCCCTGGAATTCAGCGTGCGGCTGTTCGCCAACTGATAACGTTGCATCCTGCAGGATACAGCAGC AATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTC TTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAGGGTCGGCATGGCATCTCCACCTCCTCGCGGTCCGACCTGGGCATCCGAAGGAGGACGCACGTCCACT CGGATGGCTAAGGGAGAGCCACGTTTAAACGCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACA CCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAA TGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGA TCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGG TCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGG CCTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCAGCGCTAGCGGAGTGTATACTGGCTTAC TATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCG TCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGA CTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACA GGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATC TGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGGCGGCTC CCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCT CATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCC GTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAG CACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCT AAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGC TCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGA CCAAAACGATCTCAAGAAGATCATCTTATTAAGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG GGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAA ATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTATTAGAAAAATTCATCCAGCAGACGATAA AACGCAATACGCTGGCTATCCGGTGCCGCAATGCCATACAGCACCAGAAAACGATCCGCCCATTCGCCGC CCAGTTCTTCCGCAATATCACGGGTGGCCAGCGCAATATCCTGATAACGATCCGCCACGCCCAGACGGCC GCAATCAATAAAGCCGCTAAAACGGCCATTTTCCACCATAATGTTCGGCAGGCACGCATCACCATGGGTC ACCACCAGATCTTCGCCATCCGGCATGCTCGCTTTCAGACGCGCAAACAGCTCTGCCGGTGCCAGGCCCT GATGTTCTTCATCCAGATCATCCTGATCCACCAGGCCCGCTTCCATACGGGTACGCGCACGTTCAATACG ATGTTTCGCCTGATGATCAAACGGACAGGTCGCCGGGTCCAGGGTATGCAGACGACGCATGGCATCCGCC ATAATGCTCACTTTTTCTGCCGGCGCCAGATGGCTAGACAGCAGATCCTGACCCGGCACTTCGCCCAGCA GCAGCCAATCACGGCCCGCTTCGGTCACCACATCCAGCACCGCCGCACACGGAACACCGGTGGTGGCCAG CCAGCTCAGACGCGCCGCTTCATCCTGCAGCTCGTTCAGCGCACCGCTCAGATCGGTTTTCACAAACAGC ACCGGACGACCCTGCGCGCTCAGACGAAACACCGCCGCATCAGAGCAGCCAATGGTCTGCTGCGCCCAAT CATAGCCAAACAGACGTTCCACCCACGCTGCCGGGCTACCCGCATGCAGGCCATCCTGTTCAATCATACT CTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGT ATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGT TAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATC GGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGCGCTCCCAT TCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGGTGCGGGCCTCTTCGCTATTACGCCAGCTG GCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACACGCGTAAT ACGACTCACTATAG Vetor A556: SGP-gHsol6His-SGP-gL-SGP-UL128-SGP-UL130-SGP-UL131 ATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAAATGGAGAAAGTTCACGTTGACATCGA GGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTC ACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGG ACCCATCCGACACGATCCTTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCA TTGTATCTGTCCGATGAGATGTGCGGAAGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAA AACTGTAAGGAAATAACTGATAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACC CTGACCTGGAAACTGAGACTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGT TTACCAGGATGTATACGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTC GCCTACTGGATAGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACT CTACCAACTGGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGA GCGGTCACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTTGAAACCATCCAACAATGTTCTATTCTCT GTTGGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCACT TACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAG AATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGAGGGATTC TTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAG CTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGACGCGCAAAAACTGCT GGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACACCAATACCATGAAAAATTAC CTTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAAGGAAGATCAAGAAGATGAAA GGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTGTTGGGCTTTTAGAAGGCACAAGATAAC ATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAAGTGAACAGCGATTTCCACTCATTCGTGCTG CCCAGGATAGGCAGTAACACATTGGAGATCGGGCTGAGAACAAGAATCAGGAAAATGTTAGAGGAGCACA AGGAGCCGTCACCTCTCATTACCGCCGAGGACGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGA GGTGCGTGAAGCCGAGGAGTTGCGCGCAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTG GAAGCCGATGTAGACTTGATGTTACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAA AGGTTACCAGCTACGATGGCGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAA GAGTGAAAAATTATCTTGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAA GGGCGTTATGCCGTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGG ACTTTCAAGCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCA CCATATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAG CACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAG GGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACACGACCAGC CGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTGGCATCATTAAA AGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCAGAAATTATAAGGGACG TCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACA CCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAGGTACTCTCAGAGCGCTCATAGCC ATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAACAGTGCGGTTTTTTTAACATGATGTGCC TGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCTTCCACAAAAGCATCTCTCGCCGTTGCACTAA ATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGACAAAAAAATGAGAACGACGAATCCGAAAGAGACT AAGATTGTGATTGACACTACCGGCAGTACCAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAG GGTGGGTGAAGCAGTTGCAAATAGATTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCT GACCCGTAAAGGTGTGTATGCCGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAA CATGTGAACGTCCTACTGACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGA TAAAAACACTGACTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGA TGCCATCATGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGT TGGGCCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTG TGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTT TGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACTGG GATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCAGGTACC CACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGCGCAATTATGA TCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCACCATAATGAACAC CCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTGGTGGTCGGGGAAAAGT TGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCTACCTTCAGAGCTCGGCTGGA TTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAATGTGAGGACCCCATATAAATAC CATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGTTGACCAAGAAAGCTTGTCTGCATC TGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGG TGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAAACCGAAATCCTCACTTGAAGAGACGGAAGTT CTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGTACGCACAATCCTTACAAGCTTTCATCAACCTTGA CCAACATTTATACAGGTTCCAGACTCCACGAAGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGA TATTGCCACGGCCACCGAAGGAGTGATTATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTG TGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGAC TGGTCAAAGGTGCAGCTAAACATATCATTCATGCCGTAGGACCAAACTTCAACAAAGTTTCGGAGGTTGA AGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCA GTAGCGATTCCACTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACC ATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAAT GACTCTCAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGACTCTTCAGTG ACAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACAGCA CAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGATATAGCAGA AATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCTCGGAGAAAGC ATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCACCTAGCACGCTGCCTT GCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTCACGTCCAGAACAAATTAC TGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGATCCAATGCTCCCAGCCT ATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTATCTCGTGGAAACACCACCGGTAG ACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGGGGACACCTGAACAACCACCACTTATAAC CGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCATCATCGAAGAGGAAGAAGAGGATAGCATAAGT TTGCTGTCAGATGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTAT CTAGCTCATCCTGGTCCATTCCTCATGCATCCGACTTTGATGTGGACAGTTTATCCATACTTGACACCCT GGAGGGAGCTAGCGTGACCAGCGGGGCAACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAG TTTCTGGCGCGACCGGTGCCTGCGCCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAA GAACACCGTCACTTGCACCCAGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAA TAGGGTGATCACTAGAGAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGA ACCAGCCTGGTCTCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAG CACAACAACAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACA ACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTAT GCCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTGCTA ACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTCTGCAAGG CCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTGTTCCTTTGTAT TCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAACGCCATGTTGAAAG AGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATTTGGACATGGTTGACGG AGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCAGCTTTCCAAAGAAACACTCC TATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGAACACGCTCCAGAACGTCCTGGCAG CTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAATTGCCCGTATTGGATTCGGCGGCCTTTAA TGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATATTGGGAAACGTTTAAAGAAAACCCCATCAGG CTTACTGAAGAAAACGTGGTAAATTACATTACCAAATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGA AGACACATAATTTGAATATGTTGCAGGACATACCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGT GAAAGTGACTCCAGGAACAAAACATACTGAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCG CTAGCAACAGCGTATCTGTGCGGAATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGA ACATTCATACACTGTTTGATATGTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGG GGATTGTGTTCTGGAAACTGACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCG TTAATGATTCTGGAAGACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAA TTTCATCAATACATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGATGAAATCTGGAATGTTCCT CACACTGTTTGTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGA TCACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAG ACAGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCTTA TTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCCTAAAA AGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAAGGGCATTGC ATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAGTAGAATCAAGGTA TGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAATCATTCAGC TACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGCCAA GATGAGGCCTGGCCTGCCCTCCTACCTGATCATCCTGGCCGTGTGCCTGTTCAGCCACCTGCTGTCCAGC AGATACGGCGCCGAGGCCGTGAGCGAGCCCCTGGACAAGGCTTTCCACCTGCTGCTGAACACCTACGGCA GACCCATCCGGTTTCTGCGGGAGAACACCACCCAGTGCACCTACAACAGCAGCCTGCGGAACAGCACCGT CGTGAGAGAGAACGCCATCAGCTTCAACTTTTTCCAGAGCTACAACCAGTACTACGTGTTCCACATGCCC AGATGCCTGTTTGCCGGCCCTCTGGCCGAGCAGTTCCTGAACCAGGTGGACCTGACCGAGACACTGGAAA GATACCAGCAGCGGCTGAATACCTACGCCCTGGTGTCCAAGGACCTGGCCAGCTACCGGTCCTTTAGCCA GCAGCTCAAGGCTCAGGATAGCCTCGGCGAGCAGCCTACCACCGTGCCCCCTCCCATCGACCTGAGCATC CCCCACGTGTGGATGCCTCCCCAGACCACCCCTCACGGCTGGACCGAGAGCCACACCACCTCCGGCCTGC ACAGACCCCACTTCAACCAGACCTGCATCCTGTTCGACGGCCACGACCTGCTGTTTAGCACCGTGACCCC CTGCCTGCACCAGGGCTTCTACCTGATCGACGAGCTGAGATACGTGAAGATCACCCTGACCGAGGATTTC TTCGTGGTCACCGTGTCCATCGACGACGACACCCCCATGCTGCTGATCTTCGGCCACCTGCCCAGAGTGC TGTTCAAGGCCCCCTACCAGCGGGACAACTTCATCCTGCGGCAGACCGAGAAGCACGAGCTGCTGGTGCT GGTCAAGAAGGACCAGCTGAACCGGCACTCCTACCTGAAGGACCCCGACTTCCTGGACGCCGCCCTGGAC TTCAACTACCTGGACCTGAGCGCCCTGCTGAGAAACAGCTTCCACAGATACGCCGTGGACGTGCTGAAGT CCGGACGGTGCCAGATGCTCGATCGGCGGACCGTGGAGATGGCCTTCGCCTATGCCCTCGCCCTGTTCGC CGCTGCCAGACAGGAAGAGGCTGGCGCCCAGGTGTCAGTGCCCAGAGCCCTGGATAGACAGGCCGCCCTG CTGCAGATCCAGGAATTCATGATCACCTGCCTGAGCCAGACCCCCCCTAGAACCACCCTGCTGCTGTACC CCACAGCCGTGGATCTGGCCAAGAGGGCCCTGTGGACCCCCAACCAGATCACCGACATCACAAGCCTCGT GCGGCTCGTGTACATCCTGAGCAAGCAGAACCAGCAGCACCTGATCCCCCAGTGGGCCCTGAGACAGATC GCCGACTTCGCCCTGAAGCTGCACAAGACCCATCTGGCCAGCTTTCTGAGCGCCTTCGCCAGGCAGGAAC TGTACCTGATGGGCAGCCTGGTCCACAGCATGCTGGTGCATACCACCGAGCGGCGGGAGATCTTCATCGT GGAGACAGGCCTGTGTAGCCTGGCCGAGCTGTCCCACTTTACCCAGCTGCTGGCCCACCCTCACCACGAG TACCTGAGCGACCTGTACACCCCCTGCAGCAGCAGCGGCAGACGGGACCACAGCCTGGAACGGCTGACCA GACTGTTCCCCGATGCCACCGTGCCTGCTACAGTGCCTGCCGCCCTGTCCATCCTGTCCACCATGCAGCC CAGCACCCTGGAAACCTTCCCCGACCTGTTCTGCCTGCCCCTGGGCGAGAGCTTTAGCGCCCTGACCGTG TCCGAGCACGTGTCCTACATCGTGACCAATCAGTACCTGATCAAGGGCATCAGCTACCCCGTGTCCACCA CAGTCGTGGGCCAGAGCCTGATCATCACCCAGACCGACAGCCAGACCAAGTGCGAGCTGACCCGGAACAT GCACACCACACACAGCATCACCGTGGCCCTGAACATCAGCCTGGAAAACTGCGCTTTCTGTCAGTCTGCC CTGCTGGAATACGACGATACCCAGGGCGTGATCAACATCATGTACATGCACGACAGCGACGACGTGCTGT TCGCCCTGGACCCCTACAACGAGGTGGTGGTGTCCAGCCCCCGGACCCACTACCTGATGCTGCTGAAGAA CGGCACCGTGCTGGAAGTGACCGACGTGGTGGTGGACGCCACCGACGGCAGCGGATCTGGGTCCCACCAT CACCATCACCATTGATAATCTAGAGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAG TCTAGTCCGCCAAGATGTGCAGAAGGCCCGACTGCGGCTTCAGCTTCAGCCCTGGACCCGTGATCCTGCT GTGGTGCTGCCTGCTGCTGCCTATCGTGTCCTCTGCCGCCGTGTCTGTGGCCCCTACAGCCGCCGAGAAG GTGCCAGCCGAGTGCCCCGAGCTGACCAGAAGATGCCTGCTGGGCGAGGTGTTCGAGGGCGACAAGTACG AGAGCTGGCTGCGGCCCCTGGTCAACGTGACCGGCAGAGATGGCCCCCTGAGCCAGCTGATCCGGTACAG ACCCGTGACCCCCGAGGCCGCCAATAGCGTGCTGCTGGACGAGGCCTTCCTGGATACCCTGGCCCTGCTG TACAACAACCCCGACCAGCTGAGAGCCCTGCTGACCCTGCTGTCCAGCGACACCGCCCCCAGATGGATGA CCGTGATGCGGGGCTACAGCGAGTGTGGAGATGGCAGCCCTGCCGTGTACACCTGCGTGGACGACCTGTG CAGAGGCTACGACCTGACCAGACTGAGCTACGGCCGGTCCATCTTCACAGAGCACGTGCTGGGCTTCGAG CTGGTGCCCCCCAGCCTGTTCAACGTGGTGGTGGCCATCCGGAACGAGGCCACCAGAACCAACAGAGCCG TGCGGCTGCCTGTGTCTACAGCCGCTGCACCTGAGGGCATCACACTGTTCTACGGCCTGTACAACGCCGT GAAAGAGTTCTGCCTCCGGCACCAGCTGGATCCCCCCCTGCTGAGACACCTGGACAAGTACTACGCCGGC CTGCCCCCAGAGCTGAAGCAGACCAGAGTGAACCTGCCCGCCCACAGCAGATATGGCCCTCAGGCCGTGG ACGCCAGATGATAACGCCGGCGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCT AGTCCGCCAAGATGAGCCCCAAGGACCTGACCCCCTTCCTGACAACCCTGTGGCTGCTCCTGGGCCATAG CAGAGTGCCTAGAGTGCGGGCCGAGGAATGCTGCGAGTTCATCAACGTGAACCACCCCCCCGAGCGGTGC TACGACTTCAAGATGTGCAACCGGTTCACCGTGGCCCTGAGATGCCCCGACGGCGAAGTGTGCTACAGCC CCGAGAAAACCGCCGAGATCCGGGGCATCGTGACCACCATGACCCACAGCCTGACCCGGCAGGTGGTGCA CAACAAGCTGACCAGCTGCAACTACAACCCCCTGTACCTGGAAGCCGACGGCCGGATCAGATGCGGCAAA GTGAACGACAAGGCCCAGTACCTGCTGGGAGCCGCCGGAAGCGTGCCCTACCGGTGGATCAACCTGGAAT ACGACAAGATCACCCGGATCGTGGGCCTGGACCAGTACCTGGAAAGCGTGAAGAAGCACAAGCGGCTGGA CGTGTGCAGAGCCAAGATGGGCTACATGCTGCAGTGATAAGGCGCGCCAACGTTACTGGCCGAAGCCGCT TGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAG GGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATG CAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAG CGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATA AGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAA TGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTG ATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAA CCACGGGGACGTGGTTTTCCTTTGAAAAACACGATAATATGCTGCGGCTGCTGCTGAGACACCACTTCCA CTGCCTGCTGCTGTGTGCCGTGTGGGCCACCCCTTGTCTGGCCAGCCCTTGGAGCACCCTGACCGCCAAC CAGAACCCTAGCCCCCCTTGGTCCAAGCTGACCTACAGCAAGCCCCACGACGCCGCCACCTTCTACTGCC CCTTTCTGTACCCCAGCCCTCCCAGAAGCCCCCTGCAGTTCAGCGGCTTCCAGAGAGTGTCCACCGGCCC TGAGTGCCGGAACGAGACACTGTACCTGCTGTACAACCGGGAGGGCCAGACACTGGTGGAGCGGAGCAGC ACCTGGGTGAAAAAAGTGATCTGGTATCTGAGCGGCCGGAACCAGACCATCCTGCAGCGGATGCCCAGAA CCGCCAGCAAGCCCAGCGACGGCAACGTGCAGATCAGCGTGGAGGACGCCAAAATCTTCGGAGCCCACAT GGTGCCCAAGCAGACCAAGCTGCTGAGATTCGTGGTCAACGACGGCACCAGATATCAGATGTGCGTGATG AAGCTGGAAAGCTGGGCCCACGTGTTCCGGGACTACTCCGTGAGCTTCCAGGTCCGGCTGACCTTCACCG AGGCCAACAACCAGACCTACACCTTCTGCACCCACCCCAACCTGATCGTGTGATAAGTACCTTTGTACGC CTGTTTTATACCCCCTCCCTGATTTGCAACTTAGAAGCAACGCAAACCAGATCAATAGTAGGTGTGACAT ACCAGTCGCATCTTGATCAAGCACTTCTGTATCCCCGGACCGAGTATCAATAGACTGTGCACACGGTTGA AGGAGAAAACGTCCGTTACCCGGCTAACTACTTCGAGAAGCCTAGTAACGCCATTGAAGTTGCAGAGTGT TTCGCTCAGCACTCCCCCCGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGG TGGCTGCGTTGGCGGCCTGCCTATGGGGTAACCCATAGGACGCTCTAATACGGACATGGCGTGAAGAGTC TATTGAGCTAGTTAGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACATACCCTTAA TCCAAAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCTT TTTATTCTTGTATTGGCTGCTTATGGTGACAATTAAAGAATTGTTACCATATAGCTATTGGATTGGCCAT CCAGTGTCAAACAGAGCTATTGTATATCTCTTTGTTGGATTCACACCTCTCACTCTTGAAACGTTACACA CCCTCAATTACATTATACTGCTGAACACGAAGCGCATATGCGGCTGTGCAGAGTGTGGCTGTCCGTGTGC CTGTGTGCCGTGGTGCTGGGCCAGTGCCAGAGAGAGACAGCCGAGAAGAACGACTACTACCGGGTGCCCC ACTACTGGGATGCCTGCAGCAGAGCCCTGCCCGACCAGACCCGGTACAAATACGTGGAGCAGCTCGTGGA CCTGACCCTGAACTACCACTACGACGCCAGCCACGGCCTGGACAACTTCGACGTGCTGAAGCGGATCAAC GTGACCGAGGTGTCCCTGCTGATCAGCGACTTCCGGCGGCAGAACAGAAGAGGCGGCACCAACAAGCGGA CCACCTTCAACGCCGCTGGCTCTCTGGCCCCTCACGCCAGATCCCTGGAATTCAGCGTGCGGCTGTTCGC CAACTGATAACGTTGCATCCTGCAGGATACAGCAGCAATTGGCAAGCTGCTTACATAGAACTCGCGGCGA TTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATAT TTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGTCGGCATGGCATCTCCACCTCCTCGCGGT CCGACCTGGGCATCCGAAGGAGGACGCACGTCCACTCGGATGGCTAAGGGAGAGCCACGTTTAAACGCTA GAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTT TTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCT TTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGT TCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCT CCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGG CAGACCTCAGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGC TTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCT TCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCG GAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTC CATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAG GACTATAAAGATACCAGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTT ACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCA GTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACT ATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAG AGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCC TCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCG GTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAAGG GGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTT CACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCT GACAGTTATTAGAAAAATTCATCCAGCAGACGATAAAACGCAATACGCTGGCTATCCGGTGCCGCAATGC CATACAGCACCAGAAAACGATCCGCCCATTCGCCGCCCAGTTCTTCCGCAATATCACGGGTGGCCAGCGC AATATCCTGATAACGATCCGCCACGCCCAGACGGCCGCAATCAATAAAGCCGCTAAAACGGCCATTTTCC ACCATAATGTTCGGCAGGCACGCATCACCATGGGTCACCACCAGATCTTCGCCATCCGGCATGCTCGCTT TCAGACGCGCAAACAGCTCTGCCGGTGCCAGGCCCTGATGTTCTTCATCCAGATCATCCTGATCCACCAG GCCCGCTTCCATACGGGTACGCGCACGTTCAATACGATGTTTCGCCTGATGATCAAACGGACAGGTCGCC GGGTCCAGGGTATGCAGACGACGCATGGCATCCGCCATAATGCTCACTTTTTCTGCCGGCGCCAGATGGC TAGACAGCAGATCCTGACCCGGCACTTCGCCCAGCAGCAGCCAATCACGGCCCGCTTCGGTCACCACATC CAGCACCGCCGCACACGGAACACCGGTGGTGGCCAGCCAGCTCAGACGCGCCGCTTCATCCTGCAGCTCG TTCAGCGCACCGCTCAGATCGGTTTTCACAAACAGCACCGGACGACCCTGCGCGCTCAGACGAAACACCG CCGCATCAGAGCAGCCAATGGTCTGCTGCGCCCAATCATAGCCAAACAGACGTTCCACCCACGCTGCCGG GCTACCCGCATGCAGGCCATCCTGTTCAATCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAG GGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCA CATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCG AGATAGGGTTGAGTGGCCGCTACAGGGCGCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGC GTTTCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTT GGGTAACGCCAGGGTTTTCCCAGTCACACGCGTAATACGACTCACTATAG VZV gB MFVTAVVSVSPSSFYESLQVEPTQSEDITRSAHLGDGDEIREAIHKSQDAETKPTFYVC PPPTGSTIVRLEPPRTCPDYHLGKNFTEGIAVVYKENIAAYKFKATVYYKDVIVSTAWA GSSYTQITNRYADRVPIPVSEITDTIDKFGKCSSKATYVRNNHKVEAFNEDKNPQDMPL IASKYNSVGSKAWHTTNDTYMVAGTPGTYRTGTSVNCIIEEVEARSIFPYDSFGLSTGD