KR20190127605A - Chimeric Zika virus vaccine - Google Patents

Abstract

The present invention provides a chimeric Zika virus vaccine and nucleic acid, comprising a base sequence encoding a structural protein of Zika virus and a base sequence encoding a non-structural protein of attenuated yellow fever virus.

Description

Chimeric Zika virus vaccine

The present invention relates to a vaccine, and more particularly to a vaccine capable of preventing Zika virus infection.

Zika is a disease transmitted to humans by Egyptian forest mosquitoes and is caused by the Zika virus belonging to the flavivirus. Since it was first discovered in rhesus monkeys in Uganda in 1947, human infection cases have been reported in Tanzania and Uganda in 1952. Human infections occurred mainly in Asia and Africa between 1960 and 1980, and the area of infection gradually expanded, and is now occurring in countries around the Americas and the Pacific. Zika's massive outbreak occurred on Yap Island in Micronesia in 2007, and then again on a large scale in 2013 in French Polynesia. It then began to spread in New Caledonia in 2014 and was pandemic in most countries of the Americas, including the United States and Brazil, between 2015 and 2016.

syndrome이나, 신경 이상증 등을 유발하는 것으로 보고되고 있다. Symptoms include headache, muscle / joint pain, skin rash, conjunctivitis, headache, etc. within a few days, and it is difficult to distinguish between diseases such as dengue fever and the symptoms. Most of Zika virus infections are asymptomatic or mild and can only be confirmed by molecular diagnosis. Some of the infected patients have severe disease and mortality rates are low. In particular, it is known that infection during pregnancy affects the fetal neurodevelopment and increases the likelihood of giving birth defects (approximately 8%) with microcephaly disease. Guillain-Barr, also suspected of being an autoimmune disease in infected people

It is reported to cause syndrome and neuropathy.

It is known that the mediation of the disease occurs in the blood-sucking process in the human body, and infection from human to human does not occur in general, but infection can occur through the infiltration of blood and blood, such as blood transfusion, sexual intercourse. Currently, there are no treatments and vaccines, and mosquitoes are the best way to prevent disease.

Zika virus belongs to Flaviviridae and includes dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus and tick-borne encephalitis virus. Among them, yellow fever virus has been developed and used as a vaccine for a long time. Around 1930, attenuated live vaccine was first developed and commercialized.

syndrome, 소두증(microcephaly), 신경이상, 선천성 기형 같은 중증 신경 질환을 일으킬 가능성이 있기 때문에 약독화 및 안전성 확보가 무엇보다도 중요하다.Live virus vaccines are generally known to have the highest vaccine efficacy than other vaccine types. In addition to the ability to induce antibodies, cellular immune induction is excellent, and the number of administration is usually 1 to 2 times less than other vaccine types. However, due to concerns about pathogenic reversion, the inoculation is often limited to patients with weak immunity, pregnant women, the elderly, and infants. Therefore, it is very important to secure safety in order to prepare a live virus as a preventive vaccine. In particular, in the case of live Zika vaccine, Guillain-Barr

Attenuation and ensuring safety are of paramount importance because of the potential for severe neurological disorders such as syndrome, microcephaly, neuropathy and congenital malformations.

The present invention seeks to provide an attenuated Zikavirus immunogenic composition.

The present invention is to provide an immunogenic composition capable of preventing or treating diseases, diseases, etc. caused by Zika virus infection.

In addition, the present invention is to provide a Zika virus immunity excellent method using the immunogenic composition.

The present invention aims to provide a safe and excellent Zika virus vaccine using the attenuated yellow fever virus line and the chimeric gene of Zika virus.

The present invention seeks to provide an attenuated Zikavirus immunogenic composition and a vaccine for preventing Zikavirus infection comprising the same.

The present invention seeks to provide new viral strains for the purpose of preventing, ameliorating and / or treating Zikavirus infections.

The present invention is to provide a method for producing chimeric Zika virus using reverse transcription gene technology.

It is another object of the present invention to provide a chimeric nucleic acid construct for the production of chimeric Zikavirus, which can induce an immune response against Zikavirus infection, and furthermore, to induce an Zikavirus immune response that has immunogenicity by itself. .

One embodiment of the present invention induces an immune response to Zika virus infection,

I) a gene encoding a signal peptide existing upstream of a gene encoding a pre-membrane (prM) protein of a yellow fever virus 17D vaccine strain nucleic acid molecule; ii) genes encoding pre-membrane (prM) and its transmembrane proteins; And iii) inserted into the gene region encoding envelope (E) and its transmembrane protein in place of the yellow fever virus 17D vaccine strain gene,

I) a gene encoding a signal peptide existing upstream of a gene encoding a pre-membrane (prM) protein of Zika virus; ii) genes encoding pre-membrane (prM) and its transmembrane proteins; And iii) a gene encoding the envelope (E) and its transmembrane protein, wherein some or all of the nucleic acids constituting the genes i), ii), and iii) are substituted, deleted, or inserted, Provide nucleic acid molecules. In addition, the present invention provides a use of the nucleic acid molecule to induce an immune response to Zika virus infection. In addition, to provide a composition for immune response to Zika virus infection comprising the nucleic acid molecule.

In one embodiment, the nucleic acid molecule is any one of the following sequences, and may include both a nucleic acid derived from a yellow fever virus 17D vaccine strain and a Zika virus derived nucleic acid.

a) the nucleic acid molecule of SEQ ID NO: 19;

b) the nucleic acid molecule of SEQ ID NO: 20;

c) the nucleic acid molecule of SEQ ID NO: 21;

d) the nucleic acid molecule of SEQ ID NO: 22;

e) the nucleic acid molecule of SEQ ID NO: 23; or

f) the nucleic acid molecule of SEQ ID NO: 24.

In one embodiment, the chimeric nucleic acid molecule may be any one of the following sequences.

a) the nucleic acid molecule of SEQ ID NO: 25;

b) the nucleic acid molecule of SEQ ID NO: 26;

c) the nucleic acid molecule of SEQ ID NO: 27; or

d) the nucleic acid molecule of SEQ ID NO: 28.

In one embodiment, an immunogenic composition comprising a chimeric nucleic acid molecule consisting of any one of SEQ ID NO: 19 to SEQ ID NO: 25 can be provided.

In one embodiment, the chimeric Zika virus formed by introducing a Zika virus antigen gene into the gene sequence of the attenuated yellow fever virus of SEQ ID NO: 29 may be provided.

In one embodiment the Zikavirus antigen gene is i) pre-membrane (prM) protein and its transmembrane protein gene, ii) envelope (E) protein and its transmembrane protein gene, and iii) capsid (C) protein gene. It may be a gene made.

In one embodiment the chimeric Zika virus is chimeric Zika virus for inducing an immune response to Zika virus,

I) pre-membrane (prM) protein and its transmembrane protein region of attenuated yellow fever virus, and ii) the envelope (E) protein and its transmembrane protein region

And those derived from the i) pre-membrane (prM) protein and its transmembrane protein region of wild-type Zikavirus, and ii) the envelope (E) protein and its transmembrane protein region. In addition, one embodiment may provide a use of the chimeric Zika virus to induce an immune response against Zika virus. In one embodiment the chimeric Zika virus comprises a pre-membrane (prM), and envelope (E) protein of wild type Zika virus,

The i) transmembrane protein region of the pre-membrane (prM) protein, and ii) the transmembrane protein region of the envelope (E) protein may each be derived from an attenuated yellow fever virus.

In one embodiment, the chimeric Zika virus may include a capsid (C) protein of wild type Zika virus.

In one embodiment, the chimeric Zika virus may be composed of the amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 13.

In one embodiment, the chimeric Zika virus may be a position 792 in the amino acid sequence of SEQ ID NO: 1 is substituted in S to G.

In one embodiment, the chimeric Zika virus may be substituted with GADTSVGIVGLLLTTAMA in SHDVLTVQFLILGMLLMTGG position 102-121 in the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the chimeric Zika virus may be one in which the amino acid sequence of the 104-121 amino acid sequence in SEQ ID NO: 1 is replaced by GADTSVGIVGLLLTTAMA in DVLTVQFLILGMLLMTGG.

In one embodiment, the chimeric Zika virus may be caused by the insertion of one or more amino acids after the G amino acid, the amino acid of position 121 in the amino acid sequence of SEQ ID NO: 1. Wherein the inserted amino acid is one, amino acid A.

In one embodiment, the chimeric Zika virus may be a substitution induced in the TGG amino acid of positions 119-121 in the amino acid sequence of SEQ ID NO: 1.

The chimeric Zika virus may be a TGG amino acid of positions 119-121 in the amino acid sequence of SEQ ID NO: 1 is substituted with AMA.

Chimeric zikavirus, in one embodiment administered to an animal in an effective amount to induce immunity to Zikavirus in the body of the animal; And it provides a Zika virus immunogenic composition comprising a pharmaceutically acceptable excipient.

The pharmaceutically acceptable excipient may be any one or more selected from the group consisting of water, PBS buffer solution, glycine solution, D-trehalose dehydrate, lactose, and sorbitol.

In one embodiment the animal may be a human.

The zikavirus immunogenic composition can be administered via the intramuscular route, intradermal, mucosal or subcutaneous route.

syndrome, 소두증(microcephaly), 신경이상, 선천성 기형의 예방 등에 사용될 수 있다.The immunogenic composition prevents Zika virus infection or Guillain-Barr

It can be used to prevent syndromes, microcephaly, neurological disorders, congenital malformations, etc.

One embodiment is a method for producing an immunogenic composition comprising a chimeric zika virus,

Preparing the attenuated yellow fever virus 17D vaccine strain;

Preparing a 17D-Zika chimeric genome by replacing the nucleotide sequence encoding the structural protein of the yellow fever virus 17D vaccine strain with the nucleotide sequence encoding the structural protein of the wild-type Zika virus;

Cloning the dielectric into a vector;

Introducing the cloned vector into competent cells to transform the cloned vector; And

Recovering viral DNA from the competent cells.

In one embodiment, the cloning to the vector may be a pCC1-chimeric Zika virus vector using the pCC1 vector as a vector.

The 17D-Zika chimeric genome may have any one base sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16.

The pCC1-chimeric zika virus vector was sequentially encoded by the restriction enzyme AscI coding sequence (5'-GGCGCGCC) at the 5 'end; T7 promoter sequence (TAATACGACTCACTATA); transcription start codon (GG); Nucleic acids of any one chimeric zika virus selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16; The restriction enzyme SmaI-NotI coding sequence (3'-GGGCCCCGCCGGCG) may be included at the 3 'end.

One embodiment provides a method for producing chimeric Zikavirus having immunogenicity against Zikavirus infection, comprising the following steps.

(a) preparing a YF 17D genome which is an attenuated yellow fever virus vaccine strain having SEQ ID NO: 29; And

(b) replacing the nucleic acid at the positions 482 to 2452 of the nucleotide sequence of the YF 17D genome of SEQ ID NO: 29 with the nucleotide sequence of any one of SEQ ID NOs: 19 to 28 to prepare a chimeric Zika virus gene.

The manufacturing method,

(c) adding a T7 promoter sequence (TAATACGACTCACTATA) and a transcription start codon (GG) at the 5 'end of the chimeric Zika virus gene prepared in step (b), and ascI (5`-GGCGCGCC) restriction enzymes at both ends; And SmaI-NotI (3'-GGGCCCCGCCGGCG) may be further included to prepare for cloning into a vector.

The present invention is to provide a vaccine that can prevent, prevent, treat or improve the Zika virus infection in order to solve the above problems.

The present invention provides a safe vaccine for preventing zikavirus infection and a method for producing the same, which can prevent, prevent, treat or improve Zikavirus infection.

In addition, the present invention provides a safe and low side effect vaccine for Zika virus, chimeric Zika virus of the present invention can be used to improve, prevent or treat the symptoms caused by Zika virus infection.

In one embodiment of the present invention provides a chimeric Zika virus nucleic acid comprising a base sequence encoding the structural protein of the Zika virus, and comprising a base sequence encoding a non-structural protein of the attenuated yellow fever virus.

An embodiment of the present invention provides a chimeric nucleic acid for preparing a Zika virus vaccine, wherein the nucleic acid encodes a base sequence encoding a structural protein of Zika virus and a base sequence encoding a non-structural protein of attenuated yellow fever virus. It provides a nucleic acid for producing a zika virus vaccine comprising.

In one embodiment of the present invention, a nucleic acid molecule, preferably a chimeric nucleic acid molecule, capable of directly or indirectly inducing Zikavirus immunity can be provided.

The nucleic acid molecule may be provided in a form included in an immunogenic composition capable of inducing an immune response to Zikavirus infection or inducing an immune response.

In one embodiment of the present invention, the nucleic acid molecule,

Genes encoding signal peptides upstream of the gene encoding i) pre-membrane (pr-M) of the yellow fever virus 17D vaccine strain nucleic acid molecule; ii) a gene encoding a pre-membrane (prM) and its transmembrane (TM) protein; And iii) the gene of Zika virus is inserted into any one of the gene region encoding the envelope (E) and its transmembrane protein, thereby having a form in which a structural gene of yellow fever virus and a structural gene of Zika virus exist together.

In one embodiment of the present invention i) gene encoding a signal peptide (signal peptide) existing upstream of the gene encoding the pre-membrane (prM) of Zika virus; ii) genes encoding pre-membrane (prM) and its transmembrane proteins; iii) a gene encoding the envelope (E) and its transmembrane protein may be inserted at a site corresponding to the yellow fever virus 17D vaccine strain, wherein a part or all of the nucleic acid constituting the gene is substituted, deleted, or inserted Can be.

Preferably, the nucleic acid molecule is any one of the following sequences, and may have both a nucleic acid derived from a yellow fever virus 17D vaccine strain and a Zika virus derived nucleic acid.

a) the nucleic acid molecule of SEQ ID NO: 19;

b) the nucleic acid molecule of SEQ ID NO: 20;

c) the nucleic acid molecule of SEQ ID NO: 21;

d) the nucleic acid molecule of SEQ ID NO: 22;

e) the nucleic acid molecule of SEQ ID NO: 23; or

f) the nucleic acid molecule of SEQ ID NO: 24.

In one embodiment of the invention, the chimeric nucleic acid molecule may be a TM-coding gene of the pre-membrane (prM) protein derived from the yellow fever virus 17D vaccine strain.

The chimeric nucleic acid molecule is i) a gene encoded to express the pre-membrane (prM) protein of Zika virus, ii) a gene encoded to express the TM region of the pre-membrane (prM) protein of yellow fever virus 17D vaccine strain, iii) The envelope (E) protein gene of Zika virus, iv) can be linked to the TM coding gene of the envelope (E) protein of the yellow fever virus 17D vaccine strain.

In one embodiment of the invention, the upstream of the gene encoded to express the pre-membrane (prM) protein of the chimeric nucleic acid i) Zika virus may be a gene encoding the signal peptide of the yellow fever virus 17D vaccine strain.

The chimeric nucleic acid molecule may be any one of the following sequences.

a) the nucleic acid molecule of SEQ ID NO: 25;

b) the nucleic acid molecule of SEQ ID NO: 26;

c) the nucleic acid molecule of SEQ ID NO: 27; or

d) the nucleic acid molecule of SEQ ID NO: 28.

In one embodiment of the present invention can provide an immunogenic composition comprising the chimeric nucleic acid molecule.

In one embodiment of the present invention, the attenuated yellow fever virus may be a yellow fever virus 17D vaccine strain.

In one embodiment of the present invention provides a chimeric nucleic acid molecule for preparing a Zika virus vaccine.

In the chimeric nucleic acid molecule, any one or more sequences of the base sequences 482 to 2452 of the attenuated yellow fever virus 17D vaccine strain may be substituted with the base sequence of Zika virus. Preferably, the nucleotide sequence encoding the pre-membrane (prM) and envelope (E) proteins of the wild-type Zika virus is replaced by a sequence derived from Zika virus from any one or more of the sequences 482 to 2452 of the 17D vaccine strain. The 482-2497 base sequence of the entire Zika virus sequence may be a Zika virus derived sequence.

In one embodiment of the present invention, the base sequence may be subjected to a cloning step for DNA synthesis in vitro.

In one embodiment of the present invention provides a chimeric zika virus.

In one embodiment of the present invention can provide a chimeric Zika virus formed by introducing a Zika virus antigen gene into the attenuated yellow fever virus.

The Zikavirus antigen gene consists of i) pre-membrane (prM) protein and its transmembrane protein gene, ii) envelope (E) protein and its transmembrane protein gene, and iii) capsid (C) protein gene.

In one embodiment of the invention the chimeric Zika virus is chimeric Zika virus for inducing an immune response to Zika virus,

The chimeric Zika virus is a foreign gene introduced into the genome of the attenuated yellow fever virus, i) pre-membrane (prM) protein and its transmembrane protein region of the attenuated yellow fever virus, and ii) envelope (E) protein And its transmembrane protein region may be derived from i) pre-membrane (prM) protein and its transmembrane protein region of wild type Zika virus, and ii) the envelope (E) protein and its transmembrane protein region. have. Preferably the chimeric Zika virus may have an amino acid sequence of SEQ ID NO: 1.

In one embodiment of the invention the chimeric Zika virus is chimeric Zika virus for inducing an immune response to Zika virus,

I) pre-membrane (prM) protein region of the attenuated yellow fever virus is replaced with an amino acid sequence derived from the pre-membrane (prM) protein region of the wild type zika virus, and ii of the attenuated yellow fever virus ) The envelope (E) protein region is substituted with an amino acid sequence derived from the envelope (E) protein region of the wild-type Zika virus,

The i) transmembrane protein region of the pre-membrane (prM) protein region and ii) the transmembrane protein region of the envelope (E) protein may each be derived from an attenuated yellow fever virus. Preferably the chimeric Zika virus may have an amino acid sequence of SEQ ID NO: 13.

In one embodiment of the present invention, the chimeric Zika virus may be substituted with the capsid protein of the attenuated yellow fever virus (C) protein of Zika virus.

In one embodiment of the present invention, the chimeric Zika virus may be substituted for a part of the envelope (E) protein of the structural protein, a part of the E protein may be that amino acid No. 792 of the chimeric Zika virus is substituted from S to G. have. Preferably, the chimeric Zika virus may be an amino acid of position 792 in the amino acid sequence of SEQ ID NO: 1 is substituted from S to G. Preferably it may have an amino acid sequence of SEQ ID NO: 3.

In an embodiment of the present invention, the sequence of the 102-121 amino acid position of the chimeric Zika virus may be substituted with GADTSVGIVGLLLTTAMA in SHDVLTVQFLILGMLLMTGG. Preferably, the chimeric Zika virus may be one in which amino acid position 102-121 in the amino acid sequence of SEQ ID NO: 1 is replaced with SADVLTVQFLILGMLLMTGG GADTSVGIVGLLLTTAMA. Preferably it may have an amino acid sequence of SEQ ID NO: 5.

In one embodiment of the present invention, the signal peptide present upstream of the prM of chimeric zika virus may be derived from Zika virus.

In one embodiment of the present invention, the amino acid sequence of the signal peptide, which is the sequence of the 104-121 amino acid position of the chimeric zika virus, may be substituted with GADTSVGIVGLLLTTAMA in DVLTVQFLILGMLLMTGG. Preferably, the chimeric Zika virus may be substituted for GADTSVGIVGLLLTTAMA in the position of amino acid position 104-121 amino acid sequence of SEQ ID NO: 1 from DVLTVQFLILGMLLMTGG. Preferably it may have an amino acid sequence of SEQ ID NO: 7.

In one embodiment of the present invention, the chimeric Zika virus may cause one or more amino acid insertions after G amino acid of amino acid sequence 121. Preferably, the chimeric Zika virus may have one or more amino acids inserted between the signal peptide and the pr-M protein upstream of the pr-M protein.

Preferably the inserted amino acid is one, amino acid A may be. Preferably have an amino acid sequence of SEQ ID NO: 9.

In one embodiment of the present invention can provide a chimeric Zika virus is substituted with the TGG (119-121) amino acid of the signal peptide located upstream (upstream) of the prM of chimeric Zika virus.

In one embodiment of the present invention, the TGG (119-121) amino acids of the signal peptide located upstream of the prM may be substituted with AMA. Preferably it may have an amino acid sequence of SEQ ID NO.

In one embodiment of the present invention may provide a Zika virus immunogenic composition capable of preventing, treating or ameliorating Zika virus infection.

In one embodiment of the present invention may provide a chimeric Zika virus immunogenic composition comprising the chimeric Zika virus and immunologically acceptable excipients.

In one embodiment of the present invention may be provided a method of inducing an immune response by administering the immunogenic composition to a subject.

In one embodiment of the present invention can provide a Zika virus vaccine comprising the chimeric Zika virus and a pharmaceutically acceptable excipient, which is administered to an animal to induce immunity to Zika virus in the animal body.

The animal may be a mammal, preferably human.

The Zikavirus vaccine may be administered via the intramuscular route, intradermal, mucosal or subcutaneous route, but is not limited thereto.

In one embodiment of the invention can provide a Zika virus immunogenic composition for use in the treatment or prevention of diseases associated with Zika virus infection.

In one embodiment of the present invention may be provided a method of inducing an immune response to Zika virus by administering the immunogenic composition to a subject.

In one embodiment of the present invention can provide a use of the Zika virus immunogenic composition for the treatment or prevention of diseases associated with Zika virus infection.

In one embodiment of the present invention, the use of the Zika virus immunogenic composition for inducing an immune response to Zika virus infection patients can be provided.

In one embodiment of the present invention, a method for producing an immunogenic composition comprising a chimeric zika virus,

i) preparing the attenuated yellow fever virus 17D vaccine strain,

ii) replacing the nucleotide sequence encoding the structural protein of the yellow fever virus 17D vaccine strain with the nucleotide sequence encoding the structural protein of the wild type Zika virus to prepare a 17D-Zika chimeric genome,

iii) cloning said genome into a vector, and

iv) introducing said cloned vector into competent cells to transform, and recovering viral DNA from said competent cells, a method for producing an immunogenic composition comprising a chimeric zika virus To provide.

Cloning to the vector may use a pCC1 vector as the vector, preferably pCC1BAC ™.

The 17D-Zika chimeric genome may have any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15.

The 17D-Zika chimeric genome may have any one or more base sequences selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16.

The present invention can be used as a backbone for the production of chimeric Zika virus of the yellow fever vaccine strain of the genetically attenuated flaviviruses, and heterologous antigens (such as prM, E protein of Zika virus) can induce a neutralizing immune response Attenuated virus can be produced by substituting a gene encoding a) with a sequence of an equivalent or similar site of another virus (eg, prM, E protein of 17D).

In one embodiment of the present invention, the gene of chimeric Zika virus of the present invention includes both terminal sequences of 5 ′ and 3 ′, which are non-coding sequences, in addition to the structural and nonstructural protein coding sequences.

In addition, the gene can be transcribed through in vitro transcription. The gene may include a promoter sequence, a transcription initiation codon (GG), a restriction enzyme sequence, and the like, which may be useful in development. For example, the promoter sequence may include a T7, T3, SP6 RNA polymerase promoter, but preferably a T7 promoter. In the present invention, restriction enzyme sequences AscI (5′-GGCGCGCC) and SmaI-NotI (3′-GGGCCCCGCCGGCG) may be added.

The synthesized base sequence can be cloned into a vector capable of proliferation in bacteria.

In the case of Flavivirus, it is assumed that the cDNA of the virus is cytotoxic to bacteria. Therefore, pCC1BAC ™, which can control the copy number, can be used. The cloned viral gene can be used as a template for in vitro transcription.

In order to improve the effects of antigenicity, proliferation, etc., further gene substitution and mutation may proceed. Based on the prM and E proteins, the signal peptide of each TM region and prM can be applied in various combinations. In addition, it may be possible to apply sequence mutations at the connection sites between the signal peptide of prM, pr-M protein except the signal peptide, and E protein.

The chimeric Zika virus nucleotide sequence of the present invention may be prepared into several fragments through chemical synthesis, and may be relinked to make a full-length DNA sequence to be cloned into an appropriate vector, and preferably, a copy control plasmid pCC1BAC ™ may be used. .

In one embodiment of the present invention provides a method for producing chimeric Zika virus having immunogenicity against Zika virus infection, comprising the following steps.

(a) preparing a YF 17D genome which is an attenuated yellow fever virus vaccine strain having SEQ ID NO: 29; And

(b) replacing the nucleotide sequences 482 to 2452 of the YF 17D genome with the nucleotide sequence of any one of SEQ ID NOs: 19 to 28 to produce a chimeric Zika virus gene.

Preferably, the preparation method includes (c) adding a T7 promoter sequence (TAATACGACTCACTATA) and a transcription start codon (GG) at the 5 'end of the chimeric Zikavirus gene produced in step (b), and restriction enzymes at both ends. The method may further include preparing AscI (5′-GGCGCGCC) and SmaI-NotI (3′-GGGCCCCGCCGGCG) to clone the vector.

The vector may be a pCC1BAC ™ vector.

Provided are amplification of the DNA sequence designed in the present invention and a method for producing RNA using the same for the production of the chimeric Zika virus of the present invention.

The chimeric Zika virus RNA of the present invention is inserted into a cell to provide a process for completing the production of chimeric Zika virus.

The present invention provides safety results through attenuation validation of the chimeric Zika virus vaccine produced.

The present invention provides a live vaccine that induces an effective immune response by parenteral administration of the vaccine strain to the mammal.

The present invention provides an artificial Zika sequence and amino acid sequence used for the treatment or prevention of Zika virus infection and diseases caused by Zika virus infection.

The present invention particularly provides Zikavirus vaccines.

The inventors of the present invention use the yellow fever virus 17D vaccine strain as a backbone for cloning to produce chimeric viruses, while maintaining the antigenicity of the Zika virus, while attenuating them without the need for a long time and several processes to ensure the safety of the vaccine. It was confirmed that it can be used as a live vaccine for preventing Zika virus infection.

[Definition of Terms]

“Chimera or chimeric” may refer to the presence of cells of different genetic traits in one organism, in particular “chimeric or chimeric” as used herein means a single genetic backbone. (backbone) refers to a case in which genetic information (nucleic acid) of different individuals is inserted based on a new genetic variation.

As used herein, the “jika chimera”, “chimeric zikavirus” refers to a genetic backbone by exchanging with at least the E protein of the acceptor flavivirus, or the sequence of the Zika virus corresponding to the sequences of pr-M and E proteins. By modified recipient flavivirus. Preferably the genetic backbone of the recipient flavivirus can be modified by exchanging it with a sequence of Zikavirus corresponding to a nucleotide sequence encoding both the pr-M and E proteins of the recipient flavivirus. Recipient flaviviruses may be attenuated. The recipient flavivirus may be a yellow fever (YF) virus, preferably a yellow fever virus 17D vaccine strain.

In the present invention, "chimeric Zika virus" may be understood as "chimeric YF / Zika virus". This can be understood to mean a form in which a yellow fever virus is used as a backbone and a gene capable of inducing a Zika virus immune response is inserted into a nucleic acid molecule of the yellow fever virus.

Examples of other attenuated YF strains that may be used may include, for example, attenuated YF 17D, YF 17DD and YF 17D204 (YF-VAX®) viruses, but preferably the YF17D strain may be used as the backbone. .

In terms of stability and productivity of the vaccine of the present invention, YF17D can be used as a backbone.

As used herein, the term “vaccine Zikavirus” refers to a virus capable of inducing neutralizing antibodies against Zika virus by administering the vaccine Zika virus to a subject having immune capacity. In one embodiment, the vaccine Zika virus may mean an attenuated live chimeric Zika virus.

As used herein, “genome” refers to the total sequence of a gene of an individual, and refers to a collection of all genetic information of an organism. For example, the genome of a virus is used to encompass all the genetic information sequences throughout the virus.

As used herein, a "gene" may be understood as a portion encoding a protein and may be DNA or RNA.

As used herein, the meaning of “introduction” means that a new one is added from the original one, and the introduction may cause mutation of the gene, and deletion, substitution, or insertion may occur.

For example, as used herein, "a new gene has been introduced" may mean that a part of the entire gene sequence possessed by the original individual may be substituted or inserted into a gene derived from the new individual. Due to this introduction, it may be longer, shorter, or maintained in length than the genome sequence of the original individual (eg, backbone).

Subsequent substitution of a protein of a virus herein means that the gene encoding the protein has been fully understood by those skilled in the art. For example, describing 'substituted protein A' may be understood to mean that the gene has been substituted with any of the codons encoding protein A.

[ Zika virus Immunity to infection chimera  Nucleic acid molecules]

In one embodiment of the invention may provide a chimeric nucleic acid molecule for Zika virus immunity.

The chimeric nucleic acid molecule may be provided in a form included in an immunogenic composition capable of inducing an immune response to Zikavirus infection or inducing an immune response.

The chimeric nucleic acid molecules described herein can be used separately from the chimeric zika virus.

The chimeric nucleic acid molecule includes a major antigen gene of Zika virus, but some genes may be understood as genes of yellow fever virus. The main antigen gene can be understood as a gene encoding the structural protein of Zika virus.

The chimeric Zika virus is a part or all of the gene encoding the structural protein in the genome of the yellow fever virus may have the possibility of proliferation by itself.

In one embodiment of the present invention, the chimeric nucleic acid molecule may preferably have any one of SEQ ID NOs: 19 to 24.

In one embodiment of the present invention, the chimeric nucleic acid molecule may be a TM-coding gene of the pre-membrane (prM) protein derived from the yellow fever virus 17D vaccine strain.

The chimeric nucleic acid molecule is i) a gene encoded to express the pre-membrane (prM) protein of Zika virus, starting from 5 'to 3', ii) the pre-membrane (prM) protein of yellow fever virus 17D vaccine strain. Gene encoded for expression of TM region, iii) envelope (E) protein gene of Zika virus, iv) TM encoding gene of envelope (E) protein of yellow fever virus 17D vaccine strain.

In one embodiment of the present invention, the signal peptide located 5 'upstream of the i) pre-membrane (prM) protein gene of the chimeric nucleic acid molecule may be a gene of yellow fever virus 17D vaccine strain, but is not limited thereto. May be a derived signal peptide.

The chimeric nucleic acid molecule may preferably have a nucleotide sequence of any one of SEQ ID NOs: 25 to 28.

[ Zika virus  Base sequence]

In one embodiment of the present invention provides a chimeric Zika virus nucleic acid comprising a base sequence encoding the structural protein of the Zika virus, and comprising a base sequence encoding a non-structural protein of the attenuated yellow fever virus.

In one embodiment of the present invention, the chimeric zika virus is a structural protein of Zika virus based on the 5'-non-coding sequence, the capsid protein coding sequence, the non-structural protein coding sequence, and the 3'-non-coding sequence of YF 17D. The coding sequence of the -M and E proteins can provide a chimeric virus nucleotide sequence in order.

In one embodiment of the present invention, the attenuated yellow fever virus may be a yellow fever virus 17D vaccine strain.

In one embodiment of the present invention, the base sequence encoding the structural protein of the Zika virus may be a nucleic acid encoding any one or more of pre-membrane (prM), and envelope (E).

Preferably, the sequence encoding the pr-M and E proteins of the receptor yellow fever virus 17D vaccine strain is replaced with the corresponding sequence of the Zika virus, such that the nucleic acid in which the genetic backbone of the YF virus is modified or the chimeric Zika virus into which the nucleic acid is introduced is It can be used as.

Preferably the chimeric zika virus of the present invention may have an amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15.

Preferably the chimeric zika virus of the present invention may have an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16.

At least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or at least 100% of the sequence sequences that can be used to prepare chimeric zikaviruses described herein are immunogens. It can be used for the production of chimeric Zika virus, which is used directly or as an immunogen.

[ chimera Zika virus ]

In one embodiment of the present invention, chimeric zikaviruses are non-structural with one long open reading frame (ie, the structural protein capsid (C), precursor-membrane (pr-M, precursor-Membrane) and envelope (E) proteins). Translation and the post-translational series of post-translational proteolytic cleavage.

In one embodiment of the present invention, the chimeric Zika virus of the present invention may be replaced with pr-M and E proteins of YF17D with pr-M and E proteins of Zika virus.

The preparation of chimeric zika viruses of one embodiment of the present invention may include forming a novel junction between the capsid and pre-membrane proteins of the two different flaviviruses, the envelope protein and the nonstructural region (NS1). have.

The chimeric viral base sequence of the present invention is provided in a manner similar to the general flavivirus base sequence structure, and may further substitute a structural protein or change a part of the protein sequence as appropriate for protein expression and virion production.

Additional protein sequence substitutions and mutations can be applied using various combinations, deletions, insertions, etc. of the sequences of capsid proteins, signal peptides, TM regions of prM, TM regions of E proteins.

Unless otherwise specified herein, the signal peptide is located upstream of the gene for precursor-membrane (pr-M) protein expression and is recognized by a peptidase to recognize the precursor-membrane. It can be understood to mean a site that can be cleaved with (pr-M) protein.

Such protein sequence variation may affect the level of virus proliferation, the level of attenuation of the vaccine strain.

In one embodiment of the present invention can provide a chimeric Zika virus for inducing an immune response to Zika virus.

The chimeric Zika virus is a foreign gene introduced into the genome of the attenuated yellow fever virus, i) pre-membrane (prM) protein and its transmembrane protein region of the attenuated yellow fever virus, and ii) envelope (E) protein And its transmembrane protein region is derived from the i) pre-membrane (prM) protein and its transmembrane protein region of wild-type Zika virus, and ii) the envelope (E) protein and its transmembrane protein region. .

In one embodiment of the invention said chimeric Zika virus is a transmembrane protein region is maintained of 17D YF virus. pre-membrane (prM), and envelope (E) proteins may be provided as derived from wild type Zikavirus.

In one embodiment of the present invention, the chimeric Zika virus may be substituted with the capsid protein of the attenuated yellow fever virus (C) protein of Zika virus.

The chimeric Zika virus may be substituted for a portion of the envelope (E) protein of the structural protein, a portion of the E protein may be the amino acid No. 792 of the chimeric Zika virus is substituted from S to G.

In an embodiment of the present invention, the sequence of the 102-121 amino acid position of the chimeric Zika virus may be substituted with GADTSVGIVGLLLTTAMA in SHDVLTVQFLILGMLLMTGG.

The sequence at amino acid position 104-121 of the chimeric zika virus may be substituted for GADTSVGIVGLLLTTAMA in DVLTVQFLILGMLLMTGG.

The chimeric Zika virus may be provided between the signal peptide of the chimeric Zika virus and the pr-M amino acid sequence (121-122).

The inserted amino acid may preferably be A. In particular, when the amino acid A was inserted into the site, it could be safe at the time of immunization, and less side effects can be observed.

The chimeric Zika virus of the TGG (119-121) amino acid of the signal peptide of the chimeric Zika virus can be provided.

TGG (119-121) amino acids of the signal peptide of the chimeric Zika virus may be substituted with AMA.

The inventors of the present invention confirmed that when the amino acid is substituted as described above, fewer side effects that may occur when inoculating live vaccines prepared using wild-type Zika virus were observed, and a safe immune-induced response can be expected in the body.

Immunogenic Composition

In one embodiment of the present invention may provide a Zika virus immunogenic composition capable of preventing, treating or ameliorating Zika virus infection. The immunogenic composition can be understood as a vaccine.

The present invention can provide a composition having both safety and immunogenicity. That is, the present invention may provide a composition having high safety while introducing an chimeric Zika virus according to an embodiment of the present invention into the body, while having an immuno-inducing action against Zika virus infection.

In one embodiment of the present invention may provide a chimeric Zika virus immunogenic composition comprising the chimeric Zika virus and immunologically acceptable excipients. The immunologically acceptable excipients can be used without limitation as long as the excipients used to prepare vaccines in the art.

In one embodiment of the present invention may be provided a method of inducing an immune response by administering to the subject the immunogenic composition comprising a pharmaceutically effective amount.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve a prophylactic effect against Zikavirus infection.

In one embodiment of the present invention can provide a Zika virus vaccine comprising the chimeric Zika virus and a pharmaceutically acceptable excipient, which is administered to an animal to induce immunity to Zika virus in the animal body.

The vaccine composition according to the present invention may further comprise a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers or excipients according to the invention are commonly used in pharmaceutical formulations and vaccines to improve the stability, sterilization and deliverability of the active agent and generate any secondary reactions in humans, eg allergic reactions. Any solvent, dispersion medium, or the like that is not intended. Excipients are selected based on the selected pharmaceutical form, method of administration and route. Suitable excipients and conditions associated with pharmaceutical formulations are well known to those skilled in the art.

Examples of pharmaceutically acceptable excipients may include water, PBS buffer solution and 0.3% glycine solution.

Vaccine compositions for use in the methods of the invention include pharmaceutically acceptable auxiliaries required to bring them closer to physiological conditions, such as pH adjusters and buffers, wetting agents, etc., for example sodium acetate, sodium lactate, sodium Chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, human serum albumin, essential amino acids, non-essential amino acids, L-arginine hydrochlorate, saccharose, D-trehalose dehydrate, sorbitol, tris ( Hydroxymethyl) aminomethane and / or urea. In addition, the vaccine composition may optionally include pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives.

Stabilizers can be used without limitation so long as they are generally used in vaccine compositions.

The vaccine of the present invention may be prepared in unit dose form by being formulated with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media or in the form of extracts, powders, granules, tablets or capsules, and may further include dispersants or stabilizers, but are not limited to such formulations.

Pharmaceutically acceptable carriers included in the vaccine of the present invention are conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. no. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

The animal can be used without limitation, for example can be a mammal, preferably human.

syndrome, 소두증(microcephaly), 신경이상, 선천성 기형 같은 중증 신경 질환뿐만 아니라, 발열, 발진, 관절통, 눈 충혈, 근육통, 두통, 안구통, 구토 등과 같은 지카바이러스 감염으로 인한 지카열과 연관된 증상들을 모두 포함할 수 있으며, 상기 질환에 한정되어 해석되지 않는다.In one embodiment of the invention can provide a Zika virus immunogenic composition for use in the treatment or prevention of diseases associated with Zika virus infection. Diseases associated with the Zika virus infection are Guillain-Barr

Including severe neurological disorders such as syndrome, microcephaly, neuropathy and congenital malformations, as well as symptoms associated with Zika fever from Zika virus infections such as fever, rash, arthralgia, eye redness, myalgia, headache, eye pain and vomiting It is possible to do so and is not limited to the above diseases.

In one embodiment of the present invention may be provided a method of inducing an immune response to Zika virus by administering the immunogenic composition to a subject.

As will be appreciated by those skilled in the art, the vaccine compositions of the present invention are formulated to suit the intended route of administration. Examples of suitable routes of administration may include, for example, intramuscular, transdermal, subcutaneous, mucosal or intradermal administration. Preferably, the route of administration may be administered by intramuscular route.

In one embodiment of the present invention can provide a use of the Zika virus immunogenic composition for the treatment or prevention of diseases associated with Zika virus infection.

In one embodiment of the present invention, the use of the Zika virus immunogenic composition for inducing an immune response to Zika virus infection patients can be provided.

The volume of the vaccine composition of the present invention to be administered may depend on the method of administration. For subcutaneous injection, the volume is generally 0.1 to 1.0 ml, preferably about 0.5 ml.

According to one embodiment, the invention also provides a kit comprising the vaccine composition of the invention and instructions for use of said vaccine composition in a method of protecting a human subject against Zikavirus.

The chimeric zikavirus vaccine or immunogenic composition of the present invention can be used for the prevention or treatment, preferably the prevention of various diseases caused by Zicavirus infection.

[ chimera Zika virus  Manufacturing method]

In one embodiment of the present invention provides a method for producing an immunogenic composition comprising a chimeric Zika virus.

The preparation method comprises the steps of i) preparing the attenuated yellow fever virus 17D vaccine strain,

ii) replacing the nucleotide sequence encoding the structural protein of the yellow fever virus 17D vaccine strain with the nucleotide sequence encoding the structural protein of the wild type Zika virus, to prepare a 17D- Zika chimeric genome, iii) cloning the genome into a vector Step, iv) introducing the cloned vector into a competent cell and transforming it, and v) recovering viral DNA from the competent cell.

As the vector used in the cloning to the vector, pCC1BAC ™ may be preferably used. The vector is a vector capable of copy number control. Usually, the vector can have one copy and is easy to amplify the copy in a short time.

An embodiment of the present invention may form a pCC1 (pCC1BAC ™) -chimeric Zikavirus vector using the pCC1 vector.

Since the 17D-Zika chimeric genome cloned in the vector of the present invention has a large size, it divides into several fragments and proceeds with DNA synthesis in vitro to clone the gene.

17D-Zika chimeric genome of the present invention is synthesized by arbitrarily divided into the size of, for example, 1) 3kbp, 2) 3kbp, 3) 3kbp, 4) 3kbp and the connection of 1) and 2) (A), 3) The linkage (B) and 4) and the linkage (A) and (B) can produce a total length of approximately 11 kbp, but the DNA fragment is not necessarily limited to the size.

The size of the genome can be prepared by a conventional method for cutting full length DNA and a conventional method for connecting DNA fragments to full length DNA in the art, and the method of DNA synthesis and cutting is not particularly limited.

Preferably, in one embodiment of the present invention, the 17D-Zika chimeric genome cloned into the vector comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. Can have

The pCC1-chimeric Zikavirus vector of the present invention is a restriction enzyme AscI coding sequence (5`-GGCGCGCC) at the 5 'end sequentially; T7 promoter sequence (TAATACGACTCACTATA); transcription start codon (GG); Nucleic acids of any one chimeric zika virus selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16; The restriction enzyme SmaI-NotI coding sequence (3'-GGGCCCCGCCGGCG) may be included at the 3 'end.

In one embodiment of the present invention may provide a method for producing chimeric Zika virus having immunogenicity against Zika virus infection.

(a) preparing a YF 17D genome which is an attenuated yellow fever virus vaccine having SEQ ID NO: 29, and (b) the base sequences 482 to 2452 of the YF 17D genome are selected from any one of SEQ ID NOs: 19 to 28. It may include the step of producing a chimeric Zika virus gene by replacing with a sequence.

The preparation method includes (c) adding a T7 promoter sequence (TAATACGACTCACTATA) and a transcription start codon (GG) at the 5 'end of the chimeric Zikavirus gene produced in step (b), and ascI ( 5′-GGCGCGCC) and SmaI-NotI (3′-GGGCCCCGCCGGCG) may be added to prepare for cloning into the vector.

The present invention has a prophylactic effect against Zika virus infection.

The present invention provides an attenuated chimeric zika virus vaccine.

Chimeric Zikavirus vaccines of the present invention can achieve attenuation without the need for a long time and a number of processes, particularly with respect to the attenuating problem that is particularly problematic in the case of live Zikavirus vaccines.

syndrome, 소두증(microcephaly), 신경이상, 선천성 기형 같은 중증 신경 질환을 예방할 수 있는 안전성이 뛰어난 지카바이러스 백신을 제공할 수 있다. The present invention is Guillain-Barr

It is possible to provide a highly safe Zikavirus vaccine that can prevent severe neurological diseases such as syndrome, microcephaly, neuropathy and congenital malformations.

When the chimeric Zika virus of the present invention is used as a vaccine, there are few side effects that may occur in the body, and thus it is excellent to provide a live Zika virus vaccine which has been difficult to use due to the side effects.

Vaccine of the present invention can prevent the reversion of the disease and induce a mesophase safe compared to the live vaccine prepared using wild-type Zika virus.

The present invention may be effective in the defense of disease through the induction of effective cellular immunity.

The present invention can induce the response of cytotoxic lymphocytes (CTL) can effectively prevent Zika virus infection.

In the present invention, by using the signal peptide of the YFV protein cleavage is more efficient, the productivity of the chimeric Zika virus in the culture process can be increased, and antigenicity can be increased during in vivo inoculation.

The present invention can help stabilize structural proteins of chimeric Zika virus by using TM regions of prM and E proteins of YFV. In addition, it is possible to increase the productivity and thermal stability of the chimeric Zika virus.

1 is a diagram illustrating a chimera virus production process.
Figure 2 is the gene structure of the chimeric Zika virus substituted with the yellow fever 17D vaccine strain and Zika virus major antigen gene used as a platform for chimeric virus production. Chimeric Zikavirus A corresponds to construct 1 of FIG. 3 and is the basic template of chimeric Zikavirus. Chimeric Zicavirus B is a form in which the TM region of prM and the TM region of E protein of chimeric Zicavirus A are replaced with 17D instead of Zica virus. Chimeric Zikavirus C is a form in which the capsid protein of 17D in Chimeric Zicavirus A is substituted with the capsid protein of Zica virus.
Figure 3 is an example of sequence variation of the signal peptide, prM, E protein that can be applied when the prM and E protein insertion of Zika virus in the 17D backbone. Six variants were designed from Construct 1 to 6.
Figure 4 shows the chimeric Zika virus produced through the cell insertion of the gene using immunochemical detection (A), Western blot (B), PCR amplification (C), respectively, E protein expression of Zika virus and whether the 17D gene is accompanied Is the result of checking.
5 is a result of verifying the attenuated trait of chimeric Zika virus in Vero cells compared to wild-type virus. Plaque forming (A), cytopathic effect (B) and cultured virus amount (C) were measured.
Figure 6 is a result of verifying the attenuated trait of chimeric Zika virus in MRC-5 cells compared to wild-type virus. Plaque forming (A), cytopathic effect (B) and cultured virus amount (C) were measured.
7 is a result of infecting wild type Zika virus and chimeric Zica virus by concentration in fertilized eggs and measuring mortality according to time after infection (DPI: Day Post Infection). ABC59-1, ABC59-2, and ABC59-3 show the results of ² × 10 ² pfu, 2 × 10 ³ pfu, and 5 × 10 ³ pfu injections of wild type Zikavirus PRVABC59, respectively. cZIKV-1, cZIKV-2 and cZIKV-3 show the results of ² × 10 ² pfu, 2 × 10 ³ pfu, and 5 × 10 ³ pfu injections of chimeric Zikavirus, respectively.
8 is a study measuring attenuation of chimeric zikavirus in BALB / c mice. A) is the result of measuring the body weight (BW) of the group after infection by virus concentration and B) is the result of measuring the amount of virus in blood after infection by q-PCR.
9 is a result of confirming the immune antibody formation after immunizing chimeric Zika virus in BALB / c mice. A) measured body weight change and B) measured E protein specific antibody titer by ELISA method.
Sham- VP-SFM
Immunity 1- cZIKV (chimera Zika virus) 10 ³ pfu / 200ul
Immune 2-cZIKV (chimeric zika virus) 10 ⁴ pfu / 200 ul
Immunization 3-cZIKV (chimeric zika virus) 10 ⁵ pfu / 200 ul
Immune 4- Inactivated wild type 10 ⁵ pfu / 200ul
Figure 10 shows the change in weight of Type I IFN KO mice after infection with chimeric Zikavirus A in attenuation tests in Type I IFN KO mice.
11 shows the weight change of Type I IFN KO mice after wild type Zika virus infection.
12 shows survival rate over time of mice after infection.
Figure 13 shows the extent of virus proliferation in Type I IFN KO mice. Attenuation of chimeric Zikavirus A can be confirmed.
Figure 14 is the result of confirming the immune effect in Type I IFN KO mice of chimeric Zika virus A. G1- Mock, G2- 10 ² pfu / mouse, G3- 10 ³ pfu / mouse, G4- 10 ⁴ pfu / mouse, G5- 10 ⁵ pfu / mouse
Figure 15 shows the weight change over time in Type I IFN KO mice inoculated with chimeric zikavirus A. G1- Mock, G2- 10 ² pfu / mouse, G3- 10 ³ pfu / mouse, G4- 10 ⁴ pfu / mouse, G5- 10 ⁵ pfu / mouse
FIG. 16 shows virus proliferation and survival for infection of wild type PRVABC59 in Type I IFN KO mice inoculated with chimeric zikavirus A. FIG.
17 shows the results of Vero cell culture of chimeric zikaviruses A and B. FIG.
Figure 18 shows the results of the thermostability test of chimeric Zika virus A and B.
Figure 19 shows the weight change, virus proliferation, survival rate of Type I IFN KO mice infected with chimeric Zikavirus A and B and wild type virus PRVABC59.
FIG. 20 shows survival, total and neutralizing antibody titers for wild type PRVABC59 infection in Type I IFN knock out mice immunized with chimeric Zikavirus A and B. FIG.

Hereinafter, the following examples and the like will be described to more specifically describe the present invention. However, embodiments according to the present invention can be modified in many different forms and the scope of the present invention should not be construed as being limited to the embodiments described below. Embodiments of the present invention are provided by way of example in order to facilitate a specific understanding of the present invention.

I. Chimera Zika virus production

1. Preparation of Zika Virus

ATCC VR-1843 wild-type Zika virus was prepared. The sequence can be readily obtained via Genbank No: KU365778.1 (Virus strain; BeH819015).

2. Preparation of 17D Virus, Yellow Fever Vaccine

Yellow fever vaccine strain 17D virus was prepared. It is readily available via GenBank No: X03700.1.

3. Gene Design and Synthesis of Chimeric Zika Virus

Genetic information and sequencing of 17D virus and Zika virus, a yellow fever vaccine strain, are conducted. For 17D vaccine strains, the full genome of GenBank No: X03700.1 published in NCBI was used for analysis. In general, proteins that act as major antigens in flaviviruses are known as pr-M (membrane protein) and E protein (envelope protein, envelope protein). Therefore, the gene investigation of these parts was carried out, and as a result, in the case of 17D, 482-973 nt sites were pr-M genes, and 974-2452 nt sites were E protein genes. Zika virus was used to analyze the gene of Genbank No: KU365778.1 (Virus strain; BeH819015) published in NCBI, 470 ~ 964 nt site is pr-M gene, 965 ~ 2476 nt site is E protein gene It was. Based on the identified gene structure, the 17D gene was prototyped and replaced with the homologous gene of Zika virus instead of the 17D prM and E protein genes, thereby designing a 10907 bp chimeric Zika virus gene. The T7 promoter sequence (TAATACGACTCACTATA) and the transcription start codon (GG) were inserted in sequence at the 5 'end of the virus, and the restriction enzymes AscI (5`-GGCGCGCC) and SmaI-NotI (3'-GGGCCCCGCCGGCG) were inserted at both ends. Each added. The designed sequence was a total of 10948 bp, and because of its large size, the gene was cloned by DNA synthesis in vitro by dividing into several fragments. The genes thus obtained were processed to connect the respective fragments to make a single gene, and the 10948 bp full length was finally cloned into the pCC1 vector (BAC cloning kit, Epicentre) to produce the pCC1-Chimeric ZIKV vector.