IIYMSPFFGLRDGAYREHSNYAMDRFHQFEGYRQRDLDTRALLEPAARNFLVTPHLTVG WNWKPKRTEVCSLVKWREVEDVVRDEYAHNFRFTMKTLSTTFISETNEFNLNQIHLSQC VKEEARAIINRIYTTRYNSSHVRTGDIQTYLARGGFVVVFQPLLSNSLARLYLQELVRE NTNHSPQKHPTRNTRSRRSVPVELRANRTITTTSSVEFAMLQFTYDHIQEHVNEMLARI SSSWCQLQNRERALWSGLFPINPSALASTILDQRVKARILGDVISVSNCPELGSDTRII LQNSMRVSGSTTRCYSRPLISIVSLNGSGTVEGQLGTDNELIMSRDLLEPCVANHKRYF LFGHHYVYYEDYRYVREIAVHDVGMISTYVDLNLTLLKDREFMPLQVYTRDELRDTGLL DYSEIQRRNQMHSLRFYDIDKVVQYDSGTAIMQGMAQFFQGLGTAGQAVGHVVLGATGA LLSTVHGFTTFLSNPFGALAVGLLVLAGLVAAFFAYRYVLKLKTSPMKALYPLTTKGLK QLPEGMDPFAEKPNATDTPIEEIGDSQNTEPSVNSGFDPDKFREAQEMIKYMTLVSAAE RQESKARKKNKTSALLTSRLTGLALRNRRGYSRVRTENVTGV VZV gH MFALVLAVVILPLWTTANKSYVTPTPATRSIGHMSALLREYSDRNMSLKLEAFYPTGFD EELIKSLHWGNDRKHVFLVIVKVNPTTHEGDVGLVIFPKYLLSPYHFKAEHRAPFPAGR FGFLSHPVTPDVSFFDSSFAPYLTTQHLVAFTTFPPNPLVWHLERAETAATAERPFGVS LLPARPTVPKNTILEHKAHFATWDALARHTFFSAEAIITNSTLRIHVPLFGSVWPIRYW ATGSVLLTSDSGRVEVNIGVGFMSSLISLSSGLPIELIVVPHTVKLNAVTSDTTWFQLN PPGPDPGPSYRVYLLGRGLDMNFSKHATVDICAYPEESLDYRYHLSMAHTEALRMTTKA DQHDINEESYYHIAARIATSIFALSEMGRTTEYFLLDEIVDVQYQLKFLNYILMRIGAG AHPNTISGTSDLIFADPSQLHDELSLLFGQVKPANVDYFISYDEARDQLKTAYALSRGQ DHVNALSLARRVIMSIYKGLLVKQNLNATERQALFFASMILLNFREGLENSSRVLDGRT TLLLMTSMCTAAHATQAALNIQEGLAYLNPSKHMFTIPNVYSPCMGSLRTDLTEEIHVM NLLSAIPTRPGLNEVLHTQLDESEIFDAAFKTMMIFTTWTAKDLHILHTHVPEVFTCQD AAARNGEYVLILPAVQGHSYVITRNKPQRGLVYSLADVDVYNPISVVYLSKDTCVSEHG VIETVALPHPDNLKECLYCGSVFLRYLTTGAIMDIIIIDSKDTERQLAAMGNSTIPPFN PDMHGDDSKAVLLFPNGTVVTLLGFERRQAIRMSGQYLGASLGGAFLAVVGFGIIGWML CGNSRLREYNKIPLT VZV gL MASHKWLLQMIVFLKTITIAYCLHLQDDTPLFFGAKPLSDVSLIITEPCVSSVYEAWDY AAPPVSNLSEALSGIVVKTKCPVPEVILWFKDKQMAYWTNPYVTLKGLTQSVGEEHKSG DIRDALLDALSGVWVDSTPSSTNIPENGCVWGADRLFQRVCQ VZV gI MFLIQCLISAVIFYIQVTNALIFKGDHVSLQVNSSLTSILIPMQNDNYTEIKGQLVFIG EQLPTGTNYSGTLELLYADTVAFCFRSVQVIRYDGCPRIRTSAFISCRYKHSWHYGNST DRISTEPDAGVMLKITKPGINDAGVYVLLVRLDHSRSTDGFILGVNVYTAGSHHNIHGV IYTSPSLQNGYSTRALFQQARLCDLPATPKGSGTSLFQHMLDLRAGKSLEDNPWLHEDV VTTETKSVVKEGIENHVYPTDMSTLPEKSLNDPPENLLIIIPIVASVMILTAMVIVIVI SVKRRRIKKHPIYRPNTKTRRGIQNATPESDVMLEAAIAQLATIREESPPHSVVNPFVK VZV gE MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDH AESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPT QMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVE ENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDC AENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQY LGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVG DTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGC TFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESL SGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPG TSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRVDKSPYNQSMYYAGLPVDDF EDSESTDTEEEFGNAIGGSHGGSSYTVYIDKTR VZV VEERep.SGPgB 1-ataggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttga catcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcagtttgaggtagaa gccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaac tgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgcag aatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacaga ttgtataagtatgcaactaagctgaagaaaaactgtaaggaaataactgataaggaattggaca agaaaatgaaggagctcgccgccgtcatgagcgaccctgacctggaaactgagactatgtgcct ccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggtt gacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggct ttgacaccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaactg ggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgttatggagcgg tcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaacaatgttctattct ctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctgt atttcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacggg tacgtcgttaaaagaatagctatcagtccaggcctgtatgggaagccttcaggctatgctgcta cgatgcaccgcgagggattcttgtgctgcaaagtgacagacacattgaacggggagagggtctc ttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatactggcaaca gatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacg gtcgcacccagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatt tgctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacgagat agacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatctatttataagc gcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcccaggat aggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaag gagccgtcacctctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggcta aggaggtgcgtgaagccgaggagttgcgcgcagctctaccacctttggcagctgatgttgagga gcccactctggaagccgatgtagacttgatgttacaagaggctggggccggctcagtggagaca cctcgtggcttgataaaggttaccagctacgatggcgaggacaagatcggctcttacgctgtgc tttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagt catagtgataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagta gtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtgccacca ttgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccacacatggaggagc gctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacctg tacgacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggcg agctggtggatcctcccttccatgaattcgcctacgagagtctgagaacacgaccagccgctcc ttaccaagtaccaaccataggggtgtatggcgtgccaggatcaggcaagtctggcatcattaaa agcgcagtcaccaaaaaagatctagtggtgagcgccaagaaagaaaactgtgcagaaattataa gggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaa tggatgcaaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtact ctcagagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaacagt gcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcacacaagtctt ccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgttttac gacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagta ccaaacctaagcaggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaat agattacaaaggcaacgaaataatgacggcagctgcctctcaagggctgacccgtaaaggtgtg tatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacccacctcagaacatgtgaacg tcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggataaa aacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagcat gatgccatcatgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaa acgtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgaccactga acaatggaacactgtggattattttgaaacggacaaagctcactcagcagagatagtattgaac caactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgttc cgttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaataa agaagtggtccgtcagctctctcgcaggtacccacaactgcctcgggcagttgccactggaaga gtctatgacatgaacactggtacactgcgcaattatgatccgcgcataaacctagtacctgtaa acagaagactgcctcatgctttagtcctccaccataatgaacacccacagagtgacttttcttc attcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggc aaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggca tcccaggtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaatacca tcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagcttgtctg catctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagcgaaa gcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcact tgaagagacggaagttctgtttgtattcattgggtacgatcgcaaggcccgtacgcacaatcct tacaagctttcatcaaccttgaccaacatttatacaggttccagactccacgaagccggatgtg caccctcatatcatgtggtgcgaggggatattgccacggccaccgaaggagtgattataaatgc tgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccggaa agcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacata tcattcatgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggc agaggcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattcca ctgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaaccatttgc tgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaat gactctcaaggaagcagtggctaggagagaagcagtggaggagatatgcatatccgacgactct tcagtgacagaacctgatgcagagctggtgagggtgcatccgaagagttctttggctggaagga agggctacagcacaagcgatggcaaaactttctcatatttggaagggaccaagtttcaccaggc ggccaaggatatagcagaaattaatgccatgtggcccgttgcaacggaggccaatgagcaggta tgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcgg aagcctccacaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagt acagcgcctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaag tatagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaagtgc ctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccggagcc atcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatgag accaggactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttgc tgtcagatggcccgacccaccaggtgctgcaagtcgaggcagacattcacgggccgccctctgt atctagctcatcctggtccattcctcatgcatccgactttgatgtggacagtttatccatactt gacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactcttacttcg caaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctcc acatcccgctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagc ctagtttccaccccgccaggcgtgaatagggtgatcactagagaggagctcgaggcgcttaccc cgtcacgcactcctagcaggtcggtctcgagaaccagcctggtctccaacccgccaggcgtaaa tagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatgacggtttgatgcg ggtgcatacatcttttcctccgacaccggtcaagggcatttacaacaaaaatcagtaaggcaaa cggtgctatccgaagtggtgttggagaggaccgaattggagatttcgtatgccccgcgcctcga ccaagaaaaagaagaattactacgcaagaaattacagttaaatcccacacctgctaacagaagc agataccagtccaggaaggtggagaacatgaaagccataacagctagacgtattctgcaaggcc tagggcattatttgaaggcagaaggaaaagtggagtgctaccgaaccctgcatcctgttccttt gtattcatctagtgtgaaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgcc atgttgaaagagaactttccgactgtggcttcttactgtattattccagagtacgatgcctatt tggacatggttgacggagcttcatgctgcttagacactgccagtttttgccctgcaaagctgcg cagctttccaaagaaacactcctatttggaacccacaatacgatcggcagtgccttcagcgatc cagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaatgtcacgcaaatga gagaattgcccgtattggattcggcggcctttaatgtggaatgcttcaagaaatatgcgtgtaa taatgaatattgggaaacgtttaaagaaaaccccatcaggcttactgaagaaaacgtggtaaat tacattaccaaattaaaaggaccaaaagctgctgctctttttgcgaagacacataatttgaata tgttgcaggacataccaatggacaggtttgtaatggacttaaagagagacgtgaaagtgactcc aggaacaaaacatactgaagaacggcccaaggtacaggtgatccaggctgccgatccgctagca acagcgtatctgtgcggaatccaccgagagctggttaggagattaaatgcggtcctgcttccga acattcatacactgtttgatatgtcggctgaagactttgacgctattatagccgagcacttcca gcctggggattgtgttctggaaactgacatcgcgtcgtttgataaaagtgaggacgacgccatg gctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttgacgctgattg aggcggctttcggcgaaatttcatcaatacatttgcccactaaaactaaatttaaattcggagc catgatgaaatctggaatgttcctcacactgtttgtgaacacagtcattaacattgtaatcgca agcagagtgttgagagaacggctaaccggatcaccatgtgcagcattcattggagatgacaata tcgtgaaaggagtcaaatcggacaaattaatggcagacaggtgcgccacctggttgaatatgga agtcaagattatagatgctgtggtgggcgagaaagcgccttatttctgtggagggtttattttg tgtgactccgtgaccggcacagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttg gcaaacctctggcagcagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtc aacacgctggaaccgagtgggtattctttcagagctgtgcaaggcagtagaatcaaggtatgaa accgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaatcattca gctacctgagaggggcccctataactctctacggctaacctgaatggactacgacatagtctag tcgagtctagtcgacgccaccatgttcgtgaccgccgtggtgtccgtgtcccccagcagctttt acgagagcctgcaggtcgagcccacccagagcgaggacatcacaagatctgcccacctgggcga cggcgacgagatcagagaggccatccacaagagccaggacgccgagacaaagcccaccttctac gtgtgccccccacctaccggctctacaattgtgcggctggaaccccccagaacctgccctgatt accacctgggcaagaacttcaccgagggaattgccgtggtgtacaaagagaatatcgccgccta caagttcaaggccaccgtgtactacaaggacgtgatcgtgtccaccgcctgggccggcagcagc tacacccagatcaccaacagatacgccgaccgggtgcccatccccgtgtctgagatcaccgaca ccatcgacaagttcggcaagtgcagcagcaaggccacctacgtgcggaacaaccacaaggtgga agccttcaacgaggacaagaacccccaggacatgcccctgatcgccagcaagtacaacagcgtg ggctccaaggcctggcacaccaccaacgacacctacatggtggccggcacccccggcacataca gaacaggcaccagcgtgaactgcatcatcgaggaagtggaagcccggtccatcttcccatacga cagcttcggcctgagcaccggcgacattatctacatgagccctttcttcggcctgcgggacggc gcctacagagagcacagcaactacgccatggaccggttccaccagttcgagggctacagacagc gggacctggacacaagagccctgctggaacctgccgccagaaacttcctggtcacccctcacct gaccgtgggctggaactggaagcccaagcggaccgaagtgtgcagcctggtcaagtggcgcgag gtggaagatgtcgtgcgggatgagtacgcccacaacttccggttcaccatgaagaccctgagca ccaccttcatcagcgagacaaacgagttcaacctgaaccagatccacctgagccagtgcgtgaa agaggaagccagagccatcatcaaccggatctacaccacccggtacaacagcagccacgtgcgg accggcgatatccagacctatctggctagaggcggcttcgtggtggtgtttcagcccctgctga gcaacagcctggctagactgtacctgcaggaactcgtcagagagaacaccaaccacagccccca gaagcaccccacccggaataccagatccagacgcagcgtgcccgtggaactgagagccaaccgg accatcaccaccaccagcagcgtggaattcgccatgctgcagttcacctacgaccacatccagg aacacgtgaacgagatgctggcccggatcagcagcagttggtgccagctgcagaatcgggaaag ggccctgtggtccggcctgttccccatcaatccaagcgccctggccagcaccatcctggaccag agagtgaaggccagaatcctgggggacgtgatcagcgtgtccaactgtcctgagctgggcagcg acacccggatcatcctgcagaacagcatgcgggtgtccggcagcaccaccagatgctacagcag acccctgatcagcatcgtgtccctgaacggcagcggcacagtggaaggccagctgggcaccgat aacgagctgatcatgagccgggacctgctcgaaccctgcgtggccaatcacaagcggtactttc tgttcggccaccactacgtgtactatgaggactacagatacgtgcgcgagatcgccgtgcacga cgtgggcatgatcagcacctacgtggacctgaacctgaccctgctgaaggaccgcgagttcatg ccactgcaggtctacacccgggacgagctgagagataccggcctgctggactacagcgagatcc agcggcggaaccagatgcactccctgcggttctacgacatcgacaaggtggtgcagtacgacag cggcaccgccatcatgcagggcatggcccagttctttcagggcctgggaacagccggacaggcc gtgggacatgtggtgctgggagctacaggcgccctgctgtctaccgtgcacggcttcaccacct ttctgagcaaccccttcggagccctggctgtgggactgctggtcctggctggactggtggccgc cttctttgcctaccgctacgtgctgaagctgaaaaccagccccatgaaggccctgtaccccctg accaccaagggcctgaagcagctgcctgagggcatggaccccttcgccgagaagcccaatgcca ccgacacccccatcgaggaaatcggcgacagccagaacaccgagccctccgtgaacagcggctt cgaccccgacaagtttcgcgaggcccaggaaatgatcaagtacatgaccctggtgtctgctgcc gagcggcaggaaagcaaggcccggaagaagaacaagacctccgccctgctgaccagcagactga caggactggccctgcggaacagacggggctatagcagagtgcggaccgagaatgtgaccggcgt gtaatctagacgcggccgcatacagcagcaattggcaagctgcttacatagaactcgcggcgat tggcatgccgccttaaaatttttattttatttttcttttcttttccgaatcggattttgttttt aatatttcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagggtcggcatggcatctccac ctcctcgcggtccgacctgggcatccgaaggaggacgcacgtccactcggatggctaagggaga gccacgtttaaaccagctccaattcgccctatagtgagtcgtattacgcgcgctcactggccgt cgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacat ccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgc gcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggt tacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttccct tcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggt tccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtag tgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagt ggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataag ggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaa ttttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaaccc ctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgata aatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttatt cccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaag atgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagat ccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgt ggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctc agaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaag agaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacg atcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttg atcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgt agcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaa caattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccgg ctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagc actggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaact atggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgt cagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggat ctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccac tgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaa tctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagct accaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttcta gtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgc taatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaag acgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagc ttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgc ttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcac gagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctga cttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacg cggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatc ccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccga acgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctc tccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcggg cagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacacttt atgctcccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagct atgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctgggtaccgg gcccacgcgtaatacgactcactatag_13339 VZV VEERep.SGPgH 1_ataggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttga catcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcagtttgaggtagaa gccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaac tgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgcag aatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacaga ttgtataagtatgcaactaagctgaagaaaaactgtaaggaaataactgataaggaattggaca agaaaatgaaggagctcgccgccgtcatgagcgaccctgacctggaaactgagactatgtgcct ccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggtt gacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggct ttgacaccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaactg ggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgttatggagcgg tcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaacaatgttctattct ctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctgt atttcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacggg tacgtcgttaaaagaatagctatcagtccaggcctgtatgggaagccttcaggctatgctgcta cgatgcaccgcgagggattcttgtgctgcaaagtgacagacacattgaacggggagagggtctc ttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatactggcaaca gatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacg gtcgcacccagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatt tgctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacgagat agacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatctatttataagc gcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcccaggat aggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaag gagccgtcacctctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggcta aggaggtgcgtgaagccgaggagttgcgcgcagctctaccacctttggcagctgatgttgagga gcccactctggaagccgatgtagacttgatgttacaagaggctggggccggctcagtggagaca cctcgtggcttgataaaggttaccagctacgatggcgaggacaagatcggctcttacgctgtgc tttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagt catagtgataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagta gtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtgccacca ttgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccacacatggaggagc gctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacctg tacgacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggcg agctggtggatcctcccttccatgaattcgcctacgagagtctgagaacacgaccagccgctcc ttaccaagtaccaaccataggggtgtatggcgtgccaggatcaggcaagtctggcatcattaaa agcgcagtcaccaaaaaagatctagtggtgagcgccaagaaagaaaactgtgcagaaattataa gggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaa tggatgcaaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtact ctcagagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaacagt gcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcacacaagtctt ccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgttttac gacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagta ccaaacctaagcaggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaat agattacaaaggcaacgaaataatgacggcagctgcctctcaagggctgacccgtaaaggtgtg tatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacccacctcagaacatgtgaacg tcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggataaa aacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagcat gatgccatcatgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaa acgtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgaccactga acaatggaacactgtggattattttgaaacggacaaagctcactcagcagagatagtattgaac caactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgttc cgttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaataa agaagtggtccgtcagctctctcgcaggtacccacaactgcctcgggcagttgccactggaaga gtctatgacatgaacactggtacactgcgcaattatgatccgcgcataaacctagtacctgtaa acagaagactgcctcatgctttagtcctccaccataatgaacacccacagagtgacttttcttc attcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggc aaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggca tcccaggtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaatacca tcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagcttgtctg catctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagcgaaa gcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcact tgaagagacggaagttctgtttgtattcattgggtacgatcgcaaggcccgtacgcacaatcct tacaagctttcatcaaccttgaccaacatttatacaggttccagactccacgaagccggatgtg caccctcatatcatgtggtgcgaggggatattgccacggccaccgaaggagtgattataaatgc tgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccggaa agcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacata tcattcatgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggc agaggcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattcca ctgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaaccatttgc tgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaat gactctcaaggaagcagtggctaggagagaagcagtggaggagatatgcatatccgacgactct tcagtgacagaacctgatgcagagctggtgagggtgcatccgaagagttctttggctggaagga agggctacagcacaagcgatggcaaaactttctcatatttggaagggaccaagtttcaccaggc ggccaaggatatagcagaaattaatgccatgtggcccgttgcaacggaggccaatgagcaggta tgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcgg aagcctccacaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagt acagcgcctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaag tatagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaagtgc ctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccggagcc atcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatgag accaggactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttgc tgtcagatggcccgacccaccaggtgctgcaagtcgaggcagacattcacgggccgccctctgt atctagctcatcctggtccattcctcatgcatccgactttgatgtggacagtttatccatactt gacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactcttacttcg caaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctcc acatcccgctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagc ctagtttccaccccgccaggcgtgaatagggtgatcactagagaggagctcgaggcgcttaccc cgtcacgcactcctagcaggtcggtctcgagaaccagcctggtctccaacccgccaggcgtaaa tagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatgacggtttgatgcg ggtgcatacatcttttcctccgacaccggtcaagggcatttacaacaaaaatcagtaaggcaaa cggtgctatccgaagtggtgttggagaggaccgaattggagatttcgtatgccccgcgcctcga ccaagaaaaagaagaattactacgcaagaaattacagttaaatcccacacctgctaacagaagc agataccagtccaggaaggtggagaacatgaaagccataacagctagacgtattctgcaaggcc tagggcattatttgaaggcagaaggaaaagtggagtgctaccgaaccctgcatcctgttccttt gtattcatctagtgtgaaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgcc atgttgaaagagaactttccgactgtggcttcttactgtattattccagagtacgatgcctatt tggacatggttgacggagcttcatgctgcttagacactgccagtttttgccctgcaaagctgcg cagctttccaaagaaacactcctatttggaacccacaatacgatcggcagtgccttcagcgatc cagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaatgtcacgcaaatga gagaattgcccgtattggattcggcggcctttaatgtggaatgcttcaagaaatatgcgtgtaa taatgaatattgggaaacgtttaaagaaaaccccatcaggcttactgaagaaaacgtggtaaat tacattaccaaattaaaaggaccaaaagctgctgctctttttgcgaagacacataatttgaata tgttgcaggacataccaatggacaggtttgtaatggacttaaagagagacgtgaaagtgactcc aggaacaaaacatactgaagaacggcccaaggtacaggtgatccaggctgccgatccgctagca acagcgtatctgtgcggaatccaccgagagctggttaggagattaaatgcggtcctgcttccga acattcatacactgtttgatatgtcggctgaagactttgacgctattatagccgagcacttcca gcctggggattgtgttctggaaactgacatcgcgtcgtttgataaaagtgaggacgacgccatg gctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttgacgctgattg aggcggctttcggcgaaatttcatcaatacatttgcccactaaaactaaatttaaattcggagc catgatgaaatctggaatgttcctcacactgtttgtgaacacagtcattaacattgtaatcgca agcagagtgttgagagaacggctaaccggatcaccatgtgcagcattcattggagatgacaata tcgtgaaaggagtcaaatcggacaaattaatggcagacaggtgcgccacctggttgaatatgga agtcaagattatagatgctgtggtgggcgagaaagcgccttatttctgtggagggtttattttg tgtgactccgtgaccggcacagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttg gcaaacctctggcagcagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtc aacacgctggaaccgagtgggtattctttcagagctgtgcaaggcagtagaatcaaggtatgaa accgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaatcattca gctacctgagaggggcccctataactctctacggctaacctgaatggactacgacatagtctag tcgagtctagtcgacgccaccatgttcgccctggtgctggccgtggtcatcctgcctctgtgga ccaccgccaacaagagctacgtgacccccacacccgccaccagatccatcggacacatgagcgc cctgctgagagagtacagcgaccggaacatgagcctgaagctggaagccttctaccccaccggc ttcgacgaggaactgatcaagagcctgcactggggcaacgaccggaagcacgtgttcctcgtga tcgtgaaagtgaaccccaccacccacgagggcgacgtcggcctggtcatcttccccaagtacct gctgagcccctaccacttcaaggccgagcacagagcccccttccctgctggccgctttggcttt ctgagccaccctgtgacccccgacgtgtcattcttcgacagcagcttcgccccctacctgacca cacagcacctggtggccttcaccaccttcccccccaatcctctcgtgtggcacctggaaagagc cgagacagccgccaccgccgaaagaccttttggcgtgtccctgctgcccgccagacctaccgtg cccaagaacaccatcctggaacacaaggcccacttcgccacctgggatgccctggccagacaca ccttctttagcgccgaggccatcatcaccaacagcaccctgagaatccacgtgcccctgttcgg cagcgtgtggcccatcagatactgggccacaggcagcgtgctgctgaccagcgatagcggcaga gtggaagtgaacatcggcgtgggcttcatgagcagcctgatcagcctgagcagcggcctgccca tcgagctgattgtggtgccccacaccgtgaagctgaacgccgtgaccagcgacaccacctggtt ccagctgaacccccctggccctgatcctggccctagttacagagtgtacctgctgggcagaggc ctggacatgaacttcagcaagcacgccaccgtggacatctgcgcctaccctgaggaaagcctgg actacagataccacctgagcatggcccacaccgaggccctgagaatgaccaccaaggccgacca gcacgacatcaacgaggaaagctactaccacattgccgccagaatcgccaccagcatcttcgcc ctgagcgagatgggccggaccaccgagtactttctgctggacgagatcgtggacgtgcagtacc agctgaagttcctgaactacatcctgatgcggatcggcgctggcgcccaccctaataccatcag cggcaccagcgacctgatcttcgccgatcctagccagctgcacgacgagctgagcctgctgttc ggccaggtcaaacccgccaacgtggactacttcatcagctacgacgaggcccgggaccagctga aaacagcctacgccctgtccagaggccaggatcatgtgaacgccctgtccctggccaggcgcgt gatcatgagcatctacaagggcctgctggtcaagcagaacctgaacgccaccgagcggcaggcc ctgttcttcgccagcatgatcctgctgaacttcagagagggcctggaaaacagcagccgggtgc tggatggcagaaccaccctgctgctgatgaccagcatgtgcacagccgcccatgccacacaggc cgccctgaatatccaggaaggcctggcttacctgaaccccagcaagcacatgttcaccatcccc aacgtgtacagcccctgcatgggcagcctgagaaccgacctgaccgaagagatccacgtgatga acctgctgtccgccatccccaccagacccggactgaatgaggtgctgcacacccagctggacga gtccgagatcttcgacgccgccttcaagaccatgatgatctttaccacctggaccgccaaggac ctgcacatcctgcacacacacgtgcccgaggtgttcacatgccaagatgccgccgctcggaacg gcgagtatgtgctgattctgcctgccgtgcagggccacagctacgtgatcacccggaacaagcc ccagcggggcctggtgtatagcctggctgacgtggacgtgtacaaccccatcagcgtggtgtac ctgagcaaggatacctgcgtgtccgagcacggcgtgatcgaaacagtggccctgccccaccccg acaacctgaaagagtgcctgtactgcggctccgtgttcctgcggtatctgaccaccggcgccat catggacatcatcatcatcgacagcaaggacaccgagagacagctggccgccatgggcaacagc accatcccccccttcaaccccgacatgcacggcgacgatagcaaggccgtgctgctgttcccca acggcaccgtggtcacactgctgggcttcgagcggagacaggccatcagaatgagcggccagta cctgggcgcctctctgggtggtgcctttctggccgtcgtgggctttggcatcatcggctggatg ctgtgcggcaacagcagactgcgcgagtacaacaagatccccctgacctaatctagacgcggcc gcatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaa atttttattttatttttcttttcttttccgaatcggattttgtttttaatatttcaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaagggtcggcatggcatctccacctcctcgcggtccgacc tgggcatccgaaggaggacgcacgtccactcggatggctaagggagagccacgtttaaaccagc tccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtg actgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctg gcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaa tgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccg ctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgtt cgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgcttta cggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgat agacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaac tggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcg gcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaa cgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttc taaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatatt gaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcatt ttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttg ggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgcc ccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccg tattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgag tactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctg ccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaagga gctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgt tgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggat ggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgct gataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggta agccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatag acagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactca tatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatccttt ttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgt agaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaaca aaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccga aggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttagg ccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtg gctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggata aggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgaccta caccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaag gcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggg gaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgattttt gtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttc ctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggata accgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcga gtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccg attcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaa ttaatgtgagttagctcactcattaggcaccccaggctttacactttatgctcccggctcgtat gttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgcc aagcgcgcaattaaccctcactaaagggaacaaaagctgggtaccgggcccacgcgtaatacga ctcactatag_13258 VZV VEERep.SGPgL 1_ataggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttga catcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcagtttgaggtagaa gccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaac tgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgcag aatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacaga ttgtataagtatgcaactaagctgaagaaaaactgtaaggaaataactgataaggaattggaca agaaaatgaaggagctcgccgccgtcatgagcgaccctgacctggaaactgagactatgtgcct ccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggtt gacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggct ttgacaccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaactg ggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgttatggagcgg tcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaacaatgttctattct ctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctgt atttcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacggg tacgtcgttaaaagaatagctatcagtccaggcctgtatgggaagccttcaggctatgctgcta cgatgcaccgcgagggattcttgtgctgcaaagtgacagacacattgaacggggagagggtctc ttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatactggcaaca gatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacg gtcgcacccagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatt tgctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacgagat agacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatctatttataagc gcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcccaggat aggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaag gagccgtcacctctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggcta aggaggtgcgtgaagccgaggagttgcgcgcagctctaccacctttggcagctgatgttgagga gcccactctggaagccgatgtagacttgatgttacaagaggctggggccggctcagtggagaca cctcgtggcttgataaaggttaccagctacgatggcgaggacaagatcggctcttacgctgtgc tttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagt catagtgataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagta gtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtgccacca ttgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccacacatggaggagc gctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacctg tacgacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggcg agctggtggatcctcccttccatgaattcgcctacgagagtctgagaacacgaccagccgctcc ttaccaagtaccaaccataggggtgtatggcgtgccaggatcaggcaagtctggcatcattaaa agcgcagtcaccaaaaaagatctagtggtgagcgccaagaaagaaaactgtgcagaaattataa gggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaa tggatgcaaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtact ctcagagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaacagt gcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcacacaagtctt ccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgttttac gacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagta ccaaacctaagcaggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaat agattacaaaggcaacgaaataatgacggcagctgcctctcaagggctgacccgtaaaggtgtg tatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacccacctcagaacatgtgaacg tcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggataaa aacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagcat gatgccatcatgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaa acgtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgaccactga acaatggaacactgtggattattttgaaacggacaaagctcactcagcagagatagtattgaac caactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgttc cgttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaataa agaagtggtccgtcagctctctcgcaggtacccacaactgcctcgggcagttgccactggaaga gtctatgacatgaacactggtacactgcgcaattatgatccgcgcataaacctagtacctgtaa acagaagactgcctcatgctttagtcctccaccataatgaacacccacagagtgacttttcttc attcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggc aaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggca tcccaggtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaatacca tcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagcttgtctg catctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagcgaaa gcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcact tgaagagacggaagttctgtttgtattcattgggtacgatcgcaaggcccgtacgcacaatcct tacaagctttcatcaaccttgaccaacatttatacaggttccagactccacgaagccggatgtg caccctcatatcatgtggtgcgaggggatattgccacggccaccgaaggagtgattataaatgc tgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccggaa agcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacata tcattcatgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggc agaggcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattcca ctgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaaccatttgc tgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaat gactctcaaggaagcagtggctaggagagaagcagtggaggagatatgcatatccgacgactct tcagtgacagaacctgatgcagagctggtgagggtgcatccgaagagttctttggctggaagga agggctacagcacaagcgatggcaaaactttctcatatttggaagggaccaagtttcaccaggc ggccaaggatatagcagaaattaatgccatgtggcccgttgcaacggaggccaatgagcaggta tgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcgg aagcctccacaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagt acagcgcctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaag tatagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaagtgc ctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccggagcc atcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatgag accaggactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttgc tgtcagatggcccgacccaccaggtgctgcaagtcgaggcagacattcacgggccgccctctgt atctagctcatcctggtccattcctcatgcatccgactttgatgtggacagtttatccatactt gacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactcttacttcg caaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctcc acatcccgctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagc ctagtttccaccccgccaggcgtgaatagggtgatcactagagaggagctcgaggcgcttaccc cgtcacgcactcctagcaggtcggtctcgagaaccagcctggtctccaacccgccaggcgtaaa tagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatgacggtttgatgcg ggtgcatacatcttttcctccgacaccggtcaagggcatttacaacaaaaatcagtaaggcaaa cggtgctatccgaagtggtgttggagaggaccgaattggagatttcgtatgccccgcgcctcga ccaagaaaaagaagaattactacgcaagaaattacagttaaatcccacacctgctaacagaagc agataccagtccaggaaggtggagaacatgaaagccataacagctagacgtattctgcaaggcc tagggcattatttgaaggcagaaggaaaagtggagtgctaccgaaccctgcatcctgttccttt gtattcatctagtgtgaaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgcc atgttgaaagagaactttccgactgtggcttcttactgtattattccagagtacgatgcctatt tggacatggttgacggagcttcatgctgcttagacactgccagtttttgccctgcaaagctgcg cagctttccaaagaaacactcctatttggaacccacaatacgatcggcagtgccttcagcgatc cagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaatgtcacgcaaatga gagaattgcccgtattggattcggcggcctttaatgtggaatgcttcaagaaatatgcgtgtaa taatgaatattgggaaacgtttaaagaaaaccccatcaggcttactgaagaaaacgtggtaaat tacattaccaaattaaaaggaccaaaagctgctgctctttttgcgaagacacataatttgaata tgttgcaggacataccaatggacaggtttgtaatggacttaaagagagacgtgaaagtgactcc aggaacaaaacatactgaagaacggcccaaggtacaggtgatccaggctgccgatccgctagca acagcgtatctgtgcggaatccaccgagagctggttaggagattaaatgcggtcctgcttccga acattcatacactgtttgatatgtcggctgaagactttgacgctattatagccgagcacttcca gcctggggattgtgttctggaaactgacatcgcgtcgtttgataaaagtgaggacgacgccatg gctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttgacgctgattg aggcggctttcggcgaaatttcatcaatacatttgcccactaaaactaaatttaaattcggagc catgatgaaatctggaatgttcctcacactgtttgtgaacacagtcattaacattgtaatcgca agcagagtgttgagagaacggctaaccggatcaccatgtgcagcattcattggagatgacaata tcgtgaaaggagtcaaatcggacaaattaatggcagacaggtgcgccacctggttgaatatgga agtcaagattatagatgctgtggtgggcgagaaagcgccttatttctgtggagggtttattttg tgtgactccgtgaccggcacagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttg gcaaacctctggcagcagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtc aacacgctggaaccgagtgggtattctttcagagctgtgcaaggcagtagaatcaaggtatgaa accgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaatcattca gctacctgagaggggcccctataactctctacggctaacctgaatggactacgacatagtctag tcgagtctagtcgacgccaccatggccagccacaagtggctgctgcagatgatcgtgttcctga aaaccatcacaatcgcctactgcctgcatctgcaggacgacacccctctgttcttcggcgccaa gcctctgagcgacgtgtccctgatcatcaccgagccttgcgtgtccagcgtgtacgaggcctgg gattatgccgcccctcccgtgtccaatctgagcgaagccctgagcggcatcgtggtcaagacca agtgccccgtgcccgaagtgatcctgtggttcaaggacaagcagatggcctactggaccaaccc ttacgtgaccctgaagggcctgacccagagcgtgggcgaggaacacaagagcggcgacatcaga gatgccctgctggatgccctgtccggtgtctgggtggacagcacaccctccagcaccaacatcc ccgagaacggctgtgtgtggggagccgaccggctgttccagagagtgtgtcagtaatctagacg cggccgcatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcatgccgcc ttaaaatttttattttatttttcttttcttttccgaatcggattttgtttttaatatttcaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagggtcggcatggcatctccacctcctcgcggtc cgacctgggcatccgaaggaggacgcacgtccactcggatggctaagggagagccacgtttaaa ccagctccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacg tcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgcc agctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatg gcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgt gaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgcc acgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtg ctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgcc ctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttc caaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccga tttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaat attaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttat ttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaata atattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcg gcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatc agttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttt tcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtatta tcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttgg ttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcag tgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccg aaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaac cggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaac aacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagac tggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggttta ttgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccaga tggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacga aatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagttt actcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagat cctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagac cccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgc aaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttt tccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtag ttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttac cagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttacc ggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacg acctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaaggga gaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttcc agggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcga tttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttac ggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgt ggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgc agcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgtt ggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaa cgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctcccggct cgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgatt acgccaagcgcgcaattaaccctcactaaagggaacaaaagctgggtaccgggcccacgcgtaa tacgactcactatag_11215 VZV VEERep.SGPgH-SGPgL 1_ataggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttga catcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcagtttgaggtagaa gccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaac tgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgcag aatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacaga ttgtataagtatgcaactaagctgaagaaaaactgtaaggaaataactgataaggaattggaca agaaaatgaaggagctcgccgccgtcatgagcgaccctgacctggaaactgagactatgtgcct ccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggtt gacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggct ttgacaccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaactg ggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgttatggagcgg tcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaacaatgttctattct ctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctgt atttcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacggg tacgtcgttaaaagaatagctatcagtccaggcctgtatgggaagccttcaggctatgctgcta cgatgcaccgcgagggattcttgtgctgcaaagtgacagacacattgaacggggagagggtctc ttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatactggcaaca gatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacg gtcgcacccagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatt tgctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacgagat agacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatctatttataagc gcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcccaggat aggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaag gagccgtcacctctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggcta aggaggtgcgtgaagccgaggagttgcgcgcagctctaccacctttggcagctgatgttgagga gcccactctggaagccgatgtagacttgatgttacaagaggctggggccggctcagtggagaca cctcgtggcttgataaaggttaccagctacgatggcgaggacaagatcggctcttacgctgtgc tttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagt catagtgataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagta gtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtgccacca ttgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccacacatggaggagc gctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacctg tacgacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggcg agctggtggatcctcccttccatgaattcgcctacgagagtctgagaacacgaccagccgctcc ttaccaagtaccaaccataggggtgtatggcgtgccaggatcaggcaagtctggcatcattaaa agcgcagtcaccaaaaaagatctagtggtgagcgccaagaaagaaaactgtgcagaaattataa gggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaa tggatgcaaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtact ctcagagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaacagt gcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcacacaagtctt ccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgttttac gacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagta ccaaacctaagcaggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaat agattacaaaggcaacgaaataatgacggcagctgcctctcaagggctgacccgtaaaggtgtg tatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacccacctcagaacatgtgaacg tcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggataaa aacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagcat gatgccatcatgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaa acgtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgaccactga acaatggaacactgtggattattttgaaacggacaaagctcactcagcagagatagtattgaac caactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgttc cgttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaataa agaagtggtccgtcagctctctcgcaggtacccacaactgcctcgggcagttgccactggaaga gtctatgacatgaacactggtacactgcgcaattatgatccgcgcataaacctagtacctgtaa acagaagactgcctcatgctttagtcctccaccataatgaacacccacagagtgacttttcttc attcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggc aaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggca tcccaggtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaatacca tcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagcttgtctg catctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagcgaaa gcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcact tgaagagacggaagttctgtttgtattcattgggtacgatcgcaaggcccgtacgcacaatcct tacaagctttcatcaaccttgaccaacatttatacaggttccagactccacgaagccggatgtg caccctcatatcatgtggtgcgaggggatattgccacggccaccgaaggagtgattataaatgc tgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccggaa agcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacata tcattcatgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggc agaggcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattcca ctgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaaccatttgc tgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaat gactctcaaggaagcagtggctaggagagaagcagtggaggagatatgcatatccgacgactct tcagtgacagaacctgatgcagagctggtgagggtgcatccgaagagttctttggctggaagga agggctacagcacaagcgatggcaaaactttctcatatttggaagggaccaagtttcaccaggc ggccaaggatatagcagaaattaatgccatgtggcccgttgcaacggaggccaatgagcaggta tgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcgg aagcctccacaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagt acagcgcctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaag tatagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaagtgc ctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccggagcc atcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatgag accaggactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttgc tgtcagatggcccgacccaccaggtgctgcaagtcgaggcagacattcacgggccgccctctgt atctagctcatcctggtccattcctcatgcatccgactttgatgtggacagtttatccatactt gacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactcttacttcg caaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctcc acatcccgctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagc ctagtttccaccccgccaggcgtgaatagggtgatcactagagaggagctcgaggcgcttaccc cgtcacgcactcctagcaggtcggtctcgagaaccagcctggtctccaacccgccaggcgtaaa tagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatgacggtttgatgcg ggtgcatacatcttttcctccgacaccggtcaagggcatttacaacaaaaatcagtaaggcaaa cggtgctatccgaagtggtgttggagaggaccgaattggagatttcgtatgccccgcgcctcga ccaagaaaaagaagaattactacgcaagaaattacagttaaatcccacacctgctaacagaagc agataccagtccaggaaggtggagaacatgaaagccataacagctagacgtattctgcaaggcc tagggcattatttgaaggcagaaggaaaagtggagtgctaccgaaccctgcatcctgttccttt gtattcatctagtgtgaaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgcc atgttgaaagagaactttccgactgtggcttcttactgtattattccagagtacgatgcctatt tggacatggttgacggagcttcatgctgcttagacactgccagtttttgccctgcaaagctgcg cagctttccaaagaaacactcctatttggaacccacaatacgatcggcagtgccttcagcgatc cagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaatgtcacgcaaatga gagaattgcccgtattggattcggcggcctttaatgtggaatgcttcaagaaatatgcgtgtaa taatgaatattgggaaacgtttaaagaaaaccccatcaggcttactgaagaaaacgtggtaaat tacattaccaaattaaaaggaccaaaagctgctgctctttttgcgaagacacataatttgaata tgttgcaggacataccaatggacaggtttgtaatggacttaaagagagacgtgaaagtgactcc aggaacaaaacatactgaagaacggcccaaggtacaggtgatccaggctgccgatccgctagca acagcgtatctgtgcggaatccaccgagagctggttaggagattaaatgcggtcctgcttccga acattcatacactgtttgatatgtcggctgaagactttgacgctattatagccgagcacttcca gcctggggattgtgttctggaaactgacatcgcgtcgtttgataaaagtgaggacgacgccatg gctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttgacgctgattg aggcggctttcggcgaaatttcatcaatacatttgcccactaaaactaaatttaaattcggagc catgatgaaatctggaatgttcctcacactgtttgtgaacacagtcattaacattgtaatcgca agcagagtgttgagagaacggctaaccggatcaccatgtgcagcattcattggagatgacaata tcgtgaaaggagtcaaatcggacaaattaatggcagacaggtgcgccacctggttgaatatgga agtcaagattatagatgctgtggtgggcgagaaagcgccttatttctgtggagggtttattttg tgtgactccgtgaccggcacagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttg gcaaacctctggcagcagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtc aacacgctggaaccgagtgggtattctttcagagctgtgcaaggcagtagaatcaaggtatgaa accgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaatcattca gctacctgagaggggcccctataactctctacggctaacctgaatggactacgacatagtctag tcgagtctagtcgacgccaccatgttcgccctggtgctggccgtggtcatcctgcctctgtgga ccaccgccaacaagagctacgtgacccccacacccgccaccagatccatcggacacatgagcgc cctgctgagagagtacagcgaccggaacatgagcctgaagctggaagccttctaccccaccggc ttcgacgaggaactgatcaagagcctgcactggggcaacgaccggaagcacgtgttcctcgtga tcgtgaaagtgaaccccaccacccacgagggcgacgtcggcctggtcatcttccccaagtacct gctgagcccctaccacttcaaggccgagcacagagcccccttccctgctggccgctttggcttt ctgagccaccctgtgacccccgacgtgtcattcttcgacagcagcttcgccccctacctgacca cacagcacctggtggccttcaccaccttcccccccaatcctctcgtgtggcacctggaaagagc cgagacagccgccaccgccgaaagaccttttggcgtgtccctgctgcccgccagacctaccgtg cccaagaacaccatcctggaacacaaggcccacttcgccacctgggatgccctggccagacaca ccttctttagcgccgaggccatcatcaccaacagcaccctgagaatccacgtgcccctgttcgg cagcgtgtggcccatcagatactgggccacaggcagcgtgctgctgaccagcgatagcggcaga gtggaagtgaacatcggcgtgggcttcatgagcagcctgatcagcctgagcagcggcctgccca tcgagctgattgtggtgccccacaccgtgaagctgaacgccgtgaccagcgacaccacctggtt ccagctgaacccccctggccctgatcctggccctagttacagagtgtacctgctgggcagaggc ctggacatgaacttcagcaagcacgccaccgtggacatctgcgcctaccctgaggaaagcctgg actacagataccacctgagcatggcccacaccgaggccctgagaatgaccaccaaggccgacca gcacgacatcaacgaggaaagctactaccacattgccgccagaatcgccaccagcatcttcgcc ctgagcgagatgggccggaccaccgagtactttctgctggacgagatcgtggacgtgcagtacc agctgaagttcctgaactacatcctgatgcggatcggcgctggcgcccaccctaataccatcag cggcaccagcgacctgatcttcgccgatcctagccagctgcacgacgagctgagcctgctgttc ggccaggtcaaacccgccaacgtggactacttcatcagctacgacgaggcccgggaccagctga aaacagcctacgccctgtccagaggccaggatcatgtgaacgccctgtccctggccaggcgcgt gatcatgagcatctacaagggcctgctggtcaagcagaacctgaacgccaccgagcggcaggcc ctgttcttcgccagcatgatcctgctgaacttcagagagggcctggaaaacagcagccgggtgc tggatggcagaaccaccctgctgctgatgaccagcatgtgcacagccgcccatgccacacaggc cgccctgaatatccaggaaggcctggcttacctgaaccccagcaagcacatgttcaccatcccc aacgtgtacagcccctgcatgggcagcctgagaaccgacctgaccgaagagatccacgtgatga acctgctgtccgccatccccaccagacccggactgaatgaggtgctgcacacccagctggacga gtccgagatcttcgacgccgccttcaagaccatgatgatctttaccacctggaccgccaaggac ctgcacatcctgcacacacacgtgcccgaggtgttcacatgccaagatgccgccgctcggaacg gcgagtatgtgctgattctgcctgccgtgcagggccacagctacgtgatcacccggaacaagcc ccagcggggcctggtgtatagcctggctgacgtggacgtgtacaaccccatcagcgtggtgtac ctgagcaaggatacctgcgtgtccgagcacggcgtgatcgaaacagtggccctgccccaccccg acaacctgaaagagtgcctgtactgcggctccgtgttcctgcggtatctgaccaccggcgccat catggacatcatcatcatcgacagcaaggacaccgagagacagctggccgccatgggcaacagc accatcccccccttcaaccccgacatgcacggcgacgatagcaaggccgtgctgctgttcccca acggcaccgtggtcacactgctgggcttcgagcggagacaggccatcagaatgagcggccagta cctgggcgcctctctgggtggtgcctttctggccgtcgtgggctttggcatcatcggctggatg ctgtgcggcaacagcagactgcgcgagtacaacaagatccccctgacctaatctagacgtcgcg accacccaggatccgcctataactctctacggctaacctgaatggactacgacatagtctagtc gacgccaccatggccagccacaagtggctgctgcagatgatcgtgttcctgaaaaccatcacaa tcgcctactgcctgcatctgcaggacgacacccctctgttcttcggcgccaagcctctgagcga cgtgtccctgatcatcaccgagccttgcgtgtccagcgtgtacgaggcctgggattatgccgcc cctcccgtgtccaatctgagcgaagccctgagcggcatcgtggtcaagaccaagtgccccgtgc ccgaagtgatcctgtggttcaaggacaagcagatggcctactggaccaacccttacgtgaccct gaagggcctgacccagagcgtgggcgaggaacacaagagcggcgacatcagagatgccctgctg gatgccctgtccggtgtctgggtggacagcacaccctccagcaccaacatccccgagaacggct gtgtgtggggagccgaccggctgttccagagagtgtgtcagtaatctagacgcggccgcataca gcagcaattggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaaattttta ttttatttttcttttcttttccgaatcggattttgtttttaatatttcaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaagggtcggcatggcatctccacctcctcgcggtccgacctgggcat ccgaaggaggacgcacgtccactcggatggctaagggagagccacgtttaaaccagctccaatt cgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactggga aaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaat agcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacg cgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacact tgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggc tttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacc tcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggt ttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaaca acactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctatt ggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttac aatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatac attcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaag gaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgcctt cctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcac gagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaaga acgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgac gccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcac cagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataac catgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaacc gcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatg aagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaa actattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcg gataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaat ctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctc ccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatc gctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatac tttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataa tctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaag atcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaac caccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaac tggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccac ttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctg ccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgca gcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaa ctgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggaca ggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgc ctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgc tcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggcct tttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtat taccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtg agcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcatt aatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgt gagttagctcactcattaggcaccccaggctttacactttatgctcccggctcgtatgttgtgt ggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcg caattaaccctcactaaagggaacaaaagctgggtaccgggcccacgcgtaatacgactcacta tag_13827 VZV VEERep.SGPgE 1_ataggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttga catcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcagtttgaggtagaa gccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaac tgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgcag aatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacaga ttgtataagtatgcaactaagctgaagaaaaactgtaaggaaataactgataaggaattggaca agaaaatgaaggagctcgccgccgtcatgagcgaccctgacctggaaactgagactatgtgcct ccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggtt gacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggct ttgacaccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaactg ggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgttatggagcgg tcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaacaatgttctattct ctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctgt atttcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacggg tacgtcgttaaaagaatagctatcagtccaggcctgtatgggaagccttcaggctatgctgcta cgatgcaccgcgagggattcttgtgctgcaaagtgacagacacattgaacggggagagggtctc ttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatactggcaaca gatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacg gtcgcacccagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatt tgctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacgagat agacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatctatttataagc gcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcccaggat aggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaag gagccgtcacctctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggcta aggaggtgcgtgaagccgaggagttgcgcgcagctctaccacctttggcagctgatgttgagga gcccactctggaagccgatgtagacttgatgttacaagaggctggggccggctcagtggagaca cctcgtggcttgataaaggttaccagctacgatggcgaggacaagatcggctcttacgctgtgc tttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagt catagtgataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagta gtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtgccacca ttgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccacacatggaggagc gctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacctg tacgacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggcg agctggtggatcctcccttccatgaattcgcctacgagagtctgagaacacgaccagccgctcc ttaccaagtaccaaccataggggtgtatggcgtgccaggatcaggcaagtctggcatcattaaa agcgcagtcaccaaaaaagatctagtggtgagcgccaagaaagaaaactgtgcagaaattataa gggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaa tggatgcaaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtact ctcagagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaacagt gcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcacacaagtctt ccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgttttac gacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagta ccaaacctaagcaggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaat agattacaaaggcaacgaaataatgacggcagctgcctctcaagggctgacccgtaaaggtgtg tatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacccacctcagaacatgtgaacg tcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggataaa aacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagcat gatgccatcatgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaa acgtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgaccactga acaatggaacactgtggattattttgaaacggacaaagctcactcagcagagatagtattgaac caactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgttc cgttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaataa agaagtggtccgtcagctctctcgcaggtacccacaactgcctcgggcagttgccactggaaga gtctatgacatgaacactggtacactgcgcaattatgatccgcgcataaacctagtacctgtaa acagaagactgcctcatgctttagtcctccaccataatgaacacccacagagtgacttttcttc attcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggc aaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggca tcccaggtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaatacca tcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagcttgtctg catctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagcgaaa gcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcact tgaagagacggaagttctgtttgtattcattgggtacgatcgcaaggcccgtacgcacaatcct