4. Chimeric Zika Virus Mutation Gene Design

Based on Example 1, the 17D virus and the Zika virus genes can be arranged in various combinations, or the original nucleotide sequence can be replaced with another nucleotide sequence to provide all or part amino acid sequence variations.

As an example, a 17D-Zika chimeric full length genome (10907 bp) was designed in which the gene sequence encoding the corresponding 123-794 aa sequence of Zika virus was inserted instead of the 122-778 aa portion of the 17D protein sequence (Fig. Chimeric zika virus A).

In one example, the prM and E proteins have transmembrane regions (TM, amino acid sequences that penetrate the lipid bilayer structure) at the C-terminal end. This part interacts with the capsid protein to form a viral globular structure that affects assembly and budding smoothly. Therefore, based on the chimeric Zika virus A of FIG. 2, the 17M E-protein TM was replaced with the pr-M TM sequence of 17D (NPFFAVTALTIAYLVGSNMTQRVVIALLVLAVGPAYS) instead of 253-289 aa, which is a prM TM. Chimeric Zikavirus B is designed by substitution with the sequence (LNWITKVIMGAVLIWVGINTRNMTMSMSMILVGVIMMFLSLGVGA). It has an amino acid sequence of SEQ ID NO: 13 and a nucleotide sequence of SEQ ID NO: 14.

In one example, one of the structural genes, the capsid, carries a viral genome in the spherical space and interacts with the envelope (envelope) of the virus and plays a very important role in the formation of the viral structure. Therefore, instead of the 17D capsid gene used as a backbone in flavivirus chimera gene construction, the homologous Zikavirus capsid gene can be introduced together with the prM and E genes. Therefore, based on the chimeric Zika virus A of FIG. The amino acid sequence of SEQ ID NO: 15 and the nucleotide sequence of SEQ ID NO: 16.

In one example, a flavivirus is translated from RNA into a protein that is cleaved by viral protease and host cellular protease and broken down into several proteins with separate functions. Thus, the amino acid sequence site from which each functional protein is cleaved can have a significant effect on the growth of the virus. Therefore, protease action may be affected in the hinge region where heterologous sequences are linked. Thus, sequence variation can be carried out so that the capsid and prM linkage sequences and pr-M linkage sequences facilitate cleavage of the protease.

As shown in FIG. 3, various types of variants (constructs 1 to 6) can be designed.

Construct 1 is the structure of chimeric Zika virus A of FIG. All variants can be constructed on a construct 1 basis. It has an amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO: 2.

Construct 2 is a form in which amino acid 792 of the chimeric zika virus is substituted from S to G. It has an amino acid sequence of SEQ ID NO: 3 and a nucleotide sequence of SEQ ID NO: 4.

Construct 3 is a form in which SHDVLTVQFLILGMLLMTGG, which is an amino acid sequence corresponding to a signal peptide (102-121 aa), is replaced with GADTSVGIVGLLLTTAMA. The amino acid sequence of SEQ ID NO: 5 and the nucleotide sequence of SEQ ID NO: 6.

Construct 4 is a form in which the DVLTVQFLILGMLLMTGG, the amino acid sequence corresponding to the signal peptide (104-121 aa), is replaced with GADTSVGIVGLLLTTAMA. The amino acid sequence of SEQ ID NO: 7 and the nucleotide sequence of SEQ ID NO: 8.

Construct 5 is a form in which one amino acid A is inserted between the signal peptide of the construct 1 and the pr-M amino acid sequence (121-122 aa). It has an amino acid sequence of SEQ ID NO: 9 and a nucleotide sequence of SEQ ID NO: 10.

Construct 6 is a form in which TGG (119-121 aa) of a signal peptide is substituted with three amino acids AMA. It has an amino acid sequence of SEQ ID NO: 11 and a nucleotide sequence of SEQ ID NO: 12.

5. Production of virions through chimeric Zika virus RNA production and cell insertion

To produce chimeric Zika virus gene, pCC1-Chimeric ZIKV vector is transformed into EPI300 competent cell (Epicentre) and mass produced. pCC1BAC ™ is a vector capable of copy number control. It usually has one copy and can be amplified to 10-20 copies in a short time by the addition of an induction solution. Therefore, after inoculating Chloramphenicol-containing LB medium with 5 ml of culture solution for 24 hours, inoculate 50 ml of fresh LB medium again and add induction solution (Epicentre) to recover bacteria with increased copy number of vector. From the harvested bacteria, plasmid purification is performed using midi prep kit (Qiagen).

Afterwards, chimeric Zika virus DNA is amplified by PCR using pCC1-Chimeria ZIKV vector as a template. At this time, the polymerase used can synthesize a base of 10 kb or more, and it is easy to use a product having high efficiency and accuracy (for example, PrimeSTAR GXL DNA polymerse, Clontech). In vitro transcription using T7 RNA polymerase is carried out using the produced chimeric Zika virus DNA (HiScribe T7 kit, NEB). Remove all DNA used as template by adding DNase I, precipitate RNA at -20 ℃ for 20 minutes using LiCl, wash twice with 70% ethanol and dry, and dissolve RNA in RNase free purified water. RNA is very easy to degrade, so DEPC is processed or RNase free conditions and reagents are used throughout the process.

Table 1 below shows primer information for amplifying chimeric Zika virus DNA through PCR using pCC1-Chimeria ZIKV vector as a template.

Forward 5'-TAA TAC GAC TCA CTA TAG GAG TAA ATC CTG Reverse 5'-AGT GGT TTT GTG TTT GTC ATC

Table 2 below shows the PCR compositions.

5X primeStar Buffer 10 ul dNTP (2.5 mM each) 4 ul Primer (10 pmole) -F 1 ul Primer (10 pmole) -R 1 ul Template (5 ng / ul) 2 ul Prime STAR polymerase 0.5 ul TDW 31.5 ul Total 50 ul

Table 3 below shows the in vitro transcription reaction composition.

2X ARCA / NTP mix 10 ul Template DNA 8 ul T7 polymerase mix 2 ul Total 20 ul

Vero cells are incubated in DMEM medium containing 10% FBS. For transfection, prepare cells in a 12 well plate at a cell concentration of 0.2 x 10 ⁶ cell / well.

Next day, the synthesized RNA is transfected using lipofectamine 3000. Each RNA and reagent ratio was 1: 1 and reacted for 10 minutes, and the cells were transfected. RNA used in the experiment was a concentration of 0.5ug / well. After transfection, the cells were cultured for 15-20 days to observe cell degeneration.

6. Detection and Identification of Chimeric Zika Virus

In this example, RNA carrying the chimeric Zika virus gene was transfected into Vero cells, and virus culture was harvested 20 days thereafter. By performing immunochemical detection, PCR, Western blot, and gene sequencing using the recovered culture, it was confirmed that the produced virus was a virion having both the 17D vaccine strain and the gene and protein of prM, E, the Zika virus antigen gene.

The chimeric Zika virus produced was assessed to be able to proliferate, to express the Zica virus antigen and to be accompanied by a virion with a gene sequence consistent with the inserted gene sequence. For this purpose, cell denaturation confirmation, detection of E protein, 5`UTR, 3`UTR, NS gene sequence amplification method were used.

Some of the chimeric zika virus cultures (eg 0.01-1 ml of culture) were inoculated into Vero cells and monitored for degeneration. Zika virus is known to cause cell degeneration in Vero cells.

In one example, a portion of the chimeric Zika virus culture (for example 0.01 ~ 0.1ml of the culture) is inoculated in Vero cells of 8 well chamber slide and incubated for 2 days. Infected cells were fixed at -20 ° C with 100% cold methanol and then reacted with anti-Flavivirus E antibody (D1-4G2-4-15, Merck) for 2 hours. After washing with PBS three times, the anti-mouse IgG-Alexa488 antibody (Thermo Fisher) was reacted for 1 hour. Washed five times with PBS, stained with DAPI and detected by fluorescence microscopy. As shown in A) of FIG. 4, E protein is expressed in cells infected with chimeric Zika and wild-type virus by specific antibodies. However, no signal was detected if the viral infection was not a cell.

In one example, cells are harvested on day 2 after infection with the chimeric zika virus. After cell lysis, some (eg 0.02ml) were electrophoresed using 10% acrylamide SDS gel and transferred to PVDF membrane to perform western blot. Anti-Zika Env mAb (GT871, GeneTex) was reacted for 2 hours and then anti-mouse-HRP antibody was reacted for 1 hour to detect Zikavirus E protein. ECL substrate solution is added and photosensitive to the film. As shown in B) of FIG. 4, no signal was detected in Vero cell control (lane 2), whereas E protein was detected in cells infected with chimeric virus (lane 1) and wild type virus (land 3). The molecular weight of the E protein is expected to be approximately 55KDa and detected at similar sizes.

In one example, viral RNA is purified using a portion of the chimeric Zika virus culture (eg 0.2 ml) (PureLink viral RNA / DNA kit, Thermo Fisher). Using the isolated RNA as a template, RT-PCR / PCR one-step kit is used to check whether a specific sequence is specifically amplified. As shown in C) of FIG. 4, the 5DUTR, 3UTUT, and NS genes of 17D were amplified to the expected sizes, respectively. Subsequently, the entire chimeric Zika virus genome was sequenced and found to have a gene 100% identical to that of the first designed synthetic gene (Construct 1).

II. chimera Zika virus  A (Construct 1) chimera Zika virus  Immunogenicity Check

1.In vitro attenuation test of Construct 1 chimeric Zika virus

To demonstrate the attenuated trait of chimeric zika virus by in vitro assay, attenuated trait was confirmed by infecting Vero and MRC-5 cells with wild-type Zika virus and chimeric Zica virus, respectively, and measuring cell degeneration and proliferated virus titer. It was.

To investigate the attenuated trait of chimeric zika virus, we compared it with wild-type Zika virus in terms of plaque formation, proliferative capacity, and cytopathic effects. First, a plaque forming assay is performed using Vero cells and MRC-5 cells, followed by crystal violet staining. In the results of Fig. 5A) and 6A), the wild-type virus PRVABC59 forms a clear and large plaque, whereas the chimeric Zica virus forms a faint plaque in Vero cells and no plaque in MRC-5 cells. Wild-type Zika virus is PRVABC59 virus isolated from the sera of patients from Puerto Rico in 2015 (VR-1843, ATCC).

In addition, after infecting the virus with 0.01MOI in Vero cells and MRC-5 cells, the cytopathic effect (CPE) was observed for 7 days. And 0.3 ml each of the culture solution was collected at the same time at 1 day intervals after infection and frozen and stored at -70 ℃. The virus proliferation was then analyzed using a plaque assay and Zika q-PCR kit (zika virus polyprotein gene genesig advanced kit, Genesig).

In comparison of cell degeneration of FIG. 5B and FIG. 6B), wild-type virus PRVABC59 was observed in both Vero cell (4 days culture) and MRC-5 cell (7 days culture). In the case of viral infection, cell degeneration was low or rarely observed. As a result of examining the proliferative power by measuring the virus titer in the collected culture, it was confirmed that PRVABC59, a wild-type virus, proliferated to a higher number as in C) and C) of FIG. 5.

5 and 6 show attenuation of attenuated cells using human primary lung cells as normal cells and Vero cells having good virus propagation due to no type I interferon signal as one cancer cell. In both Vero cells and primary human lung cells, chimeric Zika virus was attenuated more than wild-type virus in terms of plaque formation, cell degeneration, and virus proliferation.

2. In vivo Attenuation Test of Chimeric Zika Virus

To investigate the attenuated trait of chimeric Zika virus, egg mortality and virus proliferation in mice were measured after virus infection.

As an example, fertilized eggs were incubated in an incubator (37 ° C., 60% humidity), the shell of the air sac was opened on day 4, and viruses were injected into the amniotic cavity by concentration using a Hamilton syringe. The titers of each of the wild type Zikaviruses PRVABC59 and chimeric Zicaviruses injected were 2 × 10 ² pfu, 2 × 10 ³ pfu, and 5 × 10 ³ pfu. After infection, the opened shell was sealed with parafilm to prevent drying, hatching again in an incubator, and mortality was measured. In the result of FIG. 7A, both the wild-type PRVABC59 virus and the chimeric Zika virus showed a tendency to increase the mortality, and the wild type PRVABC59 virus had a higher mortality rate than the chimeric Zika virus.

To demonstrate the attenuated trait of the chimeric zika virus in vivo, attenuation of the chimeric zika virus was confirmed by intravenous injection of the virus into BALB / c mice and the change in weight and virus proliferation in serum.

In one example, the virus is injected into the veins of 8 week old BALB / c female mice at concentrations of 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ pfu / 200 ul. Serum was isolated for 1-6 days after injection and analyzed for proliferation using Zika q-PCR kit (Zika virus polyprotein gene genesig advanced kit, Genesig). When wild-type Zika virus is infected in BALB / c mice, it is known to be able to proliferate at a concentration of 10 ⁶ to 10 ⁷ copy / ml in serum between 2 and 5 days after infection, but the chimeric Zika virus is infected by concentration in FIG. 8 results. As a result, there was no difference in weight change among the groups, and virus proliferation was not detected in all groups.

The technique of inoculating a virus into fertilized eggs is one of the main methods used to culture the virus as in influenza or to evaluate the risk and cytotoxicity of the virus. The literature reports that early stage chicken embryos are susceptible to Zika virus. The results of FIG. 7 show that the chimeric Zika virus was attenuated by showing a low mortality rate compared to the wild type. In addition, Zika virus shows little disease in normal immune-competent mice. Unlike other mice, BALB / c mice are known to be susceptible to Zika virus and show viremia in blood upon intravenous injection (Rafael A. Larocca et al, Vaccine protection against Zika virus from Brazil, 2016). However, as can be seen in FIG. 8B), chimeric Zika virus did not proliferate at all in BALB / c mice, indicating that it was attenuated.

3. Immuno -competent mouse ( BALB / c) chimera Zika virus  Immune Antibody Formation Assay

In order to prove whether the chimeric Zika virus produced in the present invention is safe in mammals and induces an effective immune response, various concentrations of Chimeric Zica virus are injected into the muscles of BALB / c mice and serum is recovered. Check the formation. The change in weight due to viral immunity was also measured.

Four-week-old BALB / c mouse muscles were injected with concentrations of formalin inactivated wild type virus 10 ⁵ pfu / 200 ul and chimeric zika virus at 10 ³ , 10 ⁴ , 10 ⁵ pfu / 200 ul. Immunization was carried out in the same conditions at two total two-week intervals and serum was separated two weeks after the last immunization. Serum was stored at −20 ° C., and then analyzed for IgG antibodies specific to E protein using RecombiVirus ™ Zika Virus Envelop antibody ELISA kit (Alpha diagnostic). As can be seen in FIG. 9B), the E protein antibody titer was increased in a concentration-dependent manner, and a high level of antibody was induced in the group injected with live chimeric Zika virus in contrast to the same concentration of inactivated wild type virus group. In addition, as can be seen in Figure 9 A), when the weight change of the mouse was observed for 4 weeks after immunization, it was confirmed that there is no change due to the disease of the virus. The results indicate that live chimeric Zika virus proliferates smoothly in mammals to induce sufficient immunity and at the same time does not attenuate pathogenicity.

4. Immuno -compromised mouse (Type I IFN  EN) chimera Zika virus  Attenuation test

Type I IFN knock out mice are mice lacking innate immunity due to a lack of interferon (IFN) alpha and beta, which inhibit viral proliferation. Therefore, it is highly susceptible to Zika virus, making it suitable for use as an animal model. In general, immuno-competent animals are insensitive to innate immune responses.

Type I IFN KO mice were infected with chimeric Zika virus or wild type Zika virus solution by IP (Intraperitoneal Administration) at concentrations (10 ² to 10 ⁶ pfu / mouse). Serum was separated by date after infection, and changes in body weight and mortality were measured.

group Substance Dosage (pfu / head) Dosing method G1 Vehicle (VP-SFM) - IP (intraperitoneal injection) G2 Vaccine 1 (cZIKV) 1 ⅹ 10 ² G3 Vaccine 2 (cZIKV) 1 ⅹ 10 ³ G4 Vaccine 3 (cZIKV) 1 ⅹ 10 ⁴ G5 Vaccine 4 (cZIKV) 1 ⅹ 10 ⁵ G6 Vaccine 5 (cZIKV) 1 ⅹ 10 ⁶ G7 WT 1 (ABC59) 1 ⅹ 10 ² G8 WT 2 (ABC59) 1 ⅹ 10 ³ G9 WT 3 (ABC59) 1 ⅹ 10 ⁴ G10 WT 4 (ABC59) 1 ⅹ 10 ⁵ G11 WT 5 (ABC59) 1 ⅹ 10 ⁶

-G1 = Sham = Mock VP-SFM (Media) * G2 ~ G11 Virus Injection

The wild type Zika virus significantly reduced the body weight and increased the virus in the blood. The mortality rate was also high. However, when the chimeric Zika virus was infected, less weight loss, lower virus propagation and higher survival rate were observed than those of wild type virus. This shows that the chimeric Zika virus is attenuated and can safely propagate in organisms. FIG. 10 shows the weight change of mice infected with construct A virus, and FIG. 11 shows the weight change of mice infected with wild-type Zika virus. On the other hand, Figure 12 is a result of comparing the survival rate of mice infected with the construct A virus and wild-type virus, showing a higher mortality rate when wild type virus infection. This means that the chimeric zika virus is attenuated and can safely propagate in organisms.

In particular, Figure 13 confirms the virus proliferation results, it was confirmed that the cZIKV attenuated due to the relatively low virus growth compared to WT.

5. Immuno -compromised mouse (Type I IFN  EN) chimera Zika virus  Immune Antibody Formation Assay

Type I IFN KO mouse chimeric Dermalogica baeksinju (virus solution) concentrations ^{(10 2 ~ 10 5 pfu /} mouse) 2 times (2 weeks intervals) with the IM in the immunization was.

group Substance Dosage (pfu / head) Immunity Dosing method G1 Vehicle - 2 IM
intramuscular injection;
Intramuscular injection) G2 Vaccine 1 1 ⅹ 10 ² G3 Vaccine 2 1 ⅹ 10 ³ G4 Vaccine 3 1 ⅹ 10 ⁴ G5 Vaccine 4 1 ⅹ 10 ⁵

In order to prove that the chimeric Zika virus construct A produced in the present invention is safe in mammals and induces an effective immune response, various concentrations of chimeric Zica virus are injected into the muscle and serum is recovered to form antigen-specific antibodies. Check it. The change in weight due to viral immunity was also measured.

In the concentrations of Table 5, the cells were immunized under the same conditions at two total intervals of two weeks, and serum was separated two weeks after the last immunization. Serum was stored at −20 ° C., and then analyzed for IgG antibody specific for E protein using RecombiVirus ™ Zika Virus Envelop antibody ELISA kit (Alpha diagnostic). As can be seen in Figure 14, E protein antibody titer was increased in a concentration-dependent manner, showed a high level of neutralizing antibody titer.

In addition, as can be seen in Figure 15, it was confirmed that there is no weight change due to the pathology of the virus during the immune period. The survival rate and virus propagation results are shown in FIG. 16.

III. Confirmation of Immunogenicity of Chimeric Zika Virus of SEQ ID NO: 13

One. chimera Zika virus  Comparison of virus growth of A and B

(1) Experimental method

Chimeric Zikaviruses A and B are infected with Vero cells at 0.005 MOI and the cytopathic effect (CPE) is observed for 8 days. And 0.3 ml each of the culture solution was collected at the same time every day after infection and stored frozen at -70 ℃. Plaque forming assay of 0, 1, 2, 3, 4, 6, 8th day samples was carried out, crystal violet staining was performed, and plaque was counted.

(2) Experiment result

The results are shown in FIG. The titer of B was about 3 to 10 times higher than that of chimeric Zika virus A. The highest titer of the 8-day cultures was Zika virus A (4day) and Zika virus B (6day). This may be due to the increased structural stability of Zikavirus B virus, which maintains its infectivity over time. As a result of the Plaque assay, the chimer titer was found to be 4E5 and 3E6 for chimeric Zika virus A, which showed better growth of chimeric Zika virus B.

2. chimera Zika virus  Of A and B thermostability  Check and compare

(1) Experimental method

For thermostability testing, changes in infectious virus titer were measured while the same concentration of virus was stored at different temperatures of 4, 37 and 60 ° C. for 72 hours. Some samples were taken at 1, 6, 14, 24, 48 and 72h after the start of the experiment and used for analysis. Infectious virus titer was measured by plaque assay using Vero cells. Relative infectious viral titers were calculated with a titer of 100 before the temperature treatment (0hr).

(2) Experiment result

The result was shown in FIG. It was confirmed that the infectivity of chimeric Zikavirus B was better maintained than that of A virus at various temperature conditions of 4, 37 and 60 ℃. This has advantages in many aspects, including the productivity of the virus, the production of the vaccine, maintenance of effectiveness, distribution and storage of the vaccine.

3.Type I IFN  From KO mouse Vaccine  Of A and B Attenuation  Check and compare

(1) Experimental method

Four-week-old Type I IFN knock-out mice were IP-infected with chimeric Zika virus vaccines A, B, and PRVABC59 at different concentrations, and were compared with changes in body weight, virus proliferation, and lethality.

group Substance Dosage (pfu / head) Dosing method Sham Vehicle (VP-SFM) - IP ConA-E2 Chimeric Zika Virus A 1 ⅹ 10 ² ConA-E5 Chimeric Zika Virus A 1 ⅹ 10 ⁵ ConB-E2 Chimeric Zika Virus B 1 ⅹ 10 ² ConB-E5 Chimeric Zika Virus B 1 ⅹ 10 ⁵ ABC-E2 Wild-type (ABC59) 1 ⅹ 10 ² ABC-E5 Wild-type (ABC59) 1 ⅹ 10 ⁵

(2) Experiment result

The result was shown in FIG. Body weight change showed the greatest decrease of ABC59 by 30-60% compared to the initial weight, and the chimeric Zika virus A and B showed a decrease of about 10% for 5 days after injection and then recovered.

Viremia was the highest in ABC59, and chimeric Zikaviruses A and B showed similar patterns and levels.

Survival was observed only in ABC59, and chimeric Zikavirus A and B showed 100% survival.

4.Type I IFN  From KO mouse Vaccine  Check and compare the effects of A and B

(1) Experimental method

Four-week-old Type I IFN knock-out mice were immunized twice with IM every two weeks at two concentrations of chimeric Zika virus vaccine, and challenged with wild-type Zika virus PRVABC59 two weeks after the last immunization.

group Substance Dosage
(pfu / head) immune
Count administration
Way Sham Vehicle - 2 IM ConA-E2 Construct A 1 ⅹ 10 ² ConA-E5 Construct A 1 ⅹ 10 ⁵ ConB-E2 Construct B 1 ⅹ 10 ² ConB-E5 Construct B 1 ⅹ 10 ⁵

(2) Experiment result

The result was shown in FIG. The test result showed that E protein-specific total antibody showed good antibody induction depending on the dose of chimeric Zikavirus A and B. The difference in antibody concentration between virus A and B was not significant.

In the case of neutralizing antibodies, the concentrations of neutralizing antibodies were high regardless of the administration concentrations, and the results of A and B viruses showed similar values.

Survival measurements after wild-type virus infection resulted in 100% protection against PRVABC59 for all individuals immunized with chimeric Zikavirus A or B.

<110> SK bioscience Co., Ltd. <120> Chimeric Zika virus vaccine <130> P18-147 <150> KR 10-2018-0052156 <151> 2018-05-04 <160> 29 <170> KoPatentIn 3.0 <210> 1 <211> 3426 <212> PRT <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 1 amino acid sequence <400> 1 Met Ser Gly Arg Lys Ala Gln Gly Lys Thr Leu Gly Val Asn Met Val   1 5 10 15 Arg Arg Gly Val Arg Ser Leu Ser Asn Lys Ile Lys Gln Lys Thr Lys              20 25 30 Gln Ile Gly Asn Arg Pro Gly Pro Ser Arg Gly Val Gln Gly Phe Ile          35 40 45 Phe Phe Phe Leu Phe Asn Ile Leu Thr Gly Lys Lys Ile Thr Ala His      50 55 60 Leu Lys Arg Leu Trp Lys Met Leu Asp Pro Arg Gln Gly Leu Ala Val  65 70 75 80 Leu Arg Lys Val Lys Arg Val Val Ala Ser Leu Met Arg Gly Leu Ser                  85 90 95 Ser Arg Lys Arg Arg Ser His Asp Val Leu Thr Val Gln Phe Leu Ile             100 105 110 Leu Gly Met Leu Leu Met Thr Gly Gly Ala Glu Val Thr Arg Arg Gly         115 120 125 Ser Ala Tyr Tyr Met Tyr Leu Asp Arg Asn Asp Ala Gly Glu Ala Ile     130 135 140 Ser Phe Pro Thr Thr Leu Gly Met Asn Lys Cys Tyr Ile Gln Ile Met 145 150 155 160 Asp Leu Gly His Met Cys Asp Ala Thr Met Ser Tyr Glu Cys Pro Met                 165 170 175 Leu Asp Glu Gly Val Glu Pro Asp Asp Val Asp Cys Trp Cys Asn Thr             180 185 190 Thr Ser Thr Trp Val Val Tyr Gly Thr Cys His His Lys Lys Gly Glu         195 200 205 Ala Arg Arg Ser Arg Arg Ala Val Thr Leu Pro Ser His Ser Thr Arg     210 215 220 Lys Leu Gln Thr Arg Ser Gln Thr Trp Leu Glu Ser Arg Glu Tyr Thr 225 230 235 240 Lys His Leu Ile Arg Val Glu Asn Trp Ile Phe Arg Asn Pro Gly Phe                 245 250 255 Ala Leu Ala Ala Ala Ala Ile Ala Trp Leu Leu Gly Ser Ser Thr Ser             260 265 270 Gln Lys Val Ile Tyr Leu Val Met Ile Leu Leu Ile Ala Pro Ala Tyr         275 280 285 Ser Ile Arg Cys Ile Gly Val Ser Asn Arg Asp Phe Val Glu Gly Met     290 295 300 Ser Gly Gly Thr Trp Val Asp Val Val Leu Glu His Gly Gly Cys Val 305 310 315 320 Thr Val Met Ala Gln Asp Lys Pro Thr Val Asp Ile Glu Leu Val Thr                 325 330 335 Thr Thr Val Ser Asn Met Ala Glu Val Arg Ser Tyr Cys Tyr Glu Ala             340 345 350 Ser Ile Ser Asp Met Ala Ser Asp Ser Arg Cys Pro Thr Gln Gly Glu         355 360 365 Ala Tyr Leu Asp Lys Gln Ser Asp Thr Gln Tyr Val Cys Lys Arg Thr     370 375 380 Leu Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly 385 390 395 400 Ser Leu Val Thr Cys Ala Lys Phe Ala Cys Ser Lys Lys Met Thr Gly                 405 410 415 Lys Ser Ile Gln Pro Glu Asn Leu Glu Tyr Arg Ile Met Leu Ser Val             420 425 430 His Gly Ser Gln His Ser Gly Met Ile Val Asn Asp Thr Gly His Glu         435 440 445 Thr Asp Glu Asn Arg Ala Lys Val Glu Ile Thr Pro Asn Ser Pro Arg     450 455 460 Ala Glu Ala Thr Leu Gly Gly Phe Gly Ser Leu Gly Leu Asp Cys Glu 465 470 475 480 Pro Arg Thr Gly Leu Asp Phe Ser Asp Leu Tyr Tyr Leu Thr Met Asn                 485 490 495 Asn Lys His Trp Leu Val His Lys Glu Trp Phe His Asp Ile Pro Leu             500 505 510 Pro Trp His Ala Gly Ala Asp Thr Gly Thr Pro His Trp Asn Asn Lys         515 520 525 Glu Ala Leu Val Glu Phe Lys Asp Ala His Ala Lys Arg Gln Thr Val     530 535 540 Val Val Leu Gly Ser Gln Glu Gly Ala Val His Thr Ala Leu Ala Gly 545 550 555 560 Ala Leu Glu Ala Glu Met Asp Gly Ala Lys Gly Arg Leu Ser Ser Gly                 565 570 575 His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Arg Leu Lys Gly Val             580 585 590 Ser Tyr Ser Leu Cys Thr Ala Ala Phe Thr Phe Thr Lys Ile Pro Ala         595 600 605 Glu Thr Leu His Gly Thr Val Thr Val Glu Val Gln Tyr Ala Gly Thr     610 615 620 Asp Gly Pro Cys Lys Val Pro Ala Gln Met Ala Val Asp Met Gln Thr 625 630 635 640 Leu Thr Pro Val Gly Arg Leu Ile Thr Ala Asn Pro Val Ile Thr Glu                 645 650 655 Ser Thr Glu Asn Ser Lys Met Met Leu Glu Leu Asp Pro Pro Phe Gly             660 665 670 Asp Ser Tyr Ile Val Ile Gly Val Gly Glu Lys Lys Ile Thr His His         675 680 685 Trp His Arg Ser Gly Ser Thr Ile Gly Lys Ala Phe Glu Ala Thr Val     690 695 700 Arg Gly Ala Lys Arg Met Ala Val Leu Gly Asp Thr Ala Trp Asp Phe 705 710 715 720 Gly Ser Val Gly Gly Ala Leu Asn Ser Leu Gly Lys Gly Ile His Gln                 725 730 735 Ile Phe Gly Ala Ala Phe Lys Ser Leu Phe Gly Gly Met Ser Trp Phe             740 745 750 Ser Gln Ile Leu Ile Gly Thr Leu Leu Met Trp Leu Gly Leu Asn Thr         755 760 765 Lys Asn Gly Ser Ile Ser Leu Met Cys Leu Ala Leu Gly Gly Val Leu     770 775 780 Ile Phe Leu Ser Thr Ala Val Ser Ala Asp Gln Gly Cys Ala Ile Asn 785 790 795 800 Phe Gly Lys Arg Glu Leu Lys Cys Gly Asp Gly Ile Phe Ile Phe Arg                 805 810 815 Asp Ser Asp Asp Trp Leu Asn Lys Tyr Ser Tyr Tyr Pro Glu Asp Pro             820 825 830 Val Lys Leu Ala Ser Ile Val Lys Ala Ser Phe Glu Glu Gly Lys Cys         835 840 845 Gly Leu Asn Ser Val Asp Ser Leu Glu His Glu Met Trp Arg Ser Arg     850 855 860 Ala Asp Glu Ile Asn Ala Ile Phe Glu Glu Asn Glu Val Asp Ile Ser 865 870 875 880 Val Val Val Gln Asp Pro Lys Asn Val Tyr Gln Arg Gly Thr His Pro                 885 890 895 Phe Ser Arg Ile Arg Asp Gly Leu Gln Tyr Gly Trp Lys Thr Trp Gly             900 905 910 Lys Asn Leu Val Phe Ser Pro Gly Arg Lys Asn Gly Ser Phe Ile Ile         915 920 925 Asp Gly Lys Ser Arg Lys Glu Cys Pro Phe Ser Asn Arg Val Trp Asn     930 935 940 Ser Phe Gln Ile Glu Glu Phe Gly Thr Gly Val Phe Thr Thr Arg Val 945 950 955 960 Tyr Met Asp Ala Val Phe Glu Tyr Thr Ile Asp Cys Asp Gly Ser Ile                 965 970 975 Leu Gly Ala Ala Val Asn Gly Lys Lys Ser Ala His Gly Ser Pro Thr             980 985 990 Phe Trp Met Gly Ser His Glu Val Asn Gly Thr Trp Met Ile His Thr         995 1000 1005 Leu Glu Ala Leu Asp Tyr Lys Glu Cys Glu Trp Pro Leu Thr His Thr    1010 1015 1020 Ile Gly Thr Ser Val Glu Glu Ser Glu Met Phe Met Pro Arg Ser Ile 1025 1030 1035 1040 Gly Gly Pro Val Ser Ser His Asn His Ile Pro Gly Tyr Lys Val Gln                1045 1050 1055 Thr Asn Gly Pro Trp Met Gln Val Pro Leu Glu Val Lys Arg Glu Ala            1060 1065 1070 Cys Pro Gly Thr Ser Val Ile Ile Asp Gly Asn Cys Asp Gly Arg Gly        1075 1080 1085 Lys Ser Thr Arg Ser Thr Thr Asp Ser Gly Lys Val Ile Pro Glu Trp    1090 1095 1100 Cys Cys Arg Ser Cys Thr Met Pro Pro Val Ser Phe His Gly Ser Asp 1105 1110 1115 1120 Gly Cys Trp Tyr Pro Met Glu Ile Arg Pro Arg Lys Thr His Glu Ser                1125 1130 1135 His Leu Val Arg Ser Trp Val Thr Ala Gly Glu Ile His Ala Val Pro            1140 1145 1150 Phe Gly Leu Val Ser Met Met Ile Ala Met Glu Val Val Leu Arg Lys        1155 1160 1165 Arg Gln Gly Pro Lys Gln Met Leu Val Gly Gly Val Val Leu Leu Gly    1170 1175 1180 Ala Met Leu Val Gly Gln Val Thr Leu Leu Asp Leu Leu Lys Leu Thr 1185 1190 1195 1200 Val Ala Val Gly Leu His Phe His Glu Met Asn Asn Gly Gly Asp Ala                1205 1210 1215 Met Tyr Met Ala Leu Ile Ala Ala Phe Ser Ile Arg Pro Gly Leu Leu            1220 1225 1230 Ile Gly Phe Gly Leu Arg Thr Leu Trp Ser Pro Arg Glu Arg Leu Val        1235 1240 1245 Leu Thr Leu Gly Ala Ala Met Val Glu Ile Ala Leu Gly Gly Val Met    1250 1255 1260 Gly Gly Leu Trp Lys Tyr Leu Asn Ala Val Ser Leu Cys Ile Leu Thr 1265 1270 1275 1280 Ile Asn Ala Val Ala Ser Arg Lys Ala Ser Asn Thr Ile Leu Pro Leu                1285 1290 1295 Met Ala Leu Leu Thr Pro Val Thr Met Ala Glu Val Arg Leu Ala Ala            1300 1305 1310 Met Phe Phe Cys Ala Val Val Ile Gly Val Leu His Gln Asn Phe        1315 1320 1325 Lys Asp Thr Ser Met Gln Lys Thr Ile Pro Leu Val Ala Leu Thr Leu    1330 1335 1340 Thr Ser Tyr Leu Gly Leu Thr Gln Pro Phe Leu Gly Leu Cys Ala Phe 1345 1350 1355 1360 Leu Ala Thr Arg Ile Phe Gly Arg Arg Ser Ile Pro Val Asn Glu Ala                1365 1370 1375 Leu Ala Ala Ala Gly Leu Val Gly Val Leu Ala Gly Leu Ala Phe Gln            1380 1385 1390 Glu Met Glu Asn Phe Leu Gly Pro Ile Ala Val Gly Gly Leu Leu Met        1395 1400 1405 Met Leu Val Ser Val Ala Gly Arg Val Asp Gly Leu Glu Leu Lys Lys    1410 1415 1420 Leu Gly Glu Val Ser Trp Glu Glu Glu Ala Glu Ile Ser Gly Ser Ser 1425 1430 1435 1440 Ala Arg Tyr Asp Val Ala Leu Ser Glu Gln Gly Glu Phe Lys Leu Leu                1445 1450 1455 Ser Glu Glu Lys Val Pro Trp Asp Gln Val Val Met Thr Ser Leu Ala            1460 1465 1470 Leu Val Gly Ala Ala Leu His Pro Phe Ala Leu Leu Leu Val Leu Ala        1475 1480 1485 Gly Trp Leu Phe His Val Arg Gly Ala Arg Arg Ser Gly Asp Val Leu    1490 1495 1500 Trp Asp Ile Pro Thr Pro Lys Ile Ile Glu Glu Cys Glu His Leu Glu 1505 1510 1515 1520 Asp Gly Ile Tyr Gly Ile Phe Gln Ser Thr Phe Leu Gly Ala Ser Gln                1525 1530 1535 Arg Gly Val Gly Val Ala Gln Gly Gly Val Phe His Thr Met Trp His            1540 1545 1550 Val Thr Arg Gly Ala Phe Leu Val Arg Asn Gly Lys Lys Leu Ile Pro        1555 1560 1565 Ser Trp Ala Ser Val Lys Glu Asp Leu Val Ala Tyr Gly Gly Ser Trp    1570 1575 1580 Lys Leu Glu Gly Arg Trp Asp Gly Glu Glu Glu Val Gln Leu Ile Ala 1585 1590 1595 1600 Ala Val Pro Gly Lys Asn Val Val Asn Val Gln Thr Lys Pro Ser Leu                1605 1610 1615 Phe Lys Val Arg Asn Gly Gly Glu Ile Gly Ala Val Ala Leu Asp Tyr            1620 1625 1630 Pro Ser Gly Thr Ser Gly Ser Pro Ile Val Asn Arg Asn Gly Glu Val        1635 1640 1645 Ile Gly Leu Tyr Gly Asn Gly Ile Leu Val Gly Asp Asn Ser Phe Val    1650 1655 1660 Ser Ala Ile Ser Gln Thr Glu Val Lys Glu Glu Gly Lys Glu Glu Leu 1665 1670 1675 1680 Gln Glu Ile Pro Thr Met Leu Lys Lys Gly Met Thr Thr Val Leu Asp                1685 1690 1695 Phe His Pro Gly Ala Gly Lys Thr Arg Arg Phe Leu Pro Gln Ile Leu            1700 1705 1710 Ala Glu Cys Ala Arg Arg Arg Leu Arg Thr Leu Val Leu Ala Pro Thr        1715 1720 1725 Arg Val Val Leu Ser Glu Met Lys Glu Ala Phe His Gly Leu Asp Val    1730 1735 1740 Lys Phe His Thr Gln Ala Phe Ser Ala His Gly Ser Gly Arg Glu Val 1745 1750 1755 1760 Ile Asp Ala Met Cys His Ala Thr Leu Thr Tyr Arg Met Leu Glu Pro                1765 1770 1775 Thr Arg Val Val Asn Trp Glu Val Ile Met Asp Glu Ala His Phe            1780 1785 1790 Leu Asp Pro Ala Ser Ile Ala Ala Arg Gly Trp Ala Ala His Arg Ala        1795 1800 1805 Arg Ala Asn Glu Ser Ala Thr Ile Leu Met Thr Ala Thr Pro Pro Gly    1810 1815 1820 Thr Ser Asp Glu Phe Pro His Ser Asn Gly Glu Ile Glu Asp Val Gln 1825 1830 1835 1840 Thr Asp Ile Pro Ser Glu Pro Trp Asn Thr Gly His Asp Trp Ile Leu                1845 1850 1855 Ala Asp Lys Arg Pro Thr Ala Trp Phe Leu Pro Ser Ile Arg Ala Ala            1860 1865 1870 Asn Val Met Ala Ala Ser Leu Arg Lys Ala Gly Lys Ser Val Val Val        1875 1880 1885 Leu Asn Arg Lys Thr Phe Glu Arg Glu Tyr Pro Thr Ile Lys Gln Lys    1890 1895 1900 Lys Pro Asp Phe Ile Leu Ala Thr Asp Ile Ala Glu Met Gly Ala Asn 1905 1910 1915 1920 Leu Cys Val Glu Arg Val Leu Asp Cys Arg Thr Ala Phe Lys Pro Val                1925 1930 1935 Leu Val Asp Glu Gly Arg Lys Val Ala Ile Lys Gly Pro Leu Arg Ile            1940 1945 1950 Ser Ala Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn Pro        1955 1960 1965 Asn Arg Asp Gly Asp Ser Tyr Tyr Tyr Ser Glu Pro Thr Ser Glu Asn    1970 1975 1980 Asn Ala His His Val Cys Trp Leu Glu Ala Ser Met Leu Leu Asp Asn 1985 1990 1995 2000 Met Glu Val Arg Gly Gly Met Val Ala Pro Leu Tyr Gly Val Glu Gly                2005 2010 2015 Thr Lys Thr Pro Val Ser Pro Gly Glu Met Arg Leu Arg Asp Asp Gln            2020 2025 2030 Arg Lys Val Phe Arg Glu Leu Val Arg Asn Cys Asp Leu Pro Val Trp        2035 2040 2045 Leu Ser Trp Gln Val Ala Lys Ala Gly Leu Lys Thr Asn Asp Arg Lys    2050 2055 2060 Trp Cys Phe Glu Gly Pro Glu Glu His Glu Ile Leu Asn Asp Ser Gly 2065 2070 2075 2080 Glu Thr Val Lys Cys Arg Ala Pro Gly Gly Ala Lys Lys Pro Leu Arg                2085 2090 2095 Pro Arg Trp Cys Asp Glu Arg Val Ser Ser Asp Gln Ser Ala Leu Ser            2100 2105 2110 Glu Phe Ile Lys Phe Ala Glu Gly Arg Arg Gly Ala Ala Glu Val Leu        2115 2120 2125 Val Val Leu Ser Glu Leu Pro Asp Phe Leu Ala Lys Lys Gly Gly Glu    2130 2135 2140 Ala Met Asp Thr Ile Ser Val Phe Leu His Ser Glu Glu Gly Ser Arg 2145 2150 2155 2160 Ala Tyr Arg Asn Ala Leu Ser Met Met Pro Glu Ala Met Thr Ile Val                2165 2170 2175 Met Leu Phe Ile Leu Ala Gly Leu Leu Thr Ser Gly Met Val Ile Phe            2180 2185 2190 Phe Met Ser Pro Lys Gly Ile Ser Arg Met Ser Met Ala Met Gly Thr        2195 2200 2205 Met Ala Gly Cys Gly Tyr Leu Met Phe Leu Gly Gly Val Lys Pro Thr    2210 2215 2220 His Ile Ser Tyr Val Met Leu Ile Phe Phe Val Leu Met Val Val Val 2225 2230 2235 2240 Ile Pro Glu Pro Gly Gln Gln Arg Ser Ile Gln Asp Asn Gln Val Ala                2245 2250 2255 Tyr Leu Ile Ile Gly Ile Leu Thr Leu Val Ser Ala Val Ala Ala Asn            2260 2265 2270 Glu Leu Gly Met Leu Glu Lys Thr Lys Glu Asp Leu Phe Gly Lys Lys        2275 2280 2285 Asn Leu Ile Pro Ser Ser Ala Ser Pro Trp Ser Trp Pro Asp Leu Asp    2290 2295 2300 Leu Lys Pro Gly Ala Ala Trp Thr Val Tyr Val Gly Ile Val Thr Met 2305 2310 2315 2320 Leu Ser Pro Met Leu His His Trp Ile Lys Val Glu Tyr Gly Asn Leu                2325 2330 2335 Ser Leu Ser Gly Ile Ala Gln Ser Ala Ser Val Leu Ser Phe Met Asp            2340 2345 2350 Lys Gly Ile Pro Phe Met Lys Met Asn Ile Ser Val Ile Met Leu Leu        2355 2360 2365 Val Ser Gly Trp Asn Ser Ile Thr Val Met Pro Leu Leu Cys Gly Ile    2370 2375 2380 Gly Cys Ala Met Leu His Trp Ser Leu Ile Leu Pro Gly Ile Lys Ala 2385 2390 2395 2400 Gln Gln Ser Lys Leu Ala Gln Arg Arg Val Phe His Gly Val Ala Glu                2405 2410 2415 Asn Pro Val Val Asp Gly Asn Pro Thr Val Asp Ile Glu Glu Ala Pro            2420 2425 2430 Glu Met Pro Ala Leu Tyr Glu Lys Lys Leu Ala Leu Tyr Leu Leu Leu        2435 2440 2445 Ala Leu Ser Leu Ala Ser Val Ala Met Cys Arg Thr Pro Phe Ser Leu    2450 2455 2460 Ala Glu Gly Ile Val Leu Ala Ser Ala Ala Leu Gly Pro Leu Ile Glu 2465 2470 2475 2480 Gly Asn Thr Ser Leu Leu Trp Asn Gly Pro Met Ala Val Ser Met Thr                2485 2490 2495 Gly Val Met Arg Gly Asn His Tyr Ala Phe Val Gly Val Met Tyr Asn            2500 2505 2510 Leu Trp Lys Met Lys Thr Gly Arg Arg Gly Ser Ala Asn Gly Lys Thr        2515 2520 2525 Leu Gly Glu Val Trp Lys Arg Glu Leu Asn Leu Leu Asp Lys Arg Gln    2530 2535 2540 Phe Glu Leu Tyr Lys Arg Thr Asp Ile Val Glu Val Asp Arg Asp Thr 2545 2550 2555 2560 Ala Arg Arg His Leu Ala Glu Gly Lys Val Asp Thr Gly Val Ala Val                2565 2570 2575 Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe His Glu Arg Gly Tyr Val            2580 2585 2590 Lys Leu Glu Gly Arg Val Ile Asp Leu Gly Cys Gly Arg Gly Gly Trp        2595 2600 2605 Cys Tyr Tyr Ala Ala Ala Gln Lys Glu Val Ser Gly Val Lys Gly Phe    2610 2615 2620 Thr Leu Gly Arg Asp Gly His Glu Lys Pro Met Asn Val Gln Ser Leu 2625 2630 2635 2640 Gly Trp Asn Ile Ile Thr Phe Lys Asp Lys Thr Asp Ile His Arg Leu                2645 2650 2655 Glu Pro Val Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser            2660 2665 2670 Ser Ser Ser Val Thr Glu Gly Glu Arg Thr Val Arg Val Leu Asp Thr        2675 2680 2685 Val Glu Lys Trp Leu Ala Cys Gly Val Asp Asn Phe Cys Val Lys Val    2690 2695 2700 Leu Ala Pro Tyr Met Pro Asp Val Leu Glu Lys Leu Glu Leu Leu Gln 2705 2710 2715 2720 Arg Arg Phe Gly Gly Thr Val Ile Arg Asn Pro Leu Ser Arg Asn Ser                2725 2730 2735 Thr His Glu Met Tyr Tyr Val Ser Gly Ala Arg Ser Asn Val Thr Phe            2740 2745 2750 Thr Val Asn Gln Thr Ser Arg Leu Leu Met Arg Arg Met Arg Arg Pro        2755 2760 2765 Thr Gly Lys Val Thr Leu Glu Ala Asp Val Ile Leu Pro Ile Gly Thr    2770 2775 2780 Arg Ser Val Glu Thr Asp Lys Gly Pro Leu Asp Lys Glu Ala Ile Glu 2785 2790 2795 2800 Glu Arg Val Glu Arg Ile Lys Ser Glu Tyr Met Thr Ser Trp Phe Tyr                2805 2810 2815 Asp Asn Asp Asn Pro Tyr Arg Thr Trp His Tyr Cys Gly Ser Tyr Val            2820 2825 2830 Thr Lys Thr Ser Gly Ser Ala Ala Ser Met Val Asn Gly Val Ile Lys        2835 2840 2845 Ile Leu Thr Tyr Pro Trp Asp Arg Ile Glu Glu Val Thr Arg Met Ala    2850 2855 2860 Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys 2865 2870 2875 2880 Val Asp Thr Arg Ala Lys Asp Pro Pro Ala Gly Thr Arg Lys Ile Met                2885 2890 2895 Lys Val Val Asn Arg Trp Leu Phe Arg His Leu Ala Arg Glu Lys Asn            2900 2905 2910 Pro Arg Leu Cys Thr Lys Glu Glu Phe Ile Ala Lys Val Arg Ser His        2915 2920 2925 Ala Ala Ile Gly Ala Tyr Leu Glu Glu Gln Glu Gln Trp Lys Thr Ala    2930 2935 2940 Asn Glu Ala Val Gln Asp Pro Lys Phe Trp Glu Leu Val Asp Glu Glu 2945 2950 2955 2960 Arg Lys Leu His Gln Gln Gly Arg Cys Arg Thr Cys Val Tyr Asn Met                2965 2970 2975 Met Gly Lys Arg Glu Lys Lys Leu Ser Glu Phe Gly Lys Ala Lys Gly            2980 2985 2990 Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Tyr Leu Glu Phe        2995 3000 3005 Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Ala Ser Arg Glu Asn    3010 3015 3020 Ser Gly Gly Gly Val Glu Gly Ile Gly Leu Gln Tyr Leu Gly Tyr Val 3025 3030 3035 3040 Ile Arg Asp Leu Ala Ala Met Asp Gly Gly Gly Phe Tyr Ala Asp Asp                3045 3050 3055 Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Ala Asp Leu Asp Asp Glu            3060 3065 3070 Gln Glu Ile Leu Asn Tyr Met Ser Pro His His Lys Lys Leu Ala Gln        3075 3080 3085 Ala Val Met Glu Met Thr Tyr Lys Asn Lys Val Val Lys Val Leu Arg    3090 3095 3100 Pro Ala Pro Gly Gly Lys Ala Tyr Met Asp Val Ile Ser Arg Arg Asp 3105 3110 3115 3120 Gln Arg Gly Ser Gly Gln Val Val Thr Tyr Ala Leu Asn Thr Ile Thr                3125 3130 3135 Asn Leu Lys Val Gln Leu Ile Arg Met Ala Glu Ala Glu Met Val Ile            3140 3145 3150 His His Gln His Val Gln Asp Cys Asp Glu Ser Val Leu Thr Arg Leu        3155 3160 3165 Glu Ala Trp Leu Thr Glu His Gly Cys Asp Arg Leu Lys Arg Met Ala    3170 3175 3180 Val Ser Gly Asp Asp Cys Val Val Arg Pro Ile Asp Asp Arg Phe Gly 3185 3190 3195 3200 Leu Ala Leu Ser His Leu Asn Ala Met Ser Lys Val Arg Lys Asp Ile                3205 3210 3215 Ser Glu Trp Gln Pro Ser Lys Gly Trp Asn Asp Trp Glu Asn Val Pro            3220 3225 3230 Phe Cys Ser His His Phe His Glu Leu Gln Leu Lys Asp Gly Arg Arg        3235 3240 3245 Ile Val Val Pro Cys Arg Glu Gln Asp Glu Leu Ile Gly Arg Gly Arg    3250 3255 3260 Val Ser Pro Gly Asn Gly Trp Met Ile Lys Glu Thr Ala Cys Leu Ser 3265 3270 3275 3280 Lys Ala Tyr Ala Asn Met Trp Ser Leu Met Tyr Phe His Lys Arg Asp                3285 3290 3295 Met Arg Leu Leu Ser Leu Ala Val Ser Ser Ala Val Pro Thr Ser Trp            3300 3305 3310 Val Pro Gln Gly Arg Thr Thr Trp Ser Ile His Gly Lys Gly Glu Trp        3315 3320 3325 Met Thr Thr Glu Asp Met Leu Glu Val Trp Asn Arg Val Trp Ile Thr    3330 3335 3340 Asn Asn Pro His Met Gln Asp Lys Thr Met Val Lys Lys Trp Arg Asp 3345 3350 3355 3360 Val Pro Tyr Leu Thr Lys Arg Gln Asp Lys Leu Cys Gly Ser Leu Ile                3365 3370 3375 Gly Met Thr Asn Arg Ala Thr Trp Ala Ser His Ile His Leu Val Ile            3380 3385 3390 His Arg Ile Arg Thr Leu Ile Gly Gln Glu Lys Tyr Thr Asp Tyr Leu        3395 3400 3405 Thr Val Met Asp Arg Tyr Ser Val Asp Ala Asp Leu Gln Leu Gly Glu    3410 3415 3420 Leu ile 3425 <210> 2 <211> 10907 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 1 nucleic acid sequence <400> 2 agtaaatcct gtgtgctaat tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60 acacatttgg attaatttta atcgttcgtt gagcgattag cagagaactg accagaacat 120 gtctggtcgt aaagctcagg gaaaaaccct gggcgtcaat atggtacgac gaggagttcg 180 ctccttgtca aacaaaataa aacaaaaaac aaaacaaatt ggaaacagac ctggaccttc 240 aagaggtgtt caaggattta tctttttctt tttgttcaac attttgactg gaaaaaagat 300 cacagcccac ctaaagaggt tgtggaaaat gctggaccca agacaaggct tggctgttct 360 aaggaaagtc aagagagtgg tggccagttt gatgagagga ttgtcctcaa ggaaacgccg 420 ttcccatgat gttctgactg tgcaattcct aattttggga atgctgttga tgacgggtgg 480 agcggaggtc actagacgtg ggagtgcata ctatatgtac ttggacagaa acgatgctgg 540 ggaggccata tcttttccaa ccacattggg gatgaataag tgttatatac agatcatgga 600 tcttggacac atgtgtgatg ccaccatgag ctatgaatgc cctatgctgg atgagggggt 660 ggaaccagat gacgtcgatt gttggtgcaa cacgacgtca acttgggttg tgtacggaac 720 ctgccatcac aaaaaaggtg aagcacggag atctagaaga gctgtgacgc tcccctccca 780 ttccactagg aagctgcaaa cgcggtcgca aacctggttg gaatcaagag aatacacaaa 840 gcacttgatt agagtcgaaa attggatatt caggaaccct ggcttcgcgt tagcagcagc 900 tgccatcgct tggcttttgg gaagctcaac gagccaaaaa gtcatatact tggtcatgat 960 actgctgatt gccccggcat acagcatcag gtgcatagga gtcagcaata gggactttgt 1020 ggaaggtatg tcaggtggga cttgggttga tgttgtcttg gaacatggag gttgtgtcac 1080 cgtaatggca caggacaaac cgactgtcga catagagctg gttacaacaa cagtcagcaa 1140 catggcggag gtaagatcct actgctatga ggcatcaata tcagacatgg cttcggacag 1200 ccgctgccca acacaaggtg aagcctacct tgacaagcaa tcagacactc aatatgtctg 1260 caaaagaacg ttagtggaca gaggctgggg aaatggatgt ggactttttg gcaaagggag 1320 cctggtgaca tgcgctaagt ttgcatgctc caagaaaatg accgggaaga gcatccagcc 1380 agagaatctg gagtaccgga taatgctgtc agttcatggc tcccagcaca gtgggatgat 1440 cgttaatgac acaggacatg aaactgatga gaatagagcg aaagttgaga taacgcccaa 1500 ttcaccaaga gccgaagcca ccctgggggg ttttggaagc ctaggacttg attgtgaacc 1560 gaggacaggc cttgactttt cagatttgta ttacttgact atgaataaca agcactggtt 1620 ggttcacaag gagtggttcc acgacattcc attaccttgg cacgctgggg cagacaccgg 1680 aactccacac tggaacaaca aagaagcact ggtagagttc aaggacgcac atgccaaaag 1740 gcaaactgtc gtggttctag ggagtcaaga aggagcagtt cacacggccc ttgctggagc 1800 tctggaggct gagatggatg gtgcaaaggg aaggctgtcc tctggccact tgaaatgtcg 1860 cctgaaaatg gataaactta gattgaaggg cgtgtcatac tccttgtgta ctgcagcgtt 1920 cacattcacc aagatcccgg ctgaaacact gcacgggaca gtcacagtgg aggtacagta 1980 cgcagggaca gatggacctt gcaaggttcc agctcagatg gcggtggaca tgcaaactct 2040 gaccccagtt gggaggttga taaccgctaa ccccgtaatc actgaaagca ctgagaactc 2100 taagatgatg ctggaacttg atccaccatt tggggactct tacattgtca taggagtcgg 2160 ggagaagaag atcacccacc actggcacag gagtggcagc accattggaa aagcatttga 2220 agccactgtg agaggtgcca agagaatggc agtcttggga gacacagcct gggactttgg 2280 atcagttgga ggcgctctca actcattggg caagggcatc catcaaattt ttggagcagc 2340 tttcaaatca ttgtttggag gaatgtcctg gttctcacaa attctcattg gaacgttgct 2400 gatgtggttg ggtctgaaca caaagaatgg atctatttcc cttatgtgct tggccttagg 2460 gggggtgttg atcttcttat ccacagccgt ctctgctgat caaggatgcg ccatcaactt 2520 tggcaagaga gagctcaagt gcggagatgg tatcttcata tttagagact ctgatgactg 2580 gctgaacaag tactcatact atccagaaga tcctgtgaag cttgcatcaa tagtgaaagc 2640 ctcttttgaa gaagggaagt gtggcctaaa ttcagttgac tcccttgagc atgagatgtg 2700 gagaagcagg gcagatgaga tcaatgccat ttttgaggaa aacgaggtgg acatttctgt 2760 tgtcgtgcag gatccaaaga atgtttacca gagaggaact catccatttt ccagaattcg 2820 ggatggtctg cagtatggtt ggaagacttg gggtaagaac cttgtgttct ccccagggag 2880 gaagaatgga agcttcatca tagatggaaa gtccaggaaa gaatgcccgt tttcaaaccg 2940 ggtctggaat tctttccaga tagaggagtt tgggacggga gtgttcacca cacgcgtgta 3000 catggacgca gtctttgaat acaccataga ctgcgatgga tctatcttgg gtgcagcggt 3060 gaacggaaaa aagagtgccc atggctctcc aacattttgg atgggaagtc atgaagtaaa 3120 tgggacatgg atgatccaca ccttggaggc attagattac aaggagtgtg agtggccact 3180 gacacatacg attggaacat cagttgaaga gagtgaaatg ttcatgccga gatcaatcgg 3240 aggcccagtt agctctcaca atcatatccc tggatacaag gttcagacga acggaccttg 3300 gatgcaggta ccactagaag tgaagagaga agcttgccca gggactagcg tgatcattga 3360 tggcaactgt gatggacggg gaaaatcaac cagatccacc acggatagcg ggaaagttat 3420 tcctgaatgg tgttgccgct cctgcacaat gccgcctgtg agcttccatg gtagtgatgg 3480 gtgttggtat cccatggaaa ttaggccaag gaaaacgcat gaaagccatc tggtgcgctc 3540 ctgggttaca gctggagaaa tacatgctgt cccttttggt ttggtgagca tgatgatagc 3600 aatggaagtg gtcctaagga aaagacaggg accaaagcaa atgttggttg gaggagtagt 3660 gctcttggga gcaatgctgg tcgggcaagt aactctcctt gatttgctga aactcacagt 3720 ggctgtggga ttgcatttcc atgagatgaa caatggagga gacgccatgt atatggcgtt 3780 gattgctgcc ttttcaatca gaccagggct gctcatcggc tttgggctca ggaccctatg 3840 gagccctcgg gaacgccttg tgctgaccct aggagcagcc atggtggaga ttgccttggg 3900 tggcgtgatg ggcggcctgt ggaagtatct aaatgcagtt tctctctgca tcctgacaat 3960 aaatgctgtt gcttctagga aagcatcaaa taccatcttg cccctcatgg ctctgttgac 4020 acctgtcact atggctgagg tgagacttgc cgcaatgttc ttttgtgccg tggttatcat 4080 aggggtcctt caccagaatt tcaaggacac ctccatgcag aagactatac ctctggtggc 4140 cctcacactc acatcttacc tgggcttgac acaacctttt ttgggcctgt gtgcatttct 4200 ggcaacccgc atatttgggc gaaggagtat cccagtgaat gaggcactcg cagcagctgg 4260 tctagtggga gtgctggcag gactggcttt tcaggagatg gagaacttcc ttggtccgat 4320 tgcagttgga ggactcctga tgatgctggt tagcgtggct gggagggtgg atgggctaga 4380 gctcaagaag cttggtgaag tttcatggga agaggaggcg gagatcagcg ggagttccgc 4440 ccgctatgat gtggcactca gtgaacaagg ggagttcaag ctgctttctg aagagaaagt 4500 gccatgggac caggttgtga tgacctcgct ggccttggtt ggggctgccc tccatccatt 4560 tgctcttctg ctggtccttg ctgggtggct gtttcatgtc aggggagcta ggagaagtgg 4620 ggatgtcttg tgggatattc ccactcctaa gatcatcgag gaatgtgaac atctggagga 4680 tgggatttat ggcatattcc agtcaacctt cttgggggcc tcccagcgag gagtgggagt 4740 ggcacaggga ggggtgttcc acacaatgtg gcatgtcaca agaggagctt tccttgtcag 4800 gaatggcaag aagttgattc catcttgggc ttcagtaaag gaagaccttg tcgcctatgg 4860 tggctcatgg aagttggaag gcagatggga tggagaggaa gaggtccagt tgatcgcggc 4920 tgttccagga aagaacgtgg tcaacgtcca gacaaaaccg agcttgttca aagtgaggaa 4980 tgggggagaa atcggggctg tcgctcttga ctatccgagt ggcacttcag gatctcctat 5040 tgttaacagg aacggagagg tgattgggct gtacggcaat ggcatccttg tcggtgacaa 5100 ctccttcgtg tccgccatat cccagactga ggtgaaggaa gaaggaaagg aggagctcca 5160 agagatcccg acaatgctaa agaaaggaat gacaactgtc cttgattttc atcctggagc 5220 tgggaagaca agacgtttcc tcccacagat cttggccgag tgcgcacgga gacgcttgcg 5280 cactcttgtg ttggccccca ccagggttgt tctttctgaa atgaaggagg cttttcacgg 5340 cctggacgtg aaattccaca cacaggcttt ttccgctcac ggcagcggga gagaagtcat 5400 tgatgccatg tgccatgcca ccctaactta caggatgttg gaaccaacta gggttgttaa 5460 ctgggaagtg atcattatgg atgaagccca ttttttggat ccagctagca tagccgctag 5520 aggttgggca gcgcacagag ctagggcaaa tgaaagtgca acaatcttga tgacagccac 5580 accgcctggg actagtgatg aatttccaca ttcaaatggt gaaatagaag atgttcaaac 5640 ggacataccc agtgagccct ggaacacagg gcatgactgg atcctagctg acaaaaggcc 5700 cacggcatgg ttccttccat ccatcagagc tgcaaatgtc atggctgcct ctttgcgtaa 5760 ggctggaaag agtgtggtgg tcctgaacag gaaaaccttt gagagagaat accccacgat 5820 aaagcagaag aaacctgact ttatattggc cactgacata gctgaaatgg gagccaacct 5880 ttgcgtggag cgagtgctgg attgcaggac ggcttttaag cctgtgcttg tggatgaagg 5940 gaggaaggtg gcaataaaag ggccacttcg tatctccgca tcctctgctg ctcaaaggag 6000 ggggcgcatt gggagaaatc ccaacagaga tggagactca tactactatt ctgagcctac 6060 aagtgaaaat aatgcccacc acgtctgctg gttggaggcc tcaatgctct tggacaacat 6120 ggaggtgagg ggtggaatgg tcgccccact ctatggcgtt gaaggaacta aaacaccagt 6180 ttcccctggt gaaatgagac tgagggatga ccagaggaaa gtcttcagag aactagtgag 6240 gaattgtgac ctgcccgttt ggctttcgtg gcaagtggcc aaggctggtt tgaagacgaa 6300 tgatcgtaag tggtgttttg aaggccctga ggaacatgag atcttgaatg acagcggtga 6360 aacagtgaag tgcagggctc ctggaggagc aaagaagcct ctgcgcccaa ggtggtgtga 6420 tgaaagggtg tcatctgacc agagtgcgct gtctgaattt attaagtttg ctgaaggtag 6480 gaggggagct gctgaagtgc tagttgtgct gagtgaactc cctgatttcc tggctaaaaa 6540 aggtggagag gcaatggata ccatcagtgt gttcctccac tctgaggaag gctctagggc 6600 ttaccgcaat gcactatcaa tgatgcctga ggcaatgaca atagtcatgc tgtttatact 6660 ggctggacta ctgacatcgg gaatggtcat ctttttcatg tctcccaaag gcatcagtag 6720 aatgtctatg gcgatgggca caatggccgg ctgtggatat ctcatgttcc ttggaggcgt 6780 caaacccact cacatctcct atgtcatgct catattcttt gtcctgatgg tggttgtgat 6840 ccccgagcca gggcaacaaa ggtccatcca agacaaccaa gtggcatacc tcattattgg 6900 catcctgacg ctggtttcag cggtggcagc caacgagcta ggcatgctgg agaaaaccaa 6960 agaggacctc tttgggaaga agaacttaat tccatctagt gcttcaccct ggagttggcc 7020 ggatcttgac ctgaagccag gagctgcctg gacagtgtac gttggcattg ttacaatgct 7080 ctctccaatg ttgcaccact ggatcaaagt cgaatatggc aacctgtctc tgtctggaat 7140 agcccagtca gcctcagtcc tttctttcat ggacaagggg ataccattca tgaagatgaa 7200 tatctcggtc ataatgctgc tggtcagtgg ctggaattca ataacagtga tgcctctgct 7260 ctgtggcata gggtgcgcca tgctccactg gtctctcatt ttacctggaa tcaaagcgca 7320 gcagtcaaag cttgcacaga gaagggtgtt ccatggcgtt gccgagaacc ctgtggttga 7380 tgggaatcca acagttgaca ttgaggaagc tcctgaaatg cctgcccttt atgagaagaa 7440 actggctcta tatctccttc ttgctctcag cctagcttct gttgccatgt gcagaacgcc 7500 cttttcattg gctgaaggca ttgtcctagc atcagctgcc ttagggccgc tcatagaggg 7560 aaacaccagc cttctttgga atggacccat ggctgtctcc atgacaggag tcatgagggg 7620 gaatcactat gcttttgtgg gagtcatgta caatctatgg aagatgaaaa ctggacgccg 7680 ggggagcgcg aatggaaaaa ctttgggtga agtctggaag agggaactga atctgttgga 7740 caagcgacag tttgagttgt ataaaaggac cgacattgtg gaggtggatc gtgatacggc 7800 acgcaggcat ttggccgaag ggaaggtgga caccggggtg gcggtctcca gggggaccgc 7860 aaagttaagg tggttccatg agcgtggcta tgtcaagctg gaaggtaggg tgattgacct 7920 ggggtgtggc cgcggaggct ggtgttacta cgctgctgcg caaaaggaag tgagtggggt 7980 caaaggattt actcttggaa gagacggcca tgagaaaccc atgaatgtgc aaagtctggg 8040 atggaacatc atcaccttca aggacaaaac tgatatccac cgcctagaac cagtgaaatg 8100 tgacaccctt ttgtgtgaca ttggagagtc atcatcgtca tcggtcacag agggggaaag 8160 gaccgtgaga gttcttgata ctgtagaaaa atggctggct tgtggggttg acaacttctg 8220 tgtgaaggtg ttagctccat acatgccaga tgttctcgag aaactggaat tgctccaaag 8280 gaggtttggc ggaacagtga tcaggaaccc tctctccagg aattccactc atgaaatgta 8340 ctacgtgtct ggagcccgca gcaatgtcac atttactgtg aaccaaacat cccgcctcct 8400 gatgaggaga atgaggcgtc caactggaaa agtgaccctg gaggctgacg tcatcctccc 8460 aattgggaca cgcagtgttg agacagacaa gggacccctg gacaaagagg ccatagaaga 8520 aagggttgag aggataaaat ctgagtacat gacctcttgg ttttatgaca atgacaaccc 8580 ctacaggacc tggcactact gtggctccta tgtcacaaaa acctcaggaa gtgcggcgag 8640 catggtaaat ggtgttatta aaattctgac atatccatgg gacaggatag aggaggtcac 8700 aagaatggca atgactgaca caaccccttt tggacagcaa agagtgttta aagaaaaagt 8760 tgacaccaga gcaaaggatc caccagcggg aactaggaag atcatgaaag ttgtcaacag 8820 gtggctgttc cgccacctgg ccagagaaaa gaaccccaga ctgtgcacaa aggaagaatt 8880 tattgcaaaa gtccgaagtc atgcagccat tggagcttac ctggaagaac aagaacagtg 8940 gaagactgcc aatgaggctg tccaagaccc aaagttctgg gaactggtgg atgaagaaag 9000 gaagctgcac caacaaggca ggtgtcggac ttgtgtgtac aacatgatgg ggaaaagaga 9060 gaagaagctg tcagagtttg ggaaagcaaa gggaagccgt gccatatggt atatgtggct 9120 gggagcgcgg tatcttgagt ttgaggccct gggattcctg aatgaggacc attgggcttc 9180 cagggaaaac tcaggaggag gagtggaagg cattggctta caatacctag gatatgtgat 9240 cagagacctg gctgcaatgg atggtggtgg attctacgcg gatgacaccg ctggatggga 9300 cacgcgcatc acagaggcag accttgatga tgaacaggag atcttgaact acatgagccc 9360 acatcacaaa aaactggcac aagcagtgat ggaaatgaca tacaagaaca aagtggtgaa 9420 agtgttgaga ccagccccag gagggaaagc ctacatggat gtcataagtc gacgagacca 9480 gagaggatcc gggcaggtag tgacttatgc tctgaacacc atcaccaact tgaaagtcca 9540 attgatcaga atggcagaag cagagatggt gatacatcac caacatgttc aagattgtga 9600 tgaatcagtt ctgaccaggc tggaggcatg gctcactgag cacggatgtg acagactgaa 9660 gaggatggcg gtgagtggag acgactgtgt ggtccggccc atcgatgaca ggttcggcct 9720 ggccctgtcc catctcaacg ccatgtccaa ggttagaaag gacatatctg aatggcagcc 9780 atcaaaaggg tggaatgatt gggagaatgt gcccttctgt tcccaccact tccatgaact 9840 acagctgaag gatggcagga ggattgtggt gccttgccga gaacaggacg agctcattgg 9900 gagaggaagg gtgtctccag gaaacggctg gatgatcaag gaaacagctt gcctcagcaa 9960 agcctatgcc aacatgtggt cactgatgta ttttcacaaa agggacatga ggctactgtc 10020 attggctgtt tcctcagctg ttcccacctc atgggttcca caaggacgca caacatggtc 10080 gattcatggg aaaggggagt ggatgaccac ggaagacatg cttgaggtgt ggaacagagt 10140 atggataacc aacaacccac acatgcagga caagacaatg gtgaaaaaat ggagagatgt 10200 cccttatcta accaagagac aagacaagct gtgcggatca ctgattggaa tgaccaatag 10260 ggccacctgg gcctcccaca tccatttagt catccatcgt atccgaacgc tgattggaca 10320 ggagaaatac actgactacc taacagtcat ggacaggtat tctgtggatg ctgacctgca 10380 actgggtgag cttatctgaa acaccatcta acaggaataa ccgggataca aaccacgggt 10440 ggagaaccgg actccccaca acctgaaacc gggatataaa ccacggctgg agaaccgggc 10500 tccgcactta aaatgaaaca gaaaccggga taaaaactac ggatggagaa ccggactcca 10560 cacattgaga cagaagaagt tgtcagccca gaaccccaca cgagttttgc cactgctaag 10620 ctgtgaggca gtgcaggctg ggacagccga cctccaggtt gcgaaaaacc tggtttctgg 10680 gacctcccac cccagagtaa aaagaacgga gcctccgcta ccaccctccc acgtggtggt 10740 agaaagacgg ggtctagagg ttagaggaga ccctccaggg aacaaatagt gggaccatat 10800 tgacgccagg gaaagaccgg agtggttctc tgcttttcct ccagaggtct gtgagcacag 10860 tttgctcaag aataagcaga cctttggatg acaaacacaa aaccact 10907 <210> 3 <211> 3426 <212> PRT <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 2 amino acid sequence <400> 3 Met Ser Gly Arg Lys Ala Gln Gly Lys Thr Leu Gly Val Asn Met Val   1 5 10 15 Arg Arg Gly Val Arg Ser Leu Ser Asn Lys Ile Lys Gln Lys Thr Lys              20 25 30 Gln Ile Gly Asn Arg Pro Gly Pro Ser Arg Gly Val Gln Gly Phe Ile          35 40 45 Phe Phe Phe Leu Phe Asn Ile Leu Thr Gly Lys Lys Ile Thr Ala His      50 55 60 Leu Lys Arg Leu Trp Lys Met Leu Asp Pro Arg Gln Gly Leu Ala Val  65 70 75 80 Leu Arg Lys Val Lys Arg Val Val Ala Ser Leu Met Arg Gly Leu Ser                  85 90 95 Ser Arg Lys Arg Arg Ser His Asp Val Leu Thr Val Gln Phe Leu Ile             100 105 110 Leu Gly Met Leu Leu Met Thr Gly Gly Ala Glu Val Thr Arg Arg Gly         115 120 125 Ser Ala Tyr Tyr Met Tyr Leu Asp Arg Asn Asp Ala Gly Glu Ala Ile     130 135 140 Ser Phe Pro Thr Thr Leu Gly Met Asn Lys Cys Tyr Ile Gln Ile Met 145 150 155 160 Asp Leu Gly His Met Cys Asp Ala Thr Met Ser Tyr Glu Cys Pro Met                 165 170 175 Leu Asp Glu Gly Val Glu Pro Asp Asp Val Asp Cys Trp Cys Asn Thr             180 185 190 Thr Ser Thr Trp Val Val Tyr Gly Thr Cys His His Lys Lys Gly Glu         195 200 205 Ala Arg Arg Ser Arg Arg Ala Val Thr Leu Pro Ser His Ser Thr Arg     210 215 220 Lys Leu Gln Thr Arg Ser Gln Thr Trp Leu Glu Ser Arg Glu Tyr Thr 225 230 235 240 Lys His Leu Ile Arg Val Glu Asn Trp Ile Phe Arg Asn Pro Gly Phe                 245 250 255 Ala Leu Ala Ala Ala Ala Ile Ala Trp Leu Leu Gly Ser Ser Thr Ser             260 265 270 Gln Lys Val Ile Tyr Leu Val Met Ile Leu Leu Ile Ala Pro Ala Tyr         275 280 285 Ser Ile Arg Cys Ile Gly Val Ser Asn Arg Asp Phe Val Glu Gly Met     290 295 300 Ser Gly Gly Thr Trp Val Asp Val Val Leu Glu His Gly Gly Cys Val 305 310 315 320 Thr Val Met Ala Gln Asp Lys Pro Thr Val Asp Ile Glu Leu Val Thr                 325 330 335 Thr Thr Val Ser Asn Met Ala Glu Val Arg Ser Tyr Cys Tyr Glu Ala             340 345 350 Ser Ile Ser Asp Met Ala Ser Asp Ser Arg Cys Pro Thr Gln Gly Glu         355 360 365 Ala Tyr Leu Asp Lys Gln Ser Asp Thr Gln Tyr Val Cys Lys Arg Thr     370 375 380 Leu Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly 385 390 395 400 Ser Leu Val Thr Cys Ala Lys Phe Ala Cys Ser Lys Lys Met Thr Gly                 405 410 415 Lys Ser Ile Gln Pro Glu Asn Leu Glu Tyr Arg Ile Met Leu Ser Val             420 425 430 His Gly Ser Gln His Ser Gly Met Ile Val Asn Asp Thr Gly His Glu         435 440 445 Thr Asp Glu Asn Arg Ala Lys Val Glu Ile Thr Pro Asn Ser Pro Arg     450 455 460 Ala Glu Ala Thr Leu Gly Gly Phe Gly Ser Leu Gly Leu Asp Cys Glu 465 470 475 480 Pro Arg Thr Gly Leu Asp Phe Ser Asp Leu Tyr Tyr Leu Thr Met Asn                 485 490 495 Asn Lys His Trp Leu Val His Lys Glu Trp Phe His Asp Ile Pro Leu             500 505 510 Pro Trp His Ala Gly Ala Asp Thr Gly Thr Pro His Trp Asn Asn Lys         515 520 525 Glu Ala Leu Val Glu Phe Lys Asp Ala His Ala Lys Arg Gln Thr Val     530 535 540 Val Val Leu Gly Ser Gln Glu Gly Ala Val His Thr Ala Leu Ala Gly 545 550 555 560 Ala Leu Glu Ala Glu Met Asp Gly Ala Lys Gly Arg Leu Ser Ser Gly                 565 570 575 His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Arg Leu Lys Gly Val             580 585 590 Ser Tyr Ser Leu Cys Thr Ala Ala Phe Thr Phe Thr Lys Ile Pro Ala         595 600 605 Glu Thr Leu His Gly Thr Val Thr Val Glu Val Gln Tyr Ala Gly Thr     610 615 620 Asp Gly Pro Cys Lys Val Pro Ala Gln Met Ala Val Asp Met Gln Thr 625 630 635 640 Leu Thr Pro Val Gly Arg Leu Ile Thr Ala Asn Pro Val Ile Thr Glu                 645 650 655 Ser Thr Glu Asn Ser Lys Met Met Leu Glu Leu Asp Pro Pro Phe Gly             660 665 670 Asp Ser Tyr Ile Val Ile Gly Val Gly Glu Lys Lys Ile Thr His His         675 680 685 Trp His Arg Ser Gly Ser Thr Ile Gly Lys Ala Phe Glu Ala Thr Val     690 695 700 Arg Gly Ala Lys Arg Met Ala Val Leu Gly Asp Thr Ala Trp Asp Phe 705 710 715 720 Gly Ser Val Gly Gly Ala Leu Asn Ser Leu Gly Lys Gly Ile His Gln                 725 730 735 Ile Phe Gly Ala Ala Phe Lys Ser Leu Phe Gly Gly Met Ser Trp Phe             740 745 750 Ser Gln Ile Leu Ile Gly Thr Leu Leu Met Trp Leu Gly Leu Asn Thr         755 760 765 Lys Asn Gly Ser Ile Ser Leu Met Cys Leu Ala Leu Gly Gly Val Leu     770 775 780 Ile Phe Leu Ser Thr Ala Val Gly Ala Asp Gln Gly Cys Ala Ile Asn 785 790 795 800 Phe Gly Lys Arg Glu Leu Lys Cys Gly Asp Gly Ile Phe Ile Phe Arg                 805 810 815 Asp Ser Asp Asp Trp Leu Asn Lys Tyr Ser Tyr Tyr Pro Glu Asp Pro             820 825 830 Val Lys Leu Ala