tacaagctttcatcaaccttgaccaacatttatacaggttccagactccacgaagccggatgtg caccctcatatcatgtggtgcgaggggatattgccacggccaccgaaggagtgattataaatgc tgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccggaa agcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacata tcattcatgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggc agaggcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattcca ctgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaaccatttgc tgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaat gactctcaaggaagcagtggctaggagagaagcagtggaggagatatgcatatccgacgactct tcagtgacagaacctgatgcagagctggtgagggtgcatccgaagagttctttggctggaagga agggctacagcacaagcgatggcaaaactttctcatatttggaagggaccaagtttcaccaggc ggccaaggatatagcagaaattaatgccatgtggcccgttgcaacggaggccaatgagcaggta tgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcgg aagcctccacaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagt acagcgcctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaag tatagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaagtgc ctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccggagcc atcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatgag accaggactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttgc tgtcagatggcccgacccaccaggtgctgcaagtcgaggcagacattcacgggccgccctctgt atctagctcatcctggtccattcctcatgcatccgactttgatgtggacagtttatccatactt gacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactcttacttcg caaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctcc acatcccgctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagc ctagtttccaccccgccaggcgtgaatagggtgatcactagagaggagctcgaggcgcttaccc cgtcacgcactcctagcaggtcggtctcgagaaccagcctggtctccaacccgccaggcgtaaa tagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatgacggtttgatgcg ggtgcatacatcttttcctccgacaccggtcaagggcatttacaacaaaaatcagtaaggcaaa cggtgctatccgaagtggtgttggagaggaccgaattggagatttcgtatgccccgcgcctcga ccaagaaaaagaagaattactacgcaagaaattacagttaaatcccacacctgctaacagaagc agataccagtccaggaaggtggagaacatgaaagccataacagctagacgtattctgcaaggcc tagggcattatttgaaggcagaaggaaaagtggagtgctaccgaaccctgcatcctgttccttt gtattcatctagtgtgaaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgcc atgttgaaagagaactttccgactgtggcttcttactgtattattccagagtacgatgcctatt tggacatggttgacggagcttcatgctgcttagacactgccagtttttgccctgcaaagctgcg cagctttccaaagaaacactcctatttggaacccacaatacgatcggcagtgccttcagcgatc cagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaatgtcacgcaaatga gagaattgcccgtattggattcggcggcctttaatgtggaatgcttcaagaaatatgcgtgtaa taatgaatattgggaaacgtttaaagaaaaccccatcaggcttactgaagaaaacgtggtaaat tacattaccaaattaaaaggaccaaaagctgctgctctttttgcgaagacacataatttgaata tgttgcaggacataccaatggacaggtttgtaatggacttaaagagagacgtgaaagtgactcc aggaacaaaacatactgaagaacggcccaaggtacaggtgatccaggctgccgatccgctagca acagcgtatctgtgcggaatccaccgagagctggttaggagattaaatgcggtcctgcttccga acattcatacactgtttgatatgtcggctgaagactttgacgctattatagccgagcacttcca gcctggggattgtgttctggaaactgacatcgcgtcgtttgataaaagtgaggacgacgccatg gctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttgacgctgattg aggcggctttcggcgaaatttcatcaatacatttgcccactaaaactaaatttaaattcggagc catgatgaaatctggaatgttcctcacactgtttgtgaacacagtcattaacattgtaatcgca agcagagtgttgagagaacggctaaccggatcaccatgtgcagcattcattggagatgacaata tcgtgaaaggagtcaaatcggacaaattaatggcagacaggtgcgccacctggttgaatatgga agtcaagattatagatgctgtggtgggcgagaaagcgccttatttctgtggagggtttattttg tgtgactccgtgaccggcacagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttg gcaaacctctggcagcagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtc aacacgctggaaccgagtgggtattctttcagagctgtgcaaggcagtagaatcaaggtatgaa accgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaatcattca gctacctgagaggggcccctataactctctacggctaacctgaatggactacgacatagtctag tcgagtctagtcgacgccaccatgggcaccgtgaacaagcctgtcgtgggcgtgctgatgggct tcggcatcatcaccggcaccctgagaatcaccaaccctgtgcgggccagcgtgctgagatacga cgacttccacatcgacgaggacaagctggacaccaacagcgtgtacgagccctactaccacagc gaccacgccgagagcagctgggtcaacagaggcgagagcagccggaaggcctacgaccacaaca gcccctacatctggccccggaacgactacgacggcttcctggaaaacgcccacgagcaccacgg cgtgtacaatcagggcagaggcatcgacagcggcgagagactgatgcagcccacacagatgagc gcccaggaagatctgggcgacgacacaggcatccacgtgatccccaccctgaacggcgacgacc ggcacaagatcgtgaacgtggaccagcggcagtacggcgacgtgttcaagggcgacctgaaccc taagccccagggccagagactgatcgaggtgtccgtggaagagaaccaccccttcaccctgaga gcccccatccagagaatctacggcgtgcggtataccgagacttggagcttcctgcccagcctga cctgtacaggcgacgccgctcctgccatccagcacatctgcctgaagcacaccacctgtttcca ggacgtggtggtggacgtggactgcgccgagaacaccaaagaggaccagctggccgagatcagc taccggttccagggcaagaaagaggccgaccagccctggatcgtggtcaataccagcaccctgt tcgacgagctggaactggacccccccgagattgaacccggcgtgctgaaggtgctgcggaccga gaagcagtacctgggcgtgtacatctggaacatgcggggctccgacggcacctctacctacgcc accttcctggtcacatggaagggcgacgagaaaacccggaaccctacccctgccgtgacccctc agcctagaggcgccgagttccatatgtggaattaccactcccacgtgttcagcgtgggcgacac cttcagcctggccatgcatctgcagtacaagatccacgaggcccccttcgacctgctgctggaa tggctgtacgtgcccatcgaccctacctgccagcccatgcggctgtacagcacctgtctgtacc accccaacgcccctcagtgcctgagccacatgaacagcggctgcaccttcaccagccctcacct ggctcagagggtggccagcaccgtgtaccagaattgcgagcacgccgacaactacaccgcctac tgcctgggcatcagccacatggaacccagcttcggcctgatcctgcacgatggcggcaccaccc tgaagttcgtggacacacccgagagcctgagcggcctgtacgtgttcgtggtgtacttcaacgg ccacgtggaagccgtggcctacaccgtggtgtccaccgtggaccacttcgtgaacgccatcgag gaaagaggcttcccacccacagccggacagcctccagccaccaccaagcccaaagaaatcaccc ccgtgaaccccggcaccagccccctgctgagatatgctgcttggacaggcggactggccgctgt ggtgctgctgtgcctggtcatcttcctgatctgcaccgccaagcggatgagagtgaaggcctac cgggtggacaagtccccctacaaccagagcatgtactacgccggcctgcccgtggacgatttcg aggatagcgagagcaccgacaccgaggaagagttcggcaacgccatcggcggatctcacggcgg cagcagctacaccgtgtacatcgacaagaccagataatctagacgcggccgcatacagcagcaa ttggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaaatttttattttatt tttcttttcttttccgaatcggattttgtttttaatatttcaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaagggtcggcatggcatctccacctcctcgcggtccgacctgggcatccgaagg aggacgcacgtccactcggatggctaagggagagccacgtttaaaccagctccaattcgcccta tagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccct ggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaag aggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctg tagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagc gccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttcccc gtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccc caaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgc cctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactca accctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaa aaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttag gtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaa tatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagt atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgttt ttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtggg ttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgtttt ccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggc aagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcac agaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagt gataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttt tgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccat accaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactatta actggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaag ttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagc cggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatc gtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgaga taggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagat tgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatg accaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaag gatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgct accagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttc agcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaaga actctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtgg cgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcg ggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagat acctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatcc ggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtat ctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcag gggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctg gccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcc tttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgagg aagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcag ctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttag ctcactcattaggcaccccaggctttacactttatgctcccggctcgtatgttgtgtggaattg tgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaa ccctcactaaagggaacaaaagctgggtaccgggcccacgcgtaatacgactcactatag_126 04 VZV VEERep.SGPgI 1_ataggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttga catcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcagtttgaggtagaa gccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaac tgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgcag aatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacaga ttgtataagtatgcaactaagctgaagaaaaactgtaaggaaataactgataaggaattggaca agaaaatgaaggagctcgccgccgtcatgagcgaccctgacctggaaactgagactatgtgcct ccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggtt gacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggct ttgacaccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaactg ggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgttatggagcgg tcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaacaatgttctattct ctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctgt atttcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacggg tacgtcgttaaaagaatagctatcagtccaggcctgtatgggaagccttcaggctatgctgcta cgatgcaccgcgagggattcttgtgctgcaaagtgacagacacattgaacggggagagggtctc ttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatactggcaaca gatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacg gtcgcacccagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatt tgctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacgagat agacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatctatttataagc gcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcccaggat aggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaag gagccgtcacctctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggcta aggaggtgcgtgaagccgaggagttgcgcgcagctctaccacctttggcagctgatgttgagga gcccactctggaagccgatgtagacttgatgttacaagaggctggggccggctcagtggagaca cctcgtggcttgataaaggttaccagctacgatggcgaggacaagatcggctcttacgctgtgc tttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagt catagtgataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagta gtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtgccacca ttgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccacacatggaggagc gctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacctg tacgacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggcg agctggtggatcctcccttccatgaattcgcctacgagagtctgagaacacgaccagccgctcc ttaccaagtaccaaccataggggtgtatggcgtgccaggatcaggcaagtctggcatcattaaa agcgcagtcaccaaaaaagatctagtggtgagcgccaagaaagaaaactgtgcagaaattataa gggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaa tggatgcaaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtact ctcagagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaacagt gcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcacacaagtctt ccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgttttac gacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagta ccaaacctaagcaggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaat agattacaaaggcaacgaaataatgacggcagctgcctctcaagggctgacccgtaaaggtgtg tatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacccacctcagaacatgtgaacg tcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggataaa aacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagcat gatgccatcatgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaa acgtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgaccactga acaatggaacactgtggattattttgaaacggacaaagctcactcagcagagatagtattgaac caactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgttc cgttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaataa agaagtggtccgtcagctctctcgcaggtacccacaactgcctcgggcagttgccactggaaga gtctatgacatgaacactggtacactgcgcaattatgatccgcgcataaacctagtacctgtaa acagaagactgcctcatgctttagtcctccaccataatgaacacccacagagtgacttttcttc attcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggc aaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggca tcccaggtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaatacca tcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagcttgtctg catctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagcgaaa gcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcact tgaagagacggaagttctgtttgtattcattgggtacgatcgcaaggcccgtacgcacaatcct tacaagctttcatcaaccttgaccaacatttatacaggttccagactccacgaagccggatgtg caccctcatatcatgtggtgcgaggggatattgccacggccaccgaaggagtgattataaatgc tgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccggaa agcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacata tcattcatgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggc agaggcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattcca ctgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaaccatttgc tgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaat gactctcaaggaagcagtggctaggagagaagcagtggaggagatatgcatatccgacgactct tcagtgacagaacctgatgcagagctggtgagggtgcatccgaagagttctttggctggaagga agggctacagcacaagcgatggcaaaactttctcatatttggaagggaccaagtttcaccaggc ggccaaggatatagcagaaattaatgccatgtggcccgttgcaacggaggccaatgagcaggta tgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcgg aagcctccacaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagt acagcgcctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaag tatagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaagtgc ctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccggagcc atcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatgag accaggactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttgc tgtcagatggcccgacccaccaggtgctgcaagtcgaggcagacattcacgggccgccctctgt atctagctcatcctggtccattcctcatgcatccgactttgatgtggacagtttatccatactt gacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactcttacttcg caaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctcc acatcccgctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagc ctagtttccaccccgccaggcgtgaatagggtgatcactagagaggagctcgaggcgcttaccc cgtcacgcactcctagcaggtcggtctcgagaaccagcctggtctccaacccgccaggcgtaaa tagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatgacggtttgatgcg ggtgcatacatcttttcctccgacaccggtcaagggcatttacaacaaaaatcagtaaggcaaa cggtgctatccgaagtggtgttggagaggaccgaattggagatttcgtatgccccgcgcctcga ccaagaaaaagaagaattactacgcaagaaattacagttaaatcccacacctgctaacagaagc agataccagtccaggaaggtggagaacatgaaagccataacagctagacgtattctgcaaggcc tagggcattatttgaaggcagaaggaaaagtggagtgctaccgaaccctgcatcctgttccttt gtattcatctagtgtgaaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgcc atgttgaaagagaactttccgactgtggcttcttactgtattattccagagtacgatgcctatt tggacatggttgacggagcttcatgctgcttagacactgccagtttttgccctgcaaagctgcg cagctttccaaagaaacactcctatttggaacccacaatacgatcggcagtgccttcagcgatc cagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaatgtcacgcaaatga gagaattgcccgtattggattcggcggcctttaatgtggaatgcttcaagaaatatgcgtgtaa taatgaatattgggaaacgtttaaagaaaaccccatcaggcttactgaagaaaacgtggtaaat tacattaccaaattaaaaggaccaaaagctgctgctctttttgcgaagacacataatttgaata tgttgcaggacataccaatggacaggtttgtaatggacttaaagagagacgtgaaagtgactcc aggaacaaaacatactgaagaacggcccaaggtacaggtgatccaggctgccgatccgctagca acagcgtatctgtgcggaatccaccgagagctggttaggagattaaatgcggtcctgcttccga acattcatacactgtttgatatgtcggctgaagactttgacgctattatagccgagcacttcca gcctggggattgtgttctggaaactgacatcgcgtcgtttgataaaagtgaggacgacgccatg gctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttgacgctgattg aggcggctttcggcgaaatttcatcaatacatttgcccactaaaactaaatttaaattcggagc catgatgaaatctggaatgttcctcacactgtttgtgaacacagtcattaacattgtaatcgca agcagagtgttgagagaacggctaaccggatcaccatgtgcagcattcattggagatgacaata tcgtgaaaggagtcaaatcggacaaattaatggcagacaggtgcgccacctggttgaatatgga agtcaagattatagatgctgtggtgggcgagaaagcgccttatttctgtggagggtttattttg tgtgactccgtgaccggcacagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttg gcaaacctctggcagcagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtc aacacgctggaaccgagtgggtattctttcagagctgtgcaaggcagtagaatcaaggtatgaa accgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaatcattca gctacctgagaggggcccctataactctctacggctaacctgaatggactacgacatagtctag tcgagtctagtcgacgccaccatgtttctgatccagtgcctgatcagcgccgtgatcttctata ttcaagtcacaaacgccctgatctttaagggcgaccacgtgtcactgcaggtcaacagcagcct gaccagcatcctgatccccatgcagaacgacaattacaccgagatcaagggccagctggtgttc atcggcgagcagctgcccaccggcaccaattacagcggcaccctggaactgctgtacgccgata ccgtggccttctgcttcagaagcgtgcaggtcatcagatacgacggctgcccccggatcagaac cagcgccttcatcagctgccggtacaagcacagctggcactacggcaacagcaccgaccggatc agcaccgaacctgatgccggcgtgatgctgaagatcaccaagcccggcatcaacgacgccggcg tgtacgtgctgctcgtgcggctggatcacagcagaagcaccgacggcttcatcctgggcgtgaa cgtgtacaccgccggcagccaccacaacatccacggcgtgatctacaccagccccagcctgcag aacggctacagcaccagagccctgttccagcaggccagactgtgcgatctgcccgccacaccta agggcagcggcacaagcctgtttcagcacatgctggacctgagagccggcaagagcctggaaga taacccctggctgcacgaggacgtggtcaccaccgagacaaagagcgtggtcaaagagggcatc gagaaccacgtgtaccccaccgacatgagcaccctgcccgagaagtccctgaacgacccccctg agaacctgctgatcatcatccccatcgtggccagcgtgatgatcctgaccgccatggtcatcgt gatcgtgatcagcgtgaagcggcggagaatcaagaagcaccccatctaccggcccaacaccaag accagacggggcatccagaacgccacccctgagtccgacgtgatgctggaagccgccattgccc agctggccaccatcagagaggaaagcccccctcacagcgtcgtgaaccccttcgtgaagtaatc tagacgcggccgcatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcat gccgccttaaaatttttattttatttttcttttcttttccgaatcggattttgtttttaatatt tcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagggtcggcatggcatctccacctcctc gcggtccgacctgggcatccgaaggaggacgcacgtccactcggatggctaagggagagccacg tttaaaccagctccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgtttt acaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccct ttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcc tgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcg cagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttccttt ctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgat ttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggcc atcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactc ttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattt tgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaa caaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctattt gtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgct tcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttt tttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctg aagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttga gagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcg gtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatg acttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaatt atgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcgga ggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgtt gggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaat ggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaatta atagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggct ggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggg gccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggat gaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagacc aagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggt gaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcg tcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgct gcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaac tctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtag ccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcc tgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgata gttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggag cgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccg aagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgaggga gcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgag cgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcct ttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctga ttctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgacc gagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccg cgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtga gcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctc ccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgacc atgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctgggtaccgggcccac gcgtaatacgactcactatag_11797 VZV VEErep.SGPgE-SGPgI 1_ataggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttcacgttga catcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcagtttgaggtagaa gccaagcaggtcactgataatgaccatgctaatgccagagcgttttcgcatctggcttcaaaac tgatcgaaacggaggtggacccatccgacacgatccttgacattggaagtgcgcccgcccgcag aatgtattctaagcacaagtatcattgtatctgtccgatgagatgtgcggaagatccggacaga ttgtataagtatgcaactaagctgaagaaaaactgtaaggaaataactgataaggaattggaca agaaaatgaaggagctcgccgccgtcatgagcgaccctgacctggaaactgagactatgtgcct ccacgacgacgagtcgtgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggtt gacggaccgacaagtctctatcaccaagccaataagggagttagagtcgcctactggataggct ttgacaccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaactg ggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgttatggagcgg tcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaacaatgttctattct ctgttggctcgaccatctaccacgagaagagggacttactgaggagctggcacctgccgtctgt atttcacttacgtggcaagcaaaattacacatgtcggtgtgagactatagttagttgcgacggg tacgtcgttaaaagaatagctatcagtccaggcctgtatgggaagccttcaggctatgctgcta cgatgcaccgcgagggattcttgtgctgcaaagtgacagacacattgaacggggagagggtctc ttttcccgtgtgcacgtatgtgccagctacattgtgtgaccaaatgactggcatactggcaaca gatgtcagtgcggacgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacg gtcgcacccagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatt tgctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacgagat agacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatctatttataagc gcccggatacccaaaccatcatcaaagtgaacagcgatttccactcattcgtgctgcccaggat aggcagtaacacattggagatcgggctgagaacaagaatcaggaaaatgttagaggagcacaag gagccgtcacctctcattaccgccgaggacgtacaagaagctaagtgcgcagccgatgaggcta aggaggtgcgtgaagccgaggagttgcgcgcagctctaccacctttggcagctgatgttgagga gcccactctggaagccgatgtagacttgatgttacaagaggctggggccggctcagtggagaca cctcgtggcttgataaaggttaccagctacgatggcgaggacaagatcggctcttacgctgtgc tttctccgcaggctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagt catagtgataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagta gtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtgccacca ttgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccacacatggaggagc gctgaacactgatgaagaatattacaaaactgtcaagcccagcgagcacgacggcgaatacctg tacgacatcgacaggaaacagtgcgtcaagaaagaactagtcactgggctagggctcacaggcg agctggtggatcctcccttccatgaattcgcctacgagagtctgagaacacgaccagccgctcc ttaccaagtaccaaccataggggtgtatggcgtgccaggatcaggcaagtctggcatcattaaa agcgcagtcaccaaaaaagatctagtggtgagcgccaagaaagaaaactgtgcagaaattataa gggacgtcaagaaaatgaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaa tggatgcaaacaccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtact ctcagagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaacagt gcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcacacaagtctt ccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtctcaaccttgttttac gacaaaaaaatgagaacgacgaatccgaaagagactaagattgtgattgacactaccggcagta ccaaacctaagcaggacgatctcattctcacttgtttcagagggtgggtgaagcagttgcaaat agattacaaaggcaacgaaataatgacggcagctgcctctcaagggctgacccgtaaaggtgtg tatgccgttcggtacaaggtgaatgaaaatcctctgtacgcacccacctcagaacatgtgaacg tcctactgacccgcacggaggaccgcatcgtgtggaaaacactagccggcgacccatggataaa aacactgactgccaagtaccctgggaatttcactgccacgatagaggagtggcaagcagagcat gatgccatcatgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaa acgtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgaccactga acaatggaacactgtggattattttgaaacggacaaagctcactcagcagagatagtattgaac caactatgcgtgaggttctttggactcgatctggactccggtctattttctgcacccactgttc cgttatccattaggaataatcactgggataactccccgtcgcctaacatgtacgggctgaataa agaagtggtccgtcagctctctcgcaggtacccacaactgcctcgggcagttgccactggaaga gtctatgacatgaacactggtacactgcgcaattatgatccgcgcataaacctagtacctgtaa acagaagactgcctcatgctttagtcctccaccataatgaacacccacagagtgacttttcttc attcgtcagcaaattgaagggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggc aaaatggttgactggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggca tcccaggtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaatacca tcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagcttgtctg catctgaatcccggcggaacctgtgtcagcataggttatggttacgctgacagggccagcgaaa gcatcattggtgctatagcgcggcagttcaagttttcccgggtatgcaaaccgaaatcctcact tgaagagacggaagttctgtttgtattcattgggtacgatcgcaaggcccgtacgcacaatcct tacaagctttcatcaaccttgaccaacatttatacaggttccagactccacgaagccggatgtg caccctcatatcatgtggtgcgaggggatattgccacggccaccgaaggagtgattataaatgc tgctaacagcaaaggacaacctggcggaggggtgtgcggagcgctgtataagaaattcccggaa agcttcgatttacagccgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacata tcattcatgccgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggc agaggcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattcca ctgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaaccatttgc tgacagctttagacaccactgatgcagatgtagccatatactgcagggacaagaaatgggaaat gactctcaaggaagcagtggctaggagagaagcagtggaggagatatgcatatccgacgactct tcagtgacagaacctgatgcagagctggtgagggtgcatccgaagagttctttggctggaagga agggctacagcacaagcgatggcaaaactttctcatatttggaagggaccaagtttcaccaggc ggccaaggatatagcagaaattaatgccatgtggcccgttgcaacggaggccaatgagcaggta tgcatgtatatcctcggagaaagcatgagcagtattaggtcgaaatgccccgtcgaagagtcgg aagcctccacaccacctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagt acagcgcctaaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaag tatagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaagtgc ctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgagactccggagcc atcggcagagaaccaatccacagaggggacacctgaacaaccaccacttataaccgaggatgag accaggactagaacgcctgagccgatcatcatcgaagaggaagaagaggatagcataagtttgc tgtcagatggcccgacccaccaggtgctgcaagtcgaggcagacattcacgggccgccctctgt atctagctcatcctggtccattcctcatgcatccgactttgatgtggacagtttatccatactt gacaccctggagggagctagcgtgaccagcggggcaacgtcagccgagactaactcttacttcg caaagagtatggagtttctggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctcc acatcccgctccgcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagc ctagtttccaccccgccaggcgtgaatagggtgatcactagagaggagctcgaggcgcttaccc cgtcacgcactcctagcaggtcggtctcgagaaccagcctggtctccaacccgccaggcgtaaa tagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatgacggtttgatgcg ggtgcatacatcttttcctccgacaccggtcaagggcatttacaacaaaaatcagtaaggcaaa cggtgctatccgaagtggtgttggagaggaccgaattggagatttcgtatgccccgcgcctcga ccaagaaaaagaagaattactacgcaagaaattacagttaaatcccacacctgctaacagaagc agataccagtccaggaaggtggagaacatgaaagccataacagctagacgtattctgcaaggcc tagggcattatttgaaggcagaaggaaaagtggagtgctaccgaaccctgcatcctgttccttt gtattcatctagtgtgaaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgcc atgttgaaagagaactttccgactgtggcttcttactgtattattccagagtacgatgcctatt tggacatggttgacggagcttcatgctgcttagacactgccagtttttgccctgcaaagctgcg cagctttccaaagaaacactcctatttggaacccacaatacgatcggcagtgccttcagcgatc cagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaatgtcacgcaaatga gagaattgcccgtattggattcggcggcctttaatgtggaatgcttcaagaaatatgcgtgtaa taatgaatattgggaaacgtttaaagaaaaccccatcaggcttactgaagaaaacgtggtaaat tacattaccaaattaaaaggaccaaaagctgctgctctttttgcgaagacacataatttgaata tgttgcaggacataccaatggacaggtttgtaatggacttaaagagagacgtgaaagtgactcc aggaacaaaacatactgaagaacggcccaaggtacaggtgatccaggctgccgatccgctagca acagcgtatctgtgcggaatccaccgagagctggttaggagattaaatgcggtcctgcttccga acattcatacactgtttgatatgtcggctgaagactttgacgctattatagccgagcacttcca gcctggggattgtgttctggaaactgacatcgcgtcgtttgataaaagtgaggacgacgccatg gctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttgacgctgattg aggcggctttcggcgaaatttcatcaatacatttgcccactaaaactaaatttaaattcggagc catgatgaaatctggaatgttcctcacactgtttgtgaacacagtcattaacattgtaatcgca agcagagtgttgagagaacggctaaccggatcaccatgtgcagcattcattggagatgacaata tcgtgaaaggagtcaaatcggacaaattaatggcagacaggtgcgccacctggttgaatatgga agtcaagattatagatgctgtggtgggcgagaaagcgccttatttctgtggagggtttattttg tgtgactccgtgaccggcacagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttg gcaaacctctggcagcagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtc aacacgctggaaccgagtgggtattctttcagagctgtgcaaggcagtagaatcaaggtatgaa accgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaatcattca gctacctgagaggggcccctataactctctacggctaacctgaatggactacgacatagtctag tcgagtctagtcgacgccaccatgggcaccgtgaacaagcctgtcgtgggcgtgctgatgggct tcggcatcatcaccggcaccctgagaatcaccaaccctgtgcgggccagcgtgctgagatacga cgacttccacatcgacgaggacaagctggacaccaacagcgtgtacgagccctactaccacagc gaccacgccgagagcagctgggtcaacagaggcgagagcagccggaaggcctacgaccacaaca gcccctacatctggccccggaacgactacgacggcttcctggaaaacgcccacgagcaccacgg cgtgtacaatcagggcagaggcatcgacagcggcgagagactgatgcagcccacacagatgagc gcccaggaagatctgggcgacgacacaggcatccacgtgatccccaccctgaacggcgacgacc ggcacaagatcgtgaacgtggaccagcggcagtacggcgacgtgttcaagggcgacctgaaccc taagccccagggccagagactgatcgaggtgtccgtggaagagaaccaccccttcaccctgaga gcccccatccagagaatctacggcgtgcggtataccgagacttggagcttcctgcccagcctga cctgtacaggcgacgccgctcctgccatccagcacatctgcctgaagcacaccacctgtttcca ggacgtggtggtggacgtggactgcgccgagaacaccaaagaggaccagctggccgagatcagc taccggttccagggcaagaaagaggccgaccagccctggatcgtggtcaataccagcaccctgt tcgacgagctggaactggacccccccgagattgaacccggcgtgctgaaggtgctgcggaccga gaagcagtacctgggcgtgtacatctggaacatgcggggctccgacggcacctctacctacgcc accttcctggtcacatggaagggcgacgagaaaacccggaaccctacccctgccgtgacccctc agcctagaggcgccgagttccatatgtggaattaccactcccacgtgttcagcgtgggcgacac cttcagcctggccatgcatctgcagtacaagatccacgaggcccccttcgacctgctgctggaa tggctgtacgtgcccatcgaccctacctgccagcccatgcggctgtacagcacctgtctgtacc accccaacgcccctcagtgcctgagccacatgaacagcggctgcaccttcaccagccctcacct ggctcagagggtggccagcaccgtgtaccagaattgcgagcacgccgacaactacaccgcctac tgcctgggcatcagccacatggaacccagcttcggcctgatcctgcacgatggcggcaccaccc tgaagttcgtggacacacccgagagcctgagcggcctgtacgtgttcgtggtgtacttcaacgg ccacgtggaagccgtggcctacaccgtggtgtccaccgtggaccacttcgtgaacgccatcgag gaaagaggcttcccacccacagccggacagcctccagccaccaccaagcccaaagaaatcaccc ccgtgaaccccggcaccagccccctgctgagatatgctgcttggacaggcggactggccgctgt ggtgctgctgtgcctggtcatcttcctgatctgcaccgccaagcggatgagagtgaaggcctac cgggtggacaagtccccctacaaccagagcatgtactacgccggcctgcccgtggacgatttcg aggatagcgagagcaccgacaccgaggaagagttcggcaacgccatcggcggatctcacggcgg cagcagctacaccgtgtacatcgacaagaccagataatctagacgtcgcgaccacccaggatcc gcctataactctctacggctaacctgaatggactacgacatagtctagtcgacgccaccatgtt tctgatccagtgcctgatcagcgccgtgatcttctatattcaagtcacaaacgccctgatcttt aagggcgaccacgtgtcactgcaggtcaacagcagcctgaccagcatcctgatccccatgcaga acgacaattacaccgagatcaagggccagctggtgttcatcggcgagcagctgcccaccggcac caattacagcggcaccctggaactgctgtacgccgataccgtggccttctgcttcagaagcgtg caggtcatcagatacgacggctgcccccggatcagaaccagcgccttcatcagctgccggtaca agcacagctggcactacggcaacagcaccgaccggatcagcaccgaacctgatgccggcgtgat gctgaagatcaccaagcccggcatcaacgacgccggcgtgtacgtgctgctcgtgcggctggat cacagcagaagcaccgacggcttcatcctgggcgtgaacgtgtacaccgccggcagccaccaca acatccacggcgtgatctacaccagccccagcctgcagaacggctacagcaccagagccctgtt ccagcaggccagactgtgcgatctgcccgccacacctaagggcagcggcacaagcctgtttcag cacatgctggacctgagagccggcaagagcctggaagataacccctggctgcacgaggacgtgg tcaccaccgagacaaagagcgtggtcaaagagggcatcgagaaccacgtgtaccccaccgacat gagcaccctgcccgagaagtccctgaacgacccccctgagaacctgctgatcatcatccccatc gtggccagcgtgatgatcctgaccgccatggtcatcgtgatcgtgatcagcgtgaagcggcgga gaatcaagaagcaccccatctaccggcccaacaccaagaccagacggggcatccagaacgccac ccctgagtccgacgtgatgctggaagccgccattgcccagctggccaccatcagagaggaaagc ccccctcacagcgtcgtgaaccccttcgtgaagtaatctagacgcggccgcatacagcagcaat tggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaaatttttattttattt ttcttttcttttccgaatcggattttgtttttaatatttcaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaagggtcggcatggcatctccacctcctcgcggtccgacctgggcatccgaagga ggacgcacgtccactcggatggctaagggagagccacgtttaaaccagctccaattcgccctat agtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctg gcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaaga ggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgt agcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcg ccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccg tcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgacccc aaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgcc ctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaa ccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaa aatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttagg tggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaat atgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagta tgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttt tgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggt tacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttc caatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggca agagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcaca gaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtg ataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctttttt gcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccata ccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaa ctggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagt tgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagcc ggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcg tagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagat aggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagatt gatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatga ccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaagg atcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgcta ccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttca gcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaa ctctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggc gataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgg gctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagata cctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccg gtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatc tttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagg ggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgg ccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcct ttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgagga agcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagc tggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagc tcactcattaggcaccccaggctttacactttatgctcccggctcgtatgttgtgtggaattgt gagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaac cctcactaaagggaacaaaagctgggtaccgggcccacgcgtaatacgactcactatag_1377 5 VEE-based replicon encoding eGFP nsP1 ~~~~~~~~~~~~~~~~~ 1 ATAGGCGGCG CATGAGAGAA GCCCAGACCA ATTACCTACC CAAAATGGAG AAAGTTCACG nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 61 TTGACATCGA GGAAGACAGC CCATTCCTCA GAGCTTTGCA GCGGAGCTTC CCGCAGTTTG nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 121 AGGTAGAAGC CAAGCAGGTC ACTGATAATG ACCATGCTAA TGCCAGAGCG TTTTCGCATC nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 181 TGGCTTCAAA ACTGATCGAA ACGGAGGTGG ACCCATCCGA CACGATCCTT GACATTGGAA nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 241 GTGCGCCCGC CCGCAGAATG TATTCTAAGC ACAAGTATCA TTGTATCTGT CCGATGAGAT nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 301 GTGCGGAAGA TCCGGACAGA TTGTATAAGT ATGCAACTAA GCTGAAGAAA AACTGTAAGG nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 361 AAATAACTGA TAAGGAATTG GACAAGAAAA TGAAGGAGCT CGCCGCCGTC ATGAGCGACC nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 421 CTGACCTGGA AACTGAGACT ATGTGCCTCC ACGACGACGA GTCGTGTCGC TACGAAGGGC nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 481 AAGTCGCTGT TTACCAGGAT GTATACGCGG TTGACGGACC GACAAGTCTC TATCACCAAG nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 541 CCAATAAGGG AGTTAGAGTC GCCTACTGGA TAGGCTTTGA CACCACCCCT TTTATGTTTA nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 601 AGAACTTGGC TGGAGCATAT CCATCATACT CTACCAACTG GGCCGACGAA ACCGTGTTAA nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 661 CGGCTCGTAA CATAGGCCTA TGCAGCTCTG ACGTTATGGA GCGGTCACGT AGAGGGATGT nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 721 CCATTCTTAG AAAGAAGTAT TTGAAACCAT CCAACAATGT TCTATTCTCT GTTGGCTCGA nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 781 CCATCTACCA CGAGAAGAGG GACTTACTGA GGAGCTGGCA CCTGCCGTCT GTATTTCACT nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 841 TACGTGGCAA GCAAAATTAC ACATGTCGGT GTGAGACTAT AGTTAGTTGC GACGGGTACG nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 901 TCGTTAAAAG AATAGCTATC AGTCCAGGCC TGTATGGGAA GCCTTCAGGC TATGCTGCTA nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 961 CGATGCACCG CGAGGGATTC TTGTGCTGCA AAGTGACAGA CACATTGAAC GGGGAGAGGG nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1021 TCTCTTTTCC CGTGTGCACG TATGTGCCAG CTACATTGTG TGACCAAATG ACTGGCATAC nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1081 TGGCAACAGA TGTCAGTGCG GACGACGCGC AAAAACTGCT GGTTGGGCTC AACCAGCGTA nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1141 TAGTCGTCAA CGGTCGCACC CAGAGAAACA CCAATACCAT GAAAAATTAC CTTTTGCCCG nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1201 TAGTGGCCCA GGCATTTGCT AGGTGGGCAA AGGAATATAA GGAAGATCAA GAAGATGAAA nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1261 GGCCACTAGG ACTACGAGAT AGACAGTTAG TCATGGGGTG TTGTTGGGCT TTTAGAAGGC nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1321 ACAAGATAAC ATCTATTTAT AAGCGCCCGG ATACCCAAAC CATCATCAAA GTGAACAGCG nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1381 ATTTCCACTC ATTCGTGCTG CCCAGGATAG GCAGTAACAC ATTGGAGATC GGGCTGAGAA nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1441 CAAGAATCAG GAAAATGTTA GAGGAGCACA AGGAGCCGTC ACCTCTCATT ACCGCCGAGG nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1501 ACGTACAAGA AGCTAAGTGC GCAGCCGATG AGGCTAAGGA GGTGCGTGAA GCCGAGGAGT nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1561 TGCGCGCAGC TCTACCACCT TTGGCAGCTG ATGTTGAGGA GCCCACTCTG GAAGCCGATG nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1621 TAGACTTGAT GTTACAAGAG GCTGGGGCCG GCTCAGTGGA GACACCTCGT GGCTTGATAA nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1681 AGGTTACCAG CTACGATGGC GAGGACAAGA TCGGCTCTTA CGCTGTGCTT TCTCCGCAGG nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1741 CTGTACTCAA GAGTGAAAAA TTATCTTGCA TCCACCCTCT CGCTGAACAA GTCATAGTGA nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1801 TAACACACTC TGGCCGAAAA GGGCGTTATG CCGTGGAACC ATACCATGGT AAAGTAGTGG nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1861 TGCCAGAGGG ACATGCAATA CCCGTCCAGG ACTTTCAAGC TCTGAGTGAA AGTGCCACCA nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1921 TTGTGTACAA CGAACGTGAG TTCGTAAACA GGTACCTGCA CCATATTGCC ACACATGGAG nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1981 GAGCGCTGAA CACTGATGAA GAATATTACA AAACTGTCAA GCCCAGCGAG CACGACGGCG nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2041 AATACCTGTA CGACATCGAC AGGAAACAGT GCGTCAAGAA AGAACTAGTC ACTGGGCTAG nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2101 GGCTCACAGG CGAGCTGGTG GATCCTCCCT TCCATGAATT CGCCTACGAG AGTCTGAGAA nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2161 CACGACCAGC CGCTCCTTAC CAAGTACCAA