Ser Ile Val Lys Ala Ser Phe Glu Glu Gly Lys Cys         835 840 845 Gly Leu Asn Ser Val Asp Ser Leu Glu His Glu Met Trp Arg Ser Arg     850 855 860 Ala Asp Glu Ile Asn Ala Ile Phe Glu Glu Asn Glu Val Asp Ile Ser 865 870 875 880 Val Val Val Gln Asp Pro Lys Asn Val Tyr Gln Arg Gly Thr His Pro                 885 890 895 Phe Ser Arg Ile Arg Asp Gly Leu Gln Tyr Gly Trp Lys Thr Trp Gly             900 905 910 Lys Asn Leu Val Phe Ser Pro Gly Arg Lys Asn Gly Ser Phe Ile Ile         915 920 925 Asp Gly Lys Ser Arg Lys Glu Cys Pro Phe Ser Asn Arg Val Trp Asn     930 935 940 Ser Phe Gln Ile Glu Glu Phe Gly Thr Gly Val Phe Thr Thr Arg Val 945 950 955 960 Tyr Met Asp Ala Val Phe Glu Tyr Thr Ile Asp Cys Asp Gly Ser Ile                 965 970 975 Leu Gly Ala Ala Val Asn Gly Lys Lys Ser Ala His Gly Ser Pro Thr             980 985 990 Phe Trp Met Gly Ser His Glu Val Asn Gly Thr Trp Met Ile His Thr         995 1000 1005 Leu Glu Ala Leu Asp Tyr Lys Glu Cys Glu Trp Pro Leu Thr His Thr    1010 1015 1020 Ile Gly Thr Ser Val Glu Glu Ser Glu Met Phe Met Pro Arg Ser Ile 1025 1030 1035 1040 Gly Gly Pro Val Ser Ser His Asn His Ile Pro Gly Tyr Lys Val Gln                1045 1050 1055 Thr Asn Gly Pro Trp Met Gln Val Pro Leu Glu Val Lys Arg Glu Ala            1060 1065 1070 Cys Pro Gly Thr Ser Val Ile Ile Asp Gly Asn Cys Asp Gly Arg Gly        1075 1080 1085 Lys Ser Thr Arg Ser Thr Thr Asp Ser Gly Lys Val Ile Pro Glu Trp    1090 1095 1100 Cys Cys Arg Ser Cys Thr Met Pro Pro Val Ser Phe His Gly Ser Asp 1105 1110 1115 1120 Gly Cys Trp Tyr Pro Met Glu Ile Arg Pro Arg Lys Thr His Glu Ser                1125 1130 1135 His Leu Val Arg Ser Trp Val Thr Ala Gly Glu Ile His Ala Val Pro            1140 1145 1150 Phe Gly Leu Val Ser Met Met Ile Ala Met Glu Val Val Leu Arg Lys        1155 1160 1165 Arg Gln Gly Pro Lys Gln Met Leu Val Gly Gly Val Val Leu Leu Gly    1170 1175 1180 Ala Met Leu Val Gly Gln Val Thr Leu Leu Asp Leu Leu Lys Leu Thr 1185 1190 1195 1200 Val Ala Val Gly Leu His Phe His Glu Met Asn Asn Gly Gly Asp Ala                1205 1210 1215 Met Tyr Met Ala Leu Ile Ala Ala Phe Ser Ile Arg Pro Gly Leu Leu            1220 1225 1230 Ile Gly Phe Gly Leu Arg Thr Leu Trp Ser Pro Arg Glu Arg Leu Val        1235 1240 1245 Leu Thr Leu Gly Ala Ala Met Val Glu Ile Ala Leu Gly Gly Val Met    1250 1255 1260 Gly Gly Leu Trp Lys Tyr Leu Asn Ala Val Ser Leu Cys Ile Leu Thr 1265 1270 1275 1280 Ile Asn Ala Val Ala Ser Arg Lys Ala Ser Asn Thr Ile Leu Pro Leu                1285 1290 1295 Met Ala Leu Leu Thr Pro Val Thr Met Ala Glu Val Arg Leu Ala Ala            1300 1305 1310 Met Phe Phe Cys Ala Val Val Ile Gly Val Leu His Gln Asn Phe        1315 1320 1325 Lys Asp Thr Ser Met Gln Lys Thr Ile Pro Leu Val Ala Leu Thr Leu    1330 1335 1340 Thr Ser Tyr Leu Gly Leu Thr Gln Pro Phe Leu Gly Leu Cys Ala Phe 1345 1350 1355 1360 Leu Ala Thr Arg Ile Phe Gly Arg Arg Ser Ile Pro Val Asn Glu Ala                1365 1370 1375 Leu Ala Ala Ala Gly Leu Val Gly Val Leu Ala Gly Leu Ala Phe Gln            1380 1385 1390 Glu Met Glu Asn Phe Leu Gly Pro Ile Ala Val Gly Gly Leu Leu Met        1395 1400 1405 Met Leu Val Ser Val Ala Gly Arg Val Asp Gly Leu Glu Leu Lys Lys    1410 1415 1420 Leu Gly Glu Val Ser Trp Glu Glu Glu Ala Glu Ile Ser Gly Ser Ser 1425 1430 1435 1440 Ala Arg Tyr Asp Val Ala Leu Ser Glu Gln Gly Glu Phe Lys Leu Leu                1445 1450 1455 Ser Glu Glu Lys Val Pro Trp Asp Gln Val Val Met Thr Ser Leu Ala            1460 1465 1470 Leu Val Gly Ala Ala Leu His Pro Phe Ala Leu Leu Leu Val Leu Ala        1475 1480 1485 Gly Trp Leu Phe His Val Arg Gly Ala Arg Arg Ser Gly Asp Val Leu    1490 1495 1500 Trp Asp Ile Pro Thr Pro Lys Ile Ile Glu Glu Cys Glu His Leu Glu 1505 1510 1515 1520 Asp Gly Ile Tyr Gly Ile Phe Gln Ser Thr Phe Leu Gly Ala Ser Gln                1525 1530 1535 Arg Gly Val Gly Val Ala Gln Gly Gly Val Phe His Thr Met Trp His            1540 1545 1550 Val Thr Arg Gly Ala Phe Leu Val Arg Asn Gly Lys Lys Leu Ile Pro        1555 1560 1565 Ser Trp Ala Ser Val Lys Glu Asp Leu Val Ala Tyr Gly Gly Ser Trp    1570 1575 1580 Lys Leu Glu Gly Arg Trp Asp Gly Glu Glu Glu Val Gln Leu Ile Ala 1585 1590 1595 1600 Ala Val Pro Gly Lys Asn Val Val Asn Val Gln Thr Lys Pro Ser Leu                1605 1610 1615 Phe Lys Val Arg Asn Gly Gly Glu Ile Gly Ala Val Ala Leu Asp Tyr            1620 1625 1630 Pro Ser Gly Thr Ser Gly Ser Pro Ile Val Asn Arg Asn Gly Glu Val        1635 1640 1645 Ile Gly Leu Tyr Gly Asn Gly Ile Leu Val Gly Asp Asn Ser Phe Val    1650 1655 1660 Ser Ala Ile Ser Gln Thr Glu Val Lys Glu Glu Gly Lys Glu Glu Leu 1665 1670 1675 1680 Gln Glu Ile Pro Thr Met Leu Lys Lys Gly Met Thr Thr Val Leu Asp                1685 1690 1695 Phe His Pro Gly Ala Gly Lys Thr Arg Arg Phe Leu Pro Gln Ile Leu            1700 1705 1710 Ala Glu Cys Ala Arg Arg Arg Leu Arg Thr Leu Val Leu Ala Pro Thr        1715 1720 1725 Arg Val Val Leu Ser Glu Met Lys Glu Ala Phe His Gly Leu Asp Val    1730 1735 1740 Lys Phe His Thr Gln Ala Phe Ser Ala His Gly Ser Gly Arg Glu Val 1745 1750 1755 1760 Ile Asp Ala Met Cys His Ala Thr Leu Thr Tyr Arg Met Leu Glu Pro                1765 1770 1775 Thr Arg Val Val Asn Trp Glu Val Ile Met Asp Glu Ala His Phe            1780 1785 1790 Leu Asp Pro Ala Ser Ile Ala Ala Arg Gly Trp Ala Ala His Arg Ala        1795 1800 1805 Arg Ala Asn Glu Ser Ala Thr Ile Leu Met Thr Ala Thr Pro Pro Gly    1810 1815 1820 Thr Ser Asp Glu Phe Pro His Ser Asn Gly Glu Ile Glu Asp Val Gln 1825 1830 1835 1840 Thr Asp Ile Pro Ser Glu Pro Trp Asn Thr Gly His Asp Trp Ile Leu                1845 1850 1855 Ala Asp Lys Arg Pro Thr Ala Trp Phe Leu Pro Ser Ile Arg Ala Ala            1860 1865 1870 Asn Val Met Ala Ala Ser Leu Arg Lys Ala Gly Lys Ser Val Val Val        1875 1880 1885 Leu Asn Arg Lys Thr Phe Glu Arg Glu Tyr Pro Thr Ile Lys Gln Lys    1890 1895 1900 Lys Pro Asp Phe Ile Leu Ala Thr Asp Ile Ala Glu Met Gly Ala Asn 1905 1910 1915 1920 Leu Cys Val Glu Arg Val Leu Asp Cys Arg Thr Ala Phe Lys Pro Val                1925 1930 1935 Leu Val Asp Glu Gly Arg Lys Val Ala Ile Lys Gly Pro Leu Arg Ile            1940 1945 1950 Ser Ala Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn Pro        1955 1960 1965 Asn Arg Asp Gly Asp Ser Tyr Tyr Tyr Ser Glu Pro Thr Ser Glu Asn    1970 1975 1980 Asn Ala His His Val Cys Trp Leu Glu Ala Ser Met Leu Leu Asp Asn 1985 1990 1995 2000 Met Glu Val Arg Gly Gly Met Val Ala Pro Leu Tyr Gly Val Glu Gly                2005 2010 2015 Thr Lys Thr Pro Val Ser Pro Gly Glu Met Arg Leu Arg Asp Asp Gln            2020 2025 2030 Arg Lys Val Phe Arg Glu Leu Val Arg Asn Cys Asp Leu Pro Val Trp        2035 2040 2045 Leu Ser Trp Gln Val Ala Lys Ala Gly Leu Lys Thr Asn Asp Arg Lys    2050 2055 2060 Trp Cys Phe Glu Gly Pro Glu Glu His Glu Ile Leu Asn Asp Ser Gly 2065 2070 2075 2080 Glu Thr Val Lys Cys Arg Ala Pro Gly Gly Ala Lys Lys Pro Leu Arg                2085 2090 2095 Pro Arg Trp Cys Asp Glu Arg Val Ser Ser Asp Gln Ser Ala Leu Ser            2100 2105 2110 Glu Phe Ile Lys Phe Ala Glu Gly Arg Arg Gly Ala Ala Glu Val Leu        2115 2120 2125 Val Val Leu Ser Glu Leu Pro Asp Phe Leu Ala Lys Lys Gly Gly Glu    2130 2135 2140 Ala Met Asp Thr Ile Ser Val Phe Leu His Ser Glu Glu Gly Ser Arg 2145 2150 2155 2160 Ala Tyr Arg Asn Ala Leu Ser Met Met Pro Glu Ala Met Thr Ile Val                2165 2170 2175 Met Leu Phe Ile Leu Ala Gly Leu Leu Thr Ser Gly Met Val Ile Phe            2180 2185 2190 Phe Met Ser Pro Lys Gly Ile Ser Arg Met Ser Met Ala Met Gly Thr        2195 2200 2205 Met Ala Gly Cys Gly Tyr Leu Met Phe Leu Gly Gly Val Lys Pro Thr    2210 2215 2220 His Ile Ser Tyr Val Met Leu Ile Phe Phe Val Leu Met Val Val Val 2225 2230 2235 2240 Ile Pro Glu Pro Gly Gln Gln Arg Ser Ile Gln Asp Asn Gln Val Ala                2245 2250 2255 Tyr Leu Ile Ile Gly Ile Leu Thr Leu Val Ser Ala Val Ala Ala Asn            2260 2265 2270 Glu Leu Gly Met Leu Glu Lys Thr Lys Glu Asp Leu Phe Gly Lys Lys        2275 2280 2285 Asn Leu Ile Pro Ser Ser Ala Ser Pro Trp Ser Trp Pro Asp Leu Asp    2290 2295 2300 Leu Lys Pro Gly Ala Ala Trp Thr Val Tyr Val Gly Ile Val Thr Met 2305 2310 2315 2320 Leu Ser Pro Met Leu His His Trp Ile Lys Val Glu Tyr Gly Asn Leu                2325 2330 2335 Ser Leu Ser Gly Ile Ala Gln Ser Ala Ser Val Leu Ser Phe Met Asp            2340 2345 2350 Lys Gly Ile Pro Phe Met Lys Met Asn Ile Ser Val Ile Met Leu Leu        2355 2360 2365 Val Ser Gly Trp Asn Ser Ile Thr Val Met Pro Leu Leu Cys Gly Ile    2370 2375 2380 Gly Cys Ala Met Leu His Trp Ser Leu Ile Leu Pro Gly Ile Lys Ala 2385 2390 2395 2400 Gln Gln Ser Lys Leu Ala Gln Arg Arg Val Phe His Gly Val Ala Glu                2405 2410 2415 Asn Pro Val Val Asp Gly Asn Pro Thr Val Asp Ile Glu Glu Ala Pro            2420 2425 2430 Glu Met Pro Ala Leu Tyr Glu Lys Lys Leu Ala Leu Tyr Leu Leu Leu        2435 2440 2445 Ala Leu Ser Leu Ala Ser Val Ala Met Cys Arg Thr Pro Phe Ser Leu    2450 2455 2460 Ala Glu Gly Ile Val Leu Ala Ser Ala Ala Leu Gly Pro Leu Ile Glu 2465 2470 2475 2480 Gly Asn Thr Ser Leu Leu Trp Asn Gly Pro Met Ala Val Ser Met Thr                2485 2490 2495 Gly Val Met Arg Gly Asn His Tyr Ala Phe Val Gly Val Met Tyr Asn            2500 2505 2510 Leu Trp Lys Met Lys Thr Gly Arg Arg Gly Ser Ala Asn Gly Lys Thr        2515 2520 2525 Leu Gly Glu Val Trp Lys Arg Glu Leu Asn Leu Leu Asp Lys Arg Gln    2530 2535 2540 Phe Glu Leu Tyr Lys Arg Thr Asp Ile Val Glu Val Asp Arg Asp Thr 2545 2550 2555 2560 Ala Arg Arg His Leu Ala Glu Gly Lys Val Asp Thr Gly Val Ala Val                2565 2570 2575 Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe His Glu Arg Gly Tyr Val            2580 2585 2590 Lys Leu Glu Gly Arg Val Ile Asp Leu Gly Cys Gly Arg Gly Gly Trp        2595 2600 2605 Cys Tyr Tyr Ala Ala Ala Gln Lys Glu Val Ser Gly Val Lys Gly Phe    2610 2615 2620 Thr Leu Gly Arg Asp Gly His Glu Lys Pro Met Asn Val Gln Ser Leu 2625 2630 2635 2640 Gly Trp Asn Ile Ile Thr Phe Lys Asp Lys Thr Asp Ile His Arg Leu                2645 2650 2655 Glu Pro Val Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser            2660 2665 2670 Ser Ser Ser Val Thr Glu Gly Glu Arg Thr Val Arg Val Leu Asp Thr        2675 2680 2685 Val Glu Lys Trp Leu Ala Cys Gly Val Asp Asn Phe Cys Val Lys Val    2690 2695 2700 Leu Ala Pro Tyr Met Pro Asp Val Leu Glu Lys Leu Glu Leu Leu Gln 2705 2710 2715 2720 Arg Arg Phe Gly Gly Thr Val Ile Arg Asn Pro Leu Ser Arg Asn Ser                2725 2730 2735 Thr His Glu Met Tyr Tyr Val Ser Gly Ala Arg Ser Asn Val Thr Phe            2740 2745 2750 Thr Val Asn Gln Thr Ser Arg Leu Leu Met Arg Arg Met Arg Arg Pro        2755 2760 2765 Thr Gly Lys Val Thr Leu Glu Ala Asp Val Ile Leu Pro Ile Gly Thr    2770 2775 2780 Arg Ser Val Glu Thr Asp Lys Gly Pro Leu Asp Lys Glu Ala Ile Glu 2785 2790 2795 2800 Glu Arg Val Glu Arg Ile Lys Ser Glu Tyr Met Thr Ser Trp Phe Tyr                2805 2810 2815 Asp Asn Asp Asn Pro Tyr Arg Thr Trp His Tyr Cys Gly Ser Tyr Val            2820 2825 2830 Thr Lys Thr Ser Gly Ser Ala Ala Ser Met Val Asn Gly Val Ile Lys        2835 2840 2845 Ile Leu Thr Tyr Pro Trp Asp Arg Ile Glu Glu Val Thr Arg Met Ala    2850 2855 2860 Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys 2865 2870 2875 2880 Val Asp Thr Arg Ala Lys Asp Pro Pro Ala Gly Thr Arg Lys Ile Met                2885 2890 2895 Lys Val Val Asn Arg Trp Leu Phe Arg His Leu Ala Arg Glu Lys Asn            2900 2905 2910 Pro Arg Leu Cys Thr Lys Glu Glu Phe Ile Ala Lys Val Arg Ser His        2915 2920 2925 Ala Ala Ile Gly Ala Tyr Leu Glu Glu Gln Glu Gln Trp Lys Thr Ala    2930 2935 2940 Asn Glu Ala Val Gln Asp Pro Lys Phe Trp Glu Leu Val Asp Glu Glu 2945 2950 2955 2960 Arg Lys Leu His Gln Gln Gly Arg Cys Arg Thr Cys Val Tyr Asn Met                2965 2970 2975 Met Gly Lys Arg Glu Lys Lys Leu Ser Glu Phe Gly Lys Ala Lys Gly            2980 2985 2990 Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Tyr Leu Glu Phe        2995 3000 3005 Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Ala Ser Arg Glu Asn    3010 3015 3020 Ser Gly Gly Gly Val Glu Gly Ile Gly Leu Gln Tyr Leu Gly Tyr Val 3025 3030 3035 3040 Ile Arg Asp Leu Ala Ala Met Asp Gly Gly Gly Phe Tyr Ala Asp Asp                3045 3050 3055 Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Ala Asp Leu Asp Asp Glu            3060 3065 3070 Gln Glu Ile Leu Asn Tyr Met Ser Pro His His Lys Lys Leu Ala Gln        3075 3080 3085 Ala Val Met Glu Met Thr Tyr Lys Asn Lys Val Val Lys Val Leu Arg    3090 3095 3100 Pro Ala Pro Gly Gly Lys Ala Tyr Met Asp Val Ile Ser Arg Arg Asp 3105 3110 3115 3120 Gln Arg Gly Ser Gly Gln Val Val Thr Tyr Ala Leu Asn Thr Ile Thr                3125 3130 3135 Asn Leu Lys Val Gln Leu Ile Arg Met Ala Glu Ala Glu Met Val Ile            3140 3145 3150 His His Gln His Val Gln Asp Cys Asp Glu Ser Val Leu Thr Arg Leu        3155 3160 3165 Glu Ala Trp Leu Thr Glu His Gly Cys Asp Arg Leu Lys Arg Met Ala    3170 3175 3180 Val Ser Gly Asp Asp Cys Val Val Arg Pro Ile Asp Asp Arg Phe Gly 3185 3190 3195 3200 Leu Ala Leu Ser His Leu Asn Ala Met Ser Lys Val Arg Lys Asp Ile                3205 3210 3215 Ser Glu Trp Gln Pro Ser Lys Gly Trp Asn Asp Trp Glu Asn Val Pro            3220 3225 3230 Phe Cys Ser His His Phe His Glu Leu Gln Leu Lys Asp Gly Arg Arg        3235 3240 3245 Ile Val Val Pro Cys Arg Glu Gln Asp Glu Leu Ile Gly Arg Gly Arg    3250 3255 3260 Val Ser Pro Gly Asn Gly Trp Met Ile Lys Glu Thr Ala Cys Leu Ser 3265 3270 3275 3280 Lys Ala Tyr Ala Asn Met Trp Ser Leu Met Tyr Phe His Lys Arg Asp                3285 3290 3295 Met Arg Leu Leu Ser Leu Ala Val Ser Ser Ala Val Pro Thr Ser Trp            3300 3305 3310 Val Pro Gln Gly Arg Thr Thr Trp Ser Ile His Gly Lys Gly Glu Trp        3315 3320 3325 Met Thr Thr Glu Asp Met Leu Glu Val Trp Asn Arg Val Trp Ile Thr    3330 3335 3340 Asn Asn Pro His Met Gln Asp Lys Thr Met Val Lys Lys Trp Arg Asp 3345 3350 3355 3360 Val Pro Tyr Leu Thr Lys Arg Gln Asp Lys Leu Cys Gly Ser Leu Ile                3365 3370 3375 Gly Met Thr Asn Arg Ala Thr Trp Ala Ser His Ile His Leu Val Ile            3380 3385 3390 His Arg Ile Arg Thr Leu Ile Gly Gln Glu Lys Tyr Thr Asp Tyr Leu        3395 3400 3405 Thr Val Met Asp Arg Tyr Ser Val Asp Ala Asp Leu Gln Leu Gly Glu    3410 3415 3420 Leu ile 3425 <210> 4 <211> 10907 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 2 nucleic acid sequence <400> 4 agtaaatcct gtgtgctaat tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60 acacatttgg attaatttta atcgttcgtt gagcgattag cagagaactg accagaacat 120 gtctggtcgt aaagctcagg gaaaaaccct gggcgtcaat atggtacgac gaggagttcg 180 ctccttgtca aacaaaataa aacaaaaaac aaaacaaatt ggaaacagac ctggaccttc 240 aagaggtgtt caaggattta tctttttctt tttgttcaac attttgactg gaaaaaagat 300 cacagcccac ctaaagaggt tgtggaaaat gctggaccca agacaaggct tggctgttct 360 aaggaaagtc aagagagtgg tggccagttt gatgagagga ttgtcctcaa ggaaacgccg 420 ttcccatgat gttctgactg tgcaattcct aattttggga atgctgttga tgacgggtgg 480 agcggaggtc actagacgtg ggagtgcata ctatatgtac ttggacagaa acgatgctgg 540 ggaggccata tcttttccaa ccacattggg gatgaataag tgttatatac agatcatgga 600 tcttggacac atgtgtgatg ccaccatgag ctatgaatgc cctatgctgg atgagggggt 660 ggaaccagat gacgtcgatt gttggtgcaa cacgacgtca acttgggttg tgtacggaac 720 ctgccatcac aaaaaaggtg aagcacggag atctagaaga gctgtgacgc tcccctccca 780 ttccactagg aagctgcaaa cgcggtcgca aacctggttg gaatcaagag aatacacaaa 840 gcacttgatt agagtcgaaa attggatatt caggaaccct ggcttcgcgt tagcagcagc 900 tgccatcgct tggcttttgg gaagctcaac gagccaaaaa gtcatatact tggtcatgat 960 actgctgatt gccccggcat acagcatcag gtgcatagga gtcagcaata gggactttgt 1020 ggaaggtatg tcaggtggga cttgggttga tgttgtcttg gaacatggag gttgtgtcac 1080 cgtaatggca caggacaaac cgactgtcga catagagctg gttacaacaa cagtcagcaa 1140 catggcggag gtaagatcct actgctatga ggcatcaata tcagacatgg cttcggacag 1200 ccgctgccca acacaaggtg aagcctacct tgacaagcaa tcagacactc aatatgtctg 1260 caaaagaacg ttagtggaca gaggctgggg aaatggatgt ggactttttg gcaaagggag 1320 cctggtgaca tgcgctaagt ttgcatgctc caagaaaatg accgggaaga gcatccagcc 1380 agagaatctg gagtaccgga taatgctgtc agttcatggc tcccagcaca gtgggatgat 1440 cgttaatgac acaggacatg aaactgatga gaatagagcg aaagttgaga taacgcccaa 1500 ttcaccaaga gccgaagcca ccctgggggg ttttggaagc ctaggacttg attgtgaacc 1560 gaggacaggc cttgactttt cagatttgta ttacttgact atgaataaca agcactggtt 1620 ggttcacaag gagtggttcc acgacattcc attaccttgg cacgctgggg cagacaccgg 1680 aactccacac tggaacaaca aagaagcact ggtagagttc aaggacgcac atgccaaaag 1740 gcaaactgtc gtggttctag ggagtcaaga aggagcagtt cacacggccc ttgctggagc 1800 tctggaggct gagatggatg gtgcaaaggg aaggctgtcc tctggccact tgaaatgtcg 1860 cctgaaaatg gataaactta gattgaaggg cgtgtcatac tccttgtgta ctgcagcgtt 1920 cacattcacc aagatcccgg ctgaaacact gcacgggaca gtcacagtgg aggtacagta 1980 cgcagggaca gatggacctt gcaaggttcc agctcagatg gcggtggaca tgcaaactct 2040 gaccccagtt gggaggttga taaccgctaa ccccgtaatc actgaaagca ctgagaactc 2100 taagatgatg ctggaacttg atccaccatt tggggactct tacattgtca taggagtcgg 2160 ggagaagaag atcacccacc actggcacag gagtggcagc accattggaa aagcatttga 2220 agccactgtg agaggtgcca agagaatggc agtcttggga gacacagcct gggactttgg 2280 atcagttgga ggcgctctca actcattggg caagggcatc catcaaattt ttggagcagc 2340 tttcaaatca ttgtttggag gaatgtcctg gttctcacaa attctcattg gaacgttgct 2400 gatgtggttg ggtctgaaca caaagaatgg atctatttcc cttatgtgct tggccttagg 2460 gggggtgttg atcttcttat ccacagccgt tggggcggat caaggatgcg ccatcaactt 2520 tggcaagaga gagctcaagt gcggagatgg tatcttcata tttagagact ctgatgactg 2580 gctgaacaag tactcatact atccagaaga tcctgtgaag cttgcatcaa tagtgaaagc 2640 ctcttttgaa gaagggaagt gtggcctaaa ttcagttgac tcccttgagc atgagatgtg 2700 gagaagcagg gcagatgaga tcaatgccat ttttgaggaa aacgaggtgg acatttctgt 2760 tgtcgtgcag gatccaaaga atgtttacca gagaggaact catccatttt ccagaattcg 2820 ggatggtctg cagtatggtt ggaagacttg gggtaagaac cttgtgttct ccccagggag 2880 gaagaatgga agcttcatca tagatggaaa gtccaggaaa gaatgcccgt tttcaaaccg 2940 ggtctggaat tctttccaga tagaggagtt tgggacggga gtgttcacca cacgcgtgta 3000 catggacgca gtctttgaat acaccataga ctgcgatgga tctatcttgg gtgcagcggt 3060 gaacggaaaa aagagtgccc atggctctcc aacattttgg atgggaagtc atgaagtaaa 3120 tgggacatgg atgatccaca ccttggaggc attagattac aaggagtgtg agtggccact 3180 gacacatacg attggaacat cagttgaaga gagtgaaatg ttcatgccga gatcaatcgg 3240 aggcccagtt agctctcaca atcatatccc tggatacaag gttcagacga acggaccttg 3300 gatgcaggta ccactagaag tgaagagaga agcttgccca gggactagcg tgatcattga 3360 tggcaactgt gatggacggg gaaaatcaac cagatccacc acggatagcg ggaaagttat 3420 tcctgaatgg tgttgccgct cctgcacaat gccgcctgtg agcttccatg gtagtgatgg 3480 gtgttggtat cccatggaaa ttaggccaag gaaaacgcat gaaagccatc tggtgcgctc 3540 ctgggttaca gctggagaaa tacatgctgt cccttttggt ttggtgagca tgatgatagc 3600 aatggaagtg gtcctaagga aaagacaggg accaaagcaa atgttggttg gaggagtagt 3660 gctcttggga gcaatgctgg tcgggcaagt aactctcctt gatttgctga aactcacagt 3720 ggctgtggga ttgcatttcc atgagatgaa caatggagga gacgccatgt atatggcgtt 3780 gattgctgcc ttttcaatca gaccagggct gctcatcggc tttgggctca ggaccctatg 3840 gagccctcgg gaacgccttg tgctgaccct aggagcagcc atggtggaga ttgccttggg 3900 tggcgtgatg ggcggcctgt ggaagtatct aaatgcagtt tctctctgca tcctgacaat 3960 aaatgctgtt gcttctagga aagcatcaaa taccatcttg cccctcatgg ctctgttgac 4020 acctgtcact atggctgagg tgagacttgc cgcaatgttc ttttgtgccg tggttatcat 4080 aggggtcctt caccagaatt tcaaggacac ctccatgcag aagactatac ctctggtggc 4140 cctcacactc acatcttacc tgggcttgac acaacctttt ttgggcctgt gtgcatttct 4200 ggcaacccgc atatttgggc gaaggagtat cccagtgaat gaggcactcg cagcagctgg 4260 tctagtggga gtgctggcag gactggcttt tcaggagatg gagaacttcc ttggtccgat 4320 tgcagttgga ggactcctga tgatgctggt tagcgtggct gggagggtgg atgggctaga 4380 gctcaagaag cttggtgaag tttcatggga agaggaggcg gagatcagcg ggagttccgc 4440 ccgctatgat gtggcactca gtgaacaagg ggagttcaag ctgctttctg aagagaaagt 4500 gccatgggac caggttgtga tgacctcgct ggccttggtt ggggctgccc tccatccatt 4560 tgctcttctg ctggtccttg ctgggtggct gtttcatgtc aggggagcta ggagaagtgg 4620 ggatgtcttg tgggatattc ccactcctaa gatcatcgag gaatgtgaac atctggagga 4680 tgggatttat ggcatattcc agtcaacctt cttgggggcc tcccagcgag gagtgggagt 4740 ggcacaggga ggggtgttcc acacaatgtg gcatgtcaca agaggagctt tccttgtcag 4800 gaatggcaag aagttgattc catcttgggc ttcagtaaag gaagaccttg tcgcctatgg 4860 tggctcatgg aagttggaag gcagatggga tggagaggaa gaggtccagt tgatcgcggc 4920 tgttccagga aagaacgtgg tcaacgtcca gacaaaaccg agcttgttca aagtgaggaa 4980 tgggggagaa atcggggctg tcgctcttga ctatccgagt ggcacttcag gatctcctat 5040 tgttaacagg aacggagagg tgattgggct gtacggcaat ggcatccttg tcggtgacaa 5100 ctccttcgtg tccgccatat cccagactga ggtgaaggaa gaaggaaagg aggagctcca 5160 agagatcccg acaatgctaa agaaaggaat gacaactgtc cttgattttc atcctggagc 5220 tgggaagaca agacgtttcc tcccacagat cttggccgag tgcgcacgga gacgcttgcg 5280 cactcttgtg ttggccccca ccagggttgt tctttctgaa atgaaggagg cttttcacgg 5340 cctggacgtg aaattccaca cacaggcttt ttccgctcac ggcagcggga gagaagtcat 5400 tgatgccatg tgccatgcca ccctaactta caggatgttg gaaccaacta gggttgttaa 5460 ctgggaagtg atcattatgg atgaagccca ttttttggat ccagctagca tagccgctag 5520 aggttgggca gcgcacagag ctagggcaaa tgaaagtgca acaatcttga tgacagccac 5580 accgcctggg actagtgatg aatttccaca ttcaaatggt gaaatagaag atgttcaaac 5640 ggacataccc agtgagccct ggaacacagg gcatgactgg atcctagctg acaaaaggcc 5700 cacggcatgg ttccttccat ccatcagagc tgcaaatgtc atggctgcct ctttgcgtaa 5760 ggctggaaag agtgtggtgg tcctgaacag gaaaaccttt gagagagaat accccacgat 5820 aaagcagaag aaacctgact ttatattggc cactgacata gctgaaatgg gagccaacct 5880 ttgcgtggag cgagtgctgg attgcaggac ggcttttaag cctgtgcttg tggatgaagg 5940 gaggaaggtg gcaataaaag ggccacttcg tatctccgca tcctctgctg ctcaaaggag 6000 ggggcgcatt gggagaaatc ccaacagaga tggagactca tactactatt ctgagcctac 6060 aagtgaaaat aatgcccacc acgtctgctg gttggaggcc tcaatgctct tggacaacat 6120 ggaggtgagg ggtggaatgg tcgccccact ctatggcgtt gaaggaacta aaacaccagt 6180 ttcccctggt gaaatgagac tgagggatga ccagaggaaa gtcttcagag aactagtgag 6240 gaattgtgac ctgcccgttt ggctttcgtg gcaagtggcc aaggctggtt tgaagacgaa 6300 tgatcgtaag tggtgttttg aaggccctga ggaacatgag atcttgaatg acagcggtga 6360 aacagtgaag tgcagggctc ctggaggagc aaagaagcct ctgcgcccaa ggtggtgtga 6420 tgaaagggtg tcatctgacc agagtgcgct gtctgaattt attaagtttg ctgaaggtag 6480 gaggggagct gctgaagtgc tagttgtgct gagtgaactc cctgatttcc tggctaaaaa 6540 aggtggagag gcaatggata ccatcagtgt gttcctccac tctgaggaag gctctagggc 6600 ttaccgcaat gcactatcaa tgatgcctga ggcaatgaca atagtcatgc tgtttatact 6660 ggctggacta ctgacatcgg gaatggtcat ctttttcatg tctcccaaag gcatcagtag 6720 aatgtctatg gcgatgggca caatggccgg ctgtggatat ctcatgttcc ttggaggcgt 6780 caaacccact cacatctcct atgtcatgct catattcttt gtcctgatgg tggttgtgat 6840 ccccgagcca gggcaacaaa ggtccatcca agacaaccaa gtggcatacc tcattattgg 6900 catcctgacg ctggtttcag cggtggcagc caacgagcta ggcatgctgg agaaaaccaa 6960 agaggacctc tttgggaaga agaacttaat tccatctagt gcttcaccct ggagttggcc 7020 ggatcttgac ctgaagccag gagctgcctg gacagtgtac gttggcattg ttacaatgct 7080 ctctccaatg ttgcaccact ggatcaaagt cgaatatggc aacctgtctc tgtctggaat 7140 agcccagtca gcctcagtcc tttctttcat ggacaagggg ataccattca tgaagatgaa 7200 tatctcggtc ataatgctgc tggtcagtgg ctggaattca ataacagtga tgcctctgct 7260 ctgtggcata gggtgcgcca tgctccactg gtctctcatt ttacctggaa tcaaagcgca 7320 gcagtcaaag cttgcacaga gaagggtgtt ccatggcgtt gccgagaacc ctgtggttga 7380 tgggaatcca acagttgaca ttgaggaagc tcctgaaatg cctgcccttt atgagaagaa 7440 actggctcta tatctccttc ttgctctcag cctagcttct gttgccatgt gcagaacgcc 7500 cttttcattg gctgaaggca ttgtcctagc atcagctgcc ttagggccgc tcatagaggg 7560 aaacaccagc cttctttgga atggacccat ggctgtctcc atgacaggag tcatgagggg 7620 gaatcactat gcttttgtgg gagtcatgta caatctatgg aagatgaaaa ctggacgccg 7680 ggggagcgcg aatggaaaaa ctttgggtga agtctggaag agggaactga atctgttgga 7740 caagcgacag tttgagttgt ataaaaggac cgacattgtg gaggtggatc gtgatacggc 7800 acgcaggcat ttggccgaag ggaaggtgga caccggggtg gcggtctcca gggggaccgc 7860 aaagttaagg tggttccatg agcgtggcta tgtcaagctg gaaggtaggg tgattgacct 7920 ggggtgtggc cgcggaggct ggtgttacta cgctgctgcg caaaaggaag tgagtggggt 7980 caaaggattt actcttggaa gagacggcca tgagaaaccc atgaatgtgc aaagtctggg 8040 atggaacatc atcaccttca aggacaaaac tgatatccac cgcctagaac cagtgaaatg 8100 tgacaccctt ttgtgtgaca ttggagagtc atcatcgtca tcggtcacag agggggaaag 8160 gaccgtgaga gttcttgata ctgtagaaaa atggctggct tgtggggttg acaacttctg 8220 tgtgaaggtg ttagctccat acatgccaga tgttctcgag aaactggaat tgctccaaag 8280 gaggtttggc ggaacagtga tcaggaaccc tctctccagg aattccactc atgaaatgta 8340 ctacgtgtct ggagcccgca gcaatgtcac atttactgtg aaccaaacat cccgcctcct 8400 gatgaggaga atgaggcgtc caactggaaa agtgaccctg gaggctgacg tcatcctccc 8460 aattgggaca cgcagtgttg agacagacaa gggacccctg gacaaagagg ccatagaaga 8520 aagggttgag aggataaaat ctgagtacat gacctcttgg ttttatgaca atgacaaccc 8580 ctacaggacc tggcactact gtggctccta tgtcacaaaa acctcaggaa gtgcggcgag 8640 catggtaaat ggtgttatta aaattctgac atatccatgg gacaggatag aggaggtcac 8700 aagaatggca atgactgaca caaccccttt tggacagcaa agagtgttta aagaaaaagt 8760 tgacaccaga gcaaaggatc caccagcggg aactaggaag atcatgaaag ttgtcaacag 8820 gtggctgttc cgccacctgg ccagagaaaa gaaccccaga ctgtgcacaa aggaagaatt 8880 tattgcaaaa gtccgaagtc atgcagccat tggagcttac ctggaagaac aagaacagtg 8940 gaagactgcc aatgaggctg tccaagaccc aaagttctgg gaactggtgg atgaagaaag 9000 gaagctgcac caacaaggca ggtgtcggac ttgtgtgtac aacatgatgg ggaaaagaga 9060 gaagaagctg tcagagtttg ggaaagcaaa gggaagccgt gccatatggt atatgtggct 9120 gggagcgcgg tatcttgagt ttgaggccct gggattcctg aatgaggacc attgggcttc 9180 cagggaaaac tcaggaggag gagtggaagg cattggctta caatacctag gatatgtgat 9240 cagagacctg gctgcaatgg atggtggtgg attctacgcg gatgacaccg ctggatggga 9300 cacgcgcatc acagaggcag accttgatga tgaacaggag atcttgaact acatgagccc 9360 acatcacaaa aaactggcac aagcagtgat ggaaatgaca tacaagaaca aagtggtgaa 9420 agtgttgaga ccagccccag gagggaaagc ctacatggat gtcataagtc gacgagacca 9480 gagaggatcc gggcaggtag tgacttatgc tctgaacacc atcaccaact tgaaagtcca 9540 attgatcaga atggcagaag cagagatggt gatacatcac caacatgttc aagattgtga 9600 tgaatcagtt ctgaccaggc tggaggcatg gctcactgag cacggatgtg acagactgaa 9660 gaggatggcg gtgagtggag acgactgtgt ggtccggccc atcgatgaca ggttcggcct 9720 ggccctgtcc catctcaacg ccatgtccaa ggttagaaag gacatatctg aatggcagcc 9780 atcaaaaggg tggaatgatt gggagaatgt gcccttctgt tcccaccact tccatgaact 9840 acagctgaag gatggcagga ggattgtggt gccttgccga gaacaggacg agctcattgg 9900 gagaggaagg gtgtctccag gaaacggctg gatgatcaag gaaacagctt gcctcagcaa 9960 agcctatgcc aacatgtggt cactgatgta ttttcacaaa agggacatga ggctactgtc 10020 attggctgtt tcctcagctg ttcccacctc atgggttcca caaggacgca caacatggtc 10080 gattcatggg aaaggggagt ggatgaccac ggaagacatg cttgaggtgt ggaacagagt 10140 atggataacc aacaacccac acatgcagga caagacaatg gtgaaaaaat ggagagatgt 10200 cccttatcta accaagagac aagacaagct gtgcggatca ctgattggaa tgaccaatag 10260 ggccacctgg gcctcccaca tccatttagt catccatcgt atccgaacgc tgattggaca 10320 ggagaaatac actgactacc taacagtcat ggacaggtat tctgtggatg ctgacctgca 10380 actgggtgag cttatctgaa acaccatcta acaggaataa ccgggataca aaccacgggt 10440 ggagaaccgg actccccaca acctgaaacc gggatataaa ccacggctgg agaaccgggc 10500 tccgcactta aaatgaaaca gaaaccggga taaaaactac ggatggagaa ccggactcca 10560 cacattgaga cagaagaagt tgtcagccca gaaccccaca cgagttttgc cactgctaag 10620 ctgtgaggca gtgcaggctg ggacagccga cctccaggtt gcgaaaaacc tggtttctgg 10680 gacctcccac cccagagtaa aaagaacgga gcctccgcta ccaccctccc acgtggtggt 10740 agaaagacgg ggtctagagg ttagaggaga ccctccaggg aacaaatagt gggaccatat 10800 tgacgccagg gaaagaccgg agtggttctc tgcttttcct ccagaggtct gtgagcacag 10860 tttgctcaag aataagcaga cctttggatg acaaacacaa aaccact 10907 <210> 5 <211> 3424 <212> PRT <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 3 amino acid sequence <400> 5 Met Ser Gly Arg Lys Ala Gln Gly Lys Thr Leu Gly Val Asn Met Val   1 5 10 15 Arg Arg Gly Val Arg Ser Leu Ser Asn Lys Ile Lys Gln Lys Thr Lys              20 25 30 Gln Ile Gly Asn Arg Pro Gly Pro Ser Arg Gly Val Gln Gly Phe Ile          35 40 45 Phe Phe Phe Leu Phe Asn Ile Leu Thr Gly Lys Lys Ile Thr Ala His      50 55 60 Leu Lys Arg Leu Trp Lys Met Leu Asp Pro Arg Gln Gly Leu Ala Val  65 70 75 80 Leu Arg Lys Val Lys Arg Val Val Ala Ser Leu Met Arg Gly Leu Ser                  85 90 95 Ser Arg Lys Arg Arg Gly Ala Asp Thr Ser Val Gly Ile Val Gly Leu             100 105 110 Leu Leu Thr Thr Ala Met Ala Ala Glu Val Thr Arg Arg Gly Ser Ala         115 120 125 Tyr Tyr Met Tyr Leu Asp Arg Asn Asp Ala Gly Glu Ala Ile Ser Phe     130 135 140 Pro Thr Thr Leu Gly Met Asn Lys Cys Tyr Ile Gln Ile Met Asp Leu 145 150 155 160 Gly His Met Cys Asp Ala Thr Met Ser Tyr Glu Cys Pro Met Leu Asp                 165 170 175 Glu Gly Val Glu Pro Asp Asp Val Asp Cys Trp Cys Asn Thr Thr Ser             180 185 190 Thr Trp Val Val Tyr Gly Thr Cys His His Lys Lys Gly Glu Ala Arg         195 200 205 Arg Ser Arg Arg Ala Val Thr Leu Pro Ser His Ser Thr Arg Lys Leu     210 215 220 Gln Thr Arg Ser Gln Thr Trp Leu Glu Ser Arg Glu Tyr Thr Lys His 225 230 235 240 Leu Ile Arg Val Glu Asn Trp Ile Phe Arg Asn Pro Gly Phe Ala Leu                 245 250 255 Ala Ala Ala Ala Ile Ala Trp Leu Leu Gly Ser Ser Thr Ser Gln Lys             260 265 270 Val Ile Tyr Leu Val Met Ile Leu Leu Ile Ala Pro Ala Tyr Ser Ile         275 280 285 Arg Cys Ile Gly Val Ser Asn Arg Asp Phe Val Glu Gly Met Ser Gly     290 295 300 Gly Thr Trp Val Asp Val Val Leu Glu His Gly Gly Cys Val Thr Val 305 310 315 320 Met Ala Gln Asp Lys Pro Thr Val Asp Ile Glu Leu Val Thr Thr Thr                 325 330 335 Val Ser Asn Met Ala Glu Val Arg Ser Tyr Cys Tyr Glu Ala Ser Ile             340 345 350 Ser Asp Met Ala Ser Asp Ser Arg Cys Pro Thr Gln Gly Glu Ala Tyr         355 360 365 Leu Asp Lys Gln Ser Asp Thr Gln Tyr Val Cys Lys Arg Thr Leu Val     370 375 380 Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser Leu 385 390 395 400 Val Thr Cys Ala Lys Phe Ala Cys Ser Lys Lys Met Thr Gly Lys Ser                 405 410 415 Ile Gln Pro Glu Asn Leu Glu Tyr Arg Ile Met Leu Ser Val His Gly             420 425 430 Ser Gln His Ser Gly Met Ile Val Asn Asp Thr Gly His Glu Thr Asp         435 440 445 Glu Asn Arg Ala Lys Val Glu Ile Thr Pro Asn Ser Pro Arg Ala Glu     450 455 460 Ala Thr Leu Gly Gly Phe Gly Ser Leu Gly Leu Asp Cys Glu Pro Arg 465 470 475 480 Thr Gly Leu Asp Phe Ser Asp Leu Tyr Tyr Leu Thr Met Asn Asn Lys                 485 490 495 His Trp Leu Val His Lys Glu Trp Phe His Asp Ile Pro Leu Pro Trp             500 505 510 His Ala Gly Ala Asp Thr Gly Thr Pro His Trp Asn Asn Lys Glu Ala         515 520 525 Leu Val Glu Phe Lys Asp Ala His Ala Lys Arg Gln Thr Val Val Val     530 535 540 Leu Gly Ser Gln Glu Gly Ala Val His Thr Ala Leu Ala Gly Ala Leu 545 550 555 560 Glu Ala Glu Met Asp Gly Ala Lys Gly Arg Leu Ser Ser Gly His Leu                 565 570 575 Lys Cys Arg Leu Lys Met Asp Lys Leu Arg Leu Lys Gly Val Ser Tyr             580 585 590 Ser Leu Cys Thr Ala Ala Phe Thr Phe Thr Lys Ile Pro Ala Glu Thr         595 600 605 Leu His Gly Thr Val Thr Val Glu Val Gln Tyr Ala Gly Thr Asp Gly     610 615 620 Pro Cys Lys Val Pro Ala Gln Met Ala Val Asp Met Gln Thr Leu Thr 625 630 635 640 Pro Val Gly Arg Leu Ile Thr Ala Asn Pro Val Ile Thr Glu Ser Thr                 645 650 655 Glu Asn Ser Lys Met Met Leu Glu Leu Asp Pro Pro Phe Gly Asp Ser             660 665 670 Tyr Ile Val Ile Gly Val Gly Glu Lys Lys Ile Thr His His Trp His         675 680 685 Arg Ser Gly Ser Thr Ile Gly Lys Ala Phe Glu Ala Thr Val Arg Gly     690 695 700 Ala Lys Arg Met Ala Val Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser 705 710 715 720 Val Gly Gly Ala Leu Asn Ser Leu Gly Lys Gly Ile His Gln Ile Phe                 725 730 735 Gly Ala Ala Phe Lys Ser Leu Phe Gly Gly Met Ser Trp Phe Ser Gln             740 745 750 Ile Leu Ile Gly Thr Leu Leu Met Trp Leu Gly Leu Asn Thr Lys Asn         755 760 765 Gly Ser Ile Ser Leu Met Cys Leu Ala Leu Gly Gly Val Leu Ile Phe     770 775 780 Leu Ser Thr Ala Val Ser Ala Asp Gln Gly Cys Ala Ile Asn Phe Gly 785 790 795 800 Lys Arg Glu Leu Lys Cys Gly Asp Gly Ile Phe Ile Phe Arg Asp Ser                 805 810 815 Asp Asp Trp Leu Asn Lys Tyr Ser Tyr Tyr Pro Glu Asp Pro Val Lys             820 825 830 Leu Ala Ser Ile Val Lys Ala Ser Phe Glu Glu Gly Lys Cys Gly Leu         835 840 845 Asn Ser Val Asp Ser Leu Glu His Glu Met Trp Arg Ser Arg Ala Asp     850 855 860 Glu Ile Asn Ala Ile Phe Glu Glu Asn Glu Val Asp Ile Ser Val Val 865 870 875 880 Val Gln Asp Pro Lys Asn Val Tyr Gln Arg Gly Thr His Pro Phe Ser                 885 890 895 Arg Ile Arg Asp Gly Leu Gln Tyr Gly Trp Lys Thr Trp Gly Lys Asn             900 905 910 Leu Val Phe Ser Pro Gly Arg Lys Asn Gly Ser Phe Ile Ile Asp Gly         915 920 925 Lys Ser Arg Lys Glu Cys Pro Phe Ser Asn Arg Val Trp Asn Ser Phe     930 935 940 Gln Ile Glu Glu Phe Gly Thr Gly Val Phe Thr Thr Arg Val Tyr Met 945 950 955 960 Asp Ala Val Phe Glu Tyr Thr Ile Asp Cys Asp Gly Ser Ile Leu Gly                 965 970 975 Ala Ala Val Asn Gly Lys Lys Ser Ala His Gly Ser Pro Thr Phe Trp             980 985 990 Met Gly Ser His Glu Val Asn Gly Thr Trp Met Ile His Thr Leu Glu         995 1000 1005 Ala Leu Asp Tyr Lys Glu Cys Glu Trp Pro Leu Thr His Thr Ile Gly    1010 1015 1020 Thr Ser Val Glu Glu Ser Glu Met Phe Met Pro Arg Ser Ile Gly Gly 1025 1030 1035 1040 Pro Val Ser Ser His Asn His Ile Pro Gly Tyr Lys Val Gln Thr Asn                1045 1050 1055 Gly Pro Trp Met Gln Val Pro Leu Glu Val Lys Arg Glu Ala Cys Pro            1060 1065 1070 Gly Thr Ser Val Ile Ile Asp Gly Asn Cys Asp Gly Arg Gly Lys Ser        1075 1080 1085 Thr Arg Ser Thr Thr Asp Ser Gly Lys Val Ile Pro Glu Trp Cys Cys    1090 1095 1100 Arg Ser Cys Thr Met Pro Pro Val Ser Phe His Gly Ser Asp Gly Cys 1105 1110 1115 1120 Trp Tyr Pro Met Glu Ile Arg Pro Arg Lys Thr His Glu Ser His Leu                1125 1130 1135 Val Arg Ser Trp Val Thr Ala Gly Glu Ile His Ala Val Pro Phe Gly            1140 1145 1150 Leu Val Ser Met Met Ile Ala Met Glu Val Val Leu Arg Lys Arg Gln        1155 1160 1165 Gly Pro Lys Gln Met Leu Val Gly Gly Val Val Leu Leu Gly Ala Met    1170 1175 1180 Leu Val Gly Gln Val Thr Leu Leu Asp Leu Leu Lys Leu Thr Val Ala 1185 1190 1195 1200 Val Gly Leu His Phe His Glu Met Asn Asn Gly Gly Asp Ala Met Tyr                1205 1210 1215 Met Ala Leu Ile Ala Ala Phe Ser Ile Arg Pro Gly Leu Leu Ile Gly            1220 1225 1230 Phe Gly Leu Arg Thr Leu Trp Ser Pro Arg Glu Arg Leu Val Leu Thr        1235 1240 1245 Leu Gly Ala Ala Met Val Glu Ile Ala Leu Gly Gly Val Met Gly Gly    1250 1255 1260 Leu Trp Lys Tyr Leu Asn Ala Val Ser Leu Cys Ile Leu Thr Ile Asn 1265 1270 1275 1280 Ala Val Ala Ser Arg Lys Ala Ser Asn Thr Ile Leu Pro Leu Met Ala                1285 1290 1295 Leu Leu Thr Pro Val Thr Met Ala Glu Val Arg Leu Ala Ala Met Phe            1300 1305 1310 Phe Cys Ala Val Val Ile Ile Gly Val Leu His Gln Asn Phe Lys Asp        1315 1320 1325 Thr Ser Met Gln Lys Thr Ile Pro Leu Val Ala Leu Thr Leu Thr Ser    1330 1335 1340 Tyr Leu Gly Leu Thr Gln Pro Phe Leu Gly Leu Cys Ala Phe Leu Ala 1345 1350 1355 1360 Thr Arg Ile Phe Gly Arg Arg Ser Ile Pro Val Asn Glu Ala Leu Ala                1365 1370 1375 Ala Ala Gly Leu Val Gly Val Leu Ala Gly Leu Ala Phe Gln Glu Met            1380 1385 1390 Glu Asn Phe Leu Gly Pro Ile Ala Val Gly Gly Leu Leu Met Met Leu        1395 1400 1405 Val Ser Val Ala Gly Arg Val Asp Gly Leu Glu Leu Lys Lys Leu Gly    1410 1415 1420 Glu Val Ser Trp Glu Glu Glu Ala Glu Ile Ser Gly Ser Ser Ala Arg 1425 1430 1435 1440 Tyr Asp Val Ala Leu Ser Glu Gln Gly Glu Phe Lys Leu Leu Ser Glu                1445 1450 1455 Glu Lys Val Pro Trp Asp Gln Val Val Met Thr Ser Leu Ala Leu Val            1460 1465 1470 Gly Ala Ala Leu His Pro Phe Ala Leu Leu Leu Val Leu Ala Gly Trp        1475 1480 1485 Leu Phe His Val Arg Gly Ala Arg Arg Ser Gly Asp Val Leu Trp Asp    1490 1495 1500 Ile Pro Thr Pro Lys Ile Ile Glu Glu Cys Glu His Leu Glu Asp Gly 1505 1510 1515 1520 Ile Tyr Gly Ile Phe Gln Ser Thr Phe Leu Gly Ala Ser Gln Arg Gly                1525 1530 1535 Val Gly Val Ala Gln Gly Gly Val Phe His Thr Met Trp His Val Thr            1540 1545 1550 Arg Gly Ala Phe Leu Val Arg Asn Gly Lys Lys Leu Ile Pro Ser Trp        1555 1560 1565 Ala Ser Val Lys Glu Asp Leu Val Ala Tyr Gly Gly Ser Trp Lys Leu    1570 1575 1580 Glu Gly Arg Trp Asp Gly Glu Glu Glu Val Gln Leu Ile Ala Ala Val 1585 1590 1595 1600 Pro Gly Lys Asn Val Val Asn Val Gln Thr Lys Pro Ser Leu Phe Lys                1605 1610 1615 Val Arg Asn Gly Gly Glu Ile Gly Ala Val Ala Leu Asp Tyr Pro Ser            1620 1625 1630 Gly Thr Ser Gly Ser Pro Ile Val Asn Arg Asn Gly Glu Val Ile Gly        1635 1640 1645 Leu Tyr Gly Asn Gly Ile Leu Val Gly Asp Asn Ser Phe Val Ser Ala    1650 1655 1660 Ile Ser Gln Thr Glu Val Lys Glu Glu Gly Lys Glu Glu Leu Gln Glu 1665 1670 1675 1680 Ile Pro Thr Met Leu Lys Lys Gly Met Thr Thr Val Leu Asp Phe His                1685 1690 1695 Pro Gly Ala Gly Lys Thr Arg Arg Phe Leu Pro Gln Ile Leu Ala Glu            1700 1705 1710 Cys Ala Arg Arg Arg Leu Arg Thr Leu Val Leu Ala Pro Thr Arg Val        1715 1720 1725 Val Leu Ser Glu Met Lys Glu Ala Phe His Gly Leu Asp Val Lys Phe    1730 1735 1740 His Thr Gln Ala Phe Ser Ala His Gly Ser Gly Arg Glu Val Ile Asp 1745 1750 1755 1760 Ala Met Cys His Ala Thr Leu Thr Tyr Arg Met Leu Glu Pro Thr Arg                1765 1770 1775 Val Val Asn Trp Glu Val Ile Ile Met Asp Glu Ala His Phe Leu Asp            1780 1785 1790 Pro Ala Ser Ile Ala Ala Arg Gly Trp Ala Ala His Arg Ala Arg Ala        1795 1800 1805 Asn Glu Ser Ala Thr Ile Leu Met Thr Ala Thr Pro Pro Gly Thr Ser    1810 1815 1820 Asp Glu Phe Pro His Ser Asn Gly Glu Ile Glu Asp Val Gln Thr Asp 1825 1830 1835 1840 Ile Pro Ser Glu Pro Trp Asn Thr Gly His Asp Trp Ile Leu Ala Asp                1845 1850 1855 Lys Arg Pro Thr Ala Trp Phe Leu Pro Ser Ile Arg Ala Ala Asn Val            1860 1865 1870 Met Ala Ala Ser Leu Arg Lys Ala Gly Lys Ser Val Val Val Leu Asn        1875 1880 1885 Arg Lys Thr Phe Glu Arg Glu Tyr Pro Thr Ile Lys Gln Lys Lys Pro    1890 1895 1900 Asp Phe Ile Leu Ala Thr Asp Ile Ala Glu Met Gly Ala Asn Leu Cys 1905 1910 1915 1920 Val Glu Arg Val Leu Asp Cys Arg Thr Ala Phe Lys Pro Val Leu Val                1925 1930 1935 Asp Glu Gly Arg Lys Val Ala Ile Lys Gly Pro Leu Arg Ile Ser Ala            1940 1945 1950 Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn Pro Asn Arg        1955 1960 1965 Asp Gly Asp Ser Tyr Tyr Tyr Ser Glu Pro Thr Ser Glu Asn Asn Ala    1970 1975 1980 His His Val Cys Trp Leu Glu Ala Ser Met Leu Leu Asp Asn Met Glu 1985 1990 1995 2000 Val Arg Gly Gly Met Val Ala Pro Leu Tyr Gly Val Glu Gly Thr Lys                2005 2010 2015 Thr Pro Val Ser Pro Gly Glu Met Arg Leu Arg Asp Asp Gln Arg Lys            2020 2025 2030 Val Phe Arg Glu Leu Val Arg Asn Cys Asp Leu Pro Val Trp Leu Ser        2035 2040 2045 Trp Gln Val Ala Lys Ala Gly Leu Lys Thr Asn Asp Arg Lys Trp Cys    2050 2055 2060 Phe Glu Gly Pro Glu Glu His Glu Ile Leu Asn Asp Ser Gly Glu Thr 2065 2070 2075 2080 Val Lys Cys Arg Ala Pro Gly Gly Ala Lys Lys Pro Leu Arg Pro Arg                2085 2090 2095 Trp Cys Asp Glu Arg Val Ser Ser Asp Gln Ser Ala Leu Ser Glu Phe            2100 2105 2110 Ile Lys Phe Ala Glu Gly Arg Arg Gly Ala Ala Glu Val Leu Val Val        2115 2120 2125 Leu Ser Glu Leu Pro Asp Phe Leu Ala Lys Lys Gly Gly Glu Ala Met    2130 2135 2140 Asp Thr Ile Ser Val Phe Leu His Ser Glu Glu Gly Ser Arg Ala Tyr 2145 2150 2155 2160 Arg Asn Ala Leu Ser Met Met Pro Glu Ala Met Thr Ile Val Met Leu                2165 2170 2175 Phe Ile Leu Ala Gly Leu Leu Thr Ser Gly Met Val Ile Phe Phe Met            2180 2185 2190 Ser Pro Lys Gly Ile Ser Arg Met Ser Met Ala Met Gly Thr Met Ala        2195 2200 2205 Gly Cys Gly Tyr Leu Met Phe Leu Gly Gly Val Lys Pro Thr His Ile    2210 2215 2220 Ser Tyr Val Met Leu Ile Phe Phe Val Leu Met Val Val Val Ile Pro 2225 2230 2235 2240 Glu Pro Gly Gln Gln Arg Ser Ile Gln Asp Asn Gln Val Ala Tyr Leu                2245 2250 2255 Ile Ile Gly Ile Leu Thr Leu Val Ser Ala Val Ala Ala Asn Glu Leu            2260 2265 2270 Gly Met Leu Glu Lys Thr Lys Glu Asp Leu Phe Gly Lys Lys Asn Leu        2275 2280 2285 Ile Pro Ser Ser Ala Ser Pro Trp Ser Trp Pro Asp Leu Asp Leu Lys    2290 2295 2300 Pro Gly Ala Ala Trp Thr Val Tyr Val Gly Ile Val Thr Met Leu Ser 2305 2310 2315 2320 Pro Met Leu His His Trp Ile Lys Val Glu Tyr Gly Asn Leu Ser Leu                2325 2330 2335 Ser Gly Ile Ala Gln Ser Ala Ser Val Leu Ser Phe Met Asp Lys Gly            2340 2345 2350 Ile Pro Phe Met Lys Met Asn Ile Ser Val Ile Met Leu Leu Val Ser        2355 2360 2365 Gly Trp Asn Ser Ile Thr Val Met Pro Leu Leu Cys Gly Ile Gly Cys    2370 2375 2380 Ala Met Leu His Trp Ser Leu Ile Leu Pro Gly Ile Lys Ala Gln Gln 2385 2390 2395 2400 Ser Lys Leu Ala Gln Arg Arg Val Phe His Gly Val Ala Glu Asn Pro                2405 2410 2415 Val Val Asp Gly Asn Pro Thr Val Asp Ile Glu Glu Ala Pro Glu Met            2420 2425 2430 Pro Ala Leu Tyr Glu Lys Lys Leu Ala Leu Tyr Leu Leu Leu Ala Leu        2435 2440 2445 Ser Leu Ala Ser Val Ala Met Cys Arg Thr Pro Phe Ser Leu Ala Glu    2450 2455 2460 Gly Ile Val Leu Ala Ser Ala Ala Leu Gly Pro Leu Ile Glu Gly Asn 2465 2470 2475 2480 Thr Ser Leu Leu Trp Asn Gly Pro Met Ala Val Ser Met Thr Gly Val                2485 2490 2495 Met Arg Gly Asn His Tyr Ala Phe Val Gly Val Met Tyr Asn Leu Trp            2500 2505 2510 Lys Met Lys Thr Gly Arg Arg Gly Ser Ala Asn Gly Lys Thr Leu Gly        2515 2520 2525 Glu Val Trp Lys Arg Glu Leu Asn Leu Leu Asp Lys Arg Gln Phe Glu    2530 2535 2540 Leu Tyr Lys Arg Thr Asp Ile Val Glu Val Asp Arg Asp Thr Ala Arg 2545 2550 2555 2560 Arg His Leu Ala Glu Gly Lys Val Asp Thr Gly Val Ala Val Ser Arg                2565 2570 2575 Gly Thr Ala Lys Leu Arg Trp Phe His Glu Arg Gly Tyr Val Lys Leu            2580 2585 2590 Glu Gly Arg Val Ile Asp Leu Gly Cys Gly Arg Gly Gly Trp Cys Tyr        2595 2600 2605 Tyr Ala Ala Ala Gln Lys Glu Val Ser Gly Val Lys Gly Phe Thr Leu    2610 2615 2620 Gly Arg Asp Gly His Glu Lys Pro Met Asn Val Gln Ser Leu Gly Trp 2625 2630 2635 2640 Asn Ile Ile Thr Phe Lys Asp Lys Thr Asp Ile His Arg Leu Glu Pro                2645 2650 2655 Val Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Ser Ser            2660 2665 2670 Ser Val Thr Glu Gly Glu Arg Thr Val Arg Val Leu Asp Thr Val Glu        2675 2680 2685 Lys Trp Leu Ala Cys Gly Val Asp Asn Phe Cys Val Lys Val Leu Ala    2690 2695 2700 Pro Tyr Met Pro Asp Val Leu Glu Lys Leu Glu Leu Leu Gln Arg Arg 2705 2710 2715 2720 Phe Gly Gly Thr Val Ile Arg Asn Pro Leu Ser Arg Asn Ser Thr His                2725 2730 2735 Glu Met Tyr Tyr Val Ser Gly Ala Arg Ser Asn Val Thr Phe Thr Val            2740 2745 2750 Asn Gln Thr Ser Arg Leu Leu Met Arg Arg Met Arg Arg Pro Thr Gly        2755 2760 2765 Lys Val Thr Leu Glu Ala Asp Val Ile Leu Pro Ile Gly Thr Arg Ser    2770 2775 2780 Val Glu Thr Asp Lys Gly Pro Leu Asp Lys Glu Ala Ile Glu Glu Arg 2785 2790 2795 2800 Val Glu Arg Ile Lys Ser Glu Tyr Met Thr Ser Trp Phe Tyr Asp Asn                2805 2810 2815 Asp Asn Pro Tyr Arg Thr Trp His Tyr Cys Gly Ser Tyr Val Thr Lys            2820 2825 2830 Thr Ser Gly Ser Ala Ala Ser Met Val Asn Gly Val Ile Lys Ile Leu        2835 2840 2845 Thr Tyr Pro Trp Asp Arg Ile Glu Glu Val Thr Arg Met Ala Met Thr    2850 2855 2860 Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp 2865 2870 2875 2880 Thr Arg Ala Lys Asp Pro Pro Ala Gly Thr Arg Lys Ile Met Lys Val                2885 2890 2895 Val Asn Arg Trp Leu Phe Arg His Leu Ala Arg Glu Lys Asn Pro Arg            2900 2905 2910 Leu Cys Thr Lys Glu Glu Phe Ile Ala Lys Val Arg Ser His Ala Ala        2915 2920 2925 Ile Gly Ala Tyr Leu Glu Glu Gln Glu Gln Trp Lys Thr Ala Asn Glu    2930 2935 2940 Ala Val Gln Asp Pro Lys Phe Trp Glu Leu Val Asp Glu Glu Arg Lys 2945 2950 2955 2960 Leu His Gln Gln Gly Arg Cys Arg Thr Cys Val Tyr Asn Met Met Gly                2965 2970 2975 Lys Arg Glu Lys Lys Leu Ser Glu Phe Gly Lys Ala Lys Gly Ser Arg            2980 2985 2990 Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Tyr Leu Glu Phe Glu Ala        2995 3000 3005 Leu Gly Phe Leu Asn Glu Asp His Trp Ala Ser Arg Glu Asn Ser Gly    3010 3015 3020 Gly Gly Val Glu Gly Ile Gly Leu Gln Tyr Leu Gly Tyr Val Ile Arg 3025 3030 3035 3040 Asp Leu Ala Ala Met Asp Gly Gly Gly Phe Tyr Ala Asp Asp Thr Ala                3045 3050 3055 Gly Trp Asp Thr Arg Ile Thr Glu Ala Asp Leu Asp Asp Glu Gln Glu            3060 3065 3070 Ile Leu Asn Tyr Met Ser Pro His His Lys Lys Leu Ala Gln Ala Val        3075 3080 3085 Met Glu Met Thr Tyr Lys Asn Lys Val Val Lys Val Leu Arg Pro Ala    3090 3095 3100 Pro Gly Gly Lys Ala Tyr Met Asp Val Ile Ser Arg Arg Asp Gln Arg 3105 3110 3115 3120 Gly Ser Gly Gln Val Val Thr Tyr Ala Leu Asn Thr Ile Thr Asn Leu                3125 3130 3135 Lys Val Gln Leu Ile Arg Met Ala Glu Ala Glu Met Val Ile His His            3140 3145 3150 Gln His Val Gln Asp Cys Asp Glu Ser Val Leu Thr Arg Leu Glu Ala        3155 3160 3165 Trp Leu Thr Glu His Gly Cys Asp Arg Leu Lys Arg Met Ala Val Ser    3170 3175 3180 Gly Asp Asp Cys Val Val Arg Pro Ile Asp Asp Arg Phe Gly Leu Ala 3185 3190 3195 3200 Leu Ser His Leu Asn Ala Met Ser Lys Val Arg Lys Asp Ile Ser Glu                3205 3210 3215 Trp Gln Pro Ser Lys Gly Trp Asn Asp Trp Glu Asn Val Pro Phe Cys            3220 3225 3230 Ser His His Phe His Glu Leu Gln Leu Lys Asp Gly Arg Arg Ile Val        3235 3240 3245 Val Pro Cys Arg Glu Gln Asp Glu Leu Ile Gly Arg Gly Arg Val Ser    3250 3255 3260 Pro Gly Asn Gly Trp Met Ile Lys Glu Thr Ala Cys Leu Ser Lys Ala 3265 3270 3275 3280 Tyr Ala Asn Met Trp Ser Leu Met Tyr Phe His Lys Arg Asp Met Arg                3285 3290 3295 Leu Leu Ser Leu Ala Val Ser Ser Ala Val Pro Thr Ser Trp Val Pro            3300 3305 3310 Gln Gly Arg Thr Thr Trp Ser Ile His Gly Lys Gly Glu Trp Met Thr        3315 3320 3325 Thr Glu Asp Met Leu Glu Val Trp Asn Arg Val Trp Ile Thr Asn Asn    3330 3335 3340 Pro His Met Gln Asp Lys Thr Met Val Lys Lys Trp Arg Asp Val Pro 3345 3350 3355 3360 Tyr Leu Thr Lys Arg Gln Asp Lys Leu Cys Gly Ser Leu Ile Gly Met                3365 3370 3375 Thr Asn Arg Ala Thr Trp Ala Ser His Ile His Leu Val Ile His Arg            3380 3385 3390 Ile Arg Thr Leu Ile Gly Gln Glu Lys Tyr Thr Asp Tyr Leu Thr Val        3395 3400 3405 Met Asp Arg Tyr Ser Val Asp Ala Asp Leu Gln Leu Gly Glu Leu Ile    3410 3415 3420 <210> 6 <211> 10901 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 3 nucleic acid sequence <400> 6 agtaaatcct gtgtgctaat tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60 acacatttgg attaatttta atcgttcgtt gagcgattag cagagaactg accagaacat 120 gtctggtcgt aaagctcagg gaaaaaccct gggcgtcaat atggtacgac gaggagttcg 180 ctccttgtca aacaaaataa aacaaaaaac aaaacaaatt ggaaacagac ctggaccttc 240 aagaggtgtt caaggattta tctttttctt tttgttcaac attttgactg gaaaaaagat 300 cacagcccac ctaaagaggt tgtggaaaat gctggaccca agacaaggct tggctgttct 360 aaggaaagtc aagagagtgg tggccagttt gatgagagga ttgtcctcaa ggaaacgccg 420 tggcgcagat actagtgtcg gaattgttgg cctcctgctg accacagcta tggcagcgga 480 ggtcactaga cgtgggagtg catactatat gtacttggac agaaacgatg ctggggaggc 540 catatctttt ccaaccacat tggggatgaa taagtgttat atacagatca tggatcttgg 600 acacatgtgt gatgccacca tgagctatga atgccctatg ctggatgagg gggtggaacc 660 agatgacgtc gattgttggt gcaacacgac gtcaacttgg gttgtgtacg gaacctgcca 720 tcacaaaaaa ggtgaagcac ggagatctag aagagctgtg acgctcccct cccattccac 780 taggaagctg caaacgcggt cgcaaacctg gttggaatca agagaataca caaagcactt 840 gattagagtc gaaaattgga tattcaggaa ccctggcttc gcgttagcag cagctgccat 900 cgcttggctt ttgggaagct caacgagcca aaaagtcata tacttggtca tgatactgct 960 gattgccccg gcatacagca tcaggtgcat aggagtcagc aatagggact ttgtggaagg 1020 tatgtcaggt gggacttggg ttgatgttgt cttggaacat ggaggttgtg tcaccgtaat 1080 ggcacaggac aaaccgactg tcgacataga gctggttaca acaacagtca gcaacatggc 1140 ggaggtaaga tcctactgct atgaggcatc aatatcagac atggcttcgg acagccgctg 1200 cccaacacaa ggtgaagcct accttgacaa gcaatcagac actcaatatg tctgcaaaag 1260 aacgttagtg gacagaggct ggggaaatgg atgtggactt tttggcaaag ggagcctggt 1320 gacatgcgct aagtttgcat gctccaagaa aatgaccggg aagagcatcc agccagagaa 1380 tctggagtac cggataatgc tgtcagttca tggctcccag cacagtggga tgatcgttaa 1440 tgacacagga catgaaactg atgagaatag agcgaaagtt gagataacgc ccaattcacc 1500 aagagccgaa gccaccctgg ggggttttgg aagcctagga cttgattgtg aaccgaggac 1560 aggccttgac ttttcagatt tgtattactt gactatgaat aacaagcact ggttggttca 1620 caaggagtgg ttccacgaca ttccattacc ttggcacgct ggggcagaca ccggaactcc 1680 acactggaac aacaaagaag cactggtaga gttcaaggac gcacatgcca aaaggcaaac 1740 tgtcgtggtt ctagggagtc aagaaggagc agttcacacg gcccttgctg gagctctgga 1800 ggctgagatg gatggtgcaa agggaaggct gtcctctggc cacttgaaat gtcgcctgaa 1860 aatggataaa cttagattga agggcgtgtc atactccttg tgtactgcag cgttcacatt 1920 caccaagatc ccggctgaaa cactgcacgg gacagtcaca gtggaggtac agtacgcagg 1980 gacagatgga ccttgcaagg ttccagctca gatggcggtg gacatgcaaa ctctgacccc 2040 agttgggagg ttgataaccg ctaaccccgt aatcactgaa agcactgaga actctaagat 2100 gatgctggaa cttgatccac catttgggga ctcttacatt gtcataggag tcggggagaa 2160 gaagatcacc caccactggc acaggagtgg cagcaccatt ggaaaagcat ttgaagccac 2220 tgtgagaggt gccaagagaa tggcagtctt gggagacaca gcctgggact ttggatcagt 2280 tggaggcgct ctcaactcat tgggcaaggg catccatcaa atttttggag cagctttcaa 2340 atcattgttt ggaggaatgt cctggttctc acaaattctc attggaacgt tgctgatgtg 2400 gttgggtctg aacacaaaga atggatctat ttcccttatg tgcttggcct tagggggggt 2460 gttgatcttc ttatccacag ccgtctctgc tgatcaagga tgcgccatca actttggcaa 2520 gagagagctc aagtgcggag atggtatctt catatttaga gactctgatg actggctgaa 2580 caagtactca tactatccag aagatcctgt gaagcttgca tcaatagtga aagcctcttt 2640 tgaagaaggg aagtgtggcc taaattcagt tgactccctt gagcatgaga tgtggagaag 2700 cagggcagat gagatcaatg ccatttttga ggaaaacgag gtggacattt ctgttgtcgt 2760 gcaggatcca aagaatgttt accagagagg aactcatcca ttttccagaa ttcgggatgg 2820 tctgcagtat ggttggaaga cttggggtaa gaaccttgtg ttctccccag ggaggaagaa 2880 tggaagcttc atcatagatg gaaagtccag gaaagaatgc ccgttttcaa accgggtctg 2940 gaattctttc cagatagagg agtttgggac gggagtgttc accacacgcg tgtacatgga 3000 cgcagtcttt gaatacacca tagactgcga tggatctatc ttgggtgcag cggtgaacgg 3060 aaaaaagagt gcccatggct ctccaacatt ttggatggga agtcatgaag taaatgggac 3120 atggatgatc cacaccttgg aggcattaga ttacaaggag tgtgagtggc cactgacaca 3180 tacgattgga acatcagttg aagagagtga aatgttcatg ccgagatcaa tcggaggccc 3240 agttagctct cacaatcata tccctggata caaggttcag acgaacggac cttggatgca 3300 ggtaccacta gaagtgaaga gagaagcttg cccagggact agcgtgatca ttgatggcaa 3360 ctgtgatgga cggggaaaat caaccagatc caccacggat agcgggaaag ttattcctga 3420 atggtgttgc cgctcctgca caatgccgcc tgtgagcttc catggtagtg atgggtgttg 3480 gtatcccatg gaaattaggc caaggaaaac gcatgaaagc catctggtgc gctcctgggt 3540 tacagctgga gaaatacatg ctgtcccttt tggtttggtg agcatgatga tagcaatgga 3600 agtggtccta aggaaaagac agggaccaaa gcaaatgttg gttggaggag tagtgctctt 3660 gggagcaatg ctggtcgggc aagtaactct ccttgatttg ctgaaactca cagtggctgt 3720 gggattgcat ttccatgaga tgaacaatgg aggagacgcc atgtatatgg cgttgattgc 3780 tgccttttca atcagaccag ggctgctcat cggctttggg ctcaggaccc tatggagccc 3840 tcgggaacgc cttgtgctga ccctaggagc agccatggtg gagattgcct tgggtggcgt 3900 gatgggcggc ctgtggaagt atctaaatgc agtttctctc tgcatcctga caataaatgc 3960 tgttgcttct aggaaagcat caaataccat cttgcccctc atggctctgt tgacacctgt 4020 cactatggct gaggtgagac ttgccgcaat gttcttttgt gccgtggtta tcataggggt 4080 ccttcaccag aatttcaagg acacctccat gcagaagact atacctctgg tggccctcac 4140 actcacatct tacctgggct tgacacaacc ttttttgggc ctgtgtgcat ttctggcaac 4200 ccgcatattt gggcgaagga gtatcccagt gaatgaggca ctcgcagcag ctggtctagt 4260 gggagtgctg gcaggactgg cttttcagga gatggagaac ttccttggtc cgattgcagt 4320 tggaggactc ctgatgatgc tggttagcgt ggctgggagg gtggatgggc tagagctcaa 4380 gaagcttggt gaagtttcat gggaagagga ggcggagatc agcgggagtt ccgcccgcta 4440 tgatgtggca ctcagtgaac aaggggagtt caagctgctt tctgaagaga aagtgccatg 4500 ggaccaggtt gtgatgacct cgctggcctt ggttggggct gccctccatc catttgctct 4560 tctgctggtc cttgctgggt ggctgtttca tgtcagggga gctaggagaa gtggggatgt 4620 cttgtgggat attcccactc ctaagatcat cgaggaatgt gaacatctgg aggatgggat 4680 ttatggcata ttccagtcaa ccttcttggg ggcctcccag cgaggagtgg gagtggcaca 4740 gggaggggtg ttccacacaa tgtggcatgt cacaagagga gctttccttg tcaggaatgg 4800 caagaagttg attccatctt gggcttcagt aaaggaagac cttgtcgcct atggtggctc 4860 atggaagttg gaaggcagat gggatggaga ggaagaggtc cagttgatcg cggctgttcc 4920 aggaaagaac gtggtcaacg tccagacaaa accgagcttg ttcaaagtga ggaatggggg 4980 agaaatcggg gctgtcgctc ttgactatcc gagtggcact tcaggatctc ctattgttaa 5040 caggaacgga gaggtgattg ggctgtacgg caatggcatc cttgtcggtg acaactcctt 5100 cgtgtccgcc atatcccaga ctgaggtgaa ggaagaagga aaggaggagc tccaagagat 5160 cccgacaatg ctaaagaaag gaatgacaac tgtccttgat tttcatcctg gagctgggaa 5220 gacaagacgt ttcctcccac agatcttggc cgagtgcgca cggagacgct tgcgcactct 5280 tgtgttggcc cccaccaggg ttgttctttc tgaaatgaag gaggcttttc acggcctgga 5340 cgtgaaattc cacacacagg ctttttccgc tcacggcagc gggagagaag tcattgatgc 5400 catgtgccat gccaccctaa cttacaggat gttggaacca actagggttg ttaactggga 5460 agtgatcatt atggatgaag cccatttttt ggatccagct agcatagccg ctagaggttg 5520 ggcagcgcac agagctaggg caaatgaaag tgcaacaatc ttgatgacag ccacaccgcc 5580 tgggactagt gatgaatttc cacattcaaa tggtgaaata gaagatgttc aaacggacat 5640 acccagtgag ccctggaaca cagggcatga ctggatccta gctgacaaaa ggcccacggc 5700 atggttcctt ccatccatca gagctgcaaa tgtcatggct gcctctttgc gtaaggctgg 5760 aaagagtgtg gtggtcctga acaggaaaac ctttgagaga gaatacccca cgataaagca 5820 gaagaaacct gactttatat tggccactga catagctgaa atgggagcca acctttgcgt 5880 ggagcgagtg ctggattgca ggacggcttt taagcctgtg cttgtggatg aagggaggaa 5940 ggtggcaata aaagggccac ttcgtatctc cgcatcctct gctgctcaaa ggagggggcg 6000 cattgggaga aatcccaaca gagatggaga ctcatactac tattctgagc ctacaagtga 6060 aaataatgcc caccacgtct gctggttgga ggcctcaatg ctcttggaca acatggaggt 6120 gaggggtgga atggtcgccc cactctatgg cgttgaagga actaaaacac cagtttcccc 6180 tggtgaaatg agactgaggg atgaccagag gaaagtcttc agagaactag tgaggaattg 6240 tgacctgccc gtttggcttt cgtggcaagt ggccaaggct ggtttgaaga cgaatgatcg 6300 taagtggtgt tttgaaggcc ctgaggaaca tgagatcttg aatgacagcg gtgaaacagt 6360 gaagtgcagg gctcctggag gagcaaagaa gcctctgcgc ccaaggtggt gtgatgaaag 6420 ggtgtcatct gaccagagtg cgctgtctga atttattaag tttgctgaag gtaggagggg 6480 agctgctgaa gtgctagttg tgctgagtga actccctgat ttcctggcta aaaaaggtgg 6540 agaggcaatg gataccatca gtgtgttcct ccactctgag gaaggctcta gggcttaccg 6600 caatgcacta tcaatgatgc ctgaggcaat gacaatagtc atgctgttta tactggctgg 6660 actactgaca tcgggaatgg tcatcttttt catgtctccc aaaggcatca gtagaatgtc 6720 tatggcgatg ggcacaatgg ccggctgtgg atatctcatg ttccttggag gcgtcaaacc 6780 cactcacatc tcctatgtca tgctcatatt ctttgtcctg atggtggttg tgatccccga 6840 gccagggcaa caaaggtcca tccaagacaa ccaagtggca tacctcatta ttggcatcct 6900 gacgctggtt tcagcggtgg cagccaacga gctaggcatg ctggagaaaa ccaaagagga 6960 cctctttggg aagaagaact taattccatc tagtgcttca ccctggagtt ggccggatct 7020 tgacctgaag ccaggagctg cctggacagt gtacgttggc attgttacaa tgctctctcc 7080 aatgttgcac cactggatca aagtcgaata tggcaacctg tctctgtctg gaatagccca 7140 gtcagcctca gtcctttctt tcatggacaa ggggatacca ttcatgaaga tgaatatctc 7200 ggtcataatg ctgctggtca gtggctggaa ttcaataaca gtgatgcctc tgctctgtgg 7260 catagggtgc gccatgctcc actggtctct cattttacct ggaatcaaag cgcagcagtc 7320 aaagcttgca cagagaaggg tgttccatgg cgttgccgag aaccctgtgg ttgatgggaa 7380 tccaacagtt gacattgagg aagctcctga aatgcctgcc ctttatgaga agaaactggc 7440 tctatatctc cttcttgctc tcagcctagc ttctgttgcc atgtgcagaa cgcccttttc 7500 attggctgaa ggcattgtcc tagcatcagc tgccttaggg ccgctcatag agggaaacac 7560 cagccttctt tggaatggac ccatggctgt ctccatgaca ggagtcatga gggggaatca 7620 ctatgctttt gtgggagtca tgtacaatct atggaagatg aaaactggac gccgggggag 7680 cgcgaatgga aaaactttgg gtgaagtctg gaagagggaa ctgaatctgt tggacaagcg 7740 acagtttgag ttgtataaaa ggaccgacat tgtggaggtg gatcgtgata cggcacgcag 7800 gcatttggcc gaagggaagg tggacaccgg ggtggcggtc tccaggggga ccgcaaagtt 7860 aaggtggttc catgagcgtg gctatgtcaa gctggaaggt agggtgattg acctggggtg 7920 tggccgcgga ggctggtgtt actacgctgc tgcgcaaaag gaagtgagtg gggtcaaagg 7980 atttactctt ggaagagacg gccatgagaa acccatgaat gtgcaaagtc tgggatggaa 8040 catcatcacc ttcaaggaca aaactgatat ccaccgccta gaaccagtga aatgtgacac 8100 ccttttgtgt gacattggag agtcatcatc gtcatcggtc acagaggggg aaaggaccgt 8160 gagagttctt gatactgtag aaaaatggct ggcttgtggg gttgacaact tctgtgtgaa 8220 ggtgttagct ccatacatgc cagatgttct cgagaaactg gaattgctcc aaaggaggtt 8280 tggcggaaca gtgatcagga accctctctc caggaattcc actcatgaaa tgtactacgt 8340 gtctggagcc cgcagcaatg tcacatttac tgtgaaccaa acatcccgcc tcctgatgag 8400 gagaatgagg cgtccaactg gaaaagtgac cctggaggct gacgtcatcc tcccaattgg 8460 gacacgcagt gttgagacag acaagggacc cctggacaaa gaggccatag aagaaagggt 8520 tgagaggata aaatctgagt acatgacctc ttggttttat gacaatgaca acccctacag 8580 gacctggcac tactgtggct cctatgtcac aaaaacctca ggaagtgcgg cgagcatggt 8640 aaatggtgtt attaaaattc tgacatatcc atgggacagg atagaggagg tcacaagaat 8700 ggcaatgact gacacaaccc cttttggaca gcaaagagtg tttaaagaaa aagttgacac 8760 cagagcaaag gatccaccag cgggaactag gaagatcatg aaagttgtca acaggtggct 8820 gttccgccac ctggccagag aaaagaaccc cagactgtgc acaaaggaag aatttattgc 8880 aaaagtccga agtcatgcag ccattggagc ttacctggaa gaacaagaac agtggaagac 8940 tgccaatgag gctgtccaag acccaaagtt ctgggaactg gtggatgaag aaaggaagct 9000 gcaccaacaa ggcaggtgtc ggacttgtgt gtacaacatg atggggaaaa gagagaagaa 9060 gctgtcagag tttgggaaag caaagggaag ccgtgccata tggtatatgt ggctgggagc 9120 gcggtatctt gagtttgagg ccctgggatt cctgaatgag gaccattggg cttccaggga 9180 aaactcagga ggaggagtgg aaggcattgg cttacaatac ctaggatatg tgatcagaga 9240 cctggctgca atggatggtg gtggattcta cgcggatgac accgctggat gggacacgcg 9300 catcacagag gcagaccttg atgatgaaca ggagatcttg aactacatga gcccacatca 9360 caaaaaactg gcacaagcag tgatggaaat gacatacaag aacaaagtgg tgaaagtgtt 9420 gagaccagcc ccaggaggga aagcctacat ggatgtcata agtcgacgag accagagagg 9480 atccgggcag gtagtgactt atgctctgaa caccatcacc aacttgaaag tccaattgat 9540 cagaatggca gaagcagaga tggtgataca tcaccaacat gttcaagatt gtgatgaatc 9600 agttctgacc aggctggagg catggctcac tgagcacgga tgtgacagac tgaagaggat 9660 ggcggtgagt ggagacgact gtgtggtccg gcccatcgat gacaggttcg gcctggccct 9720 gtcccatctc aacgccatgt ccaaggttag aaaggacata tctgaatggc agccatcaaa 9780 agggtggaat gattgggaga atgtgccctt ctgttcccac cacttccatg aactacagct 9840 gaaggatggc aggaggattg tggtgccttg ccgagaacag gacgagctca ttgggagagg 9900 aagggtgtct ccaggaaacg gctggatgat caaggaaaca gcttgcctca gcaaagccta 9960 tgccaacatg tggtcactga tgtattttca caaaagggac atgaggctac tgtcattggc 10020 tgtttcctca gctgttccca cctcatgggt tccacaagga cgcacaacat ggtcgattca 10080 tgggaaaggg gagtggatga ccacggaaga catgcttgag gtgtggaaca gagtatggat 10140 aaccaacaac ccacacatgc aggacaagac aatggtgaaa aaatggagag atgtccctta 10200 tctaaccaag agacaagaca agctgtgcgg atcactgatt ggaatgacca atagggccac 10260 ctgggcctcc cacatccatt tagtcatcca tcgtatccga acgctgattg gacaggagaa 10320 atacactgac tacctaacag tcatggacag gtattctgtg gatgctgacc tgcaactggg 10380 tgagcttatc tgaaacacca tctaacagga ataaccggga tacaaaccac gggtggagaa 10440 ccggactccc cacaacctga aaccgggata taaaccacgg ctggagaacc gggctccgca 10500 cttaaaatga aacagaaacc gggataaaaa ctacggatgg agaaccggac tccacacatt 10560 gagacagaag aagttgtcag cccagaaccc cacacgagtt ttgccactgc taagctgtga 10620 ggcagtgcag gctgggacag ccgacctcca ggttgcgaaa aacctggttt ctgggacctc 10680 ccaccccaga gtaaaaagaa cggagcctcc gctaccaccc tcccacgtgg tggtagaaag 10740 acggggtcta gaggttagag gagaccctcc agggaacaaa tagtgggacc atattgacgc 10800 cagggaaaga ccggagtggt tctctgcttt tcctccagag gtctgtgagc acagtttgct 10860 caagaataag cagacctttg gatgacaaac acaaaaccac t 10901 <210> 7 <211> 3426 <212> PRT <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 4 amino acid sequence <400> 7 Met Ser Gly Arg Lys Ala Gln Gly Lys Thr Leu Gly Val Asn Met Val   1 5 10 15 Arg Arg Gly Val Arg Ser Leu Ser Asn Lys Ile Lys Gln Lys Thr Lys              20 25 30 Gln Ile Gly Asn Arg Pro Gly Pro Ser Arg Gly Val Gln Gly Phe Ile          35 40 45 Phe Phe Phe Leu Phe Asn Ile Leu Thr Gly Lys Lys Ile Thr Ala His      50 55 60 Leu Lys Arg Leu Trp Lys Met Leu Asp Pro Arg Gln Gly Leu Ala Val  65 70 75 80 Leu Arg Lys Val Lys Arg Val Val Ala Ser Leu Met Arg Gly Leu Ser                  85 90 95 Ser Arg Lys Arg Arg Ser His Gly Ala Asp Thr Ser Val Gly Ile Val             100 105 110 Gly Leu Leu Leu Thr Thr Ala Met Ala Ala Glu Val Thr Arg Arg Gly         115 120 125 Ser Ala Tyr Tyr Met Tyr Leu Asp Arg Asn Asp Ala Gly Glu Ala Ile     130 135 140 Ser Phe Pro Thr Thr Leu Gly Met Asn Lys Cys Tyr Ile Gln Ile Met 145 150 155 160 Asp Leu Gly His Met Cys Asp Ala Thr Met Ser Tyr Glu Cys Pro Met                 165 170 175 Leu Asp Glu Gly Val Glu Pro Asp Asp Val Asp Cys Trp Cys Asn Thr             180 185 190 Thr Ser Thr Trp Val Val Tyr Gly Thr Cys His His Lys Lys Gly Glu         195 200 205 Ala Arg Arg Ser Arg Arg Ala Val Thr Leu Pro Ser His Ser Thr Arg     210 215 220 Lys Leu Gln Thr Arg Ser Gln Thr Trp Leu Glu Ser Arg Glu Tyr Thr 225 230 235 240 Lys His Leu Ile Arg Val Glu Asn Trp Ile Phe Arg Asn Pro Gly Phe                 245 250 255 Ala Leu Ala Ala Ala Ala Ile Ala Trp Leu Leu Gly Ser Ser Thr Ser             260 265 270 Gln Lys Val Ile Tyr Leu Val Met Ile Leu Leu Ile Ala Pro Ala Tyr         275 280 285 Ser Ile Arg Cys Ile Gly Val Ser Asn Arg Asp Phe Val Glu Gly Met     290 295 300 Ser Gly Gly Thr Trp Val Asp Val Val Leu Glu His Gly Gly Cys Val 305 310 315 320 Thr Val Met Ala Gln Asp Lys Pro Thr Val Asp Ile Glu Leu Val Thr                 325 330 335 Thr Thr Val Ser Asn Met Ala Glu Val Arg Ser Tyr Cys Tyr Glu Ala             340 345 350 Ser Ile Ser Asp Met Ala Ser Asp Ser Arg Cys Pro Thr Gln Gly Glu         355 360 365 Ala Tyr Leu Asp Lys Gln Ser Asp Thr Gln Tyr Val Cys Lys Arg Thr     370 375 380 Leu Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly 385 390 395 400 Ser Leu Val Thr Cys Ala Lys Phe Ala Cys Ser Lys Lys Met Thr Gly                 405 410 415 Lys Ser Ile Gln Pro Glu Asn Leu Glu Tyr Arg Ile Met Leu Ser Val             420 425 430 His Gly Ser Gln His Ser Gly Met Ile Val Asn Asp Thr Gly His Glu         435 440 445 Thr Asp Glu Asn Arg Ala Lys Val Glu Ile Thr Pro Asn Ser Pro Arg     450 455 460 Ala Glu Ala Thr Leu Gly Gly Phe Gly Ser Leu Gly Leu Asp Cys Glu 465 470 475 480 Pro Arg Thr Gly Leu Asp Phe Ser Asp Leu Tyr Tyr Leu Thr Met Asn                 485 490 495 Asn Lys His Trp Leu Val His Lys Glu Trp Phe His Asp Ile Pro Leu             500 505 510 Pro Trp His Ala Gly Ala Asp Thr Gly Thr Pro His Trp Asn Asn Lys         515 520 525 Glu Ala Leu Val Glu Phe Lys Asp Ala His Ala Lys Arg Gln Thr Val     530 535 540 Val Val Leu Gly Ser Gln Glu Gly Ala Val His Thr Ala Leu Ala Gly 545 550 555 560 Ala Leu Glu Ala Glu Met Asp Gly Ala Lys Gly Arg Leu Ser Ser Gly                 565 570 575 His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Arg Leu Lys Gly Val             580 585 590 Ser Tyr Ser Leu Cys Thr Ala Ala Phe Thr Phe Thr Lys Ile Pro Ala         595 600 605 Glu Thr Leu His Gly Thr Val Thr Val Glu Val Gln Tyr Ala Gly Thr     610 615 620 Asp Gly Pro Cys Lys Val Pro Ala Gln Met Ala Val Asp Met Gln Thr 625 630 635 640 Leu Thr Pro Val Gly Arg Leu Ile Thr Ala Asn Pro Val Ile Thr Glu                 645 650 655 Ser Thr Glu Asn Ser Lys Met Met Leu Glu Leu Asp Pro Pro Phe Gly             660 665 670 Asp Ser Tyr Ile Val Ile Gly Val Gly Glu Lys Lys Ile Thr His His         675 680 685 Trp His Arg Ser Gly Ser Thr Ile Gly Lys Ala Phe Glu Ala Thr Val     690 695 700 Arg Gly Ala Lys Arg Met Ala Val Leu Gly Asp Thr Ala Trp Asp Phe 705 710 715 720 Gly Ser Val Gly Gly Ala Leu Asn Ser Leu Gly Lys Gly Ile His Gln                 725 730 735 Ile Phe Gly Ala Ala Phe Lys Ser Leu Phe Gly Gly Met Ser Trp Phe             740 745 750 Ser Gln Ile Leu Ile Gly Thr Leu Leu Met Trp Leu Gly Leu Asn Thr         755 760 765 Lys Asn Gly Ser Ile Ser Leu Met Cys Leu Ala Leu Gly Gly Val Leu     770 775 780 Ile Phe Leu Ser Thr Ala Val Ser Ala Asp Gln Gly Cys Ala Ile Asn 785 790 795 800 Phe Gly Lys Arg Glu Leu Lys Cys Gly Asp Gly Ile Phe Ile Phe Arg                 805 810 815 Asp Ser Asp Asp Trp Leu Asn Lys Tyr Ser Tyr Tyr Pro Glu Asp Pro             820 825 830 Val Lys Leu Ala Ser Ile Val Lys Ala Ser Phe Glu Glu Gly Lys Cys         835 840 845 Gly Leu Asn Ser Val Asp Ser Leu Glu His Glu Met Trp Arg Ser Arg     850 855 860 Ala Asp Glu Ile Asn Ala Ile Phe Glu Glu Asn Glu Val Asp Ile Ser 865 870 875 880 Val Val Val Gln Asp Pro Lys Asn Val Tyr Gln Arg Gly Thr His Pro                 885 890 895 Phe Ser Arg Ile Arg Asp Gly Leu Gln Tyr Gly Trp Lys Thr Trp Gly             900 905 910 Lys Asn Leu Val Phe Ser Pro Gly Arg Lys Asn Gly Ser Phe Ile Ile         915 920 925 Asp Gly Lys Ser Arg Lys Glu Cys Pro Phe Ser Asn Arg Val Trp Asn     930 935 940 Ser Phe Gln Ile Glu Glu Phe Gly Thr Gly Val Phe Thr Thr Arg Val 945 950 955 960 Tyr Met Asp Ala Val Phe Glu Tyr Thr Ile Asp Cys Asp Gly Ser Ile                 965 970 975 Leu Gly Ala Ala Val Asn Gly Lys Lys Ser Ala His Gly Ser Pro Thr             980 985 990 Phe Trp Met Gly Ser His Glu Val Asn Gly Thr Trp Met Ile His Thr         995 1000 1005 Leu Glu Ala Leu Asp Tyr Lys Glu Cys Glu Trp Pro Leu Thr His Thr    1010 1015 1020 Ile Gly Thr Ser Val Glu Glu Ser Glu Met Phe Met Pro Arg Ser Ile 1025 1030 1035 1040 Gly Gly Pro Val Ser Ser His Asn His Ile Pro Gly Tyr Lys Val Gln                1045 1050 1055 Thr Asn Gly Pro Trp Met Gln Val Pro Leu Glu Val Lys Arg Glu Ala            1060 1065 1070 Cys Pro Gly Thr Ser Val Ile Ile Asp Gly Asn Cys Asp Gly Arg Gly        1075 1080 1085 Lys Ser Thr Arg Ser Thr Thr Asp Ser Gly Lys Val Ile Pro Glu Trp    1090 1095 1100 Cys Cys Arg Ser Cys Thr Met Pro Pro Val Ser Phe His Gly Ser Asp 1105 1110 1115 1120 Gly Cys Trp Tyr Pro Met Glu Ile Arg Pro Arg Lys Thr His Glu Ser                1125 1130 1135 His Leu Val Arg Ser Trp Val Thr Ala Gly Glu Ile His Ala Val Pro            1140 1145 1150 Phe Gly Leu Val Ser Met Met Ile Ala Met Glu Val Val Leu Arg Lys        1155 1160 1165 Arg Gln Gly Pro Lys Gln Met Leu Val Gly Gly Val Val Leu Leu Gly    1170 1175 1180 Ala Met Leu Val Gly Gln Val Thr Leu Leu Asp Leu Leu Lys Leu Thr 1185 1190 1195 1200 Val Ala Val Gly Leu His Phe His Glu Met Asn Asn Gly Gly Asp Ala                1205 1210 1215 Met Tyr Met Ala Leu Ile Ala Ala Phe Ser Ile Arg Pro Gly Leu Leu            1220 1225 1230 Ile Gly Phe Gly Leu Arg Thr Leu Trp Ser Pro Arg Glu Arg Leu Val        1235 1240 1245 Leu Thr Leu Gly Ala Ala Met Val Glu Ile Ala Leu Gly Gly Val Met    1250 1255 1260 Gly Gly Leu Trp Lys Tyr Leu Asn Ala Val Ser Leu Cys Ile Leu Thr 1265 1270 1275 1280 Ile Asn Ala Val Ala Ser Arg Lys Ala Ser Asn Thr Ile Leu Pro Leu                1285 1290 1295 Met Ala Leu Leu Thr Pro Val Thr Met Ala Glu Val Arg Leu Ala Ala            1300 1305 1310 Met Phe Phe Cys Ala Val Val Ile Gly Val Leu His Gln Asn Phe        1315 1320 1325 Lys Asp Thr Ser Met Gln Lys Thr Ile Pro Leu Val Ala Leu Thr Leu    1330 1335 1340 Thr Ser Tyr Leu Gly Leu Thr Gln Pro Phe Leu Gly Leu Cys Ala Phe 1345 1350 1355 1360 Leu Ala Thr Arg Ile Phe Gly Arg Arg Ser Ile Pro Val Asn Glu Ala                1365 1370 1375 Leu Ala Ala Ala Gly Leu Val Gly Val Leu Ala Gly Leu Ala Phe Gln            1380 1385 1390 Glu Met Glu Asn Phe Leu Gly Pro Ile Ala Val Gly Gly Leu Leu Met        1395 1400 1405 Met Leu Val Ser Val Ala Gly Arg Val Asp Gly Leu Glu Leu Lys Lys    1410 1415 1420 Leu Gly Glu Val Ser Trp Glu Glu Glu Ala Glu Ile Ser Gly Ser Ser 1425 1430 1435 1440 Ala Arg Tyr Asp Val Ala Leu Ser Glu Gln Gly Glu Phe Lys Leu Leu                1445 1450 1455 Ser Glu Glu Lys Val Pro Trp Asp Gln Val Val Met Thr Ser Leu Ala            1460 1465 1470 Leu Val Gly Ala Ala Leu His Pro Phe Ala Leu Leu Leu Val Leu Ala        1475 1480 1485 Gly Trp Leu Phe His Val Arg Gly Ala Arg Arg Ser Gly Asp Val Leu    1490 1495 1500 Trp Asp Ile Pro Thr Pro Lys Ile Ile Glu Glu Cys Glu His Leu Glu 1505 1510 1515 1520 Asp Gly Ile Tyr Gly Ile Phe Gln Ser Thr Phe Leu Gly Ala Ser Gln                1525 1530 1535 Arg Gly Val Gly Val Ala Gln Gly Gly Val Phe His Thr Met Trp His            1540 1545 1550 Val Thr Arg Gly Ala Phe Leu Val Arg Asn Gly Lys Lys Leu Ile Pro        1555 1560 1565 Ser Trp Ala Ser Val Lys Glu Asp Leu Val Ala Tyr Gly Gly Ser Trp    1570 1575 1580 Lys Leu Glu Gly Arg Trp Asp Gly Glu Glu Glu Val Gln Leu Ile Ala 1585 1590 1595 1600 Ala Val Pro Gly Lys Asn Val Val Asn Val Gln Thr Lys Pro Ser Leu                1605 1610 1615 Phe Lys Val Arg Asn Gly Gly Glu Ile Gly Ala Val Ala Leu Asp Tyr            1620 1625 1630 Pro Ser Gly Thr Ser Gly Ser Pro Ile Val Asn Arg Asn Gly Glu Val        1635 1640 1645 Ile Gly Leu Tyr Gly Asn Gly Ile Leu Val Gly Asp Asn Ser Phe Val    1650 1655 1660 Ser Ala Ile Ser Gln Thr Glu Val Lys Glu Glu Gly Lys Glu Glu Leu 1665 1670 1675 1680 Gln Glu Ile Pro Thr Met Leu Lys Lys Gly Met Thr Thr Val Leu Asp                1685 1690 1695 Phe His Pro Gly Ala Gly Lys Thr Arg Arg Phe Leu Pro Gln Ile Leu            1700 1705 1710 Ala Glu Cys Ala Arg Arg Arg Leu Arg Thr Leu Val Leu Ala Pro Thr        1715 1720 1725 Arg Val Val Leu Ser Glu Met Lys Glu Ala Phe His Gly Leu Asp Val    1730 1735 1740 Lys Phe His Thr Gln Ala Phe Ser Ala His Gly Ser Gly Arg Glu Val 1745 1750 1755 1760 Ile Asp Ala Met Cys His Ala Thr Leu Thr Tyr Arg Met Leu Glu Pro                1765 1770 1775 Thr Arg Val Val Asn Trp Glu Val Ile Met Asp Glu Ala His Phe            1780 1785 1790 Leu Asp Pro Ala Ser Ile Ala Ala Arg Gly Trp Ala Ala His Arg Ala        1795 1800 1805 Arg Ala Asn Glu Ser Ala Thr Ile Leu Met Thr Ala Thr Pro Pro Gly    1810 1815 1820 Thr Ser Asp Glu Phe Pro His Ser Asn Gly Glu Ile Glu Asp Val Gln 1825 1830 1835 1840 Thr Asp Ile Pro Ser Glu Pro Trp Asn Thr Gly His Asp Trp Ile Leu                1845 1850 1855 Ala Asp Lys Arg Pro Thr Ala Trp Phe Leu Pro Ser Ile Arg Ala Ala            1860 1865 1870 Asn Val Met Ala Ala Ser Leu Arg Lys Ala Gly Lys Ser Val Val Val        1875 1880 1885 Leu Asn Arg Lys Thr Phe Glu Arg Glu Tyr Pro Thr Ile Lys Gln Lys    1890 1895 1900 Lys Pro Asp Phe Ile Leu Ala Thr Asp Ile Ala Glu Met Gly Ala Asn 1905 1910 1915 1920 Leu Cys Val Glu Arg Val Leu Asp Cys Arg Thr Ala Phe Lys Pro Val                1925 1930 1935 Leu Val Asp Glu Gly Arg Lys Val Ala Ile Lys Gly Pro Leu Arg Ile            1940 1945 1950 Ser Ala Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn Pro        1955 1960 1965 Asn Arg Asp Gly Asp Ser Tyr Tyr Tyr Ser Glu Pro Thr Ser Glu Asn    1970 1975 1980 Asn Ala His His Val Cys Trp Leu Glu Ala Ser Met Leu Leu Asp Asn 1985 1990 1995 2000 Met Glu Val Arg Gly Gly Met Val Ala Pro Leu Tyr Gly Val Glu Gly                2005 2010 2015 Thr Lys Thr Pro Val Ser Pro Gly Glu Met Arg Leu Arg Asp Asp Gln            2020 2025 2030 Arg Lys Val Phe Arg Glu Leu Val Arg Asn Cys Asp Leu Pro Val Trp        2035 2040 2045 Leu Ser Trp Gln Val Ala Lys Ala Gly Leu Lys Thr Asn Asp Arg Lys    2050 2055 2060 Trp Cys Phe Glu Gly Pro Glu Glu His Glu Ile Leu Asn Asp Ser Gly 2065 2070 2075 2080 Glu Thr Val Lys Cys Arg Ala Pro Gly Gly Ala Lys Lys Pro Leu Arg                2085 2090 2095 Pro Arg Trp Cys Asp Glu Arg Val Ser Ser Asp Gln Ser Ala Leu Ser            2100 2105 2110 Glu Phe Ile Lys Phe Ala Glu Gly Arg Arg Gly Ala Ala Glu Val Leu        2115 2120 2125 Val Val Leu Ser Glu Leu Pro Asp Phe Leu Ala Lys Lys Gly Gly Glu    2130 2135 2140 Ala Met Asp Thr Ile Ser Val Phe Leu His Ser Glu Glu Gly Ser Arg 2145 2150 2155 2160 Ala Tyr Arg Asn Ala Leu Ser Met Met Pro Glu Ala Met Thr Ile Val                2165 2170 2175 Met Leu Phe Ile Leu Ala Gly Leu Leu Thr Ser Gly Met Val Ile Phe            2180 2185 2190 Phe Met Ser Pro Lys Gly Ile Ser Arg Met Ser Met Ala Met Gly Thr        2195 2200 2205 Met Ala Gly Cys Gly Tyr Leu Met Phe Leu Gly Gly Val Lys Pro Thr    2210 2215 2220 His Ile Ser Tyr Val Met Leu Ile Phe Phe Val Leu Met Val Val Val 2225 2230 2235 2240 Ile Pro Glu Pro Gly Gln Gln Arg Ser Ile Gln Asp Asn Gln Val Ala                2245 2250 2255 Tyr Leu Ile Ile Gly Ile Leu Thr Leu Val Ser Ala Val Ala Ala Asn            2260 2265 2270 Glu Leu Gly Met Leu Glu Lys Thr Lys Glu Asp Leu Phe Gly Lys Lys        2275 2280 2285 Asn Leu Ile Pro Ser Ser Ala Ser Pro Trp Ser Trp Pro Asp Leu Asp    2290 2295 2300 Leu Lys Pro Gly Ala Ala Trp Thr Val Tyr Val Gly Ile Val Thr Met 2305 2310 2315 2320 Leu Ser Pro Met Leu His His Trp Ile Lys Val Glu Tyr Gly Asn Leu                2325 2330 2335 Ser Leu Ser Gly Ile Ala Gln Ser Ala Ser Val Leu Ser Phe Met Asp            2340 2345 2350 Lys Gly Ile Pro Phe Met Lys Met Asn Ile Ser Val Ile Met Leu Leu        2355 2360 2365 Val Ser Gly Trp Asn Ser Ile Thr Val Met Pro Leu Leu Cys Gly Ile    2370 2375 2380 Gly Cys Ala Met Leu His Trp Ser Leu Ile Leu Pro Gly Ile Lys Ala 2385 2390 2395 2400 Gln Gln Ser Lys Leu Ala Gln Arg Arg Val Phe His Gly Val Ala Glu                2405 2410 2415 Asn Pro Val Val Asp Gly Asn Pro Thr Val Asp Ile Glu Glu Ala Pro            2420 2425 2430 Glu Met Pro Ala Leu Tyr Glu Lys Lys Leu Ala Leu Tyr Leu Leu Leu        2435 2440 2445 Ala Leu Ser Leu Ala Ser Val Ala Met Cys Arg Thr Pro Phe Ser Leu    2450 2455 2460 Ala Glu Gly Ile Val Leu Ala Ser Ala Ala Leu Gly Pro Leu Ile Glu 2465 2470 2475 2480 Gly Asn Thr Ser Leu Leu Trp Asn Gly Pro Met Ala Val Ser Met Thr                2485 2490 2495 Gly Val Met Arg Gly Asn His Tyr Ala Phe Val Gly Val Met Tyr Asn            2500 2505 2510 Leu Trp Lys Met Lys Thr Gly Arg Arg Gly Ser Ala Asn Gly Lys Thr        2515 2520 2525 Leu Gly Glu Val Trp Lys Arg Glu Leu Asn Leu Leu Asp Lys Arg Gln    2530 2535 2540 Phe Glu Leu Tyr Lys Arg Thr Asp Ile Val Glu Val Asp Arg Asp Thr 2545 2550 2555 2560 Ala Arg Arg His Leu Ala Glu Gly Lys