CCATAGGGGT GTATGGCGTG CCAGGATCAG nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2221 GCAAGTCTGG CATCATTAAA AGCGCAGTCA CCAAAAAAGA TCTAGTGGTG AGCGCCAAGA nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2281 AAGAAAACTG TGCAGAAATT ATAAGGGACG TCAAGAAAAT GAAAGGGCTG GACGTCAATG nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2341 CCAGAACTGT GGACTCAGTG CTCTTGAATG GATGCAAACA CCCCGTAGAG ACCCTGTATA nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2401 TTGACGAAGC TTTTGCTTGT CATGCAGGTA CTCTCAGAGC GCTCATAGCC ATTATAAGAC nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2461 CTAAAAAGGC AGTGCTCTGC GGGGATCCCA AACAGTGCGG TTTTTTTAAC ATGATGTGCC nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2521 TGAAAGTGCA TTTTAACCAC GAGATTTGCA CACAAGTCTT CCACAAAAGC ATCTCTCGCC nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2581 GTTGCACTAA ATCTGTGACT TCGGTCGTCT CAACCTTGTT TTACGACAAA AAAATGAGAA nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2641 CGACGAATCC GAAAGAGACT AAGATTGTGA TTGACACTAC CGGCAGTACC AAACCTAAGC nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2701 AGGACGATCT CATTCTCACT TGTTTCAGAG GGTGGGTGAA GCAGTTGCAA ATAGATTACA nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2761 AAGGCAACGA AATAATGACG GCAGCTGCCT CTCAAGGGCT GACCCGTAAA GGTGTGTATG nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2821 CCGTTCGGTA CAAGGTGAAT GAAAATCCTC TGTACGCACC CACCTCAGAA CATGTGAACG nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2881 TCCTACTGAC CCGCACGGAG GACCGCATCG TGTGGAAAAC ACTAGCCGGC GACCCATGGA nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2941 TAAAAACACT GACTGCCAAG TACCCTGGGA ATTTCACTGC CACGATAGAG GAGTGGCAAG nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3001 CAGAGCATGA TGCCATCATG AGGCACATCT TGGAGAGACC GGACCCTACC GACGTCTTCC nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3061 AGAATAAGGC AAACGTGTGT TGGGCCAAGG CTTTAGTGCC GGTGCTGAAG ACCGCTGGCA nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3121 TAGACATGAC CACTGAACAA TGGAACACTG TGGATTATTT TGAAACGGAC AAAGCTCACT nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3181 CAGCAGAGAT AGTATTGAAC CAACTATGCG TGAGGTTCTT TGGACTCGAT CTGGACTCCG nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3241 GTCTATTTTC TGCACCCACT GTTCCGTTAT CCATTAGGAA TAATCACTGG GATAACTCCC nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3301 CGTCGCCTAA CATGTACGGG CTGAATAAAG AAGTGGTCCG TCAGCTCTCT CGCAGGTACC nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3361 CACAACTGCC TCGGGCAGTT GCCACTGGAA GAGTCTATGA CATGAACACT GGTACACTGC nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3421 GCAATTATGA TCCGCGCATA AACCTAGTAC CTGTAAACAG AAGACTGCCT CATGCTTTAG nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3481 TCCTCCACCA TAATGAACAC CCACAGAGTG ACTTTTCTTC ATTCGTCAGC AAATTGAAGG nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3541 GCAGAACTGT CCTGGTGGTC GGGGAAAAGT TGTCCGTCCC AGGCAAAATG GTTGACTGGT nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3601 TGTCAGACCG GCCTGAGGCT ACCTTCAGAG CTCGGCTGGA TTTAGGCATC CCAGGTGATG nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3661 TGCCCAAATA TGACATAATA TTTGTTAATG TGAGGACCCC ATATAAATAC CATCACTATC nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3721 AGCAGTGTGA AGACCATGCC ATTAAGCTTA GCATGTTGAC CAAGAAAGCT TGTCTGCATC nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3781 TGAATCCCGG CGGAACCTGT GTCAGCATAG GTTATGGTTA CGCTGACAGG GCCAGCGAAA nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3841 GCATCATTGG TGCTATAGCG CGGCAGTTCA AGTTTTCCCG GGTATGCAAA CCGAAATCCT nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3901 CACTTGAAGA GACGGAAGTT CTGTTTGTAT TCATTGGGTA CGATCGCAAG GCCCGTACGC nsP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3961 ACAATCCTTA CAAGCTTTCA TCAACCTTGA CCAACATTTA TACAGGTTCC AGACTCCACG nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ nsP2 ~~~~~~~~~~~~ 4021 AAGCCGGATG TGCACCCTCA TATCATGTGG TGCGAGGGGA TATTGCCACG GCCACCGAAG nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4081 GAGTGATTAT AAATGCTGCT AACAGCAAAG GACAACCTGG CGGAGGGGTG TGCGGAGCGC nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4141 TGTATAAGAA ATTCCCGGAA AGCTTCGATT TACAGCCGAT CGAAGTAGGA AAAGCGCGAC nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4201 TGGTCAAAGG TGCAGCTAAA CATATCATTC ATGCCGTAGG ACCAAACTTC AACAAAGTTT nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4261 CGGAGGTTGA AGGTGACAAA CAGTTGGCAG AGGCTTATGA GTCCATCGCT AAGATTGTCA nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4321 ACGATAACAA TTACAAGTCA GTAGCGATTC CACTGTTGTC CACCGGCATC TTTTCCGGGA nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4381 ACAAAGATCG ACTAACCCAA TCATTGAACC ATTTGCTGAC AGCTTTAGAC ACCACTGATG nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4441 CAGATGTAGC CATATACTGC AGGGACAAGA AATGGGAAAT GACTCTCAAG GAAGCAGTGG nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4501 CTAGGAGAGA AGCAGTGGAG GAGATATGCA TATCCGACGA CTCTTCAGTG ACAGAACCTG nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4561 ATGCAGAGCT GGTGAGGGTG CATCCGAAGA GTTCTTTGGC TGGAAGGAAG GGCTACAGCA nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4621 CAAGCGATGG CAAAACTTTC TCATATTTGG AAGGGACCAA GTTTCACCAG GCGGCCAAGG nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4681 ATATAGCAGA AATTAATGCC ATGTGGCCCG TTGCAACGGA GGCCAATGAG CAGGTATGCA nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4741 TGTATATCCT CGGAGAAAGC ATGAGCAGTA TTAGGTCGAA ATGCCCCGTC GAAGAGTCGG nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4801 AAGCCTCCAC ACCACCTAGC ACGCTGCCTT GCTTGTGCAT CCATGCCATG ACTCCAGAAA nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4861 GAGTACAGCG CCTAAAAGCC TCACGTCCAG AACAAATTAC TGTGTGCTCA TCCTTTCCAT nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4921 TGCCGAAGTA TAGAATCACT GGTGTGCAGA AGATCCAATG CTCCCAGCCT ATATTGTTCT nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4981 CACCGAAAGT GCCTGCGTAT ATTCATCCAA GGAAGTATCT CGTGGAAACA CCACCGGTAG nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5041 ACGAGACTCC GGAGCCATCG GCAGAGAACC AATCCACAGA GGGGACACCT GAACAACCAC nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5101 CACTTATAAC CGAGGATGAG ACCAGGACTA GAACGCCTGA GCCGATCATC ATCGAAGAGG nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5161 AAGAAGAGGA TAGCATAAGT TTGCTGTCAG ATGGCCCGAC CCACCAGGTG CTGCAAGTCG nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5221 AGGCAGACAT TCACGGGCCG CCCTCTGTAT CTAGCTCATC CTGGTCCATT CCTCATGCAT nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5281 CCGACTTTGA TGTGGACAGT TTATCCATAC TTGACACCCT GGAGGGAGCT AGCGTGACCA nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5341 GCGGGGCAAC GTCAGCCGAG ACTAACTCTT ACTTCGCAAA GAGTATGGAG TTTCTGGCGC nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5401 GACCGGTGCC TGCGCCTCGA ACAGTATTCA GGAACCCTCC ACATCCCGCT CCGCGCACAA nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5461 GAACACCGTC ACTTGCACCC AGCAGGGCCT GCTCGAGAAC CAGCCTAGTT TCCACCCCGC nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5521 CAGGCGTGAA TAGGGTGATC ACTAGAGAGG AGCTCGAGGC GCTTACCCCG TCACGCACTC nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5581 CTAGCAGGTC GGTCTCGAGA ACCAGCCTGG TCTCCAACCC GCCAGGCGTA AATAGGGTGA nsP4 ~~~~~~~~~~~~~~~~~~~~ nsP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5641 TTACAAGAGA GGAGTTTGAG GCGTTCGTAG CACAACAACA ATGACGGTTT GATGCGGGTG nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5701 CATACATCTT TTCCTCCGAC ACCGGTCAAG GGCATTTACA ACAAAAATCA GTAAGGCAAA nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5761 CGGTGCTATC CGAAGTGGTG TTGGAGAGGA CCGAATTGGA GATTTCGTAT GCCCCGCGCC nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5821 TCGACCAAGA AAAAGAAGAA TTACTACGCA AGAAATTACA GTTAAATCCC ACACCTGCTA nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5881 ACAGAAGCAG ATACCAGTCC AGGAAGGTGG AGAACATGAA AGCCATAACA GCTAGACGTA nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 5941 TTCTGCAAGG CCTAGGGCAT TATTTGAAGG CAGAAGGAAA AGTGGAGTGC TACCGAACCC nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6001 TGCATCCTGT TCCTTTGTAT TCATCTAGTG TGAACCGTGC CTTTTCAAGC CCCAAGGTCG nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6061 CAGTGGAAGC CTGTAACGCC ATGTTGAAAG AGAACTTTCC GACTGTGGCT TCTTACTGTA nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6121 TTATTCCAGA GTACGATGCC TATTTGGACA TGGTTGACGG AGCTTCATGC TGCTTAGACA nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6181 CTGCCAGTTT TTGCCCTGCA AAGCTGCGCA GCTTTCCAAA GAAACACTCC TATTTGGAAC nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6241 CCACAATACG ATCGGCAGTG CCTTCAGCGA TCCAGAACAC GCTCCAGAAC GTCCTGGCAG nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6301 CTGCCACAAA AAGAAATTGC AATGTCACGC AAATGAGAGA ATTGCCCGTA TTGGATTCGG nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6361 CGGCCTTTAA TGTGGAATGC TTCAAGAAAT ATGCGTGTAA TAATGAATAT TGGGAAACGT nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6421 TTAAAGAAAA CCCCATCAGG CTTACTGAAG AAAACGTGGT AAATTACATT ACCAAATTAA nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6481 AAGGACCAAA AGCTGCTGCT CTTTTTGCGA AGACACATAA TTTGAATATG TTGCAGGACA nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6541 TACCAATGGA CAGGTTTGTA ATGGACTTAA AGAGAGACGT GAAAGTGACT CCAGGAACAA nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6601 AACATACTGA AGAACGGCCC AAGGTACAGG TGATCCAGGC TGCCGATCCG CTAGCAACAG nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6661 CGTATCTGTG CGGAATCCAC CGAGAGCTGG TTAGGAGATT AAATGCGGTC CTGCTTCCGA nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6721 ACATTCATAC ACTGTTTGAT ATGTCGGCTG AAGACTTTGA CGCTATTATA GCCGAGCACT nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6781 TCCAGCCTGG GGATTGTGTT CTGGAAACTG ACATCGCGTC GTTTGATAAA AGTGAGGACG nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6841 ACGCCATGGC TCTGACCGCG TTAATGATTC TGGAAGACTT AGGTGTGGAC GCAGAGCTGT nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6901 TGACGCTGAT TGAGGCGGCT TTCGGCGAAA TTTCATCAAT ACATTTGCCC ACTAAAACTA nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 6961 AATTTAAATT CGGAGCCATG ATGAAATCTG GAATGTTCCT CACACTGTTT GTGAACACAG nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7021 TCATTAACAT TGTAATCGCA AGCAGAGTGT TGAGAGAACG GCTAACCGGA TCACCATGTG nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7081 CAGCATTCAT TGGAGATGAC AATATCGTGA AAGGAGTCAA ATCGGACAAA TTAATGGCAG nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7141 ACAGGTGCGC CACCTGGTTG AATATGGAAG TCAAGATTAT AGATGCTGTG GTGGGCGAGA nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7201 AAGCGCCTTA TTTCTGTGGA GGGTTTATTT TGTGTGACTC CGTGACCGGC ACAGCGTGCC nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7261 GTGTGGCAGA CCCCCTAAAA AGGCTGTTTA AGCTTGGCAA ACCTCTGGCA GCAGACGATG nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7321 AACATGATGA TGACAGGAGA AGGGCATTGC ATGAAGAGTC AACACGCTGG AACCGAGTGG nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7381 GTATTCTTTC AGAGCTGTGC AAGGCAGTAG AATCAAGGTA TGAAACCGTA GGAACTTCCA nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7441 TCATAGTTAT GGCCATGACT ACTCTAGCTA GCAGTGTTAA ATCATTCAGC TACCTGAGAG subgenomic promoter ~~~~~~~~~~~~~~~~~~~~~~~~~~ nsP4 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7501 GGGCCCCTAT AACTCTCTAC GGCTAACCTG AATGGACTAC GACATAGTCT AGTCGACGCC eGFP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7561 ACCATGGTGA GCAAGGGCGA GGAGCTGTTC ACCGGGGTGG TGCCCATCCT GGTCGAGCTG eGFP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7621 GACGGCGACG TAAACGGCCA CAAGTTCAGC GTGTCCGGCG AGGGCGAGGG CGATGCCACC eGFP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7681 TACGGCAAGC TGACCCTGAA GTTCATCTGC ACCACCGGCA AGCTGCCCGT GCCCTGGCCC eGFP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7741 ACCCTCGTGA CCACCCTGAC CTACGGCGTG CAGTGCTTCA GCCGCTACCC CGACCACATG eGFP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7801 AAGCAGCACG ACTTCTTCAA GTCCGCCATG CCCGAAGGCT ACGTCCAGGA GCGCACCATC eGFP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7861 TTCTTCAAGG ACGACGGCAA CTACAAGACC CGCGCCGAGG TGAAGTTCGA GGGCGACACC eGFP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7921 CTGGTGAACC GCATCGAGCT GAAGGGCATC GACTTCAAGG AGGACGGCAA CATCCTGGGG eGFP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7981 CACAAGCTGG AGTACAACTA CAACAGCCAC AACGTCTATA TCATGGCCGA CAAGCAGAAG eGFP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 8041 AACGGCATCA AGGTGAACTT CAAGATCCGC CACAACATCG AGGACGGCAG CGTGCAGCTC eGFP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 8101 GCCGACCACT ACCAGCAGAA CACCCCCATC GGCGACGGCC CCGTGCTGCT GCCCGACAAC eGFP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 8161 CACTACCTGA GCACCCAGTC CGCCCTGAGC AAAGACCCCA ACGAGAAGCG CGATCACATG eGFP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 8221 GTCCTGCTGG AGTTCGTGAC CGCCGCCGGG ATCACTCTCG GCATGGACGA GCTGTACAAG eGFP 3'UTR ~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~ 8281 TGATAATCTA GACGGCGCGC CCACCCAGCG GCCGCATACA GCAGCAATTG GCAAGCTGCT 3'UTR ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 8341 TACATAGAAC TCGCGGCGAT TGGCATGCCG CCTTAAAATT TTTATTTTAT TTTTCTTTTC 3'UTR ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 8401 TTTTCCGAAT CGGATTTTGT TTTTAATATT TCAAAAAAAA AAAAAAAAAA AAAAAAAAAA HDV ribozyme ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 8461 AAAAAAAGGG TCGGCATGGC ATCTCCACCT CCTCGCGGTC CGACCTGGGC ATCCGAAGGA HDV ribozyme ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 8521 GGACGCACGT CCACTCGGAT GGCTAAGGGA GAGCCACGTT TAAACCAGCT CCAATTCGCC 8581 CTATAGTGAG TCGTATTACG CGCGCTCACT GGCCGTCGTT TTACAACGTC GTGACTGGGA 8641 AAACCCTGGC GTTACCCAAC TTAATCGCCT TGCAGCACAT CCCCCTTTCG CCAGCTGGCG 8701 TAATAGCGAA GAGGCCCGCA CCGATCGCCC TTCCCAACAG TTGCGCAGCC TGAATGGCGA 8761 ATGGGACGCG CCCTGTAGCG GCGCATTAAG CGCGGCGGGT GTGGTGGTTA CGCGCAGCGT 8821 GACCGCTACA CTTGCCAGCG CCCTAGCGCC CGCTCCTTTC GCTTTCTTCC CTTCCTTTCT 8881 CGCCACGTTC GCCGGCTTTC CCCGTCAAGC TCTAAATCGG GGGCTCCCTT TAGGGTTCCG 8941 ATTTAGTGCT TTACGGCACC TCGACCCCAA AAAACTTGAT TAGGGTGATG GTTCACGTAG 9001 TGGGCCATCG CCCTGATAGA CGGTTTTTCG CCCTTTGACG TTGGAGTCCA CGTTCTTTAA 9061 TAGTGGACTC TTGTTCCAAA CTGGAACAAC ACTCAACCCT ATCTCGGTCT ATTCTTTTGA 9121 TTTATAAGGG ATTTTGCCGA TTTCGGCCTA TTGGTTAAAA AATGAGCTGA TTTAACAAAA 9181 ATTTAACGCG AATTTTAACA AAATATTAAC GCTTACAATT TAGGTGGCAC TTTTCGGGGA 9241 AATGTGCGCG GAACCCCTAT TTGTTTATTT TTCTAAATAC ATTCAAATAT GTATCCGCTC bla ~~~~~~~~~ 9301 ATGAGACAAT AACCCTGATA AATGCTTCAA TAATATTGAA AAAGGAAGAG TATGAGTATT bla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 9361 CAACATTTCC GTGTCGCCCT TATTCCCTTT TTTGCGGCAT TTTGCCTTCC TGTTTTTGCT bla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 9421 CACCCAGAAA CGCTGGTGAA AGTAAAAGAT GCTGAAGATC AGTTGGGTGC ACGAGTGGGT bla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 9481 TACATCGAAC TGGATCTCAA CAGCGGTAAG ATCCTTGAGA GTTTTCGCCC CGAAGAACGT bla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 9541 TTTCCAATGA TGAGCACTTT TAAAGTTCTG CTATGTGGCG CGGTATTATC CCGTATTGAC bla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 9601 GCCGGGCAAG AGCAACTCGG TCGCCGCATA CACTATTCTC AGAATGACTT GGTTGAGTAC bla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 9661 TCACCAGTCA CAGAAAAGCA TCTTACGGAT GGCATGACAG TAAGAGAATT ATGCAGTGCT bla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 9721 GCCATAACCA TGAGTGATAA CACTGCGGCC AACTTACTTC TGACAACGAT CGGAGGACCG bla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 9781 AAGGAGCTAA CCGCTTTTTT GCACAACATG GGGGATCATG TAACTCGCCT TGATCGTTGG bla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 9841 GAACCGGAGC TGAATGAAGC CATACCAAAC GACGAGCGTG ACACCACGAT GCCTGTAGCA bla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 9901 ATGGCAACAA CGTTGCGCAA ACTATTAACT GGCGAACTAC TTACTCTAGC TTCCCGGCAA bla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 9961 CAATTAATAG ACTGGATGGA GGCGGATAAA GTTGCAGGAC CACTTCTGCG CTCGGCCCTT bla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10021 CCGGCTGGCT GGTTTATTGC TGATAAATCT GGAGCCGGTG AGCGTGGGTC TCGCGGTATC bla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10081 ATTGCAGCAC TGGGGCCAGA TGGTAAGCCC TCCCGTATCG TAGTTATCTA CACGACGGGG bla ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10141 AGTCAGGCAA CTATGGATGA ACGAAATAGA CAGATCGCTG AGATAGGTGC CTCACTGATT bla ~~~~~~~~~ 10201 AAGCATTGGT AACTGTCAGA CCAAGTTTAC TCATATATAC TTTAGATTGA TTTAAAACTT 10261 CATTTTTAAT TTAAAAGGAT CTAGGTGAAG ATCCTTTTTG ATAATCTCAT GACCAAAATC 10321 CCTTAACGTG AGTTTTCGTT CCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT 10381 TCTTGAGATC CTTTTTTTCT GCGCGTAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA 10441 CCAGCGGTGG TTTGTTTGCC GGATCAAGAG CTACCAACTC TTTTTCCGAA GGTAACTGGC 10501 TTCAGCAGAG CGCAGATACC AAATACTGTT CTTCTAGTGT AGCCGTAGTT AGGCCACCAC 10561 TTCAAGAACT CTGTAGCACC GCCTACATAC CTCGCTCTGC TAATCCTGTT ACCAGTGGCT 10621 GCTGCCAGTG GCGATAAGTC GTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT 10681 AAGGCGCAGC GGTCGGGCTG AACGGGGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG 10741 ACCTACACCG AACTGAGATA CCTACAGCGT GAGCTATGAG AAAGCGCCAC GCTTCCCGAA 10801 GGGAGAAAGG CGGACAGGTA TCCGGTAAGC GGCAGGGTCG GAACAGGAGA GCGCACGAGG 10861 GAGCTTCCAG GGGGAAACGC CTGGTATCTT TATAGTCCTG TCGGGTTTCG CCACCTCTGA 10921 CTTGAGCGTC GATTTTTGTG ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC 10981 AACGCGGCCT TTTTACGGTT CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT 11041 GCGTTATCCC CTGATTCTGT GGATAACCGT ATTACCGCCT TTGAGTGAGC TGATACCGCT 11101 CGCCGCAGCC GAACGACCGA GCGCAGCGAG TCAGTGAGCG AGGAAGCGGA AGAGCGCCCA 11161 ATACGCAAAC CGCCTCTCCC CGCGCGTTGG CCGATTCATT AATGCAGCTG GCACGACAGG 11221 TTTCCCGACT GGAAAGCGGG CAGTGAGCGC AACGCAATTA ATGTGAGTTA GCTCACTCAT 11281 TAGGCACCCC AGGCTTTACA CTTTATGCTC CCGGCTCGTA TGTTGTGTGG AATTGTGAGC 11341 GGATAACAAT TTCACACAGG AAACAGCTAT GACCATGATT ACGCCAAGCG CGCAATTAAC 11401 CCTCACTAAA GGGAACAAAA GCTGGGTACC GGGCCCACGC GTAATACGAC TCACTATAG VEE cap helper 5'UTR ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ nsP1 ~~~~~~~~~~~~~~~~~ 1 ATAGGCGGCG CATGAGAGAA GCCCAGACCA ATTACCTACC CAAATAGGAG AAAGTTCACG nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 61 TTGACATCGA GGAAGACAGC CCATTCCTCA GAGCTTTGCA GCGGAGCTTC CCGCAGTTTG nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 121 AGGTAGAAGC CAAGCAGGTC ACTGATAATG ACCATGCTAA TGCCAGAGCG TTTTCGCATC nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 181 TGGCTTCAAA ACTGATCGAA ACGGAGGTGG ACCCATCCGA CACGATCCTT GACATTGGAC VEECAP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 241 GGACCGACCA TGTTCCCGTT CCAGCCAATG TATCCGATGC AGCCAATGCC CTATCGCAAC VEECAP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 301 CCGTTCGCGG CCCCGCGCAG GCCCTGGTTC CCCAGAACCG ACCCTTTTCT GGCGATGCAG VEECAP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 361 GTGCAGGAAT TAACCCGCTC GATGGCTAAC CTGACGTTCA AGCAACGCCG GGACGCGCCA VEECAP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 421 CCTGAGGGGC CATCCGCTAA GAAACCGAAG AAGGAGGCCT CGCAAAAACA GAAAGGGGGA VEECAP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 481 GGCCAAGGGA AGAAGAAGAA GAACCAAGGG AAGAAGAAGG CTAAGACAGG GCCGCCTAAT VEECAP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 541 CCGAAGGCAC AGAATGGAAA CAAGAAGAAG ACCAACAAGA AACCAGGCAA GAGACAGCGC VEECAP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 601 ATGGTCATGA AATTGGAATC TGACAAGACG TTCCCAATCA TGTTGGAAGG GAAGATAAAC VEECAP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ H152G ~~~ 661 GGCTACGCTT GTGTGGTCGG AGGGAAGTTA TTCAGGCCGA TGGGTGTGGA AGGCAAGATC VEECAP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 721 GACAACGACG TTCTGGCCGC GCTTAAGACG AAGAAAGCAT CCAAATACGA TCTTGAGTAT VEECAP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 