Val Asp Thr Gly Val Ala Val                2565 2570 2575 Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe His Glu Arg Gly Tyr Val            2580 2585 2590 Lys Leu Glu Gly Arg Val Ile Asp Leu Gly Cys Gly Arg Gly Gly Trp        2595 2600 2605 Cys Tyr Tyr Ala Ala Ala Gln Lys Glu Val Ser Gly Val Lys Gly Phe    2610 2615 2620 Thr Leu Gly Arg Asp Gly His Glu Lys Pro Met Asn Val Gln Ser Leu 2625 2630 2635 2640 Gly Trp Asn Ile Ile Thr Phe Lys Asp Lys Thr Asp Ile His Arg Leu                2645 2650 2655 Glu Pro Val Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser            2660 2665 2670 Ser Ser Ser Val Thr Glu Gly Glu Arg Thr Val Arg Val Leu Asp Thr        2675 2680 2685 Val Glu Lys Trp Leu Ala Cys Gly Val Asp Asn Phe Cys Val Lys Val    2690 2695 2700 Leu Ala Pro Tyr Met Pro Asp Val Leu Glu Lys Leu Glu Leu Leu Gln 2705 2710 2715 2720 Arg Arg Phe Gly Gly Thr Val Ile Arg Asn Pro Leu Ser Arg Asn Ser                2725 2730 2735 Thr His Glu Met Tyr Tyr Val Ser Gly Ala Arg Ser Asn Val Thr Phe            2740 2745 2750 Thr Val Asn Gln Thr Ser Arg Leu Leu Met Arg Arg Met Arg Arg Pro        2755 2760 2765 Thr Gly Lys Val Thr Leu Glu Ala Asp Val Ile Leu Pro Ile Gly Thr    2770 2775 2780 Arg Ser Val Glu Thr Asp Lys Gly Pro Leu Asp Lys Glu Ala Ile Glu 2785 2790 2795 2800 Glu Arg Val Glu Arg Ile Lys Ser Glu Tyr Met Thr Ser Trp Phe Tyr                2805 2810 2815 Asp Asn Asp Asn Pro Tyr Arg Thr Trp His Tyr Cys Gly Ser Tyr Val            2820 2825 2830 Thr Lys Thr Ser Gly Ser Ala Ala Ser Met Val Asn Gly Val Ile Lys        2835 2840 2845 Ile Leu Thr Tyr Pro Trp Asp Arg Ile Glu Glu Val Thr Arg Met Ala    2850 2855 2860 Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys 2865 2870 2875 2880 Val Asp Thr Arg Ala Lys Asp Pro Pro Ala Gly Thr Arg Lys Ile Met                2885 2890 2895 Lys Val Val Asn Arg Trp Leu Phe Arg His Leu Ala Arg Glu Lys Asn            2900 2905 2910 Pro Arg Leu Cys Thr Lys Glu Glu Phe Ile Ala Lys Val Arg Ser His        2915 2920 2925 Ala Ala Ile Gly Ala Tyr Leu Glu Glu Gln Glu Gln Trp Lys Thr Ala    2930 2935 2940 Asn Glu Ala Val Gln Asp Pro Lys Phe Trp Glu Leu Val Asp Glu Glu 2945 2950 2955 2960 Arg Lys Leu His Gln Gln Gly Arg Cys Arg Thr Cys Val Tyr Asn Met                2965 2970 2975 Met Gly Lys Arg Glu Lys Lys Leu Ser Glu Phe Gly Lys Ala Lys Gly            2980 2985 2990 Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Tyr Leu Glu Phe        2995 3000 3005 Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Ala Ser Arg Glu Asn    3010 3015 3020 Ser Gly Gly Gly Val Glu Gly Ile Gly Leu Gln Tyr Leu Gly Tyr Val 3025 3030 3035 3040 Ile Arg Asp Leu Ala Ala Met Asp Gly Gly Gly Phe Tyr Ala Asp Asp                3045 3050 3055 Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Ala Asp Leu Asp Asp Glu            3060 3065 3070 Gln Glu Ile Leu Asn Tyr Met Ser Pro His His Lys Lys Leu Ala Gln        3075 3080 3085 Ala Val Met Glu Met Thr Tyr Lys Asn Lys Val Val Lys Val Leu Arg    3090 3095 3100 Pro Ala Pro Gly Gly Lys Ala Tyr Met Asp Val Ile Ser Arg Arg Asp 3105 3110 3115 3120 Gln Arg Gly Ser Gly Gln Val Val Thr Tyr Ala Leu Asn Thr Ile Thr                3125 3130 3135 Asn Leu Lys Val Gln Leu Ile Arg Met Ala Glu Ala Glu Met Val Ile            3140 3145 3150 His His Gln His Val Gln Asp Cys Asp Glu Ser Val Leu Thr Arg Leu        3155 3160 3165 Glu Ala Trp Leu Thr Glu His Gly Cys Asp Arg Leu Lys Arg Met Ala    3170 3175 3180 Val Ser Gly Asp Asp Cys Val Val Arg Pro Ile Asp Asp Arg Phe Gly 3185 3190 3195 3200 Leu Ala Leu Ser His Leu Asn Ala Met Ser Lys Val Arg Lys Asp Ile                3205 3210 3215 Ser Glu Trp Gln Pro Ser Lys Gly Trp Asn Asp Trp Glu Asn Val Pro            3220 3225 3230 Phe Cys Ser His His Phe His Glu Leu Gln Leu Lys Asp Gly Arg Arg        3235 3240 3245 Ile Val Val Pro Cys Arg Glu Gln Asp Glu Leu Ile Gly Arg Gly Arg    3250 3255 3260 Val Ser Pro Gly Asn Gly Trp Met Ile Lys Glu Thr Ala Cys Leu Ser 3265 3270 3275 3280 Lys Ala Tyr Ala Asn Met Trp Ser Leu Met Tyr Phe His Lys Arg Asp                3285 3290 3295 Met Arg Leu Leu Ser Leu Ala Val Ser Ser Ala Val Pro Thr Ser Trp            3300 3305 3310 Val Pro Gln Gly Arg Thr Thr Trp Ser Ile His Gly Lys Gly Glu Trp        3315 3320 3325 Met Thr Thr Glu Asp Met Leu Glu Val Trp Asn Arg Val Trp Ile Thr    3330 3335 3340 Asn Asn Pro His Met Gln Asp Lys Thr Met Val Lys Lys Trp Arg Asp 3345 3350 3355 3360 Val Pro Tyr Leu Thr Lys Arg Gln Asp Lys Leu Cys Gly Ser Leu Ile                3365 3370 3375 Gly Met Thr Asn Arg Ala Thr Trp Ala Ser His Ile His Leu Val Ile            3380 3385 3390 His Arg Ile Arg Thr Leu Ile Gly Gln Glu Lys Tyr Thr Asp Tyr Leu        3395 3400 3405 Thr Val Met Asp Arg Tyr Ser Val Asp Ala Asp Leu Gln Leu Gly Glu    3410 3415 3420 Leu ile 3425 <210> 8 <211> 10907 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 4 nucleic acid sequence <400> 8 agtaaatcct gtgtgctaat tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60 acacatttgg attaatttta atcgttcgtt gagcgattag cagagaactg accagaacat 120 gtctggtcgt aaagctcagg gaaaaaccct gggcgtcaat atggtacgac gaggagttcg 180 ctccttgtca aacaaaataa aacaaaaaac aaaacaaatt ggaaacagac ctggaccttc 240 aagaggtgtt caaggattta tctttttctt tttgttcaac attttgactg gaaaaaagat 300 cacagcccac ctaaagaggt tgtggaaaat gctggaccca agacaaggct tggctgttct 360 aaggaaagtc aagagagtgg tggccagttt gatgagagga ttgtcctcaa ggaaacgccg 420 ttcccatggc gcagatacta gtgtcggaat tgttggcctc ctgctgacca cagctatggc 480 agcggaggtc actagacgtg ggagtgcata ctatatgtac ttggacagaa acgatgctgg 540 ggaggccata tcttttccaa ccacattggg gatgaataag tgttatatac agatcatgga 600 tcttggacac atgtgtgatg ccaccatgag ctatgaatgc cctatgctgg atgagggggt 660 ggaaccagat gacgtcgatt gttggtgcaa cacgacgtca acttgggttg tgtacggaac 720 ctgccatcac aaaaaaggtg aagcacggag atctagaaga gctgtgacgc tcccctccca 780 ttccactagg aagctgcaaa cgcggtcgca aacctggttg gaatcaagag aatacacaaa 840 gcacttgatt agagtcgaaa attggatatt caggaaccct ggcttcgcgt tagcagcagc 900 tgccatcgct tggcttttgg gaagctcaac gagccaaaaa gtcatatact tggtcatgat 960 actgctgatt gccccggcat acagcatcag gtgcatagga gtcagcaata gggactttgt 1020 ggaaggtatg tcaggtggga cttgggttga tgttgtcttg gaacatggag gttgtgtcac 1080 cgtaatggca caggacaaac cgactgtcga catagagctg gttacaacaa cagtcagcaa 1140 catggcggag gtaagatcct actgctatga ggcatcaata tcagacatgg cttcggacag 1200 ccgctgccca acacaaggtg aagcctacct tgacaagcaa tcagacactc aatatgtctg 1260 caaaagaacg ttagtggaca gaggctgggg aaatggatgt ggactttttg gcaaagggag 1320 cctggtgaca tgcgctaagt ttgcatgctc caagaaaatg accgggaaga gcatccagcc 1380 agagaatctg gagtaccgga taatgctgtc agttcatggc tcccagcaca gtgggatgat 1440 cgttaatgac acaggacatg aaactgatga gaatagagcg aaagttgaga taacgcccaa 1500 ttcaccaaga gccgaagcca ccctgggggg ttttggaagc ctaggacttg attgtgaacc 1560 gaggacaggc cttgactttt cagatttgta ttacttgact atgaataaca agcactggtt 1620 ggttcacaag gagtggttcc acgacattcc attaccttgg cacgctgggg cagacaccgg 1680 aactccacac tggaacaaca aagaagcact ggtagagttc aaggacgcac atgccaaaag 1740 gcaaactgtc gtggttctag ggagtcaaga aggagcagtt cacacggccc ttgctggagc 1800 tctggaggct gagatggatg gtgcaaaggg aaggctgtcc tctggccact tgaaatgtcg 1860 cctgaaaatg gataaactta gattgaaggg cgtgtcatac tccttgtgta ctgcagcgtt 1920 cacattcacc aagatcccgg ctgaaacact gcacgggaca gtcacagtgg aggtacagta 1980 cgcagggaca gatggacctt gcaaggttcc agctcagatg gcggtggaca tgcaaactct 2040 gaccccagtt gggaggttga taaccgctaa ccccgtaatc actgaaagca ctgagaactc 2100 taagatgatg ctggaacttg atccaccatt tggggactct tacattgtca taggagtcgg 2160 ggagaagaag atcacccacc actggcacag gagtggcagc accattggaa aagcatttga 2220 agccactgtg agaggtgcca agagaatggc agtcttggga gacacagcct gggactttgg 2280 atcagttgga ggcgctctca actcattggg caagggcatc catcaaattt ttggagcagc 2340 tttcaaatca ttgtttggag gaatgtcctg gttctcacaa attctcattg gaacgttgct 2400 gatgtggttg ggtctgaaca caaagaatgg atctatttcc cttatgtgct tggccttagg 2460 gggggtgttg atcttcttat ccacagccgt ctctgctgat caaggatgcg ccatcaactt 2520 tggcaagaga gagctcaagt gcggagatgg tatcttcata tttagagact ctgatgactg 2580 gctgaacaag tactcatact atccagaaga tcctgtgaag cttgcatcaa tagtgaaagc 2640 ctcttttgaa gaagggaagt gtggcctaaa ttcagttgac tcccttgagc atgagatgtg 2700 gagaagcagg gcagatgaga tcaatgccat ttttgaggaa aacgaggtgg acatttctgt 2760 tgtcgtgcag gatccaaaga atgtttacca gagaggaact catccatttt ccagaattcg 2820 ggatggtctg cagtatggtt ggaagacttg gggtaagaac cttgtgttct ccccagggag 2880 gaagaatgga agcttcatca tagatggaaa gtccaggaaa gaatgcccgt tttcaaaccg 2940 ggtctggaat tctttccaga tagaggagtt tgggacggga gtgttcacca cacgcgtgta 3000 catggacgca gtctttgaat acaccataga ctgcgatgga tctatcttgg gtgcagcggt 3060 gaacggaaaa aagagtgccc atggctctcc aacattttgg atgggaagtc atgaagtaaa 3120 tgggacatgg atgatccaca ccttggaggc attagattac aaggagtgtg agtggccact 3180 gacacatacg attggaacat cagttgaaga gagtgaaatg ttcatgccga gatcaatcgg 3240 aggcccagtt agctctcaca atcatatccc tggatacaag gttcagacga acggaccttg 3300 gatgcaggta ccactagaag tgaagagaga agcttgccca gggactagcg tgatcattga 3360 tggcaactgt gatggacggg gaaaatcaac cagatccacc acggatagcg ggaaagttat 3420 tcctgaatgg tgttgccgct cctgcacaat gccgcctgtg agcttccatg gtagtgatgg 3480 gtgttggtat cccatggaaa ttaggccaag gaaaacgcat gaaagccatc tggtgcgctc 3540 ctgggttaca gctggagaaa tacatgctgt cccttttggt ttggtgagca tgatgatagc 3600 aatggaagtg gtcctaagga aaagacaggg accaaagcaa atgttggttg gaggagtagt 3660 gctcttggga gcaatgctgg tcgggcaagt aactctcctt gatttgctga aactcacagt 3720 ggctgtggga ttgcatttcc atgagatgaa caatggagga gacgccatgt atatggcgtt 3780 gattgctgcc ttttcaatca gaccagggct gctcatcggc tttgggctca ggaccctatg 3840 gagccctcgg gaacgccttg tgctgaccct aggagcagcc atggtggaga ttgccttggg 3900 tggcgtgatg ggcggcctgt ggaagtatct aaatgcagtt tctctctgca tcctgacaat 3960 aaatgctgtt gcttctagga aagcatcaaa taccatcttg cccctcatgg ctctgttgac 4020 acctgtcact atggctgagg tgagacttgc cgcaatgttc ttttgtgccg tggttatcat 4080 aggggtcctt caccagaatt tcaaggacac ctccatgcag aagactatac ctctggtggc 4140 cctcacactc acatcttacc tgggcttgac acaacctttt ttgggcctgt gtgcatttct 4200 ggcaacccgc atatttgggc gaaggagtat cccagtgaat gaggcactcg cagcagctgg 4260 tctagtggga gtgctggcag gactggcttt tcaggagatg gagaacttcc ttggtccgat 4320 tgcagttgga ggactcctga tgatgctggt tagcgtggct gggagggtgg atgggctaga 4380 gctcaagaag cttggtgaag tttcatggga agaggaggcg gagatcagcg ggagttccgc 4440 ccgctatgat gtggcactca gtgaacaagg ggagttcaag ctgctttctg aagagaaagt 4500 gccatgggac caggttgtga tgacctcgct ggccttggtt ggggctgccc tccatccatt 4560 tgctcttctg ctggtccttg ctgggtggct gtttcatgtc aggggagcta ggagaagtgg 4620 ggatgtcttg tgggatattc ccactcctaa gatcatcgag gaatgtgaac atctggagga 4680 tgggatttat ggcatattcc agtcaacctt cttgggggcc tcccagcgag gagtgggagt 4740 ggcacaggga ggggtgttcc acacaatgtg gcatgtcaca agaggagctt tccttgtcag 4800 gaatggcaag aagttgattc catcttgggc ttcagtaaag gaagaccttg tcgcctatgg 4860 tggctcatgg aagttggaag gcagatggga tggagaggaa gaggtccagt tgatcgcggc 4920 tgttccagga aagaacgtgg tcaacgtcca gacaaaaccg agcttgttca aagtgaggaa 4980 tgggggagaa atcggggctg tcgctcttga ctatccgagt ggcacttcag gatctcctat 5040 tgttaacagg aacggagagg tgattgggct gtacggcaat ggcatccttg tcggtgacaa 5100 ctccttcgtg tccgccatat cccagactga ggtgaaggaa gaaggaaagg aggagctcca 5160 agagatcccg acaatgctaa agaaaggaat gacaactgtc cttgattttc atcctggagc 5220 tgggaagaca agacgtttcc tcccacagat cttggccgag tgcgcacgga gacgcttgcg 5280 cactcttgtg ttggccccca ccagggttgt tctttctgaa atgaaggagg cttttcacgg 5340 cctggacgtg aaattccaca cacaggcttt ttccgctcac ggcagcggga gagaagtcat 5400 tgatgccatg tgccatgcca ccctaactta caggatgttg gaaccaacta gggttgttaa 5460 ctgggaagtg atcattatgg atgaagccca ttttttggat ccagctagca tagccgctag 5520 aggttgggca gcgcacagag ctagggcaaa tgaaagtgca acaatcttga tgacagccac 5580 accgcctggg actagtgatg aatttccaca ttcaaatggt gaaatagaag atgttcaaac 5640 ggacataccc agtgagccct ggaacacagg gcatgactgg atcctagctg acaaaaggcc 5700 cacggcatgg ttccttccat ccatcagagc tgcaaatgtc atggctgcct ctttgcgtaa 5760 ggctggaaag agtgtggtgg tcctgaacag gaaaaccttt gagagagaat accccacgat 5820 aaagcagaag aaacctgact ttatattggc cactgacata gctgaaatgg gagccaacct 5880 ttgcgtggag cgagtgctgg attgcaggac ggcttttaag cctgtgcttg tggatgaagg 5940 gaggaaggtg gcaataaaag ggccacttcg tatctccgca tcctctgctg ctcaaaggag 6000 ggggcgcatt gggagaaatc ccaacagaga tggagactca tactactatt ctgagcctac 6060 aagtgaaaat aatgcccacc acgtctgctg gttggaggcc tcaatgctct tggacaacat 6120 ggaggtgagg ggtggaatgg tcgccccact ctatggcgtt gaaggaacta aaacaccagt 6180 ttcccctggt gaaatgagac tgagggatga ccagaggaaa gtcttcagag aactagtgag 6240 gaattgtgac ctgcccgttt ggctttcgtg gcaagtggcc aaggctggtt tgaagacgaa 6300 tgatcgtaag tggtgttttg aaggccctga ggaacatgag atcttgaatg acagcggtga 6360 aacagtgaag tgcagggctc ctggaggagc aaagaagcct ctgcgcccaa ggtggtgtga 6420 tgaaagggtg tcatctgacc agagtgcgct gtctgaattt attaagtttg ctgaaggtag 6480 gaggggagct gctgaagtgc tagttgtgct gagtgaactc cctgatttcc tggctaaaaa 6540 aggtggagag gcaatggata ccatcagtgt gttcctccac tctgaggaag gctctagggc 6600 ttaccgcaat gcactatcaa tgatgcctga ggcaatgaca atagtcatgc tgtttatact 6660 ggctggacta ctgacatcgg gaatggtcat ctttttcatg tctcccaaag gcatcagtag 6720 aatgtctatg gcgatgggca caatggccgg ctgtggatat ctcatgttcc ttggaggcgt 6780 caaacccact cacatctcct atgtcatgct catattcttt gtcctgatgg tggttgtgat 6840 ccccgagcca gggcaacaaa ggtccatcca agacaaccaa gtggcatacc tcattattgg 6900 catcctgacg ctggtttcag cggtggcagc caacgagcta ggcatgctgg agaaaaccaa 6960 agaggacctc tttgggaaga agaacttaat tccatctagt gcttcaccct ggagttggcc 7020 ggatcttgac ctgaagccag gagctgcctg gacagtgtac gttggcattg ttacaatgct 7080 ctctccaatg ttgcaccact ggatcaaagt cgaatatggc aacctgtctc tgtctggaat 7140 agcccagtca gcctcagtcc tttctttcat ggacaagggg ataccattca tgaagatgaa 7200 tatctcggtc ataatgctgc tggtcagtgg ctggaattca ataacagtga tgcctctgct 7260 ctgtggcata gggtgcgcca tgctccactg gtctctcatt ttacctggaa tcaaagcgca 7320 gcagtcaaag cttgcacaga gaagggtgtt ccatggcgtt gccgagaacc ctgtggttga 7380 tgggaatcca acagttgaca ttgaggaagc tcctgaaatg cctgcccttt atgagaagaa 7440 actggctcta tatctccttc ttgctctcag cctagcttct gttgccatgt gcagaacgcc 7500 cttttcattg gctgaaggca ttgtcctagc atcagctgcc ttagggccgc tcatagaggg 7560 aaacaccagc cttctttgga atggacccat ggctgtctcc atgacaggag tcatgagggg 7620 gaatcactat gcttttgtgg gagtcatgta caatctatgg aagatgaaaa ctggacgccg 7680 ggggagcgcg aatggaaaaa ctttgggtga agtctggaag agggaactga atctgttgga 7740 caagcgacag tttgagttgt ataaaaggac cgacattgtg gaggtggatc gtgatacggc 7800 acgcaggcat ttggccgaag ggaaggtgga caccggggtg gcggtctcca gggggaccgc 7860 aaagttaagg tggttccatg agcgtggcta tgtcaagctg gaaggtaggg tgattgacct 7920 ggggtgtggc cgcggaggct ggtgttacta cgctgctgcg caaaaggaag tgagtggggt 7980 caaaggattt actcttggaa gagacggcca tgagaaaccc atgaatgtgc aaagtctggg 8040 atggaacatc atcaccttca aggacaaaac tgatatccac cgcctagaac cagtgaaatg 8100 tgacaccctt ttgtgtgaca ttggagagtc atcatcgtca tcggtcacag agggggaaag 8160 gaccgtgaga gttcttgata ctgtagaaaa atggctggct tgtggggttg acaacttctg 8220 tgtgaaggtg ttagctccat acatgccaga tgttctcgag aaactggaat tgctccaaag 8280 gaggtttggc ggaacagtga tcaggaaccc tctctccagg aattccactc atgaaatgta 8340 ctacgtgtct ggagcccgca gcaatgtcac atttactgtg aaccaaacat cccgcctcct 8400 gatgaggaga atgaggcgtc caactggaaa agtgaccctg gaggctgacg tcatcctccc 8460 aattgggaca cgcagtgttg agacagacaa gggacccctg gacaaagagg ccatagaaga 8520 aagggttgag aggataaaat ctgagtacat gacctcttgg ttttatgaca atgacaaccc 8580 ctacaggacc tggcactact gtggctccta tgtcacaaaa acctcaggaa gtgcggcgag 8640 catggtaaat ggtgttatta aaattctgac atatccatgg gacaggatag aggaggtcac 8700 aagaatggca atgactgaca caaccccttt tggacagcaa agagtgttta aagaaaaagt 8760 tgacaccaga gcaaaggatc caccagcggg aactaggaag atcatgaaag ttgtcaacag 8820 gtggctgttc cgccacctgg ccagagaaaa gaaccccaga ctgtgcacaa aggaagaatt 8880 tattgcaaaa gtccgaagtc atgcagccat tggagcttac ctggaagaac aagaacagtg 8940 gaagactgcc aatgaggctg tccaagaccc aaagttctgg gaactggtgg atgaagaaag 9000 gaagctgcac caacaaggca ggtgtcggac ttgtgtgtac aacatgatgg ggaaaagaga 9060 gaagaagctg tcagagtttg ggaaagcaaa gggaagccgt gccatatggt atatgtggct 9120 gggagcgcgg tatcttgagt ttgaggccct gggattcctg aatgaggacc attgggcttc 9180 cagggaaaac tcaggaggag gagtggaagg cattggctta caatacctag gatatgtgat 9240 cagagacctg gctgcaatgg atggtggtgg attctacgcg gatgacaccg ctggatggga 9300 cacgcgcatc acagaggcag accttgatga tgaacaggag atcttgaact acatgagccc 9360 acatcacaaa aaactggcac aagcagtgat ggaaatgaca tacaagaaca aagtggtgaa 9420 agtgttgaga ccagccccag gagggaaagc ctacatggat gtcataagtc gacgagacca 9480 gagaggatcc gggcaggtag tgacttatgc tctgaacacc atcaccaact tgaaagtcca 9540 attgatcaga atggcagaag cagagatggt gatacatcac caacatgttc aagattgtga 9600 tgaatcagtt ctgaccaggc tggaggcatg gctcactgag cacggatgtg acagactgaa 9660 gaggatggcg gtgagtggag acgactgtgt ggtccggccc atcgatgaca ggttcggcct 9720 ggccctgtcc catctcaacg ccatgtccaa ggttagaaag gacatatctg aatggcagcc 9780 atcaaaaggg tggaatgatt gggagaatgt gcccttctgt tcccaccact tccatgaact 9840 acagctgaag gatggcagga ggattgtggt gccttgccga gaacaggacg agctcattgg 9900 gagaggaagg gtgtctccag gaaacggctg gatgatcaag gaaacagctt gcctcagcaa 9960 agcctatgcc aacatgtggt cactgatgta ttttcacaaa agggacatga ggctactgtc 10020 attggctgtt tcctcagctg ttcccacctc atgggttcca caaggacgca caacatggtc 10080 gattcatggg aaaggggagt ggatgaccac ggaagacatg cttgaggtgt ggaacagagt 10140 atggataacc aacaacccac acatgcagga caagacaatg gtgaaaaaat ggagagatgt 10200 cccttatcta accaagagac aagacaagct gtgcggatca ctgattggaa tgaccaatag 10260 ggccacctgg gcctcccaca tccatttagt catccatcgt atccgaacgc tgattggaca 10320 ggagaaatac actgactacc taacagtcat ggacaggtat tctgtggatg ctgacctgca 10380 actgggtgag cttatctgaa acaccatcta acaggaataa ccgggataca aaccacgggt 10440 ggagaaccgg actccccaca acctgaaacc gggatataaa ccacggctgg agaaccgggc 10500 tccgcactta aaatgaaaca gaaaccggga taaaaactac ggatggagaa ccggactcca 10560 cacattgaga cagaagaagt tgtcagccca gaaccccaca cgagttttgc cactgctaag 10620 ctgtgaggca gtgcaggctg ggacagccga cctccaggtt gcgaaaaacc tggtttctgg 10680 gacctcccac cccagagtaa aaagaacgga gcctccgcta ccaccctccc acgtggtggt 10740 agaaagacgg ggtctagagg ttagaggaga ccctccaggg aacaaatagt gggaccatat 10800 tgacgccagg gaaagaccgg agtggttctc tgcttttcct ccagaggtct gtgagcacag 10860 tttgctcaag aataagcaga cctttggatg acaaacacaa aaccact 10907 <210> 9 <211> 3427 <212> PRT <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 5 amino acid sequence <400> 9 Met Ser Gly Arg Lys Ala Gln Gly Lys Thr Leu Gly Val Asn Met Val   1 5 10 15 Arg Arg Gly Val Arg Ser Leu Ser Asn Lys Ile Lys Gln Lys Thr Lys              20 25 30 Gln Ile Gly Asn Arg Pro Gly Pro Ser Arg Gly Val Gln Gly Phe Ile          35 40 45 Phe Phe Phe Leu Phe Asn Ile Leu Thr Gly Lys Lys Ile Thr Ala His      50 55 60 Leu Lys Arg Leu Trp Lys Met Leu Asp Pro Arg Gln Gly Leu Ala Val  65 70 75 80 Leu Arg Lys Val Lys Arg Val Val Ala Ser Leu Met Arg Gly Leu Ser                  85 90 95 Ser Arg Lys Arg Arg Ser His Asp Val Leu Thr Val Gln Phe Leu Ile             100 105 110 Leu Gly Met Leu Leu Met Thr Gly Gly Ala Ala Glu Val Thr Arg Arg         115 120 125 Gly Ser Ala Tyr Tyr Met Tyr Leu Asp Arg Asn Asp Ala Gly Glu Ala     130 135 140 Ile Ser Phe Pro Thr Thr Leu Gly Met Asn Lys Cys Tyr Ile Gln Ile 145 150 155 160 Met Asp Leu Gly His Met Cys Asp Ala Thr Met Ser Tyr Glu Cys Pro                 165 170 175 Met Leu Asp Glu Gly Val Glu Pro Asp Asp Val Asp Cys Trp Cys Asn             180 185 190 Thr Thr Ser Thr Trp Val Val Tyr Gly Thr Cys His His Lys Lys Gly         195 200 205 Glu Ala Arg Arg Ser Arg Arg Ala Val Thr Leu Pro Ser His Ser Thr     210 215 220 Arg Lys Leu Gln Thr Arg Ser Gln Thr Trp Leu Glu Ser Arg Glu Tyr 225 230 235 240 Thr Lys His Leu Ile Arg Val Glu Asn Trp Ile Phe Arg Asn Pro Gly                 245 250 255 Phe Ala Leu Ala Ala Ala Ala Ile Ala Trp Leu Leu Gly Ser Ser Thr             260 265 270 Ser Gln Lys Val Ile Tyr Leu Val Met Ile Leu Leu Ile Ala Pro Ala         275 280 285 Tyr Ser Ile Arg Cys Ile Gly Val Ser Asn Arg Asp Phe Val Glu Gly     290 295 300 Met Ser Gly Gly Thr Trp Val Asp Val Val Leu Glu His Gly Gly Cys 305 310 315 320 Val Thr Val Met Ala Gln Asp Lys Pro Thr Val Asp Ile Glu Leu Val                 325 330 335 Thr Thr Thr Val Ser Asn Met Ala Glu Val Arg Ser Tyr Cys Tyr Glu             340 345 350 Ala Ser Ile Ser Asp Met Ala Ser Asp Ser Arg Cys Pro Thr Gln Gly         355 360 365 Glu Ala Tyr Leu Asp Lys Gln Ser Asp Thr Gln Tyr Val Cys Lys Arg     370 375 380 Thr Leu Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys 385 390 395 400 Gly Ser Leu Val Thr Cys Ala Lys Phe Ala Cys Ser Lys Lys Met Thr                 405 410 415 Gly Lys Ser Ile Gln Pro Glu Asn Leu Glu Tyr Arg Ile Met Leu Ser             420 425 430 Val His Gly Ser Gln His Ser Gly Met Ile Val Asn Asp Thr Gly His         435 440 445 Glu Thr Asp Glu Asn Arg Ala Lys Val Glu Ile Thr Pro Asn Ser Pro     450 455 460 Arg Ala Glu Ala Thr Leu Gly Gly Phe Gly Ser Leu Gly Leu Asp Cys 465 470 475 480 Glu Pro Arg Thr Gly Leu Asp Phe Ser Asp Leu Tyr Tyr Leu Thr Met                 485 490 495 Asn Asn Lys His Trp Leu Val His Lys Glu Trp Phe His Asp Ile Pro             500 505 510 Leu Pro Trp His Ala Gly Ala Asp Thr Gly Thr Pro His Trp Asn Asn         515 520 525 Lys Glu Ala Leu Val Glu Phe Lys Asp Ala His Ala Lys Arg Gln Thr     530 535 540 Val Val Val Leu Gly Ser Gln Glu Gly Ala Val His Thr Ala Leu Ala 545 550 555 560 Gly Ala Leu Glu Ala Glu Met Asp Gly Ala Lys Gly Arg Leu Ser Ser                 565 570 575 Gly His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Arg Leu Lys Gly             580 585 590 Val Ser Tyr Ser Leu Cys Thr Ala Ala Phe Thr Phe Thr Lys Ile Pro         595 600 605 Ala Glu Thr Leu His Gly Thr Val Thr Val Glu Val Gln Tyr Ala Gly     610 615 620 Thr Asp Gly Pro Cys Lys Val Pro Ala Gln Met Ala Val Asp Met Gln 625 630 635 640 Thr Leu Thr Pro Val Gly Arg Leu Ile Thr Ala Asn Pro Val Ile Thr                 645 650 655 Glu Ser Thr Glu Asn Ser Lys Met Met Leu Glu Leu Asp Pro Pro Phe             660 665 670 Gly Asp Ser Tyr Ile Val Ile Gly Val Gly Glu Lys Lys Ile Thr His         675 680 685 His Trp His Arg Ser Gly Ser Thr Ile Gly Lys Ala Phe Glu Ala Thr     690 695 700 Val Arg Gly Ala Lys Arg Met Ala Val Leu Gly Asp Thr Ala Trp Asp 705 710 715 720 Phe Gly Ser Val Gly Gly Ala Leu Asn Ser Leu Gly Lys Gly Ile His                 725 730 735 Gln Ile Phe Gly Ala Ala Phe Lys Ser Leu Phe Gly Gly Met Ser Trp             740 745 750 Phe Ser Gln Ile Leu Ile Gly Thr Leu Leu Met Trp Leu Gly Leu Asn         755 760 765 Thr Lys Asn Gly Ser Ile Ser Leu Met Cys Leu Ala Leu Gly Gly Val     770 775 780 Leu Ile Phe Leu Ser Thr Ala Val Ser Ala Asp Gln Gly Cys Ala Ile 785 790 795 800 Asn Phe Gly Lys Arg Glu Leu Lys Cys Gly Asp Gly Ile Phe Ile Phe                 805 810 815 Arg Asp Ser Asp Asp Trp Leu Asn Lys Tyr Ser Tyr Tyr Pro Glu Asp             820 825 830 Pro Val Lys Leu Ala Ser Ile Val Lys Ala Ser Phe Glu Glu Gly Lys         835 840 845 Cys Gly Leu Asn Ser Val Asp Ser Leu Glu His Glu Met Trp Arg Ser     850 855 860 Arg Ala Asp Glu Ile Asn Ala Ile Phe Glu Glu Asn Glu Val Asp Ile 865 870 875 880 Ser Val Val Val Gln Asp Pro Lys Asn Val Tyr Gln Arg Gly Thr His                 885 890 895 Pro Phe Ser Arg Ile Arg Asp Gly Leu Gln Tyr Gly Trp Lys Thr Trp             900 905 910 Gly Lys Asn Leu Val Phe Ser Pro Gly Arg Lys Asn Gly Ser Phe Ile         915 920 925 Ile Asp Gly Lys Ser Arg Lys Glu Cys Pro Phe Ser Asn Arg Val Trp     930 935 940 Asn Ser Phe Gln Ile Glu Glu Phe Gly Thr Gly Val Phe Thr Thr Arg 945 950 955 960 Val Tyr Met Asp Ala Val Phe Glu Tyr Thr Ile Asp Cys Asp Gly Ser                 965 970 975 Ile Leu Gly Ala Ala Val Asn Gly Lys Lys Ser Ala His Gly Ser Pro             980 985 990 Thr Phe Trp Met Gly Ser His Glu Val Asn Gly Thr Trp Met Ile His         995 1000 1005 Thr Leu Glu Ala Leu Asp Tyr Lys Glu Cys Glu Trp Pro Leu Thr His    1010 1015 1020 Thr Ile Gly Thr Ser Val Glu Glu Ser Glu Met Phe Met Pro Arg Ser 1025 1030 1035 1040 Ile Gly Gly Pro Val Ser Ser His Asn His Ile Pro Gly Tyr Lys Val                1045 1050 1055 Gln Thr Asn Gly Pro Trp Met Gln Val Pro Leu Glu Val Lys Arg Glu            1060 1065 1070 Ala Cys Pro Gly Thr Ser Val Ile Asp Gly Asn Cys Asp Gly Arg        1075 1080 1085 Gly Lys Ser Thr Arg Ser Thr Thr Asp Ser Gly Lys Val Ile Pro Glu    1090 1095 1100 Trp Cys Cys Arg Ser Cys Thr Met Pro Pro Val Ser Phe His Gly Ser 1105 1110 1115 1120 Asp Gly Cys Trp Tyr Pro Met Glu Ile Arg Pro Arg Lys Thr His Glu                1125 1130 1135 Ser His Leu Val Arg Ser Trp Val Thr Ala Gly Glu Ile His Ala Val            1140 1145 1150 Pro Phe Gly Leu Val Ser Met Met Ile Ala Met Glu Val Val Leu Arg        1155 1160 1165 Lys Arg Gln Gly Pro Lys Gln Met Leu Val Gly Gly Val Val Leu Leu    1170 1175 1180 Gly Ala Met Leu Val Gly Gln Val Thr Leu Leu Asp Leu Leu Lys Leu 1185 1190 1195 1200 Thr Val Ala Val Gly Leu His Phe His Glu Met Asn Asn Gly Gly Asp                1205 1210 1215 Ala Met Tyr Met Ala Leu Ile Ala Ala Phe Ser Ile Arg Pro Gly Leu            1220 1225 1230 Leu Ile Gly Phe Gly Leu Arg Thr Leu Trp Ser Pro Arg Glu Arg Leu        1235 1240 1245 Val Leu Thr Leu Gly Ala Ala Met Val Glu Ile Ala Leu Gly Gly Val    1250 1255 1260 Met Gly Gly Leu Trp Lys Tyr Leu Asn Ala Val Ser Leu Cys Ile Leu 1265 1270 1275 1280 Thr Ile Asn Ala Val Ala Ser Arg Lys Ala Ser Asn Thr Ile Leu Pro                1285 1290 1295 Leu Met Ala Leu Leu Thr Pro Val Thr Met Ala Glu Val Arg Leu Ala            1300 1305 1310 Ala Met Phe Phe Cys Ala Val Val Ile Gly Val Leu His Gln Asn        1315 1320 1325 Phe Lys Asp Thr Ser Met Gln Lys Thr Ile Pro Leu Val Ala Leu Thr    1330 1335 1340 Leu Thr Ser Tyr Leu Gly Leu Thr Gln Pro Phe Leu Gly Leu Cys Ala 1345 1350 1355 1360 Phe Leu Ala Thr Arg Ile Phe Gly Arg Arg Ser Ile Pro Val Asn Glu                1365 1370 1375 Ala Leu Ala Ala Ala Gly Leu Val Gly Val Leu Ala Gly Leu Ala Phe            1380 1385 1390 Gln Glu Met Glu Asn Phe Leu Gly Pro Ile Ala Val Gly Gly Leu Leu        1395 1400 1405 Met Met Leu Val Ser Val Ala Gly Arg Val Asp Gly Leu Glu Leu Lys    1410 1415 1420 Lys Leu Gly Glu Val Ser Trp Glu Glu Glu Ala Glu Ile Ser Gly Ser 1425 1430 1435 1440 Ser Ala Arg Tyr Asp Val Ala Leu Ser Glu Gln Gly Glu Phe Lys Leu                1445 1450 1455 Leu Ser Glu Glu Lys Val Pro Trp Asp Gln Val Val Met Thr Ser Leu            1460 1465 1470 Ala Leu Val Gly Ala Ala Leu His Pro Phe Ala Leu Leu Leu Val Leu        1475 1480 1485 Ala Gly Trp Leu Phe His Val Arg Gly Ala Arg Arg Ser Gly Asp Val    1490 1495 1500 Leu Trp Asp Ile Pro Thr Pro Lys Ile Ile Glu Glu Cys Glu His Leu 1505 1510 1515 1520 Glu Asp Gly Ile Tyr Gly Ile Phe Gln Ser Thr Phe Leu Gly Ala Ser                1525 1530 1535 Gln Arg Gly Val Gly Val Ala Gln Gly Gly Val Phe His Thr Met Trp            1540 1545 1550 His Val Thr Arg Gly Ala Phe Leu Val Arg Asn Gly Lys Lys Leu Ile        1555 1560 1565 Pro Ser Trp Ala Ser Val Lys Glu Asp Leu Val Ala Tyr Gly Gly Ser    1570 1575 1580 Trp Lys Leu Glu Gly Arg Trp Asp Gly Glu Glu Glu Val Gln Leu Ile 1585 1590 1595 1600 Ala Ala Val Pro Gly Lys Asn Val Val Asn Val Gln Thr Lys Pro Ser                1605 1610 1615 Leu Phe Lys Val Arg Asn Gly Gly Glu Ile Gly Ala Val Ala Leu Asp            1620 1625 1630 Tyr Pro Ser Gly Thr Ser Gly Ser Pro Ile Val Asn Arg Asn Gly Glu        1635 1640 1645 Val Ile Gly Leu Tyr Gly Asn Gly Ile Leu Val Gly Asp Asn Ser Phe    1650 1655 1660 Val Ser Ala Ile Ser Gln Thr Glu Val Lys Glu Glu Gly Lys Glu Glu 1665 1670 1675 1680 Leu Gln Glu Ile Pro Thr Met Leu Lys Lys Gly Met Thr Thr Val Leu                1685 1690 1695 Asp Phe His Pro Gly Ala Gly Lys Thr Arg Arg Phe Leu Pro Gln Ile            1700 1705 1710 Leu Ala Glu Cys Ala Arg Arg Arg Leu Arg Thr Leu Val Leu Ala Pro        1715 1720 1725 Thr Arg Val Val Leu Ser Glu Met Lys Glu Ala Phe His Gly Leu Asp    1730 1735 1740 Val Lys Phe His Thr Gln Ala Phe Ser Ala His Gly Ser Gly Arg Glu 1745 1750 1755 1760 Val Ile Asp Ala Met Cys His Ala Thr Leu Thr Tyr Arg Met Leu Glu                1765 1770 1775 Pro Thr Arg Val Val Asn Trp Glu Val Ile Ile Met Asp Glu Ala His            1780 1785 1790 Phe Leu Asp Pro Ala Ser Ile Ala Ala Arg Gly Trp Ala Ala His Arg        1795 1800 1805 Ala Arg Ala Asn Glu Ser Ala Thr Ile Leu Met Thr Ala Thr Pro Pro    1810 1815 1820 Gly Thr Ser Asp Glu Phe Pro His Ser Asn Gly Glu Ile Glu Asp Val 1825 1830 1835 1840 Gln Thr Asp Ile Pro Ser Glu Pro Trp Asn Thr Gly His Asp Trp Ile                1845 1850 1855 Leu Ala Asp Lys Arg Pro Thr Ala Trp Phe Leu Pro Ser Ile Arg Ala            1860 1865 1870 Ala Asn Val Met Ala Ala Ser Leu Arg Lys Ala Gly Lys Ser Val Val        1875 1880 1885 Val Leu Asn Arg Lys Thr Phe Glu Arg Glu Tyr Pro Thr Ile Lys Gln    1890 1895 1900 Lys Lys Pro Asp Phe Ile Leu Ala Thr Asp Ile Ala Glu Met Gly Ala 1905 1910 1915 1920 Asn Leu Cys Val Glu Arg Val Leu Asp Cys Arg Thr Ala Phe Lys Pro                1925 1930 1935 Val Leu Val Asp Glu Gly Arg Lys Val Ala Ile Lys Gly Pro Leu Arg            1940 1945 1950 Ile Ser Ala Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn        1955 1960 1965 Pro Asn Arg Asp Gly Asp Ser Tyr Tyr Tyr Ser Glu Pro Thr Ser Glu    1970 1975 1980 Asn Asn Ala His His Val Cys Trp Leu Glu Ala Ser Met Leu Leu Asp 1985 1990 1995 2000 Asn Met Glu Val Arg Gly Gly Met Val Ala Pro Leu Tyr Gly Val Glu                2005 2010 2015 Gly Thr Lys Thr Pro Val Ser Pro Gly Glu Met Arg Leu Arg Asp Asp            2020 2025 2030 Gln Arg Lys Val Phe Arg Glu Leu Val Arg Asn Cys Asp Leu Pro Val        2035 2040 2045 Trp Leu Ser Trp Gln Val Ala Lys Ala Gly Leu Lys Thr Asn Asp Arg    2050 2055 2060 Lys Trp Cys Phe Glu Gly Pro Glu Glu His Glu Ile Leu Asn Asp Ser 2065 2070 2075 2080 Gly Glu Thr Val Lys Cys Arg Ala Pro Gly Gly Ala Lys Lys Pro Leu                2085 2090 2095 Arg Pro Arg Trp Cys Asp Glu Arg Val Ser Ser Asp Gln Ser Ala Leu            2100 2105 2110 Ser Glu Phe Ile Lys Phe Ala Glu Gly Arg Arg Gly Ala Ala Glu Val        2115 2120 2125 Leu Val Val Leu Ser Glu Leu Pro Asp Phe Leu Ala Lys Lys Gly Gly    2130 2135 2140 Glu Ala Met Asp Thr Ile Ser Val Phe Leu His Ser Glu Glu Gly Ser 2145 2150 2155 2160 Arg Ala Tyr Arg Asn Ala Leu Ser Met Met Pro Glu Ala Met Thr Ile                2165 2170 2175 Val Met Leu Phe Ile Leu Ala Gly Leu Leu Thr Ser Gly Met Val Ile            2180 2185 2190 Phe Phe Met Ser Pro Lys Gly Ile Ser Arg Met Ser Met Ala Met Gly        2195 2200 2205 Thr Met Ala Gly Cys Gly Tyr Leu Met Phe Leu Gly Gly Val Lys Pro    2210 2215 2220 Thr His Ile Ser Tyr Val Met Leu Ile Phe Phe Val Leu Met Val Val 2225 2230 2235 2240 Val Ile Pro Glu Pro Gly Gln Gln Arg Ser Ile Gln Asp Asn Gln Val                2245 2250 2255 Ala Tyr Leu Ile Ile Gly Ile Leu Thr Leu Val Ser Ala Val Ala Ala            2260 2265 2270 Asn Glu Leu Gly Met Leu Glu Lys Thr Lys Glu Asp Leu Phe Gly Lys        2275 2280 2285 Lys Asn Leu Ile Pro Ser Ser Ala Ser Pro Trp Ser Trp Pro Asp Leu    2290 2295 2300 Asp Leu Lys Pro Gly Ala Ala Trp Thr Val Tyr Val Gly Ile Val Thr 2305 2310 2315 2320 Met Leu Ser Pro Met Leu His His Trp Ile Lys Val Glu Tyr Gly Asn                2325 2330 2335 Leu Ser Leu Ser Gly Ile Ala Gln Ser Ala Ser Val Leu Ser Phe Met            2340 2345 2350 Asp Lys Gly Ile Pro Phe Met Lys Met Asn Ile Ser Val Ile Met Leu        2355 2360 2365 Leu Val Ser Gly Trp Asn Ser Ile Thr Val Met Pro Leu Leu Cys Gly    2370 2375 2380 Ile Gly Cys Ala Met Leu His Trp Ser Leu Ile Leu Pro Gly Ile Lys 2385 2390 2395 2400 Ala Gln Gln Ser Lys Leu Ala Gln Arg Arg Val Phe His Gly Val Ala                2405 2410 2415 Glu Asn Pro Val Val Asp Gly Asn Pro Thr Val Asp Ile Glu Glu Ala            2420 2425 2430 Pro Glu Met Pro Ala Leu Tyr Glu Lys Lys Leu Ala Leu Tyr Leu Leu        2435 2440 2445 Leu Ala Leu Ser Leu Ala Ser Val Ala Met Cys Arg Thr Pro Phe Ser    2450 2455 2460 Leu Ala Glu Gly Ile Val Leu Ala Ser Ala Ala Leu Gly Pro Leu Ile 2465 2470 2475 2480 Glu Gly Asn Thr Ser Leu Leu Trp Asn Gly Pro Met Ala Val Ser Met                2485 2490 2495 Thr Gly Val Met Arg Gly Asn His Tyr Ala Phe Val Gly Val Met Tyr            2500 2505 2510 Asn Leu Trp Lys Met Lys Thr Gly Arg Arg Gly Ser Ala Asn Gly Lys        2515 2520 2525 Thr Leu Gly Glu Val Trp Lys Arg Glu Leu Asn Leu Leu Asp Lys Arg    2530 2535 2540 Gln Phe Glu Leu Tyr Lys Arg Thr Asp Ile Val Glu Val Asp Arg Asp 2545 2550 2555 2560 Thr Ala Arg Arg His Leu Ala Glu Gly Lys Val Asp Thr Gly Val Ala                2565 2570 2575 Val Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe His Glu Arg Gly Tyr            2580 2585 2590 Val Lys Leu Glu Gly Arg Val Ile Asp Leu Gly Cys Gly Arg Gly Gly        2595 2600 2605 Trp Cys Tyr Tyr Ala Ala Ala Gln Lys Glu Val Ser Gly Val Lys Gly    2610 2615 2620 Phe Thr Leu Gly Arg Asp Gly His Glu Lys Pro Met Asn Val Gln Ser 2625 2630 2635 2640 Leu Gly Trp Asn Ile Ile Thr Phe Lys Asp Lys Thr Asp Ile His Arg                2645 2650 2655 Leu Glu Pro Val Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser            2660 2665 2670 Ser Ser Ser Ser Val Thr Glu Gly Glu Arg Thr Val Arg Val Leu Asp        2675 2680 2685 Thr Val Glu Lys Trp Leu Ala Cys Gly Val Asp Asn Phe Cys Val Lys    2690 2695 2700 Val Leu Ala Pro Tyr Met Pro Asp Val Leu Glu Lys Leu Glu Leu Leu 2705 2710 2715 2720 Gln Arg Arg Phe Gly Gly Thr Val Ile Arg Asn Pro Leu Ser Arg Asn                2725 2730 2735 Ser Thr His Glu Met Tyr Tyr Val Ser Gly Ala Arg Ser Asn Val Thr            2740 2745 2750 Phe Thr Val Asn Gln Thr Ser Arg Leu Leu Met Arg Arg Met Arg Arg        2755 2760 2765 Pro Thr Gly Lys Val Thr Leu Glu Ala Asp Val Ile Leu Pro Ile Gly    2770 2775 2780 Thr Arg Ser Val Glu Thr Asp Lys Gly Pro Leu Asp Lys Glu Ala Ile 2785 2790 2795 2800 Glu Glu Arg Val Glu Arg Ile Lys Ser Glu Tyr Met Thr Ser Trp Phe                2805 2810 2815 Tyr Asp Asn Asp Asn Pro Tyr Arg Thr Trp His Tyr Cys Gly Ser Tyr            2820 2825 2830 Val Thr Lys Thr Ser Gly Ser Ala Ala Ser Met Val Asn Gly Val Ile        2835 2840 2845 Lys Ile Leu Thr Tyr Pro Trp Asp Arg Ile Glu Glu Val Thr Arg Met    2850 2855 2860 Ala Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu 2865 2870 2875 2880 Lys Val Asp Thr Arg Ala Lys Asp Pro Pro Ala Gly Thr Arg Lys Ile                2885 2890 2895 Met Lys Val Val Asn Arg Trp Leu Phe Arg His Leu Ala Arg Glu Lys            2900 2905 2910 Asn Pro Arg Leu Cys Thr Lys Glu Glu Phe Ile Ala Lys Val Arg Ser        2915 2920 2925 His Ala Ala Ile Gly Ala Tyr Leu Glu Glu Gln Glu Gln Trp Lys Thr    2930 2935 2940 Ala Asn Glu Ala Val Gln Asp Pro Lys Phe Trp Glu Leu Val Asp Glu 2945 2950 2955 2960 Glu Arg Lys Leu His Gln Gln Gly Arg Cys Arg Thr Cys Val Tyr Asn                2965 2970 2975 Met Met Gly Lys Arg Glu Lys Lys Leu Ser Glu Phe Gly Lys Ala Lys            2980 2985 2990 Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Tyr Leu Glu        2995 3000 3005 Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Ala Ser Arg Glu    3010 3015 3020 Asn Ser Gly Gly Gly Val Glu Gly Ile Gly Leu Gln Tyr Leu Gly Tyr 3025 3030 3035 3040 Val Ile Arg Asp Leu Ala Ala Met Asp Gly Gly Gly Phe Tyr Ala Asp                3045 3050 3055 Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Ala Asp Leu Asp Asp            3060 3065 3070 Glu Gln Glu Ile Leu Asn Tyr Met Ser Pro His His Lys Lys Leu Ala        3075 3080 3085 Gln Ala Val Met Glu Met Thr Tyr Lys Asn Lys Val Val Lys Val Leu    3090 3095 3100 Arg Pro Ala Pro Gly Gly Lys Ala Tyr Met Asp Val Ile Ser Arg Arg 3105 3110 3115 3120 Asp Gln Arg Gly Ser Gly Gln Val Val Thr Tyr Ala Leu Asn Thr Ile                3125 3130 3135 Thr Asn Leu Lys Val Gln Leu Ile Arg Met Ala Glu Ala Glu Met Val            3140 3145 3150 Ile His His Gln His Val Gln Asp Cys Asp Glu Ser Val Leu Thr Arg        3155 3160 3165 Leu Glu Ala Trp Leu Thr Glu His Gly Cys Asp Arg Leu Lys Arg Met    3170 3175 3180 Ala Val Ser Gly Asp Asp Cys Val Val Arg Pro Ile Asp Asp Arg Phe 3185 3190 3195 3200 Gly Leu Ala Leu Ser His Leu Asn Ala Met Ser Lys Val Arg Lys Asp                3205 3210 3215 Ile Ser Glu Trp Gln Pro Ser Lys Gly Trp Asn Asp Trp Glu Asn Val            3220 3225 3230 Pro Phe Cys Ser His His Phe His Glu Leu Gln Leu Lys Asp Gly Arg        3235 3240 3245 Arg Ile Val Val Pro Cys Arg Glu Gln Asp Glu Leu Ile Gly Arg Gly    3250 3255 3260 Arg Val Ser Pro Gly Asn Gly Trp Met Ile Lys Glu Thr Ala Cys Leu 3265 3270 3275 3280 Ser Lys Ala Tyr Ala Asn Met Trp Ser Leu Met Tyr Phe His Lys Arg                3285 3290 3295 Asp Met Arg Leu Leu Ser Leu Ala Val Ser Ser Ala Val Pro Thr Ser            3300 3305 3310 Trp Val Pro Gln Gly Arg Thr Thr Trp Ser Ile His Gly Lys Gly Glu        3315 3320 3325 Trp Met Thr Thr Glu Asp Met Leu Glu Val Trp Asn Arg Val Trp Ile    3330 3335 3340 Thr Asn Asn Pro His Met Gln Asp Lys Thr Met Val Lys Lys Trp Arg 3345 3350 3355 3360 Asp Val Pro Tyr Leu Thr Lys Arg Gln Asp Lys Leu Cys Gly Ser Leu                3365 3370 3375 Ile Gly Met Thr Asn Arg Ala Thr Trp Ala Ser His Ile His Leu Val            3380 3385 3390 Ile His Arg Ile Arg Thr Leu Ile Gly Gln Glu Lys Tyr Thr Asp Tyr        3395 3400 3405 Leu Thr Val Met Asp Arg Tyr Ser Val Asp Ala Asp Leu Gln Leu Gly    3410 3415 3420 Glu leu ile 3425 <210> 10 <211> 10910 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 5 nucleic acid sequence <400> 10 agtaaatcct gtgtgctaat tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60 acacatttgg attaatttta atcgttcgtt gagcgattag cagagaactg accagaacat 120 gtctggtcgt aaagctcagg gaaaaaccct gggcgtcaat atggtacgac gaggagttcg 180 ctccttgtca aacaaaataa aacaaaaaac aaaacaaatt ggaaacagac ctggaccttc 240 aagaggtgtt caaggattta tctttttctt tttgttcaac attttgactg gaaaaaagat 300 cacagcccac ctaaagaggt tgtggaaaat gctggaccca agacaaggct tggctgttct 360 aaggaaagtc aagagagtgg tggccagttt gatgagagga ttgtcctcaa ggaaacgccg 420 ttcccatgat gttctgactg tgcaattcct aattttggga atgctgttga tgacgggtgg 480 agcagcggag gtcactagac gtgggagtgc atactatatg tacttggaca gaaacgatgc 540 tggggaggcc atatcttttc caaccacatt ggggatgaat aagtgttata tacagatcat 600 ggatcttgga cacatgtgtg atgccaccat gagctatgaa tgccctatgc tggatgaggg 660 ggtggaacca gatgacgtcg attgttggtg caacacgacg tcaacttggg ttgtgtacgg 720 aacctgccat cacaaaaaag gtgaagcacg gagatctaga agagctgtga cgctcccctc 780 ccattccact aggaagctgc aaacgcggtc gcaaacctgg ttggaatcaa gagaatacac 840 aaagcacttg attagagtcg aaaattggat attcaggaac cctggcttcg cgttagcagc 900 agctgccatc gcttggcttt tgggaagctc aacgagccaa aaagtcatat acttggtcat 960 gatactgctg attgccccgg catacagcat caggtgcata ggagtcagca atagggactt 1020 tgtggaaggt atgtcaggtg ggacttgggt tgatgttgtc ttggaacatg gaggttgtgt 1080 caccgtaatg gcacaggaca aaccgactgt cgacatagag ctggttacaa caacagtcag 1140 caacatggcg gaggtaagat cctactgcta tgaggcatca atatcagaca tggcttcgga 1200 cagccgctgc ccaacacaag gtgaagccta ccttgacaag caatcagaca ctcaatatgt 1260 ctgcaaaaga acgttagtgg acagaggctg gggaaatgga tgtggacttt ttggcaaagg 1320 gagcctggtg acatgcgcta agtttgcatg ctccaagaaa atgaccggga agagcatcca 1380 gccagagaat ctggagtacc ggataatgct gtcagttcat ggctcccagc acagtgggat 1440 gatcgttaat gacacaggac atgaaactga tgagaataga gcgaaagttg agataacgcc 1500 caattcacca agagccgaag ccaccctggg gggttttgga agcctaggac ttgattgtga 1560 accgaggaca ggccttgact tttcagattt gtattacttg actatgaata acaagcactg 1620 gttggttcac aaggagtggt tccacgacat tccattacct tggcacgctg gggcagacac 1680 cggaactcca cactggaaca acaaagaagc actggtagag ttcaaggacg cacatgccaa 1740 aaggcaaact gtcgtggttc tagggagtca agaaggagca gttcacacgg cccttgctgg 1800 agctctggag gctgagatgg atggtgcaaa gggaaggctg tcctctggcc acttgaaatg 1860 tcgcctgaaa atggataaac ttagattgaa gggcgtgtca tactccttgt gtactgcagc 1920 gttcacattc accaagatcc cggctgaaac actgcacggg acagtcacag tggaggtaca 1980 gtacgcaggg acagatggac cttgcaaggt tccagctcag atggcggtgg acatgcaaac 2040 tctgacccca gttgggaggt tgataaccgc taaccccgta atcactgaaa gcactgagaa 2100 ctctaagatg atgctggaac ttgatccacc atttggggac tcttacattg tcataggagt 2160 cggggagaag aagatcaccc accactggca caggagtggc agcaccattg gaaaagcatt 2220 tgaagccact gtgagaggtg ccaagagaat ggcagtcttg ggagacacag cctgggactt 2280 tggatcagtt ggaggcgctc tcaactcatt gggcaagggc atccatcaaa tttttggagc 2340 agctttcaaa tcattgtttg gaggaatgtc ctggttctca caaattctca ttggaacgtt 2400 gctgatgtgg ttgggtctga acacaaagaa tggatctatt tcccttatgt gcttggcctt 2460 agggggggtg ttgatcttct tatccacagc cgtctctgct gatcaaggat gcgccatcaa 2520 ctttggcaag agagagctca agtgcggaga tggtatcttc atatttagag actctgatga 2580 ctggctgaac aagtactcat actatccaga agatcctgtg aagcttgcat caatagtgaa 2640 agcctctttt gaagaaggga agtgtggcct aaattcagtt gactcccttg agcatgagat 2700 gtggagaagc agggcagatg agatcaatgc catttttgag gaaaacgagg tggacatttc 2760 tgttgtcgtg caggatccaa agaatgttta ccagagagga actcatccat tttccagaat 2820 tcgggatggt ctgcagtatg gttggaagac ttggggtaag aaccttgtgt tctccccagg 2880 gaggaagaat ggaagcttca tcatagatgg aaagtccagg aaagaatgcc cgttttcaaa 2940 ccgggtctgg aattctttcc agatagagga gtttgggacg ggagtgttca ccacacgcgt 3000 gtacatggac gcagtctttg aatacaccat agactgcgat ggatctatct tgggtgcagc 3060 ggtgaacgga aaaaagagtg cccatggctc tccaacattt tggatgggaa gtcatgaagt 3120 aaatgggaca tggatgatcc acaccttgga ggcattagat tacaaggagt gtgagtggcc 3180 actgacacat acgattggaa catcagttga agagagtgaa atgttcatgc cgagatcaat 3240 cggaggccca gttagctctc acaatcatat ccctggatac aaggttcaga cgaacggacc 3300 ttggatgcag gtaccactag aagtgaagag agaagcttgc ccagggacta gcgtgatcat 3360 tgatggcaac tgtgatggac ggggaaaatc aaccagatcc accacggata gcgggaaagt 3420 tattcctgaa tggtgttgcc gctcctgcac aatgccgcct gtgagcttcc atggtagtga 3480 tgggtgttgg tatcccatgg aaattaggcc aaggaaaacg catgaaagcc atctggtgcg 3540 ctcctgggtt acagctggag aaatacatgc tgtccctttt ggtttggtga gcatgatgat 3600 agcaatggaa gtggtcctaa ggaaaagaca gggaccaaag caaatgttgg ttggaggagt 3660 agtgctcttg ggagcaatgc tggtcgggca agtaactctc cttgatttgc tgaaactcac 3720 agtggctgtg ggattgcatt tccatgagat gaacaatgga ggagacgcca tgtatatggc 3780 gttgattgct gccttttcaa tcagaccagg gctgctcatc ggctttgggc tcaggaccct 3840 atggagccct cgggaacgcc ttgtgctgac cctaggagca gccatggtgg agattgcctt 3900 gggtggcgtg atgggcggcc tgtggaagta tctaaatgca gtttctctct gcatcctgac 3960 aataaatgct gttgcttcta ggaaagcatc aaataccatc ttgcccctca tggctctgtt 4020 gacacctgtc actatggctg aggtgagact tgccgcaatg ttcttttgtg ccgtggttat 4080 cataggggtc cttcaccaga atttcaagga cacctccatg cagaagacta tacctctggt 4140 ggccctcaca ctcacatctt acctgggctt gacacaacct tttttgggcc tgtgtgcatt 4200 tctggcaacc cgcatatttg ggcgaaggag tatcccagtg aatgaggcac tcgcagcagc 4260 tggtctagtg ggagtgctgg caggactggc ttttcaggag atggagaact tccttggtcc 4320 gattgcagtt ggaggactcc tgatgatgct ggttagcgtg gctgggaggg tggatgggct 4380 agagctcaag aagcttggtg aagtttcatg ggaagaggag gcggagatca gcgggagttc 4440 cgcccgctat gatgtggcac tcagtgaaca aggggagttc aagctgcttt ctgaagagaa 4500 agtgccatgg gaccaggttg tgatgacctc gctggccttg gttggggctg ccctccatcc 4560 atttgctctt ctgctggtcc ttgctgggtg gctgtttcat gtcaggggag ctaggagaag 4620 tggggatgtc ttgtgggata ttcccactcc taagatcatc gaggaatgtg aacatctgga 4680 ggatgggatt tatggcatat tccagtcaac cttcttgggg gcctcccagc gaggagtggg 4740 agtggcacag ggaggggtgt tccacacaat gtggcatgtc acaagaggag ctttccttgt 4800 caggaatggc aagaagttga ttccatcttg ggcttcagta aaggaagacc ttgtcgccta 4860 tggtggctca tggaagttgg aaggcagatg ggatggagag gaagaggtcc agttgatcgc 4920 ggctgttcca ggaaagaacg tggtcaacgt ccagacaaaa ccgagcttgt tcaaagtgag 4980 gaatggggga gaaatcgggg ctgtcgctct tgactatccg agtggcactt caggatctcc 5040 tattgttaac aggaacggag aggtgattgg gctgtacggc aatggcatcc ttgtcggtga 5100 caactccttc gtgtccgcca tatcccagac tgaggtgaag gaagaaggaa aggaggagct 5160 ccaagagatc ccgacaatgc taaagaaagg aatgacaact gtccttgatt ttcatcctgg 5220 agctgggaag acaagacgtt tcctcccaca gatcttggcc gagtgcgcac ggagacgctt 5280 gcgcactctt gtgttggccc ccaccagggt tgttctttct gaaatgaagg aggcttttca 5340 cggcctggac gtgaaattcc acacacaggc tttttccgct cacggcagcg ggagagaagt 5400 cattgatgcc atgtgccatg ccaccctaac ttacaggatg ttggaaccaa ctagggttgt 5460 taactgggaa gtgatcatta tggatgaagc ccattttttg gatccagcta gcatagccgc 5520 tagaggttgg gcagcgcaca gagctagggc aaatgaaagt gcaacaatct tgatgacagc 5580 cacaccgcct gggactagtg atgaatttcc acattcaaat ggtgaaatag aagatgttca 5640 aacggacata cccagtgagc cctggaacac agggcatgac tggatcctag ctgacaaaag 5700 gcccacggca tggttccttc catccatcag agctgcaaat gtcatggctg cctctttgcg 5760 taaggctgga aagagtgtgg tggtcctgaa caggaaaacc tttgagagag aataccccac 5820 gataaagcag aagaaacctg actttatatt ggccactgac atagctgaaa tgggagccaa 5880 cctttgcgtg gagcgagtgc tggattgcag gacggctttt aagcctgtgc ttgtggatga 5940 agggaggaag gtggcaataa aagggccact tcgtatctcc gcatcctctg ctgctcaaag 6000 gagggggcgc attgggagaa atcccaacag agatggagac tcatactact attctgagcc 6060 tacaagtgaa aataatgccc accacgtctg ctggttggag gcctcaatgc tcttggacaa 6120 catggaggtg aggggtggaa tggtcgcccc actctatggc gttgaaggaa ctaaaacacc 6180 agtttcccct ggtgaaatga gactgaggga tgaccagagg aaagtcttca gagaactagt 6240 gaggaattgt gacctgcccg tttggctttc gtggcaagtg gccaaggctg gtttgaagac 6300 gaatgatcgt aagtggtgtt ttgaaggccc tgaggaacat gagatcttga atgacagcgg 6360 tgaaacagtg aagtgcaggg ctcctggagg agcaaagaag cctctgcgcc caaggtggtg 6420 tgatgaaagg gtgtcatctg accagagtgc gctgtctgaa tttattaagt ttgctgaagg 6480 taggagggga gctgctgaag tgctagttgt gctgagtgaa ctccctgatt tcctggctaa 6540 aaaaggtgga gaggcaatgg ataccatcag tgtgttcctc cactctgagg aaggctctag 6600 ggcttaccgc aatgcactat caatgatgcc tgaggcaatg acaatagtca tgctgtttat 6660 actggctgga ctactgacat cgggaatggt catctttttc atgtctccca aaggcatcag 6720 tagaatgtct atggcgatgg gcacaatggc cggctgtgga tatctcatgt tccttggagg 6780 cgtcaaaccc actcacatct cctatgtcat gctcatattc tttgtcctga tggtggttgt 6840 gatccccgag ccagggcaac aaaggtccat ccaagacaac caagtggcat acctcattat 6900 tggcatcctg acgctggttt cagcggtggc agccaacgag ctaggcatgc tggagaaaac 6960 caaagaggac ctctttggga agaagaactt aattccatct agtgcttcac cctggagttg 7020 gccggatctt gacctgaagc caggagctgc ctggacagtg tacgttggca ttgttacaat 7080 gctctctcca atgttgcacc actggatcaa agtcgaatat ggcaacctgt ctctgtctgg 7140 aatagcccag tcagcctcag tcctttcttt catggacaag gggataccat tcatgaagat 7200 gaatatctcg gtcataatgc tgctggtcag tggctggaat tcaataacag tgatgcctct 7260 gctctgtggc atagggtgcg ccatgctcca ctggtctctc attttacctg gaatcaaagc 7320 gcagcagtca aagcttgcac agagaagggt gttccatggc gttgccgaga accctgtggt 7380 tgatgggaat ccaacagttg acattgagga agctcctgaa atgcctgccc tttatgagaa 7440 gaaactggct ctatatctcc ttcttgctct cagcctagct tctgttgcca tgtgcagaac 7500 gcccttttca ttggctgaag gcattgtcct agcatcagct gccttagggc cgctcataga 7560 gggaaacacc agccttcttt ggaatggacc catggctgtc tccatgacag gagtcatgag 7620 ggggaatcac tatgcttttg tgggagtcat gtacaatcta tggaagatga aaactggacg 7680 ccgggggagc gcgaatggaa aaactttggg tgaagtctgg aagagggaac tgaatctgtt 7740 ggacaagcga cagtttgagt tgtataaaag gaccgacatt gtggaggtgg atcgtgatac 7800 ggcacgcagg catttggccg aagggaaggt ggacaccggg gtggcggtct ccagggggac 7860 cgcaaagtta aggtggttcc atgagcgtgg ctatgtcaag ctggaaggta gggtgattga 7920 cctggggtgt ggccgcggag gctggtgtta ctacgctgct gcgcaaaagg aagtgagtgg 7980 ggtcaaagga tttactcttg gaagagacgg ccatgagaaa cccatgaatg tgcaaagtct 8040 gggatggaac atcatcacct tcaaggacaa aactgatatc caccgcctag aaccagtgaa 8100 atgtgacacc cttttgtgtg acattggaga gtcatcatcg tcatcggtca cagaggggga 8160 aaggaccgtg agagttcttg atactgtaga aaaatggctg gcttgtgggg ttgacaactt 8220 ctgtgtgaag gtgttagctc catacatgcc agatgttctc gagaaactgg aattgctcca 8280 aaggaggttt ggcggaacag tgatcaggaa ccctctctcc aggaattcca ctcatgaaat 8340 gtactacgtg tctggagccc gcagcaatgt cacatttact gtgaaccaaa catcccgcct 8400 cctgatgagg agaatgaggc gtccaactgg aaaagtgacc ctggaggctg acgtcatcct 8460 cccaattggg acacgcagtg ttgagacaga caagggaccc ctggacaaag aggccataga 8520 agaaagggtt gagaggataa aatctgagta catgacctct tggttttatg acaatgacaa 8580 cccctacagg acctggcact actgtggctc ctatgtcaca aaaacctcag gaagtgcggc 8640 gagcatggta aatggtgtta ttaaaattct gacatatcca tgggacagga tagaggaggt 8700 cacaagaatg gcaatgactg acacaacccc ttttggacag caaagagtgt ttaaagaaaa 8760 agttgacacc agagcaaagg atccaccagc gggaactagg aagatcatga aagttgtcaa 8820 caggtggctg ttccgccacc tggccagaga aaagaacccc agactgtgca caaaggaaga 8880 atttattgca aaagtccgaa gtcatgcagc cattggagct tacctggaag aacaagaaca 8940 gtggaagact gccaatgagg ctgtccaaga cccaaagttc tgggaactgg tggatgaaga 9000 aaggaagctg caccaacaag gcaggtgtcg gacttgtgtg tacaacatga tggggaaaag 9060 agagaagaag ctgtcagagt ttgggaaagc aaagggaagc cgtgccatat ggtatatgtg 9120 gctgggagcg cggtatcttg agtttgaggc cctgggattc ctgaatgagg accattgggc 9180 ttccagggaa aactcaggag gaggagtgga aggcattggc ttacaatacc taggatatgt 9240 gatcagagac ctggctgcaa tggatggtgg tggattctac gcggatgaca ccgctggatg 9300 ggacacgcgc atcacagagg cagaccttga tgatgaacag gagatcttga actacatgag 9360 cccacatcac aaaaaactgg cacaagcagt gatggaaatg acatacaaga acaaagtggt 9420 gaaagtgttg agaccagccc caggagggaa agcctacatg gatgtcataa gtcgacgaga 9480 ccagagagga tccgggcagg tagtgactta tgctctgaac accatcacca acttgaaagt 9540 ccaattgatc agaatggcag aagcagagat ggtgatacat caccaacatg ttcaagattg 9600 tgatgaatca gttctgacca ggctggaggc atggctcact gagcacggat gtgacagact 9660 gaagaggatg gcggtgagtg gagacgactg tgtggtccgg cccatcgatg acaggttcgg 9720 cctggccctg tcccatctca acgccatgtc caaggttaga aaggacatat ctgaatggca 9780 gccatcaaaa gggtggaatg attgggagaa tgtgcccttc tgttcccacc acttccatga 9840 actacagctg aaggatggca ggaggattgt ggtgccttgc cgagaacagg acgagctcat 9900 tgggagagga agggtgtctc caggaaacgg ctggatgatc aaggaaacag cttgcctcag 9960 caaagcctat gccaacatgt ggtcactgat gtattttcac aaaagggaca tgaggctact 10020 gtcattggct gtttcctcag ctgttcccac ctcatgggtt ccacaaggac gcacaacatg 10080 gtcgattcat gggaaagggg agtggatgac cacggaagac atgcttgagg tgtggaacag 10140 agtatggata accaacaacc cacacatgca ggacaagaca atggtgaaaa aatggagaga 10200 tgtcccttat ctaaccaaga gacaagacaa gctgtgcgga tcactgattg gaatgaccaa 10260 tagggccacc tgggcctccc acatccattt agtcatccat cgtatccgaa cgctgattgg 10320 acaggagaaa tacactgact acctaacagt catggacagg tattctgtgg atgctgacct 10380 gcaactgggt gagcttatct gaaacaccat ctaacaggaa taaccgggat acaaaccacg 10440 ggtggagaac cggactcccc acaacctgaa accgggatat aaaccacggc tggagaaccg 10500 ggctccgcac ttaaaatgaa acagaaaccg ggataaaaac tacggatgga gaaccggact 10560 ccacacattg agacagaaga agttgtcagc ccagaacccc acacgagttt tgccactgct 10620 aagctgtgag gcagtgcagg ctgggacagc cgacctccag gttgcgaaaa acctggtttc 10680 tgggacctcc caccccagag taaaaagaac ggagcctccg ctaccaccct cccacgtggt 10740 ggtagaaaga cggggtctag aggttagagg agaccctcca gggaacaaat agtgggacca 10800 tattgacgcc agggaaagac cggagtggtt ctctgctttt cctccagagg tctgtgagca 10860 cagtttgctc aagaataagc agacctttgg atgacaaaca caaaaccact 10910 <210> 11 <211> 3426 <212> PRT <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 6 amino acid sequence <400> 11 Met Ser Gly Arg Lys Ala Gln Gly Lys Thr Leu Gly Val Asn Met Val   1 5 10 15 Arg Arg Gly Val Arg Ser Leu Ser Asn Lys Ile Lys Gln Lys Thr Lys              20 25 30 Gln Ile Gly Asn Arg Pro Gly Pro Ser Arg Gly Val Gln Gly Phe Ile          35 40 45 Phe Phe Phe Leu Phe Asn Ile Leu Thr Gly Lys Lys Ile Thr Ala His      50 55 60 Leu Lys Arg Leu Trp Lys Met Leu Asp Pro Arg Gln Gly Leu Ala Val  65 70 75 80 Leu Arg Lys Val Lys Arg Val Val Ala Ser Leu Met Arg Gly Leu Ser                  85 90 95 Ser Arg Lys Arg Arg Ser His Asp Val Leu Thr Val Gln Phe Leu Ile             100 105 110 Leu Gly Met Leu Leu Met Ala Met Ala Ala Glu Val Thr Arg Arg Gly         115 120 125 Ser Ala Tyr Tyr Met Tyr Leu Asp Arg Asn Asp Ala Gly Glu Ala Ile     130 135 140 Ser Phe Pro Thr Thr Leu Gly Met Asn Lys Cys Tyr Ile Gln Ile Met 145 150 155 160 Asp Leu Gly His Met Cys Asp Ala Thr Met Ser Tyr Glu Cys Pro Met                 165 170 175 Leu Asp Glu Gly Val Glu Pro Asp Asp Val Asp Cys Trp Cys Asn Thr             180 185 190 Thr Ser Thr Trp Val Val Tyr Gly Thr Cys His His Lys Lys Gly Glu         195 200 205 Ala Arg Arg Ser Arg Arg Ala Val Thr Leu Pro Ser His Ser Thr Arg     210 215 220 Lys Leu Gln Thr Arg Ser Gln Thr Trp Leu Glu Ser Arg Glu Tyr Thr 225 230 235 240 Lys His Leu Ile Arg Val Glu Asn Trp Ile Phe Arg Asn Pro Gly Phe                 245 250 255 Ala Leu Ala Ala Ala Ala Ile Ala Trp Leu Leu Gly Ser Ser Thr Ser             260 265 270 Gln Lys Val Ile Tyr Leu Val Met Ile Leu Leu Ile Ala Pro Ala Tyr         275 280 285 Ser Ile Arg Cys Ile Gly Val Ser Asn Arg Asp Phe Val Glu Gly Met     290 295 300 Ser Gly Gly Thr Trp Val Asp Val Val Leu Glu His Gly Gly Cys Val 305 310 315 320 Thr Val Met Ala Gln Asp Lys Pro Thr Val Asp Ile Glu Leu Val Thr                 325 330 335 Thr Thr Val Ser Asn Met Ala Glu Val Arg Ser Tyr Cys Tyr Glu Ala             340 345 350 Ser Ile Ser Asp Met Ala Ser Asp Ser Arg Cys Pro Thr Gln Gly Glu         355 360 365 Ala Tyr Leu Asp Lys Gln Ser Asp Thr Gln Tyr Val Cys Lys Arg Thr     370 375 380 Leu Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly 385 390 395 400 Ser Leu Val Thr Cys Ala Lys Phe Ala Cys Ser Lys Lys Met Thr Gly                 405 410 415 Lys Ser Ile Gln Pro Glu Asn Leu Glu Tyr Arg Ile Met Leu Ser Val             420 425 430 His Gly Ser Gln His Ser Gly Met Ile Val Asn Asp Thr Gly His Glu         435 440 445 Thr Asp Glu Asn Arg Ala Lys Val Glu Ile Thr Pro Asn Ser Pro Arg     450 455 460 Ala Glu Ala Thr Leu Gly Gly Phe Gly Ser Leu Gly Leu Asp Cys Glu 465 470 475 480 Pro Arg Thr Gly Leu Asp Phe Ser Asp Leu Tyr Tyr Leu Thr Met Asn                 485 490 495 Asn Lys His Trp Leu Val His Lys Glu Trp Phe His Asp Ile Pro Leu             500 505 510 Pro Trp His Ala Gly Ala Asp Thr Gly Thr Pro His Trp Asn Asn Lys         515 520 525 Glu Ala Leu Val Glu Phe Lys Asp Ala His Ala Lys Arg Gln Thr Val     530 535 540 Val Val Leu Gly Ser Gln Glu Gly Ala Val His Thr Ala Leu Ala Gly 545 550 555 560 Ala Leu Glu Ala Glu Met Asp Gly Ala Lys Gly Arg Leu Ser Ser Gly                 565 570 575 His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Arg Leu Lys Gly Val             580 585 590 Ser Tyr Ser Leu Cys Thr Ala Ala Phe Thr Phe Thr Lys Ile Pro Ala         595 600 605 Glu Thr Leu His Gly Thr Val Thr Val Glu Val Gln Tyr Ala Gly Thr     610 615 620 Asp Gly Pro Cys Lys Val Pro Ala Gln Met Ala Val Asp Met Gln Thr 625 630 635 640 Leu Thr Pro Val Gly Arg Leu Ile Thr Ala Asn Pro Val Ile Thr Glu                 645 650 655 Ser Thr Glu Asn Ser Lys Met Met Leu Glu Leu Asp Pro Pro Phe Gly             660 665 670 Asp Ser Tyr Ile Val Ile Gly Val Gly Glu Lys Lys Ile Thr His His         675 680 685 Trp His Arg Ser Gly Ser Thr Ile Gly Lys Ala Phe Glu Ala Thr Val     690 695 700 Arg Gly Ala Lys Arg Met Ala Val Leu Gly Asp Thr Ala Trp Asp Phe 705 710 715 720 Gly Ser Val Gly Gly Ala Leu Asn Ser Leu Gly Lys Gly Ile His Gln                 725 730 735 Ile Phe Gly Ala Ala Phe Lys Ser Leu Phe Gly Gly Met Ser Trp Phe             740 745 750 Ser Gln Ile Leu Ile Gly Thr Leu Leu Met Trp Leu Gly Leu Asn Thr         755 760 765 Lys Asn Gly Ser Ile Ser Leu Met Cys Leu Ala Leu Gly Gly Val Leu     770 775 780 Ile Phe Leu Ser Thr Ala Val Ser Ala Asp Gln Gly Cys Ala Ile Asn 785 790 795 800 Phe Gly Lys Arg Glu Leu Lys Cys Gly Asp Gly Ile Phe Ile Phe Arg                 805 810 815 Asp Ser Asp Asp Trp Leu Asn Lys Tyr Ser Tyr Tyr Pro Glu Asp Pro             820 825 830 Val Lys Leu Ala Ser Ile Val Lys Ala Ser Phe Glu Glu Gly Lys Cys         835 840 845 Gly Leu Asn Ser Val Asp Ser Leu Glu His Glu Met Trp Arg Ser Arg     850 855 860 Ala Asp Glu Ile Asn Ala Ile Phe Glu Glu Asn Glu Val Asp Ile Ser 865 870 875 880 Val Val Val Gln Asp Pro Lys Asn Val Tyr Gln Arg Gly Thr His Pro                 885 890 895 Phe Ser Arg Ile Arg Asp Gly Leu Gln Tyr Gly Trp Lys Thr Trp Gly             900 905 910 Lys Asn Leu Val Phe Ser Pro Gly Arg Lys Asn Gly Ser Phe Ile Ile         915 920 925 Asp Gly Lys Ser Arg Lys Glu Cys Pro Phe Ser Asn Arg Val Trp Asn     930 935 940 Ser Phe Gln Ile Glu Glu Phe Gly Thr Gly Val Phe Thr Thr Arg Val 945 950 955 960 Tyr Met Asp Ala Val Phe Glu Tyr Thr Ile Asp Cys Asp Gly Ser Ile                 965 970 975 Leu Gly Ala Ala Val Asn Gly Lys Lys Ser Ala His Gly Ser Pro Thr             980 985 990 Phe Trp Met Gly Ser His Glu Val Asn Gly Thr Trp Met Ile His Thr         995 1000 1005 Leu Glu Ala Leu Asp Tyr Lys Glu Cys Glu Trp Pro Leu Thr His Thr    1010 1015 1020 Ile Gly Thr Ser Val Glu Glu Ser Glu Met Phe Met Pro Arg Ser Ile 1025 1030 1035 1040 Gly Gly Pro Val Ser Ser His Asn His Ile Pro Gly Tyr Lys Val Gln                1045 1050 1055 Thr Asn Gly Pro Trp Met Gln Val Pro Leu Glu Val Lys Arg Glu Ala            1060 1065 1070 Cys Pro Gly Thr Ser Val Ile Ile Asp Gly Asn Cys Asp Gly Arg Gly        1075 1080 1085 Lys Ser Thr Arg Ser Thr Thr Asp Ser Gly Lys Val Ile Pro Glu Trp    1090 1095 1100 Cys Cys Arg Ser Cys Thr Met Pro Pro Val Ser Phe His Gly Ser Asp 1105 1110 1115 1120 Gly Cys Trp Tyr Pro Met Glu Ile Arg Pro Arg Lys Thr His Glu Ser                1125 1130 1135 His Leu Val Arg Ser Trp Val Thr Ala Gly Glu Ile His Ala Val Pro            1140 1145 1150 Phe Gly Leu Val Ser Met Met Ile Ala Met Glu Val Val Leu Arg Lys        1155 1160 1165 Arg Gln Gly Pro Lys Gln Met Leu Val Gly Gly Val Val Leu Leu Gly    1170 1175 1180 Ala Met Leu Val Gly Gln Val Thr Leu Leu Asp Leu Leu Lys Leu Thr 1185 1190 1195 1200 Val Ala Val Gly Leu His Phe His Glu Met Asn Asn Gly Gly Asp Ala                1205 1210 1215 Met Tyr Met Ala Leu Ile Ala Ala Phe Ser Ile Arg Pro Gly Leu Leu            1220 1225 1230 Ile Gly Phe Gly Leu Arg Thr Leu Trp Ser Pro Arg Glu Arg Leu Val        1235 1240 1245 Leu Thr Leu Gly Ala Ala Met Val Glu Ile Ala Leu Gly Gly Val Met    1250 1255 1260 Gly Gly Leu Trp Lys Tyr Leu Asn Ala Val Ser Leu Cys Ile Leu Thr 1265 1270 1275 1280 Ile Asn Ala Val Ala Ser Arg Lys Ala Ser Asn Thr Ile Leu Pro Leu                1285 1290 1295 Met Ala Leu Leu Thr Pro Val Thr Met Ala Glu Val Arg Leu Ala Ala            1300 1305 1310 Met Phe Phe Cys Ala Val Val Ile Gly Val Leu His Gln Asn Phe        1315 1320 1325 Lys Asp Thr Ser Met Gln Lys Thr Ile Pro Leu Val Ala Leu Thr Leu    1330 1335 1340 Thr Ser Tyr Leu Gly Leu Thr Gln Pro Phe Leu Gly Leu Cys Ala Phe 1345 1350 1355 1360 Leu Ala Thr Arg Ile Phe Gly Arg Arg Ser Ile Pro Val Asn Glu Ala                1365 1370 1375 Leu Ala Ala Ala Gly Leu Val Gly Val Leu Ala Gly Leu Ala Phe Gln            1380 1385 1390 Glu Met Glu Asn Phe Leu Gly Pro Ile Ala Val Gly Gly Leu Leu Met        1395 1400 1405 Met Leu Val Ser Val Ala Gly Arg Val Asp Gly Leu Glu Leu Lys Lys    1410 1415 1420 Leu Gly Glu Val Ser Trp Glu Glu Glu Ala Glu Ile Ser Gly Ser Ser 1425 1430 1435 1440 Ala Arg Tyr Asp Val Ala Leu Ser Glu Gln Gly Glu Phe Lys Leu Leu                1445 1450 1455 Ser Glu Glu Lys Val Pro Trp Asp Gln Val Val Met Thr Ser Leu Ala            1460 1465 1470 Leu Val Gly Ala Ala Leu His Pro Phe Ala Leu Leu Leu Val Leu Ala        1475 1480 1485 Gly Trp Leu Phe His Val Arg Gly Ala Arg Arg Ser Gly Asp Val Leu    1490 1495 1500 Trp Asp Ile Pro Thr Pro Lys Ile Ile Glu Glu Cys Glu His Leu Glu 1505 1510 1515 1520 Asp Gly Ile Tyr Gly Ile Phe Gln Ser Thr Phe Leu Gly Ala Ser Gln                1525 1530 1535 Arg Gly Val Gly Val Ala Gln Gly Gly Val Phe His Thr Met Trp His            1540 1545 1550 Val Thr Arg Gly Ala Phe Leu Val Arg Asn Gly Lys Lys Leu Ile Pro        1555 1560 1565 Ser Trp Ala Ser Val Lys Glu Asp Leu Val Ala Tyr Gly Gly Ser Trp    1570 1575 1580 Lys Leu Glu Gly Arg Trp Asp Gly Glu Glu Glu Val Gln Leu Ile Ala 1585 1590 1595 1600 Ala Val Pro Gly Lys Asn Val Val Asn Val Gln Thr Lys Pro Ser Leu                1605 1610 1615 Phe Lys Val Arg Asn Gly Gly Glu Ile Gly Ala Val Ala Leu Asp Tyr            1620 1625 1630 Pro Ser Gly Thr Ser Gly Ser Pro Ile Val Asn Arg Asn Gly Glu Val        1635 1640 1645 Ile Gly Leu Tyr Gly Asn Gly Ile Leu Val Gly Asp Asn Ser Phe Val    1650 1655 1660 Ser Ala Ile Ser Gln Thr Glu Val Lys Glu Glu Gly Lys Glu Glu Leu 1665 1670 1675 1680 Gln Glu Ile Pro Thr Met Leu Lys Lys Gly Met Thr Thr Val Leu Asp                1685 1690 1695 Phe His Pro Gly Ala Gly Lys Thr Arg Arg Phe Leu Pro Gln Ile Leu            1700 1705 1710 Ala Glu Cys Ala Arg Arg Arg Leu Arg Thr Leu Val Leu Ala Pro Thr        1715 1720 1725 Arg Val Val Leu Ser Glu Met Lys Glu Ala Phe His Gly Leu Asp Val    1730 1735 1740 Lys Phe His Thr Gln Ala Phe Ser Ala His Gly Ser Gly Arg Glu Val 1745 1750 1755 1760 Ile Asp Ala Met Cys His Ala Thr Leu Thr Tyr Arg Met Leu Glu Pro                1765 1770 1775 Thr Arg Val Val Asn Trp Glu Val Ile Met Asp Glu Ala His Phe            1780 1785 1790 Leu Asp Pro Ala Ser Ile Ala Ala Arg Gly Trp Ala Ala His Arg Ala        1795 1800 1805 Arg Ala Asn Glu Ser Ala Thr Ile Leu Met Thr Ala Thr Pro Pro Gly    1810 1815 1820 Thr Ser Asp Glu Phe Pro His Ser Asn Gly Glu Ile Glu Asp Val Gln 1825 1830 1835 1840 Thr Asp Ile Pro Ser Glu Pro Trp Asn Thr Gly His Asp Trp Ile Leu                1845 1850 1855 Ala Asp Lys Arg Pro Thr Ala Trp Phe Leu Pro Ser Ile Arg Ala Ala            1860 1865 1870 Asn Val Met Ala Ala Ser Leu Arg Lys Ala Gly Lys Ser Val Val Val        1875 1880 1885 Leu Asn Arg Lys Thr Phe Glu Arg Glu Tyr Pro Thr Ile Lys Gln Lys    1890 1895 1900 Lys Pro Asp Phe Ile Leu Ala Thr Asp Ile Ala Glu Met Gly Ala Asn 1905 1910 1915 1920 Leu Cys Val Glu Arg Val Leu Asp Cys Arg Thr Ala Phe Lys Pro Val                1925 1930 1935 Leu Val Asp Glu Gly Arg Lys Val Ala Ile Lys Gly Pro Leu Arg Ile            1940 1945 1950 Ser Ala Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn Pro        1955 1960 1965 Asn Arg Asp Gly Asp Ser Tyr Tyr Tyr Ser Glu Pro Thr Ser Glu Asn    1970 1975 1980 Asn Ala His His Val Cys Trp Leu Glu Ala Ser Met Leu Leu Asp Asn 1985 1990 1995 2000 Met Glu Val Arg Gly Gly Met Val Ala Pro Leu Tyr Gly Val Glu Gly                2005 2010 2015 Thr Lys Thr Pro Val Ser Pro Gly Glu Met Arg Leu Arg Asp Asp Gln            2020 2025 2030 Arg Lys Val Phe Arg Glu Leu Val Arg Asn Cys Asp Leu Pro Val Trp        2035 2040 2045 Leu Ser Trp Gln Val Ala Lys Ala Gly Leu Lys Thr Asn Asp Arg Lys    2050 2055 2060 Trp Cys Phe Glu Gly Pro Glu Glu His Glu Ile Leu Asn Asp Ser Gly 2065 2070 2075 2080 Glu Thr Val Lys Cys Arg Ala Pro Gly Gly Ala Lys Lys Pro Leu Arg                2085 2090 2095 Pro Arg Trp Cys Asp Glu Arg Val Ser Ser Asp Gln Ser Ala Leu Ser            2100 2105 2110 Glu Phe Ile Lys Phe Ala Glu Gly Arg Arg Gly Ala Ala Glu Val Leu        2115 2120 2125 Val Val Leu Ser Glu Leu Pro Asp Phe Leu Ala Lys Lys Gly Gly Glu    2130 2135 2140 Ala Met Asp Thr Ile Ser Val Phe Leu His Ser Glu Glu Gly Ser Arg 2145 2150 2155 2160 Ala Tyr Arg Asn Ala Leu Ser Met Met Pro Glu Ala Met Thr Ile Val                2165 2170 2175 Met Leu Phe Ile Leu Ala Gly Leu Leu Thr Ser Gly Met Val Ile Phe            2180 2185 2190 Phe Met Ser Pro Lys Gly Ile Ser Arg Met Ser Met Ala Met Gly Thr        2195 2200 2205 Met Ala Gly Cys Gly Tyr Leu Met Phe Leu Gly Gly Val Lys Pro Thr    2210 2215 2220 His Ile Ser Tyr Val Met Leu Ile Phe Phe Val Leu Met Val Val Val 2225 2230 2235 2240 Ile Pro Glu Pro Gly Gln Gln Arg Ser Ile Gln Asp Asn Gln Val Ala                2245 2250 2255 Tyr Leu Ile Ile Gly Ile Leu Thr Leu Val Ser Ala Val Ala Ala Asn            2260 2265 2270 Glu Leu Gly Met Leu Glu Lys Thr Lys Glu Asp Leu Phe Gly Lys Lys        2275 2280 2285 Asn Leu Ile Pro Ser Ser Ala Ser Pro Trp Ser Trp Pro Asp Leu Asp    2290 2295 2300 Leu Lys Pro Gly Ala Ala Trp Thr Val Tyr Val Gly Ile Val Thr Met 2305 2310 2315 2320 Leu Ser Pro Met Leu His His Trp Ile Lys Val Glu Tyr Gly Asn Leu                2325 2330 2335 Ser Leu Ser Gly Ile Ala Gln Ser Ala Ser Val Leu Ser Phe Met Asp            2340 2345 2350 Lys Gly Ile Pro Phe Met Lys Met Asn Ile Ser Val Ile Met Leu Leu        2355 2360 2365 Val Ser Gly Trp Asn Ser Ile Thr Val Met Pro Leu Leu Cys Gly Ile    2370 2375 2380 Gly Cys Ala Met Leu His Trp Ser Leu Ile Leu Pro Gly Ile Lys Ala 2385 2390 2395 2400 Gln Gln Ser Lys Leu Ala Gln Arg Arg Val Phe His Gly Val Ala Glu                2405 2410 2415 Asn Pro Val Val Asp Gly Asn Pro Thr Val Asp Ile Glu Glu Ala Pro            2420 2425 2430 Glu Met Pro Ala Leu Tyr Glu Lys Lys Leu Ala Leu Tyr Leu Leu Leu        2435 2440 2445 Ala Leu Ser Leu Ala Ser Val Ala Met Cys Arg Thr Pro Phe Ser Leu    2450 2455 2460 Ala Glu Gly Ile Val Leu Ala Ser Ala Ala Leu Gly Pro Leu Ile Glu 2465 2470 2475 2480 Gly Asn Thr Ser Leu Leu Trp Asn Gly Pro Met Ala Val Ser Met Thr                2485 2490 2495 Gly Val Met Arg Gly Asn His Tyr Ala Phe Val Gly Val Met Tyr Asn            2500 2505 2510 Leu Trp Lys Met Lys Thr Gly Arg Arg Gly Ser Ala Asn Gly Lys Thr        2515 2520 2525 Leu Gly Glu Val Trp Lys Arg Glu Leu Asn Leu Leu Asp Lys Arg Gln    2530 2535 2540 Phe Glu Leu Tyr Lys Arg Thr Asp Ile Val Glu Val Asp Arg Asp Thr 2545 2550 2555 2560 Ala Arg Arg His Leu Ala Glu Gly Lys Val Asp Thr Gly Val Ala Val                2565 2570 2575 Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe His Glu Arg Gly Tyr Val            2580 2585 2590 Lys Leu Glu Gly Arg Val Ile Asp Leu Gly Cys Gly Arg Gly Gly Trp        2595 2600 2605 Cys Tyr Tyr Ala Ala Ala Gln Lys Glu Val Ser Gly Val Lys Gly Phe    2610 2615 2620 Thr Leu Gly Arg Asp Gly His Glu Lys Pro Met Asn Val Gln Ser Leu 2625 2630 2635 2640 Gly Trp Asn Ile Ile Thr Phe Lys Asp Lys Thr Asp Ile His Arg Leu                2645 2650 2655 Glu Pro Val Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser            2660 2665 2670 Ser Ser Ser Val Thr Glu Gly Glu Arg Thr Val Arg Val Leu Asp Thr        2675 2680 2685 Val Glu Lys Trp Leu Ala Cys Gly Val Asp Asn Phe Cys Val Lys Val    2690 2695 2700 Leu Ala Pro Tyr Met Pro Asp Val Leu Glu Lys Leu Glu Leu Leu Gln 2705 2710 2715 2720 Arg Arg Phe Gly Gly Thr Val Ile Arg Asn Pro Leu Ser Arg Asn Ser                2725 2730 2735 Thr His Glu Met Tyr Tyr Val Ser Gly Ala Arg Ser Asn Val Thr Phe            2740 2745 2750 Thr Val Asn Gln Thr Ser Arg Leu Leu Met Arg Arg Met Arg Arg Pro        2755 2760 2765 Thr Gly Lys Val Thr Leu Glu Ala Asp Val Ile Leu Pro Ile Gly Thr    2770 2775 2780 Arg Ser Val Glu Thr Asp Lys Gly Pro Leu Asp Lys Glu Ala Ile Glu 2785 2790 2795 2800 Glu Arg Val Glu Arg Ile Lys Ser Glu Tyr Met Thr Ser Trp Phe Tyr                2805 2810 2815 Asp Asn Asp Asn Pro Tyr Arg Thr Trp His Tyr Cys Gly Ser Tyr Val            2820 2825 2830 Thr Lys Thr Ser Gly Ser Ala Ala Ser Met Val Asn Gly Val Ile Lys        2835 2840 2845 Ile Leu Thr Tyr Pro Trp Asp Arg Ile Glu Glu Val Thr Arg Met Ala    2850 2855 2860 Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys 2865 2870 2875 2880 Val Asp Thr Arg Ala Lys Asp Pro Pro Ala Gly Thr Arg Lys Ile Met                2885 2890 2895 Lys Val Val Asn Arg Trp Leu Phe Arg His Leu Ala Arg Glu Lys Asn            2900 2905 2910 Pro Arg Leu Cys Thr Lys Glu Glu Phe Ile Ala Lys Val Arg Ser His        2915 2920 2925 Ala Ala Ile Gly Ala Tyr Leu Glu Glu Gln Glu Gln Trp Lys Thr Ala    2930 2935 2940 Asn Glu Ala Val Gln Asp Pro Lys Phe Trp Glu Leu Val Asp Glu Glu 2945 2950 2955 2960 Arg Lys Leu His Gln Gln Gly Arg Cys Arg Thr Cys Val Tyr Asn Met                2965 2970 2975 Met Gly Lys Arg Glu Lys Lys Leu Ser Glu Phe Gly Lys Ala Lys Gly            2980 2985 2990 Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Tyr Leu Glu Phe        2995 3000 3005 Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Ala Ser Arg Glu Asn    3010 3015 3020 Ser Gly Gly Gly Val Glu Gly Ile Gly Leu Gln Tyr Leu Gly Tyr Val 3025 3030 3035 3040 Ile Arg Asp Leu Ala Ala Met Asp Gly Gly Gly Phe Tyr Ala Asp Asp                3045 3050 3055 Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Ala Asp Leu Asp Asp Glu            3060 3065 3070 Gln Glu Ile Leu Asn Tyr Met Ser Pro His His Lys Lys Leu Ala Gln        3075 3080 3085 Ala Val Met Glu Met Thr Tyr Lys Asn Lys Val Val Lys Val Leu Arg    3090 3095 3100 Pro Ala Pro Gly Gly Lys Ala Tyr Met Asp Val Ile Ser Arg Arg Asp 3105 3110 3115 3120 Gln Arg Gly Ser Gly Gln Val Val Thr Tyr Ala Leu Asn Thr Ile Thr                3125 3130 3135 Asn Leu Lys Val Gln Leu Ile Arg Met Ala Glu Ala Glu Met Val Ile            3140 3145 3150 His His Gln His Val Gln Asp Cys Asp Glu Ser Val Leu Thr Arg Leu        3155 3160 3165 Glu Ala Trp Leu Thr Glu His Gly Cys Asp Arg Leu Lys Arg Met Ala    3170 3175 3180 Val Ser Gly Asp Asp Cys Val Val Arg Pro Ile Asp Asp Arg Phe Gly 3185 3190 3195 3200 Leu Ala Leu Ser His Leu Asn Ala Met Ser Lys Val Arg Lys Asp Ile                3205 3210 3215 Ser Glu Trp Gln Pro Ser Lys Gly Trp Asn Asp Trp Glu Asn Val Pro            3220 3225 3230 Phe Cys Ser His His Phe His Glu Leu Gln Leu Lys Asp Gly Arg Arg        3235 3240 3245 Ile Val Val Pro Cys Arg Glu Gln Asp Glu Leu Ile Gly Arg Gly Arg    3250 3255 3260 Val Ser Pro Gly Asn Gly Trp Met Ile Lys Glu Thr Ala Cys Leu Ser 3265 3270 3275 3280 Lys Ala Tyr Ala Asn Met Trp Ser Leu Met Tyr Phe His Lys Arg Asp                3285 3290 3295 Met Arg Leu Leu Ser Leu Ala Val Ser Ser Ala Val Pro Thr Ser Trp            3300 3305 3310 Val Pro Gln Gly Arg Thr Thr Trp Ser Ile His Gly Lys Gly Glu Trp        3315 3320 3325 Met Thr Thr Glu Asp Met Leu Glu Val Trp Asn Arg Val Trp Ile Thr    3330 3335 3340 Asn Asn Pro His Met Gln Asp Lys Thr Met Val Lys Lys Trp Arg Asp 3345 3350 3355 3360 Val Pro Tyr Leu Thr Lys Arg Gln Asp Lys Leu Cys Gly Ser Leu Ile                3365 3370 3375 Gly Met Thr Asn Arg Ala Thr Trp Ala Ser His Ile His Leu Val Ile            3380 3385 3390 His Arg Ile Arg Thr Leu Ile Gly Gln Glu Lys Tyr Thr Asp Tyr Leu        3395 3400 3405 Thr Val Met Asp Arg Tyr Ser Val Asp Ala Asp Leu Gln Leu Gly Glu    3410 3415 3420 Leu ile 3425 <210> 12 <211> 10907 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 6 nucleic acid sequence <400> 12 agtaaatcct gtgtgctaat tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60 acacatttgg attaatttta atcgttcgtt gagcgattag cagagaactg accagaacat 120 gtctggtcgt aaagctcagg gaaaaaccct gggcgtcaat atggtacgac gaggagttcg 180 ctccttgtca aacaaaataa aacaaaaaac aaaacaaatt ggaaacagac ctggaccttc 240 aagaggtgtt caaggattta tctttttctt tttgttcaac attttgactg gaaaaaagat 300 cacagcccac ctaaagaggt tgtggaaaat gctggaccca agacaaggct tggctgttct 360 aaggaaagtc aagagagtgg tggccagttt gatgagagga ttgtcctcaa ggaaacgccg 420 ttcccatgat gttctgactg tgcaattcct aattttggga atgctgttga tggctatggc 480 agcggaggtc actagacgtg ggagtgcata ctatatgtac ttggacagaa acgatgctgg 540 ggaggccata tcttttccaa ccacattggg gatgaataag tgttatatac agatcatgga 600 tcttggacac atgtgtgatg ccaccatgag ctatgaatgc cctatgctgg atgagggggt 660 ggaaccagat gacgtcgatt gttggtgcaa cacgacgtca acttgggttg tgtacggaac 720 ctgccatcac aaaaaaggtg aagcacggag atctagaaga gctgtgacgc tcccctccca 780 ttccactagg aagctgcaaa cgcggtcgca aacctggttg gaatcaagag aatacacaaa 840 gcacttgatt agagtcgaaa attggatatt caggaaccct ggcttcgcgt tagcagcagc 900 tgccatcgct tggcttttgg gaagctcaac gagccaaaaa gtcatatact tggtcatgat 960 actgctgatt gccccggcat acagcatcag gtgcatagga gtcagcaata gggactttgt 1020 ggaaggtatg tcaggtggga cttgggttga tgttgtcttg gaacatggag gttgtgtcac 1080 cgtaatggca caggacaaac cgactgtcga catagagctg gttacaacaa cagtcagcaa 1140 catggcggag gtaagatcct actgctatga ggcatcaata tcagacatgg cttcggacag 1200 ccgctgccca acacaaggtg aagcctacct tgacaagcaa tcagacactc aatatgtctg 1260 caaaagaacg ttagtggaca gaggctgggg aaatggatgt ggactttttg gcaaagggag 1320 cctggtgaca tgcgctaagt ttgcatgctc caagaaaatg accgggaaga gcatccagcc 1380 agagaatctg gagtaccgga taatgctgtc agttcatggc tcccagcaca gtgggatgat 1440 cgttaatgac acaggacatg aaactgatga gaatagagcg aaagttgaga taacgcccaa 1500 ttcaccaaga gccgaagcca ccctgggggg ttttggaagc ctaggacttg attgtgaacc 1560 gaggacaggc cttgactttt cagatttgta ttacttgact atgaataaca agcactggtt 1620 ggttcacaag gagtggttcc acgacattcc attaccttgg cacgctgggg cagacaccgg 1680 aactccacac tggaacaaca aagaagcact ggtagagttc aaggacgcac atgccaaaag 1740 gcaaactgtc gtggttctag ggagtcaaga aggagcagtt cacacggccc ttgctggagc 1800 tctggaggct gagatggatg gtgcaaaggg aaggctgtcc tctggccact tgaaatgtcg 1860 cctgaaaatg gataaactta gattgaaggg cgtgtcatac tccttgtgta ctgcagcgtt 1920 cacattcacc aagatcccgg ctgaaacact gcacgggaca gtcacagtgg aggtacagta 1980 cgcagggaca gatggacctt gcaaggttcc agctcagatg gcggtggaca tgcaaactct 2040 gaccccagtt gggaggttga taaccgctaa ccccgtaatc actgaaagca ctgagaactc 2100 taagatgatg ctggaacttg atccaccatt tggggactct tacattgtca taggagtcgg 2160 ggagaagaag atcacccacc actggcacag gagtggcagc accattggaa aagcatttga 2220 agccactgtg agaggtgcca agagaatggc agtcttggga gacacagcct gggactttgg 2280 atcagttgga ggcgctctca actcattggg caagggcatc catcaaattt ttggagcagc 2340 tttcaaatca ttgtttggag gaatgtcctg gttctcacaa attctcattg gaacgttgct 2400 gatgtggttg ggtctgaaca caaagaatgg atctatttcc cttatgtgct tggccttagg 2460 gggggtgttg atcttcttat ccacagccgt ctctgctgat caaggatgcg ccatcaactt 2520 tggcaagaga gagctcaagt gcggagatgg tatcttcata tttagagact ctgatgactg 2580 gctgaacaag tactcatact atccagaaga tcctgtgaag cttgcatcaa tagtgaaagc 2640 ctcttttgaa gaagggaagt gtggcctaaa ttcagttgac tcccttgagc atgagatgtg 2700 gagaagcagg gcagatgaga tcaatgccat ttttgaggaa aacgaggtgg acatttctgt 2760 tgtcgtgcag gatccaaaga atgtttacca gagaggaact catccatttt ccagaattcg 2820 ggatggtctg cagtatggtt ggaagacttg gggtaagaac cttgtgttct ccccagggag 2880 gaagaatgga agcttcatca tagatggaaa gtccaggaaa gaatgcccgt tttcaaaccg 2940 ggtctggaat tctttccaga tagaggagtt tgggacggga gtgttcacca cacgcgtgta 3000 catggacgca gtctttgaat acaccataga ctgcgatgga tctatcttgg gtgcagcggt 3060 gaacggaaaa aagagtgccc atggctctcc aacattttgg atgggaagtc atgaagtaaa 3120 tgggacatgg atgatccaca ccttggaggc attagattac aaggagtgtg agtggccact 3180 gacacatacg attggaacat cagttgaaga gagtgaaatg ttcatgccga gatcaatcgg 3240 aggcccagtt agctctcaca atcatatccc tggatacaag gttcagacga acggaccttg 3300 gatgcaggta ccactagaag tgaagagaga agcttgccca gggactagcg tgatcattga 3360 tggcaactgt gatggacggg gaaaatcaac cagatccacc acggatagcg ggaaagttat 3420 tcctgaatgg tgttgccgct cctgcacaat gccgcctgtg agcttccatg gtagtgatgg 3480 gtgttggtat cccatggaaa ttaggccaag gaaaacgcat gaaagccatc tggtgcgctc 3540 ctgggttaca gctggagaaa tacatgctgt cccttttggt ttggtgagca tgatgatagc 3600 aatggaagtg gtcctaagga aaagacaggg accaaagcaa atgttggttg gaggagtagt 3660 gctcttggga gcaatgctgg tcgggcaagt aactctcctt gatttgctga aactcacagt 3720 ggctgtggga ttgcatttcc atgagatgaa caatggagga gacgccatgt atatggcgtt 3780 gattgctgcc ttttcaatca gaccagggct gctcatcggc