781 GCAGATGTGC CACAGAACAT GCGGGCCGAT ACATTCAAAT ACACCCATGA GAAACCCCAA VEECAP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 841 GGCTATTACA GCTGGCATCA TGGAGCAGTC CAATATGAAA ATGGGCGTTT CACGGTGCCG VEECAP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 901 AAAGGAGTTG GGGCCAAGGG AGACAGCGGA CGACCCATTC TGGATAACCA GGGACGGGTG VEECAP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 961 GTCGCTATTG TGCTGGGAGG TGTGAATGAA GGATCTAGGA CAGCCCTTTC AGTCGTCATG VEECAP ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1021 TGGAACGAGA AGGGAGTTAC CGTGAAGTAT ACTCCGGAGA ACTGCGAGCA ATGGTAATAG VEECAP 3'UTR ~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1081 TAAGCGGCCG CATACAGCAG CAATTGGCAA GCTGCTTACA TAGAACTCGC GGCGATTGGC 3'UTR ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1141 ATGCCGCCTT AAAATTTTTA TTTTATTTTT CTTTTCTTTT CCGAATCGGA TTTTGTTTTT 3'UTR HDV ribozyme ~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 1201 AATATTTCAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAGGGTCGG CATGGCATCT HDV ribozyme ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1261 CCACCTCCTC GCGGTCCGAC CTGGGCATCC GAAGGAGGAC GCACGTCCAC TCGGATGGCT HDV ribozyme ~~~~~~~~~~~~~~ 1321 AAGGGAGAGC CACGTTTAAA CACGTGATAT CTGGCCTCAT GGGCCTTCCT TTCACTGCCC 1381 GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAC ATGGTCATAG CTGTTTCCTT 1441 GCGTATTGGG CGCTCTCCGC TTCCTCGCTC ACTGACTCGC TGCGCTCGGT CGTTCGGGTA colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1501 AAGCCTGGGG TGCCTAATGA GCAAAAGGCC AGCAAAAGGC CAGGAACCGT AAAAAGGCCG colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1561 CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1621 CAAGTCAGAG GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1681 GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1741 TCCCTTCGGG AAGCGTGGCG CTTTCTCATA GCTCACGCTG TAGGTATCTC AGTTCGGTGT colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1801 AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1861 CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1921 CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1981 TGAAGTGGTG GCCTAACTAC GGCTACACTA GAAGAACAGT ATTTGGTATC TGCGCTCTGC colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2041 TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2101 CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2161 AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT 2221 AAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA 2281 AATGAAGTTT TAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTATTAGA ~~~ KanR 2341 AAAATTCATC CAGCAGACGA TAAAACGCAA TACGCTGGCT ATCCGGTGCC GCAATGCCAT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 2401 ACAGCACCAG AAAACGATCC GCCCATTCGC CGCCCAGTTC TTCCGCAATA TCACGGGTGG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 2461 CCAGCGCAAT ATCCTGATAA CGATCCGCCA CGCCCAGACG GCCGCAATCA ATAAAGCCGC ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 2521 TAAAACGGCC ATTTTCCACC ATAATGTTCG GCAGGCACGC ATCACCATGG GTCACCACCA ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 2581 GATCTTCGCC ATCCGGCATG CTCGCTTTCA GACGCGCAAA CAGCTCTGCC GGTGCCAGGC ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 2641 CCTGATGTTC TTCATCCAGA TCATCCTGAT CCACCAGGCC CGCTTCCATA CGGGTACGCG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 2701 CACGTTCAAT ACGATGTTTC GCCTGATGAT CAAACGGACA GGTCGCCGGG TCCAGGGTAT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 2761 GCAGACGACG CATGGCATCC GCCATAATGC TCACTTTTTC TGCCGGCGCC AGATGGCTAG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 2821 ACAGCAGATC CTGACCCGGC ACTTCGCCCA GCAGCAGCCA ATCACGGCCC GCTTCGGTCA ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 2881 CCACATCCAG CACCGCCGCA CACGGAACAC CGGTGGTGGC CAGCCAGCTC AGACGCGCCG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 2941 CTTCATCCTG CAGCTCGTTC AGCGCACCGC TCAGATCGGT TTTCACAAAC AGCACCGGAC ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 3001 GACCCTGCGC GCTCAGACGA AACACCGCCG CATCAGAGCA GCCAATGGTC TGCTGCGCCC ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 3061 AATCATAGCC AAACAGACGT TCCACCCACG CTGCCGGGCT ACCCGCATGC AGGCCATCCT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 3121 GTTCAATCAT ACTCTTCCTT TTTCAATATT ATTGAAGCAT TTATCAGGGT TATTGTCTCA ~~~~~~~~~~~ KanR 3181 TGAGCGGATA CATATTTGAA TGTATTTAGA AAAATAAACA AATAGGGGTT CCGCGCACAT 3241 TTCCCCGAAA AGTGCCACCT AAATTGTAAG CGTTAATATT TTGTTAAAAT TCGCGTTAAA 3301 TTTTTGTTAA ATCAGCTCAT TTTTTAACCA ATAGGCCGAA ATCGGCAAAA TCCCTTATAA 3361 ATCAAAAGAA TAGACCGAGA TAGGGTTGAG TGGCCGCTAC AGGGCGCTCC CATTCGCCAT 3421 TCAGGCTGCG CAACTGTTGG GAAGGGCGTT TCGGTGCGGG CCTCTTCGCT ATTACGCCAG 3481 CTGGCGAAAG GGGGATGTGC TGCAAGGCGA TTAAGTTGGG TAACGCCAGG GTTTTCCCAG T7 promoter ~~~~~~~~~~~~~~~~~~~~ 3541 TCACACGCGT AATACGACTC ACTATAG VEE gly helper 5'UTR ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ nsP1 ~~~~~~~~~~~~~~~~~ 1 ATAGGCGGCG CATGAGAGAA GCCCAGACCA ATTACCTACC CAAATAGGAG AAAGTTCACG nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 61 TTGACATCGA GGAAGACAGC CCATTCCTCA GAGCTTTGCA GCGGAGCTTC CCGCAGTTTG nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 121 AGGTAGAAGC CAAGCAGGTC ACTGATAATG ACCATGCTAA TGCCAGAGCG TTTTCGCATC nsP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 181 TGGCTTCAAA ACTGATCGAA ACGGAGGTGG ACCCATCCGA CACGATCCTT GACATTGGAC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 241 GGACCGACCA TGTCACTAGT GACCACCATG TGTCTGCTCG CCAATGTGAC GTTCCCATGT VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 301 GCTCAACCAC CAATTTGCTA CGACAGAAAA CCAGCAGAGA CTTTGGCCAT GCTCAGCGTT VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 361 AACGTTGACA ACCCGGGCTA CGATGAGCTG CTGGAAGCAG CTGTTAAGTG CCCCGGAAGG VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 421 AAAAGGAGAT CCACCGAGGA GCTGTTTAAT GAGTATAAGC TAACGCGCCC TTACATGGCC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 481 AGATGCATCA GATGTGCAGT TGGGAGCTGC CATAGTCCAA TAGCAATCGA GGCAGTAAAG VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 541 AGCGACGGGC ACGACGGTTA TGTTAGACTT CAGACTTCCT CGCAGTATGG CCTGGATTCC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 601 TCCGGCAACT TAAAGGGCAG GACCATGCGG TATGACATGC ACGGGACCAT TAAAGAGATA VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 661 CCACTACATC AAGTGTCACT CTATACATCT CGCCCGTGTC ACATTGTGGA TGGGCACGGT VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 721 TATTTCCTGC TTGCCAGGTG CCCGGCAGGG GACTCCATCA CCATGGAATT TAAGAAAGAT VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 781 TCCGTCAGAC ACTCCTGCTC GGTGCCGTAT GAAGTGAAAT TTAATCCTGT AGGCAGAGAA VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 841 CTCTATACTC ATCCCCCAGA ACACGGAGTA GAGCAAGCGT GCCAAGTCTA CGCACATGAT VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 901 GCACAGAACA GAGGAGCTTA TGTCGAGATG CACCTCCCGG GCTCAGAAGT GGACAGCAGT VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 961 TTGGTTTCCT TGAGCGGCAG TTCAGTCACC GTGACACCTC CTGATGGGAC TAGCGCCCTG VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1021 GTGGAATGCG AGTGTGGCGG CACAAAGATC TCCGAGACCA TCAACAAGAC AAAACAGTTC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1081 AGCCAGTGCA CAAAGAAGGA GCAGTGCAGA GCATATCGGC TGCAGAACGA TAAGTGGGTG VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1141 TATAATTCTG ACAAACTGCC CAAAGCAGCG GGAGCCACCT TAAAAGGAAA ACTGCATGTC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1201 CCATTCTTGC TGGCAGACGG CAAATGCACC GTGCCTCTAG CACCAGAACC TATGATAACC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1261 TTCGGTTTCA GATCAGTGTC ACTGAAACTG CACCCTAAGA ATCCCACATA TCTAATCACC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1321 CGCCAACTTG CTGATGAGCC TCACTACACG CACGAGCTCA TATCTGAACC AGCTGTTAGG VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1381 AATTTTACCG TCACCGAAAA AGGGTGGGAG TTTGTATGGG GAAACCACCC GCCGAAAAGG VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1441 TTTTGGGCAC AGGAAACAGC ACCCGGAAAT CCACATGGGC TACCGCACGA GGTGATAACT VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1501 CATTATTACC ACAGATACCC TATGTCCACC ATCCTGGGTT TGTCAATTTG TGCCGCCATT VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1561 GCAACCGTTT CCGTTGCAGC GTCTACCTGG CTGTTTTGCA GATCTAGAGT TGCGTGCCTA VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1621 ACTCCTTACC GGCTAACACC TAACGCTAGG ATACCATTTT GTCTGGCTGT GCTTTGCTGC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1681 GCCCGCACTG CCCGGGCCGA GACCACCTGG GAGTCCTTGG ATCACCTATG GAACAATAAC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1741 CAACAGATGT TCTGGATTCA ATTGCTGATC CCTCTGGCCG CCTTGATCGT AGTGACTCGC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1801 CTGCTCAGGT GCGTGTGCTG TGTCGTGCCT TTTTTAGTCA TGGCCGGCGC CGCAGGCGCC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1861 GGCGCCTACG AGCACGCGAC CACGATGCCG AGCCAAGCGG GAATCTCGTA TAACACTATA VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1921 GTCAACAGAG CAGGCTACGC ACCACTCCCT ATCAGCATAA CACCAACAAA GATCAAGCTG VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1981 ATACCTACAG TGAACTTGGA GTACGTCACC TGCCACTACA AAACAGGAAT GGATTCACCA VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2041 GCCATCAAAT GCTGCGGATC TCAGGAATGC ACTCCAACTT ACAGGCCTGA TGAACAGTGC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2101 AAAGTCTTCA CAGGGGTTTA CCCGTTCATG TGGGGTGGTG CATATTGCTT TTGCGACACT VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2161 GAGAACACCC AAGTCAGCAA GGCCTACGTA ATGAAATCTG ACGACTGCCT TGCGGATCAT VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2221 GCTGAAGCAT ATAAAGCGCA CACAGCCTCA GTGCAGGCGT TCCTCAACAT CACAGTGGGA VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2281 GAACACTCTA TTGTGACTAC CGTGTATGTG AATGGAGAAA CTCCTGTGAA TTTCAATGGG VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2341 GTCAAAATAA CTGCAGGTCC GCTTTCCACA GCTTGGACAC CCTTTGATCG CAAAATCGTG VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2401 CAGTATGCCG GGGAGATCTA TAATTATGAT TTTCCTGAGT ATGGGGCAGG ACAACCAGGA VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2461 GCATTTGGAG ATATACAATC CAGAACAGTC TCAAGCTCTG ATCTGTATGC CAATACCAAC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2521 CTAGTGCTGC AGAGACCCAA AGCAGGAGCG ATCCACGTGC CATACACTCA GGCACCTTCG VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2581 GGTTTTGAGC AATGGAAGAA AGATAAAGCT CCATCATTGA AATTTACCGC CCCTTTCGGA VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2641 TGCGAAATAT ATACAAACCC CATTCGCGCC GAAAACTGTG CTGTAGGGTC AATTCCATTA VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2701 GCCTTTGACA TTCCCGACGC CTTGTTCACC AGGGTGTCAG AAACACCGAC ACTTTCAGCG VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2761 GCCGAATGCA CTCTTAACGA GTGCGTGTAT TCTTCCGACT TTGGTGGGAT CGCCACGGTC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2821 AAGTACTCGG CCAGCAAGTC AGGCAAGTGC GCAGTCCATG TGCCATCAGG GACTGCTACC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2881 CTAAAAGAAG CAGCAGTCGA GCTAACCGAG CAAGGGTCGG CGACTATCCA TTTCTCGACC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2941 GCAAATATCC ACCCGGAGTT CAGGCTCCAA ATATGCACAT CATATGTTAC GTGCAAAGGT VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3001 GATTGTCACC CCCCGAAAGA CCATATTGTG ACACACCCTC AGTATCACGC CCAAACATTT VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3061 ACAGCCGCGG TGTCAAAAAC CGCGTGGACG TGGTTAACAT CCCTGCTGGG AGGATCAGCC VEE GLY ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3121 GTAATTATTA TAATTGGCTT GGTGCTGGCT ACTATTGTGG CCATGTACGT GCTGACCAAC VEE GLY 3'UTR ~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3181 CAGAAACATA ATTAATAGTA AGCGGCCGCA TACAGCAGCA ATTGGCAAGC TGCTTACATA 3'UTR ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3241 GAACTCGCGG CGATTGGCAT GCCGCCTTAA AATTTTTATT TTATTTTTCT TTTCTTTTCC 3'UTR ~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3301 GAATCGGATT TTGTTTTTAA TATTTCAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA HDV ribozyme ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3361 AGGGTCGGCA TGGCATCTCC ACCTCCTCGC GGTCCGACCT GGGCATCCGA AGGAGGACGC HDV ribozyme ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3421 ACGTCCACTC GGATGGCTAA GGGAGAGCCA CGTTTAAACA CGTGATATCT GGCCTCATGG 3481 GCCTTCCTTT CACTGCCCGC TTTCCAGTCG GGAAACCTGT CGTGCCAGCT GCATTAACAT 3541 GGTCATAGCT GTTTCCTTGC GTATTGGGCG CTCTCCGCTT CCTCGCTCAC TGACTCGCTG colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3601 CGCTCGGTCG TTCGGGTAAA GCCTGGGGTG CCTAATGAGC AAAAGGCCAG CAAAAGGCCA colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3661 GGAACCGTAA AAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CCTGACGAGC colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3721 ATCACAAAAA TCGACGCTCA AGTCAGAGGT GGCGAAACCC GACAGGACTA TAAAGATACC colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3781 AGGCGTTTCC CCCTGGAAGC TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CCGCTTACCG colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3841 GATACCTGTC CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT TTCTCATAGC TCACGCTGTA colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3901 GGTATCTCAG TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3961 TTCAGCCCGA CCGCTGCGCC TTATCCGGTA ACTATCGTCT TGAGTCCAAC CCGGTAAGAC colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4021 ACGACTTATC GCCACTGGCA GCAGCCACTG GTAACAGGAT TAGCAGAGCG AGGTATGTAG colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4081 GCGGTGCTAC AGAGTTCTTG AAGTGGTGGC CTAACTACGG CTACACTAGA AGAACAGTAT colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4141 TTGGTATCTG CGCTCTGCTG AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4201 CCGGCAAACA AACCACCGCT GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG CAGATTACGC colE1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4261 GCAGAAAAAA AGGATCTCAA GAAGATCCTT TGATCTTTTC TACGGGGTCT GACGCTCAGT 4321 GGAACGAAAA CTCACGTTAA GGGATTTTGG TCATGAGATT ATCAAAAAGG ATCTTCACCT 4381 AGATCCTTTT AAATTAAAAA TGAAGTTTTA AATCAATCTA AAGTATATAT GAGTAAACTT 4441 GGTCTGACAG TTATTAGAAA AATTCATCCA GCAGACGATA AAACGCAATA CGCTGGCTAT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 4501 CCGGTGCCGC AATGCCATAC AGCACCAGAA AACGATCCGC CCATTCGCCG CCCAGTTCTT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 4561 CCGCAATATC ACGGGTGGCC AGCGCAATAT CCTGATAACG ATCCGCCACG CCCAGACGGC ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 4621 CGCAATCAAT AAAGCCGCTA AAACGGCCAT TTTCCACCAT AATGTTCGGC AGGCACGCAT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 4681 CACCATGGGT CACCACCAGA TCTTCGCCAT CCGGCATGCT CGCTTTCAGA CGCGCAAACA ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 4741 GCTCTGCCGG TGCCAGGCCC TGATGTTCTT CATCCAGATC ATCCTGATCC ACCAGGCCCG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 4801 CTTCCATACG GGTACGCGCA CGTTCAATAC GATGTTTCGC CTGATGATCA AACGGACAGG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 4861 TCGCCGGGTC CAGGGTATGC AGACGACGCA TGGCATCCGC CATAATGCTC ACTTTTTCTG ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 4921 CCGGCGCCAG ATGGCTAGAC AGCAGATCCT GACCCGGCAC TTCGCCCAGC AGCAGCCAAT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 4981 CACGGCCCGC TTCGGTCACC ACATCCAGCA CCGCCGCACA CGGAACACCG GTGGTGGCCA ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 5041 GCCAGCTCAG ACGCGCCGCT TCATCCTGCA GCTCGTTCAG CGCACCGCTC AGATCGGTTT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 5101 TCACAAACAG CACCGGACGA CCCTGCGCGC TCAGACGAAA CACCGCCGCA TCAGAGCAGC ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 5161 CAATGGTCTG CTGCGCCCAA TCATAGCCAA ACAGACGTTC CACCCACGCT GCCGGGCTAC ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 5221 CCGCATGCAG GCCATCCTGT TCAATCATAC TCTTCCTTTT TCAATATTAT TGAAGCATTT ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ KanR 5281 ATCAGGGTTA TTGTCTCATG AGCGGATACA TATTTGAATG TATTTAGAAA AATAAACAAA 5341 TAGGGGTTCC GCGCACATTT CCCCGAAAAG TGCCACCTAA ATTGTAAGCG TTAATATTTT 5401 GTTAAAATTC GCGTTAAATT TTTGTTAAAT CAGCTCATTT TTTAACCAAT AGGCCGAAAT 5461 CGGCAAAATC CCTTATAAAT CAAAAGAATA GACCGAGATA GGGTTGAGTG GCCGCTACAG 5521 GGCGCTCCCA TTCGCCATTC AGGCTGCGCA ACTGTTGGGA AGGGCGTTTC GGTGCGGGCC 5581 TCTTCGCTAT TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA T7 promoter ~~~~~~~~~~~~~~~~~~~~ 5641 ACGCCAGGGT TTTCCCAGTC ACACGCGTAA TACGACTCAC TATAG

Claims (14)

1.Molécula de RNA auto-replicante caracterizada pelo fato de que compreende um polinucleotídeo que é codificado porumamolécula deDNA que compreende: a.asequênciagH daSEQ IDNO:31ou33; b.a sequênciagL da SEQ ID NO: 35; c.asequênciaUL128da SEQIDNO:43; d.asequênciaUL130da SEQIDNO:45;e e.a sequênciaUL131 da SEQ ID NO:47 em que a sequência gH, a sequência gL, a sequência 128, a sequência UL130 e a sequência UL131 estão ligadas operacionalmente a um ou mais elementos de controle de modo que, quando a molécula de RNA auto-replicante é introduzida em uma célula adequada, as proteínas gH, gL, UL128, UL130 e UL131 são produzidas em uma quantidade suficiente para formar um complexo na célula que contém as proteínas gH, gL, UL128,UL130 e UL131.
2.Molécula de RNA auto-replicante, de acordo com a reivindicação 1, caracterizada pelo fato de que cada um dos elementos de controle é selecionado independentemente do grupo que consiste em um promotor subgenômico, um IRES e um sítio viral 2A.
3.Molécula de RNA auto-replicante, de acordo com a reivindicação 1 ou 2, caracterizada pelo fato de que o herpesvírus é citomegalovírus (CMV).
4.Molécula de RNA auto-replicante, de acordo com a reivindicação 3, caracterizada pelo fato de que a primeira proteína ou fragmento é gH ou um fragmento da mesma, a segunda proteína ou fragmento é gL ou um fragmento da mesma, a terceira proteína ou fragmento é UL128 ou um fragmento da mesma, a quarta proteína ou fragmento é UL130 ou um fragmento da mesma e a quinta proteína ou fragmento é UL131 ou um fragmento da mesma.
5.Molécula de RNA auto-replicante, de acordo com qualquer uma das reivindicações 1 a 4, caracterizada pelo fato de que a molécula de RNA auto-replicante é um replicon de alfavírus.
6.Partícula de replicon de alfavírus (VRP) caracterizada pelo fato de que compreende o replicon de alfavírus conforme definido na reivindicação 5.
7.Composição caracterizada pelo fato de que compreende uma VRP, conforme definida na reivindicação 6 e um veículo farmaceuticamente aceitável.
8.Composição, de acordo com a reivindicação 7, caracterizada pelo fato de que ainda compreende um adjuvante.
9.Composição caracterizada pelo fato de que compreende o RNA auto-replicante, conforme definido em qualquer uma das reivindicações 1 a 5 e um veículo farmaceuticamente aceitável.
10.Composição, de acordo com a reivindicação 9, caracterizada pelo fato de que ainda compreende um sistema de liberação de RNA.
11.Composição, de acordo com a reivindicação 10, caracterizada pelo fato de que o sistema de liberação de RNA compreende:(i) um lipossomo, (ii) uma nanopartícula polimérica, (iii) uma nanoemulsão óleo-em-água catiônica, ou (iv) qualquer combinação dos mesmos.
12.Composição, de acordo com a reivindicação 11, caracterizada pelo fato de que o sistema de liberação compreende uma nanoemulsão óleo-em-água catiônica.
13.Molécula de DNA recombinante caracterizada pelo fato de que codifica a molécula de RNA auto-replicante conforme definida em qualquer uma das reivindicações 1 a 5.
14.Uso da molécula de RNA auto-replicante, conforme definida em qualquer uma das reivindicações 1 a 5, ou da VRP conforme definida na reivindicação 6, ou da composição conforme definida em qualquer uma das reivindicações 7 a 12, caracterizado pelo fato de ser para preparar um medicamento para induzir uma resposta imune em um indivíduo contra o herpesvírus.
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