tttgggctca ggaccctatg 3840 gagccctcgg gaacgccttg tgctgaccct aggagcagcc atggtggaga ttgccttggg 3900 tggcgtgatg ggcggcctgt ggaagtatct aaatgcagtt tctctctgca tcctgacaat 3960 aaatgctgtt gcttctagga aagcatcaaa taccatcttg cccctcatgg ctctgttgac 4020 acctgtcact atggctgagg tgagacttgc cgcaatgttc ttttgtgccg tggttatcat 4080 aggggtcctt caccagaatt tcaaggacac ctccatgcag aagactatac ctctggtggc 4140 cctcacactc acatcttacc tgggcttgac acaacctttt ttgggcctgt gtgcatttct 4200 ggcaacccgc atatttgggc gaaggagtat cccagtgaat gaggcactcg cagcagctgg 4260 tctagtggga gtgctggcag gactggcttt tcaggagatg gagaacttcc ttggtccgat 4320 tgcagttgga ggactcctga tgatgctggt tagcgtggct gggagggtgg atgggctaga 4380 gctcaagaag cttggtgaag tttcatggga agaggaggcg gagatcagcg ggagttccgc 4440 ccgctatgat gtggcactca gtgaacaagg ggagttcaag ctgctttctg aagagaaagt 4500 gccatgggac caggttgtga tgacctcgct ggccttggtt ggggctgccc tccatccatt 4560 tgctcttctg ctggtccttg ctgggtggct gtttcatgtc aggggagcta ggagaagtgg 4620 ggatgtcttg tgggatattc ccactcctaa gatcatcgag gaatgtgaac atctggagga 4680 tgggatttat ggcatattcc agtcaacctt cttgggggcc tcccagcgag gagtgggagt 4740 ggcacaggga ggggtgttcc acacaatgtg gcatgtcaca agaggagctt tccttgtcag 4800 gaatggcaag aagttgattc catcttgggc ttcagtaaag gaagaccttg tcgcctatgg 4860 tggctcatgg aagttggaag gcagatggga tggagaggaa gaggtccagt tgatcgcggc 4920 tgttccagga aagaacgtgg tcaacgtcca gacaaaaccg agcttgttca aagtgaggaa 4980 tgggggagaa atcggggctg tcgctcttga ctatccgagt ggcacttcag gatctcctat 5040 tgttaacagg aacggagagg tgattgggct gtacggcaat ggcatccttg tcggtgacaa 5100 ctccttcgtg tccgccatat cccagactga ggtgaaggaa gaaggaaagg aggagctcca 5160 agagatcccg acaatgctaa agaaaggaat gacaactgtc cttgattttc atcctggagc 5220 tgggaagaca agacgtttcc tcccacagat cttggccgag tgcgcacgga gacgcttgcg 5280 cactcttgtg ttggccccca ccagggttgt tctttctgaa atgaaggagg cttttcacgg 5340 cctggacgtg aaattccaca cacaggcttt ttccgctcac ggcagcggga gagaagtcat 5400 tgatgccatg tgccatgcca ccctaactta caggatgttg gaaccaacta gggttgttaa 5460 ctgggaagtg atcattatgg atgaagccca ttttttggat ccagctagca tagccgctag 5520 aggttgggca gcgcacagag ctagggcaaa tgaaagtgca acaatcttga tgacagccac 5580 accgcctggg actagtgatg aatttccaca ttcaaatggt gaaatagaag atgttcaaac 5640 ggacataccc agtgagccct ggaacacagg gcatgactgg atcctagctg acaaaaggcc 5700 cacggcatgg ttccttccat ccatcagagc tgcaaatgtc atggctgcct ctttgcgtaa 5760 ggctggaaag agtgtggtgg tcctgaacag gaaaaccttt gagagagaat accccacgat 5820 aaagcagaag aaacctgact ttatattggc cactgacata gctgaaatgg gagccaacct 5880 ttgcgtggag cgagtgctgg attgcaggac ggcttttaag cctgtgcttg tggatgaagg 5940 gaggaaggtg gcaataaaag ggccacttcg tatctccgca tcctctgctg ctcaaaggag 6000 ggggcgcatt gggagaaatc ccaacagaga tggagactca tactactatt ctgagcctac 6060 aagtgaaaat aatgcccacc acgtctgctg gttggaggcc tcaatgctct tggacaacat 6120 ggaggtgagg ggtggaatgg tcgccccact ctatggcgtt gaaggaacta aaacaccagt 6180 ttcccctggt gaaatgagac tgagggatga ccagaggaaa gtcttcagag aactagtgag 6240 gaattgtgac ctgcccgttt ggctttcgtg gcaagtggcc aaggctggtt tgaagacgaa 6300 tgatcgtaag tggtgttttg aaggccctga ggaacatgag atcttgaatg acagcggtga 6360 aacagtgaag tgcagggctc ctggaggagc aaagaagcct ctgcgcccaa ggtggtgtga 6420 tgaaagggtg tcatctgacc agagtgcgct gtctgaattt attaagtttg ctgaaggtag 6480 gaggggagct gctgaagtgc tagttgtgct gagtgaactc cctgatttcc tggctaaaaa 6540 aggtggagag gcaatggata ccatcagtgt gttcctccac tctgaggaag gctctagggc 6600 ttaccgcaat gcactatcaa tgatgcctga ggcaatgaca atagtcatgc tgtttatact 6660 ggctggacta ctgacatcgg gaatggtcat ctttttcatg tctcccaaag gcatcagtag 6720 aatgtctatg gcgatgggca caatggccgg ctgtggatat ctcatgttcc ttggaggcgt 6780 caaacccact cacatctcct atgtcatgct catattcttt gtcctgatgg tggttgtgat 6840 ccccgagcca gggcaacaaa ggtccatcca agacaaccaa gtggcatacc tcattattgg 6900 catcctgacg ctggtttcag cggtggcagc caacgagcta ggcatgctgg agaaaaccaa 6960 agaggacctc tttgggaaga agaacttaat tccatctagt gcttcaccct ggagttggcc 7020 ggatcttgac ctgaagccag gagctgcctg gacagtgtac gttggcattg ttacaatgct 7080 ctctccaatg ttgcaccact ggatcaaagt cgaatatggc aacctgtctc tgtctggaat 7140 agcccagtca gcctcagtcc tttctttcat ggacaagggg ataccattca tgaagatgaa 7200 tatctcggtc ataatgctgc tggtcagtgg ctggaattca ataacagtga tgcctctgct 7260 ctgtggcata gggtgcgcca tgctccactg gtctctcatt ttacctggaa tcaaagcgca 7320 gcagtcaaag cttgcacaga gaagggtgtt ccatggcgtt gccgagaacc ctgtggttga 7380 tgggaatcca acagttgaca ttgaggaagc tcctgaaatg cctgcccttt atgagaagaa 7440 actggctcta tatctccttc ttgctctcag cctagcttct gttgccatgt gcagaacgcc 7500 cttttcattg gctgaaggca ttgtcctagc atcagctgcc ttagggccgc tcatagaggg 7560 aaacaccagc cttctttgga atggacccat ggctgtctcc atgacaggag tcatgagggg 7620 gaatcactat gcttttgtgg gagtcatgta caatctatgg aagatgaaaa ctggacgccg 7680 ggggagcgcg aatggaaaaa ctttgggtga agtctggaag agggaactga atctgttgga 7740 caagcgacag tttgagttgt ataaaaggac cgacattgtg gaggtggatc gtgatacggc 7800 acgcaggcat ttggccgaag ggaaggtgga caccggggtg gcggtctcca gggggaccgc 7860 aaagttaagg tggttccatg agcgtggcta tgtcaagctg gaaggtaggg tgattgacct 7920 ggggtgtggc cgcggaggct ggtgttacta cgctgctgcg caaaaggaag tgagtggggt 7980 caaaggattt actcttggaa gagacggcca tgagaaaccc atgaatgtgc aaagtctggg 8040 atggaacatc atcaccttca aggacaaaac tgatatccac cgcctagaac cagtgaaatg 8100 tgacaccctt ttgtgtgaca ttggagagtc atcatcgtca tcggtcacag agggggaaag 8160 gaccgtgaga gttcttgata ctgtagaaaa atggctggct tgtggggttg acaacttctg 8220 tgtgaaggtg ttagctccat acatgccaga tgttctcgag aaactggaat tgctccaaag 8280 gaggtttggc ggaacagtga tcaggaaccc tctctccagg aattccactc atgaaatgta 8340 ctacgtgtct ggagcccgca gcaatgtcac atttactgtg aaccaaacat cccgcctcct 8400 gatgaggaga atgaggcgtc caactggaaa agtgaccctg gaggctgacg tcatcctccc 8460 aattgggaca cgcagtgttg agacagacaa gggacccctg gacaaagagg ccatagaaga 8520 aagggttgag aggataaaat ctgagtacat gacctcttgg ttttatgaca atgacaaccc 8580 ctacaggacc tggcactact gtggctccta tgtcacaaaa acctcaggaa gtgcggcgag 8640 catggtaaat ggtgttatta aaattctgac atatccatgg gacaggatag aggaggtcac 8700 aagaatggca atgactgaca caaccccttt tggacagcaa agagtgttta aagaaaaagt 8760 tgacaccaga gcaaaggatc caccagcggg aactaggaag atcatgaaag ttgtcaacag 8820 gtggctgttc cgccacctgg ccagagaaaa gaaccccaga ctgtgcacaa aggaagaatt 8880 tattgcaaaa gtccgaagtc atgcagccat tggagcttac ctggaagaac aagaacagtg 8940 gaagactgcc aatgaggctg tccaagaccc aaagttctgg gaactggtgg atgaagaaag 9000 gaagctgcac caacaaggca ggtgtcggac ttgtgtgtac aacatgatgg ggaaaagaga 9060 gaagaagctg tcagagtttg ggaaagcaaa gggaagccgt gccatatggt atatgtggct 9120 gggagcgcgg tatcttgagt ttgaggccct gggattcctg aatgaggacc attgggcttc 9180 cagggaaaac tcaggaggag gagtggaagg cattggctta caatacctag gatatgtgat 9240 cagagacctg gctgcaatgg atggtggtgg attctacgcg gatgacaccg ctggatggga 9300 cacgcgcatc acagaggcag accttgatga tgaacaggag atcttgaact acatgagccc 9360 acatcacaaa aaactggcac aagcagtgat ggaaatgaca tacaagaaca aagtggtgaa 9420 agtgttgaga ccagccccag gagggaaagc ctacatggat gtcataagtc gacgagacca 9480 gagaggatcc gggcaggtag tgacttatgc tctgaacacc atcaccaact tgaaagtcca 9540 attgatcaga atggcagaag cagagatggt gatacatcac caacatgttc aagattgtga 9600 tgaatcagtt ctgaccaggc tggaggcatg gctcactgag cacggatgtg acagactgaa 9660 gaggatggcg gtgagtggag acgactgtgt ggtccggccc atcgatgaca ggttcggcct 9720 ggccctgtcc catctcaacg ccatgtccaa ggttagaaag gacatatctg aatggcagcc 9780 atcaaaaggg tggaatgatt gggagaatgt gcccttctgt tcccaccact tccatgaact 9840 acagctgaag gatggcagga ggattgtggt gccttgccga gaacaggacg agctcattgg 9900 gagaggaagg gtgtctccag gaaacggctg gatgatcaag gaaacagctt gcctcagcaa 9960 agcctatgcc aacatgtggt cactgatgta ttttcacaaa agggacatga ggctactgtc 10020 attggctgtt tcctcagctg ttcccacctc atgggttcca caaggacgca caacatggtc 10080 gattcatggg aaaggggagt ggatgaccac ggaagacatg cttgaggtgt ggaacagagt 10140 atggataacc aacaacccac acatgcagga caagacaatg gtgaaaaaat ggagagatgt 10200 cccttatcta accaagagac aagacaagct gtgcggatca ctgattggaa tgaccaatag 10260 ggccacctgg gcctcccaca tccatttagt catccatcgt atccgaacgc tgattggaca 10320 ggagaaatac actgactacc taacagtcat ggacaggtat tctgtggatg ctgacctgca 10380 actgggtgag cttatctgaa acaccatcta acaggaataa ccgggataca aaccacgggt 10440 ggagaaccgg actccccaca acctgaaacc gggatataaa ccacggctgg agaaccgggc 10500 tccgcactta aaatgaaaca gaaaccggga taaaaactac ggatggagaa ccggactcca 10560 cacattgaga cagaagaagt tgtcagccca gaaccccaca cgagttttgc cactgctaag 10620 ctgtgaggca gtgcaggctg ggacagccga cctccaggtt gcgaaaaacc tggtttctgg 10680 gacctcccac cccagagtaa aaagaacgga gcctccgcta ccaccctccc acgtggtggt 10740 agaaagacgg ggtctagagg ttagaggaga ccctccaggg aacaaatagt gggaccatat 10800 tgacgccagg gaaagaccgg agtggttctc tgcttttcct ccagaggtct gtgagcacag 10860 tttgctcaag aataagcaga cctttggatg acaaacacaa aaccact 10907 <210> 13 <211> 3426 <212> PRT <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct B amino acid sequence <400> 13 Met Ser Gly Arg Lys Ala Gln Gly Lys Thr Leu Gly Val Asn Met Val   1 5 10 15 Arg Arg Gly Val Arg Ser Leu Ser Asn Lys Ile Lys Gln Lys Thr Lys              20 25 30 Gln Ile Gly Asn Arg Pro Gly Pro Ser Arg Gly Val Gln Gly Phe Ile          35 40 45 Phe Phe Phe Leu Phe Asn Ile Leu Thr Gly Lys Lys Ile Thr Ala His      50 55 60 Leu Lys Arg Leu Trp Lys Met Leu Asp Pro Arg Gln Gly Leu Ala Val  65 70 75 80 Leu Arg Lys Val Lys Arg Val Val Ala Ser Leu Met Arg Gly Leu Ser                  85 90 95 Ser Arg Lys Arg Arg Ser His Asp Val Leu Thr Val Gln Phe Leu Ile             100 105 110 Leu Gly Met Leu Leu Met Thr Gly Gly Ala Glu Val Thr Arg Arg Gly         115 120 125 Ser Ala Tyr Tyr Met Tyr Leu Asp Arg Asn Asp Ala Gly Glu Ala Ile     130 135 140 Ser Phe Pro Thr Thr Leu Gly Met Asn Lys Cys Tyr Ile Gln Ile Met 145 150 155 160 Asp Leu Gly His Met Cys Asp Ala Thr Met Ser Tyr Glu Cys Pro Met                 165 170 175 Leu Asp Glu Gly Val Glu Pro Asp Asp Val Asp Cys Trp Cys Asn Thr             180 185 190 Thr Ser Thr Trp Val Val Tyr Gly Thr Cys His His Lys Lys Gly Glu         195 200 205 Ala Arg Arg Ser Arg Arg Ala Val Thr Leu Pro Ser His Ser Thr Arg     210 215 220 Lys Leu Gln Thr Arg Ser Gln Thr Trp Leu Glu Ser Arg Glu Tyr Thr 225 230 235 240 Lys His Leu Ile Arg Val Glu Asn Trp Ile Phe Arg Asn Pro Phe Phe                 245 250 255 Ala Val Thr Ala Leu Thr Ile Ala Tyr Leu Val Gly Ser Asn Met Thr             260 265 270 Gln Arg Val Val Ile Ala Leu Leu Val Leu Ala Val Gly Pro Ala Tyr         275 280 285 Ser Ile Arg Cys Ile Gly Val Ser Asn Arg Asp Phe Val Glu Gly Met     290 295 300 Ser Gly Gly Thr Trp Val Asp Val Val Leu Glu His Gly Gly Cys Val 305 310 315 320 Thr Val Met Ala Gln Asp Lys Pro Thr Val Asp Ile Glu Leu Val Thr                 325 330 335 Thr Thr Val Ser Asn Met Ala Glu Val Arg Ser Tyr Cys Tyr Glu Ala             340 345 350 Ser Ile Ser Asp Met Ala Ser Asp Ser Arg Cys Pro Thr Gln Gly Glu         355 360 365 Ala Tyr Leu Asp Lys Gln Ser Asp Thr Gln Tyr Val Cys Lys Arg Thr     370 375 380 Leu Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly 385 390 395 400 Ser Leu Val Thr Cys Ala Lys Phe Ala Cys Ser Lys Lys Met Thr Gly                 405 410 415 Lys Ser Ile Gln Pro Glu Asn Leu Glu Tyr Arg Ile Met Leu Ser Val             420 425 430 His Gly Ser Gln His Ser Gly Met Ile Val Asn Asp Thr Gly His Glu         435 440 445 Thr Asp Glu Asn Arg Ala Lys Val Glu Ile Thr Pro Asn Ser Pro Arg     450 455 460 Ala Glu Ala Thr Leu Gly Gly Phe Gly Ser Leu Gly Leu Asp Cys Glu 465 470 475 480 Pro Arg Thr Gly Leu Asp Phe Ser Asp Leu Tyr Tyr Leu Thr Met Asn                 485 490 495 Asn Lys His Trp Leu Val His Lys Glu Trp Phe His Asp Ile Pro Leu             500 505 510 Pro Trp His Ala Gly Ala Asp Thr Gly Thr Pro His Trp Asn Asn Lys         515 520 525 Glu Ala Leu Val Glu Phe Lys Asp Ala His Ala Lys Arg Gln Thr Val     530 535 540 Val Val Leu Gly Ser Gln Glu Gly Ala Val His Thr Ala Leu Ala Gly 545 550 555 560 Ala Leu Glu Ala Glu Met Asp Gly Ala Lys Gly Arg Leu Ser Ser Gly                 565 570 575 His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Arg Leu Lys Gly Val             580 585 590 Ser Tyr Ser Leu Cys Thr Ala Ala Phe Thr Phe Thr Lys Ile Pro Ala         595 600 605 Glu Thr Leu His Gly Thr Val Thr Val Glu Val Gln Tyr Ala Gly Thr     610 615 620 Asp Gly Pro Cys Lys Val Pro Ala Gln Met Ala Val Asp Met Gln Thr 625 630 635 640 Leu Thr Pro Val Gly Arg Leu Ile Thr Ala Asn Pro Val Ile Thr Glu                 645 650 655 Ser Thr Glu Asn Ser Lys Met Met Leu Glu Leu Asp Pro Pro Phe Gly             660 665 670 Asp Ser Tyr Ile Val Ile Gly Val Gly Glu Lys Lys Ile Thr His His         675 680 685 Trp His Arg Ser Gly Ser Thr Ile Gly Lys Ala Phe Glu Ala Thr Val     690 695 700 Arg Gly Ala Lys Arg Met Ala Val Leu Gly Asp Thr Ala Trp Asp Phe 705 710 715 720 Gly Ser Val Gly Gly Ala Leu Asn Ser Leu Gly Lys Gly Ile His Gln                 725 730 735 Ile Phe Gly Ala Ala Phe Lys Ser Leu Phe Gly Gly Leu Asn Trp Ile             740 745 750 Thr Lys Val Ile Met Gly Ala Val Leu Ile Trp Val Gly Ile Asn Thr         755 760 765 Arg Asn Met Thr Met Ser Met Ser Met Ile Leu Val Gly Val Ile Met     770 775 780 Met Phe Leu Ser Leu Gly Val Gly Ala Asp Gln Gly Cys Ala Ile Asn 785 790 795 800 Phe Gly Lys Arg Glu Leu Lys Cys Gly Asp Gly Ile Phe Ile Phe Arg                 805 810 815 Asp Ser Asp Asp Trp Leu Asn Lys Tyr Ser Tyr Tyr Pro Glu Asp Pro             820 825 830 Val Lys Leu Ala Ser Ile Val Lys Ala Ser Phe Glu Glu Gly Lys Cys         835 840 845 Gly Leu Asn Ser Val Asp Ser Leu Glu His Glu Met Trp Arg Ser Arg     850 855 860 Ala Asp Glu Ile Asn Ala Ile Phe Glu Glu Asn Glu Val Asp Ile Ser 865 870 875 880 Val Val Val Gln Asp Pro Lys Asn Val Tyr Gln Arg Gly Thr His Pro                 885 890 895 Phe Ser Arg Ile Arg Asp Gly Leu Gln Tyr Gly Trp Lys Thr Trp Gly             900 905 910 Lys Asn Leu Val Phe Ser Pro Gly Arg Lys Asn Gly Ser Phe Ile Ile         915 920 925 Asp Gly Lys Ser Arg Lys Glu Cys Pro Phe Ser Asn Arg Val Trp Asn     930 935 940 Ser Phe Gln Ile Glu Glu Phe Gly Thr Gly Val Phe Thr Thr Arg Val 945 950 955 960 Tyr Met Asp Ala Val Phe Glu Tyr Thr Ile Asp Cys Asp Gly Ser Ile                 965 970 975 Leu Gly Ala Ala Val Asn Gly Lys Lys Ser Ala His Gly Ser Pro Thr             980 985 990 Phe Trp Met Gly Ser His Glu Val Asn Gly Thr Trp Met Ile His Thr         995 1000 1005 Leu Glu Ala Leu Asp Tyr Lys Glu Cys Glu Trp Pro Leu Thr His Thr    1010 1015 1020 Ile Gly Thr Ser Val Glu Glu Ser Glu Met Phe Met Pro Arg Ser Ile 1025 1030 1035 1040 Gly Gly Pro Val Ser Ser His Asn His Ile Pro Gly Tyr Lys Val Gln                1045 1050 1055 Thr Asn Gly Pro Trp Met Gln Val Pro Leu Glu Val Lys Arg Glu Ala            1060 1065 1070 Cys Pro Gly Thr Ser Val Ile Ile Asp Gly Asn Cys Asp Gly Arg Gly        1075 1080 1085 Lys Ser Thr Arg Ser Thr Thr Asp Ser Gly Lys Val Ile Pro Glu Trp    1090 1095 1100 Cys Cys Arg Ser Cys Thr Met Pro Pro Val Ser Phe His Gly Ser Asp 1105 1110 1115 1120 Gly Cys Trp Tyr Pro Met Glu Ile Arg Pro Arg Lys Thr His Glu Ser                1125 1130 1135 His Leu Val Arg Ser Trp Val Thr Ala Gly Glu Ile His Ala Val Pro            1140 1145 1150 Phe Gly Leu Val Ser Met Met Ile Ala Met Glu Val Val Leu Arg Lys        1155 1160 1165 Arg Gln Gly Pro Lys Gln Met Leu Val Gly Gly Val Val Leu Leu Gly    1170 1175 1180 Ala Met Leu Val Gly Gln Val Thr Leu Leu Asp Leu Leu Lys Leu Thr 1185 1190 1195 1200 Val Ala Val Gly Leu His Phe His Glu Met Asn Asn Gly Gly Asp Ala                1205 1210 1215 Met Tyr Met Ala Leu Ile Ala Ala Phe Ser Ile Arg Pro Gly Leu Leu            1220 1225 1230 Ile Gly Phe Gly Leu Arg Thr Leu Trp Ser Pro Arg Glu Arg Leu Val        1235 1240 1245 Leu Thr Leu Gly Ala Ala Met Val Glu Ile Ala Leu Gly Gly Val Met    1250 1255 1260 Gly Gly Leu Trp Lys Tyr Leu Asn Ala Val Ser Leu Cys Ile Leu Thr 1265 1270 1275 1280 Ile Asn Ala Val Ala Ser Arg Lys Ala Ser Asn Thr Ile Leu Pro Leu                1285 1290 1295 Met Ala Leu Leu Thr Pro Val Thr Met Ala Glu Val Arg Leu Ala Ala            1300 1305 1310 Met Phe Phe Cys Ala Val Val Ile Gly Val Leu His Gln Asn Phe        1315 1320 1325 Lys Asp Thr Ser Met Gln Lys Thr Ile Pro Leu Val Ala Leu Thr Leu    1330 1335 1340 Thr Ser Tyr Leu Gly Leu Thr Gln Pro Phe Leu Gly Leu Cys Ala Phe 1345 1350 1355 1360 Leu Ala Thr Arg Ile Phe Gly Arg Arg Ser Ile Pro Val Asn Glu Ala                1365 1370 1375 Leu Ala Ala Ala Gly Leu Val Gly Val Leu Ala Gly Leu Ala Phe Gln            1380 1385 1390 Glu Met Glu Asn Phe Leu Gly Pro Ile Ala Val Gly Gly Leu Leu Met        1395 1400 1405 Met Leu Val Ser Val Ala Gly Arg Val Asp Gly Leu Glu Leu Lys Lys    1410 1415 1420 Leu Gly Glu Val Ser Trp Glu Glu Glu Ala Glu Ile Ser Gly Ser Ser 1425 1430 1435 1440 Ala Arg Tyr Asp Val Ala Leu Ser Glu Gln Gly Glu Phe Lys Leu Leu                1445 1450 1455 Ser Glu Glu Lys Val Pro Trp Asp Gln Val Val Met Thr Ser Leu Ala            1460 1465 1470 Leu Val Gly Ala Ala Leu His Pro Phe Ala Leu Leu Leu Val Leu Ala        1475 1480 1485 Gly Trp Leu Phe His Val Arg Gly Ala Arg Arg Ser Gly Asp Val Leu    1490 1495 1500 Trp Asp Ile Pro Thr Pro Lys Ile Ile Glu Glu Cys Glu His Leu Glu 1505 1510 1515 1520 Asp Gly Ile Tyr Gly Ile Phe Gln Ser Thr Phe Leu Gly Ala Ser Gln                1525 1530 1535 Arg Gly Val Gly Val Ala Gln Gly Gly Val Phe His Thr Met Trp His            1540 1545 1550 Val Thr Arg Gly Ala Phe Leu Val Arg Asn Gly Lys Lys Leu Ile Pro        1555 1560 1565 Ser Trp Ala Ser Val Lys Glu Asp Leu Val Ala Tyr Gly Gly Ser Trp    1570 1575 1580 Lys Leu Glu Gly Arg Trp Asp Gly Glu Glu Glu Val Gln Leu Ile Ala 1585 1590 1595 1600 Ala Val Pro Gly Lys Asn Val Val Asn Val Gln Thr Lys Pro Ser Leu                1605 1610 1615 Phe Lys Val Arg Asn Gly Gly Glu Ile Gly Ala Val Ala Leu Asp Tyr            1620 1625 1630 Pro Ser Gly Thr Ser Gly Ser Pro Ile Val Asn Arg Asn Gly Glu Val        1635 1640 1645 Ile Gly Leu Tyr Gly Asn Gly Ile Leu Val Gly Asp Asn Ser Phe Val    1650 1655 1660 Ser Ala Ile Ser Gln Thr Glu Val Lys Glu Glu Gly Lys Glu Glu Leu 1665 1670 1675 1680 Gln Glu Ile Pro Thr Met Leu Lys Lys Gly Met Thr Thr Val Leu Asp                1685 1690 1695 Phe His Pro Gly Ala Gly Lys Thr Arg Arg Phe Leu Pro Gln Ile Leu            1700 1705 1710 Ala Glu Cys Ala Arg Arg Arg Leu Arg Thr Leu Val Leu Ala Pro Thr        1715 1720 1725 Arg Val Val Leu Ser Glu Met Lys Glu Ala Phe His Gly Leu Asp Val    1730 1735 1740 Lys Phe His Thr Gln Ala Phe Ser Ala His Gly Ser Gly Arg Glu Val 1745 1750 1755 1760 Ile Asp Ala Met Cys His Ala Thr Leu Thr Tyr Arg Met Leu Glu Pro                1765 1770 1775 Thr Arg Val Val Asn Trp Glu Val Ile Met Asp Glu Ala His Phe            1780 1785 1790 Leu Asp Pro Ala Ser Ile Ala Ala Arg Gly Trp Ala Ala His Arg Ala        1795 1800 1805 Arg Ala Asn Glu Ser Ala Thr Ile Leu Met Thr Ala Thr Pro Pro Gly    1810 1815 1820 Thr Ser Asp Glu Phe Pro His Ser Asn Gly Glu Ile Glu Asp Val Gln 1825 1830 1835 1840 Thr Asp Ile Pro Ser Glu Pro Trp Asn Thr Gly His Asp Trp Ile Leu                1845 1850 1855 Ala Asp Lys Arg Pro Thr Ala Trp Phe Leu Pro Ser Ile Arg Ala Ala            1860 1865 1870 Asn Val Met Ala Ala Ser Leu Arg Lys Ala Gly Lys Ser Val Val Val        1875 1880 1885 Leu Asn Arg Lys Thr Phe Glu Arg Glu Tyr Pro Thr Ile Lys Gln Lys    1890 1895 1900 Lys Pro Asp Phe Ile Leu Ala Thr Asp Ile Ala Glu Met Gly Ala Asn 1905 1910 1915 1920 Leu Cys Val Glu Arg Val Leu Asp Cys Arg Thr Ala Phe Lys Pro Val                1925 1930 1935 Leu Val Asp Glu Gly Arg Lys Val Ala Ile Lys Gly Pro Leu Arg Ile            1940 1945 1950 Ser Ala Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn Pro        1955 1960 1965 Asn Arg Asp Gly Asp Ser Tyr Tyr Tyr Ser Glu Pro Thr Ser Glu Asn    1970 1975 1980 Asn Ala His His Val Cys Trp Leu Glu Ala Ser Met Leu Leu Asp Asn 1985 1990 1995 2000 Met Glu Val Arg Gly Gly Met Val Ala Pro Leu Tyr Gly Val Glu Gly                2005 2010 2015 Thr Lys Thr Pro Val Ser Pro Gly Glu Met Arg Leu Arg Asp Asp Gln            2020 2025 2030 Arg Lys Val Phe Arg Glu Leu Val Arg Asn Cys Asp Leu Pro Val Trp        2035 2040 2045 Leu Ser Trp Gln Val Ala Lys Ala Gly Leu Lys Thr Asn Asp Arg Lys    2050 2055 2060 Trp Cys Phe Glu Gly Pro Glu Glu His Glu Ile Leu Asn Asp Ser Gly 2065 2070 2075 2080 Glu Thr Val Lys Cys Arg Ala Pro Gly Gly Ala Lys Lys Pro Leu Arg                2085 2090 2095 Pro Arg Trp Cys Asp Glu Arg Val Ser Ser Asp Gln Ser Ala Leu Ser            2100 2105 2110 Glu Phe Ile Lys Phe Ala Glu Gly Arg Arg Gly Ala Ala Glu Val Leu        2115 2120 2125 Val Val Leu Ser Glu Leu Pro Asp Phe Leu Ala Lys Lys Gly Gly Glu    2130 2135 2140 Ala Met Asp Thr Ile Ser Val Phe Leu His Ser Glu Glu Gly Ser Arg 2145 2150 2155 2160 Ala Tyr Arg Asn Ala Leu Ser Met Met Pro Glu Ala Met Thr Ile Val                2165 2170 2175 Met Leu Phe Ile Leu Ala Gly Leu Leu Thr Ser Gly Met Val Ile Phe            2180 2185 2190 Phe Met Ser Pro Lys Gly Ile Ser Arg Met Ser Met Ala Met Gly Thr        2195 2200 2205 Met Ala Gly Cys Gly Tyr Leu Met Phe Leu Gly Gly Val Lys Pro Thr    2210 2215 2220 His Ile Ser Tyr Val Met Leu Ile Phe Phe Val Leu Met Val Val Val 2225 2230 2235 2240 Ile Pro Glu Pro Gly Gln Gln Arg Ser Ile Gln Asp Asn Gln Val Ala                2245 2250 2255 Tyr Leu Ile Ile Gly Ile Leu Thr Leu Val Ser Ala Val Ala Ala Asn            2260 2265 2270 Glu Leu Gly Met Leu Glu Lys Thr Lys Glu Asp Leu Phe Gly Lys Lys        2275 2280 2285 Asn Leu Ile Pro Ser Ser Ala Ser Pro Trp Ser Trp Pro Asp Leu Asp    2290 2295 2300 Leu Lys Pro Gly Ala Ala Trp Thr Val Tyr Val Gly Ile Val Thr Met 2305 2310 2315 2320 Leu Ser Pro Met Leu His His Trp Ile Lys Val Glu Tyr Gly Asn Leu                2325 2330 2335 Ser Leu Ser Gly Ile Ala Gln Ser Ala Ser Val Leu Ser Phe Met Asp            2340 2345 2350 Lys Gly Ile Pro Phe Met Lys Met Asn Ile Ser Val Ile Met Leu Leu        2355 2360 2365 Val Ser Gly Trp Asn Ser Ile Thr Val Met Pro Leu Leu Cys Gly Ile    2370 2375 2380 Gly Cys Ala Met Leu His Trp Ser Leu Ile Leu Pro Gly Ile Lys Ala 2385 2390 2395 2400 Gln Gln Ser Lys Leu Ala Gln Arg Arg Val Phe His Gly Val Ala Glu                2405 2410 2415 Asn Pro Val Val Asp Gly Asn Pro Thr Val Asp Ile Glu Glu Ala Pro            2420 2425 2430 Glu Met Pro Ala Leu Tyr Glu Lys Lys Leu Ala Leu Tyr Leu Leu Leu        2435 2440 2445 Ala Leu Ser Leu Ala Ser Val Ala Met Cys Arg Thr Pro Phe Ser Leu    2450 2455 2460 Ala Glu Gly Ile Val Leu Ala Ser Ala Ala Leu Gly Pro Leu Ile Glu 2465 2470 2475 2480 Gly Asn Thr Ser Leu Leu Trp Asn Gly Pro Met Ala Val Ser Met Thr                2485 2490 2495 Gly Val Met Arg Gly Asn His Tyr Ala Phe Val Gly Val Met Tyr Asn            2500 2505 2510 Leu Trp Lys Met Lys Thr Gly Arg Arg Gly Ser Ala Asn Gly Lys Thr        2515 2520 2525 Leu Gly Glu Val Trp Lys Arg Glu Leu Asn Leu Leu Asp Lys Arg Gln    2530 2535 2540 Phe Glu Leu Tyr Lys Arg Thr Asp Ile Val Glu Val Asp Arg Asp Thr 2545 2550 2555 2560 Ala Arg Arg His Leu Ala Glu Gly Lys Val Asp Thr Gly Val Ala Val                2565 2570 2575 Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe His Glu Arg Gly Tyr Val            2580 2585 2590 Lys Leu Glu Gly Arg Val Ile Asp Leu Gly Cys Gly Arg Gly Gly Trp        2595 2600 2605 Cys Tyr Tyr Ala Ala Ala Gln Lys Glu Val Ser Gly Val Lys Gly Phe    2610 2615 2620 Thr Leu Gly Arg Asp Gly His Glu Lys Pro Met Asn Val Gln Ser Leu 2625 2630 2635 2640 Gly Trp Asn Ile Ile Thr Phe Lys Asp Lys Thr Asp Ile His Arg Leu                2645 2650 2655 Glu Pro Val Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser            2660 2665 2670 Ser Ser Ser Val Thr Glu Gly Glu Arg Thr Val Arg Val Leu Asp Thr        2675 2680 2685 Val Glu Lys Trp Leu Ala Cys Gly Val Asp Asn Phe Cys Val Lys Val    2690 2695 2700 Leu Ala Pro Tyr Met Pro Asp Val Leu Glu Lys Leu Glu Leu Leu Gln 2705 2710 2715 2720 Arg Arg Phe Gly Gly Thr Val Ile Arg Asn Pro Leu Ser Arg Asn Ser                2725 2730 2735 Thr His Glu Met Tyr Tyr Val Ser Gly Ala Arg Ser Asn Val Thr Phe            2740 2745 2750 Thr Val Asn Gln Thr Ser Arg Leu Leu Met Arg Arg Met Arg Arg Pro        2755 2760 2765 Thr Gly Lys Val Thr Leu Glu Ala Asp Val Ile Leu Pro Ile Gly Thr    2770 2775 2780 Arg Ser Val Glu Thr Asp Lys Gly Pro Leu Asp Lys Glu Ala Ile Glu 2785 2790 2795 2800 Glu Arg Val Glu Arg Ile Lys Ser Glu Tyr Met Thr Ser Trp Phe Tyr                2805 2810 2815 Asp Asn Asp Asn Pro Tyr Arg Thr Trp His Tyr Cys Gly Ser Tyr Val            2820 2825 2830 Thr Lys Thr Ser Gly Ser Ala Ala Ser Met Val Asn Gly Val Ile Lys        2835 2840 2845 Ile Leu Thr Tyr Pro Trp Asp Arg Ile Glu Glu Val Thr Arg Met Ala    2850 2855 2860 Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys 2865 2870 2875 2880 Val Asp Thr Arg Ala Lys Asp Pro Pro Ala Gly Thr Arg Lys Ile Met                2885 2890 2895 Lys Val Val Asn Arg Trp Leu Phe Arg His Leu Ala Arg Glu Lys Asn            2900 2905 2910 Pro Arg Leu Cys Thr Lys Glu Glu Phe Ile Ala Lys Val Arg Ser His        2915 2920 2925 Ala Ala Ile Gly Ala Tyr Leu Glu Glu Gln Glu Gln Trp Lys Thr Ala    2930 2935 2940 Asn Glu Ala Val Gln Asp Pro Lys Phe Trp Glu Leu Val Asp Glu Glu 2945 2950 2955 2960 Arg Lys Leu His Gln Gln Gly Arg Cys Arg Thr Cys Val Tyr Asn Met                2965 2970 2975 Met Gly Lys Arg Glu Lys Lys Leu Ser Glu Phe Gly Lys Ala Lys Gly            2980 2985 2990 Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Tyr Leu Glu Phe        2995 3000 3005 Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Ala Ser Arg Glu Asn    3010 3015 3020 Ser Gly Gly Gly Val Glu Gly Ile Gly Leu Gln Tyr Leu Gly Tyr Val 3025 3030 3035 3040 Ile Arg Asp Leu Ala Ala Met Asp Gly Gly Gly Phe Tyr Ala Asp Asp                3045 3050 3055 Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Ala Asp Leu Asp Asp Glu            3060 3065 3070 Gln Glu Ile Leu Asn Tyr Met Ser Pro His His Lys Lys Leu Ala Gln        3075 3080 3085 Ala Val Met Glu Met Thr Tyr Lys Asn Lys Val Val Lys Val Leu Arg    3090 3095 3100 Pro Ala Pro Gly Gly Lys Ala Tyr Met Asp Val Ile Ser Arg Arg Asp 3105 3110 3115 3120 Gln Arg Gly Ser Gly Gln Val Val Thr Tyr Ala Leu Asn Thr Ile Thr                3125 3130 3135 Asn Leu Lys Val Gln Leu Ile Arg Met Ala Glu Ala Glu Met Val Ile            3140 3145 3150 His His Gln His Val Gln Asp Cys Asp Glu Ser Val Leu Thr Arg Leu        3155 3160 3165 Glu Ala Trp Leu Thr Glu His Gly Cys Asp Arg Leu Lys Arg Met Ala    3170 3175 3180 Val Ser Gly Asp Asp Cys Val Val Arg Pro Ile Asp Asp Arg Phe Gly 3185 3190 3195 3200 Leu Ala Leu Ser His Leu Asn Ala Met Ser Lys Val Arg Lys Asp Ile                3205 3210 3215 Ser Glu Trp Gln Pro Ser Lys Gly Trp Asn Asp Trp Glu Asn Val Pro            3220 3225 3230 Phe Cys Ser His His Phe His Glu Leu Gln Leu Lys Asp Gly Arg Arg        3235 3240 3245 Ile Val Val Pro Cys Arg Glu Gln Asp Glu Leu Ile Gly Arg Gly Arg    3250 3255 3260 Val Ser Pro Gly Asn Gly Trp Met Ile Lys Glu Thr Ala Cys Leu Ser 3265 3270 3275 3280 Lys Ala Tyr Ala Asn Met Trp Ser Leu Met Tyr Phe His Lys Arg Asp                3285 3290 3295 Met Arg Leu Leu Ser Leu Ala Val Ser Ser Ala Val Pro Thr Ser Trp            3300 3305 3310 Val Pro Gln Gly Arg Thr Thr Trp Ser Ile His Gly Lys Gly Glu Trp        3315 3320 3325 Met Thr Thr Glu Asp Met Leu Glu Val Trp Asn Arg Val Trp Ile Thr    3330 3335 3340 Asn Asn Pro His Met Gln Asp Lys Thr Met Val Lys Lys Trp Arg Asp 3345 3350 3355 3360 Val Pro Tyr Leu Thr Lys Arg Gln Asp Lys Leu Cys Gly Ser Leu Ile                3365 3370 3375 Gly Met Thr Asn Arg Ala Thr Trp Ala Ser His Ile His Leu Val Ile            3380 3385 3390 His Arg Ile Arg Thr Leu Ile Gly Gln Glu Lys Tyr Thr Asp Tyr Leu        3395 3400 3405 Thr Val Met Asp Arg Tyr Ser Val Asp Ala Asp Leu Gln Leu Gly Glu    3410 3415 3420 Leu ile 3425 <210> 14 <211> 10907 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct B nucleic acid sequence <400> 14 agtaaatcct gtgtgctaat tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60 acacatttgg attaatttta atcgttcgtt gagcgattag cagagaactg accagaacat 120 gtctggtcgt aaagctcagg gaaaaaccct gggcgtcaat atggtacgac gaggagttcg 180 ctccttgtca aacaaaataa aacaaaaaac aaaacaaatt ggaaacagac ctggaccttc 240 aagaggtgtt caaggattta tctttttctt tttgttcaac attttgactg gaaaaaagat 300 cacagcccac ctaaagaggt tgtggaaaat gctggaccca agacaaggct tggctgttct 360 aaggaaagtc aagagagtgg tggccagttt gatgagagga ttgtcctcaa ggaaacgccg 420 ttcccatgat gttctgactg tgcaattcct aattttggga atgctgttga tgacgggtgg 480 agcggaggtc actagacgtg ggagtgcata ctatatgtac ttggacagaa acgatgctgg 540 ggaggccata tcttttccaa ccacattggg gatgaataag tgttatatac agatcatgga 600 tcttggacac atgtgtgatg ccaccatgag ctatgaatgc cctatgctgg atgagggggt 660 ggaaccagat gacgtcgatt gttggtgcaa cacgacgtca acttgggttg tgtacggaac 720 ctgccatcac aaaaaaggtg aagcacggag atctagaaga gctgtgacgc tcccctccca 780 ttccactagg aagctgcaaa cgcggtcgca aacctggttg gaatcaagag aatacacaaa 840 gcacttgatt agagtcgaaa attggatatt caggaacccc ttttttgcag tgacggctct 900 gaccattgcc taccttgtgg gaagcaacat gacgcaacga gtcgtgattg ccctactggt 960 cttggctgtt ggtccggcct actcaatcag gtgcatagga gtcagcaata gggactttgt 1020 ggaaggtatg tcaggtggga cttgggttga tgttgtcttg gaacatggag gttgtgtcac 1080 cgtaatggca caggacaaac cgactgtcga catagagctg gttacaacaa cagtcagcaa 1140 catggcggag gtaagatcct actgctatga ggcatcaata tcagacatgg cttcggacag 1200 ccgctgccca acacaaggtg aagcctacct tgacaagcaa tcagacactc aatatgtctg 1260 caaaagaacg ttagtggaca gaggctgggg aaatggatgt ggactttttg gcaaagggag 1320 cctggtgaca tgcgctaagt ttgcatgctc caagaaaatg accgggaaga gcatccagcc 1380 agagaatctg gagtaccgga taatgctgtc agttcatggc tcccagcaca gtgggatgat 1440 cgttaatgac acaggacatg aaactgatga gaatagagcg aaagttgaga taacgcccaa 1500 ttcaccaaga gccgaagcca ccctgggggg ttttggaagc ctaggacttg attgtgaacc 1560 gaggacaggc cttgactttt cagatttgta ttacttgact atgaataaca agcactggtt 1620 ggttcacaag gagtggttcc acgacattcc attaccttgg cacgctgggg cagacaccgg 1680 aactccacac tggaacaaca aagaagcact ggtagagttc aaggacgcac atgccaaaag 1740 gcaaactgtc gtggttctag ggagtcaaga aggagcagtt cacacggccc ttgctggagc 1800 tctggaggct gagatggatg gtgcaaaggg aaggctgtcc tctggccact tgaaatgtcg 1860 cctgaaaatg gataaactta gattgaaggg cgtgtcatac tccttgtgta ctgcagcgtt 1920 cacattcacc aagatcccgg ctgaaacact gcacgggaca gtcacagtgg aggtacagta 1980 cgcagggaca gatggacctt gcaaggttcc agctcagatg gcggtggaca tgcaaactct 2040 gaccccagtt gggaggttga taaccgctaa ccccgtaatc actgaaagca ctgagaactc 2100 taagatgatg ctggaacttg atccaccatt tggggactct tacattgtca taggagtcgg 2160 ggagaagaag atcacccacc actggcacag gagtggcagc accattggaa aagcatttga 2220 agccactgtg agaggtgcca agagaatggc agtcttggga gacacagcct gggactttgg 2280 atcagttgga ggcgctctca actcattggg caagggcatc catcaaattt ttggagcagc 2340 tttcaaatca ttgtttggag gattgaactg gataacaaag gtcatcatgg gggcggtact 2400 tatatgggtt ggcatcaaca caagaaacat gacaatgtcc atgagcatga tcttggtagg 2460 agtgatcatg atgtttttgt ctctaggagt tggggcggat caaggatgcg ccatcaactt 2520 tggcaagaga gagctcaagt gcggagatgg tatcttcata tttagagact ctgatgactg 2580 gctgaacaag tactcatact atccagaaga tcctgtgaag cttgcatcaa tagtgaaagc 2640 ctcttttgaa gaagggaagt gtggcctaaa ttcagttgac tcccttgagc atgagatgtg 2700 gagaagcagg gcagatgaga tcaatgccat ttttgaggaa aacgaggtgg acatttctgt 2760 tgtcgtgcag gatccaaaga atgtttacca gagaggaact catccatttt ccagaattcg 2820 ggatggtctg cagtatggtt ggaagacttg gggtaagaac cttgtgttct ccccagggag 2880 gaagaatgga agcttcatca tagatggaaa gtccaggaaa gaatgcccgt tttcaaaccg 2940 ggtctggaat tctttccaga tagaggagtt tgggacggga gtgttcacca cacgcgtgta 3000 catggacgca gtctttgaat acaccataga ctgcgatgga tctatcttgg gtgcagcggt 3060 gaacggaaaa aagagtgccc atggctctcc aacattttgg atgggaagtc atgaagtaaa 3120 tgggacatgg atgatccaca ccttggaggc attagattac aaggagtgtg agtggccact 3180 gacacatacg attggaacat cagttgaaga gagtgaaatg ttcatgccga gatcaatcgg 3240 aggcccagtt agctctcaca atcatatccc tggatacaag gttcagacga acggaccttg 3300 gatgcaggta ccactagaag tgaagagaga agcttgccca gggactagcg tgatcattga 3360 tggcaactgt gatggacggg gaaaatcaac cagatccacc acggatagcg ggaaagttat 3420 tcctgaatgg tgttgccgct cctgcacaat gccgcctgtg agcttccatg gtagtgatgg 3480 gtgttggtat cccatggaaa ttaggccaag gaaaacgcat gaaagccatc tggtgcgctc 3540 ctgggttaca gctggagaaa tacatgctgt cccttttggt ttggtgagca tgatgatagc 3600 aatggaagtg gtcctaagga aaagacaggg accaaagcaa atgttggttg gaggagtagt 3660 gctcttggga gcaatgctgg tcgggcaagt aactctcctt gatttgctga aactcacagt 3720 ggctgtggga ttgcatttcc atgagatgaa caatggagga gacgccatgt atatggcgtt 3780 gattgctgcc ttttcaatca gaccagggct gctcatcggc tttgggctca ggaccctatg 3840 gagccctcgg gaacgccttg tgctgaccct aggagcagcc atggtggaga ttgccttggg 3900 tggcgtgatg ggcggcctgt ggaagtatct aaatgcagtt tctctctgca tcctgacaat 3960 aaatgctgtt gcttctagga aagcatcaaa taccatcttg cccctcatgg ctctgttgac 4020 acctgtcact atggctgagg tgagacttgc cgcaatgttc ttttgtgccg tggttatcat 4080 aggggtcctt caccagaatt tcaaggacac ctccatgcag aagactatac ctctggtggc 4140 cctcacactc acatcttacc tgggcttgac acaacctttt ttgggcctgt gtgcatttct 4200 ggcaacccgc atatttgggc gaaggagtat cccagtgaat gaggcactcg cagcagctgg 4260 tctagtggga gtgctggcag gactggcttt tcaggagatg gagaacttcc ttggtccgat 4320 tgcagttgga ggactcctga tgatgctggt tagcgtggct gggagggtgg atgggctaga 4380 gctcaagaag cttggtgaag tttcatggga agaggaggcg gagatcagcg ggagttccgc 4440 ccgctatgat gtggcactca gtgaacaagg ggagttcaag ctgctttctg aagagaaagt 4500 gccatgggac caggttgtga tgacctcgct ggccttggtt ggggctgccc tccatccatt 4560 tgctcttctg ctggtccttg ctgggtggct gtttcatgtc aggggagcta ggagaagtgg 4620 ggatgtcttg tgggatattc ccactcctaa gatcatcgag gaatgtgaac atctggagga 4680 tgggatttat ggcatattcc agtcaacctt cttgggggcc tcccagcgag gagtgggagt 4740 ggcacaggga ggggtgttcc acacaatgtg gcatgtcaca agaggagctt tccttgtcag 4800 gaatggcaag aagttgattc catcttgggc ttcagtaaag gaagaccttg tcgcctatgg 4860 tggctcatgg aagttggaag gcagatggga tggagaggaa gaggtccagt tgatcgcggc 4920 tgttccagga aagaacgtgg tcaacgtcca gacaaaaccg agcttgttca aagtgaggaa 4980 tgggggagaa atcggggctg tcgctcttga ctatccgagt ggcacttcag gatctcctat 5040 tgttaacagg aacggagagg tgattgggct gtacggcaat ggcatccttg tcggtgacaa 5100 ctccttcgtg tccgccatat cccagactga ggtgaaggaa gaaggaaagg aggagctcca 5160 agagatcccg acaatgctaa agaaaggaat gacaactgtc cttgattttc atcctggagc 5220 tgggaagaca agacgtttcc tcccacagat cttggccgag tgcgcacgga gacgcttgcg 5280 cactcttgtg ttggccccca ccagggttgt tctttctgaa atgaaggagg cttttcacgg 5340 cctggacgtg aaattccaca cacaggcttt ttccgctcac ggcagcggga gagaagtcat 5400 tgatgccatg tgccatgcca ccctaactta caggatgttg gaaccaacta gggttgttaa 5460 ctgggaagtg atcattatgg atgaagccca ttttttggat ccagctagca tagccgctag 5520 aggttgggca gcgcacagag ctagggcaaa tgaaagtgca acaatcttga tgacagccac 5580 accgcctggg actagtgatg aatttccaca ttcaaatggt gaaatagaag atgttcaaac 5640 ggacataccc agtgagccct ggaacacagg gcatgactgg atcctagctg acaaaaggcc 5700 cacggcatgg ttccttccat ccatcagagc tgcaaatgtc atggctgcct ctttgcgtaa 5760 ggctggaaag agtgtggtgg tcctgaacag gaaaaccttt gagagagaat accccacgat 5820 aaagcagaag aaacctgact ttatattggc cactgacata gctgaaatgg gagccaacct 5880 ttgcgtggag cgagtgctgg attgcaggac ggcttttaag cctgtgcttg tggatgaagg 5940 gaggaaggtg gcaataaaag ggccacttcg tatctccgca tcctctgctg ctcaaaggag 6000 ggggcgcatt gggagaaatc ccaacagaga tggagactca tactactatt ctgagcctac 6060 aagtgaaaat aatgcccacc acgtctgctg gttggaggcc tcaatgctct tggacaacat 6120 ggaggtgagg ggtggaatgg tcgccccact ctatggcgtt gaaggaacta aaacaccagt 6180 ttcccctggt gaaatgagac tgagggatga ccagaggaaa gtcttcagag aactagtgag 6240 gaattgtgac ctgcccgttt ggctttcgtg gcaagtggcc aaggctggtt tgaagacgaa 6300 tgatcgtaag tggtgttttg aaggccctga ggaacatgag atcttgaatg acagcggtga 6360 aacagtgaag tgcagggctc ctggaggagc aaagaagcct ctgcgcccaa ggtggtgtga 6420 tgaaagggtg tcatctgacc agagtgcgct gtctgaattt attaagtttg ctgaaggtag 6480 gaggggagct gctgaagtgc tagttgtgct gagtgaactc cctgatttcc tggctaaaaa 6540 aggtggagag gcaatggata ccatcagtgt gttcctccac tctgaggaag gctctagggc 6600 ttaccgcaat gcactatcaa tgatgcctga ggcaatgaca atagtcatgc tgtttatact 6660 ggctggacta ctgacatcgg gaatggtcat ctttttcatg tctcccaaag gcatcagtag 6720 aatgtctatg gcgatgggca caatggccgg ctgtggatat ctcatgttcc ttggaggcgt 6780 caaacccact cacatctcct atgtcatgct catattcttt gtcctgatgg tggttgtgat 6840 ccccgagcca gggcaacaaa ggtccatcca agacaaccaa gtggcatacc tcattattgg 6900 catcctgacg ctggtttcag cggtggcagc caacgagcta ggcatgctgg agaaaaccaa 6960 agaggacctc tttgggaaga agaacttaat tccatctagt gcttcaccct ggagttggcc 7020 ggatcttgac ctgaagccag gagctgcctg gacagtgtac gttggcattg ttacaatgct 7080 ctctccaatg ttgcaccact ggatcaaagt cgaatatggc aacctgtctc tgtctggaat 7140 agcccagtca gcctcagtcc tttctttcat ggacaagggg ataccattca tgaagatgaa 7200 tatctcggtc ataatgctgc tggtcagtgg ctggaattca ataacagtga tgcctctgct 7260 ctgtggcata gggtgcgcca tgctccactg gtctctcatt ttacctggaa tcaaagcgca 7320 gcagtcaaag cttgcacaga gaagggtgtt ccatggcgtt gccgagaacc ctgtggttga 7380 tgggaatcca acagttgaca ttgaggaagc tcctgaaatg cctgcccttt atgagaagaa 7440 actggctcta tatctccttc ttgctctcag cctagcttct gttgccatgt gcagaacgcc 7500 cttttcattg gctgaaggca ttgtcctagc atcagctgcc ttagggccgc tcatagaggg 7560 aaacaccagc cttctttgga atggacccat ggctgtctcc atgacaggag tcatgagggg 7620 gaatcactat gcttttgtgg gagtcatgta caatctatgg aagatgaaaa ctggacgccg 7680 ggggagcgcg aatggaaaaa ctttgggtga agtctggaag agggaactga atctgttgga 7740 caagcgacag tttgagttgt ataaaaggac cgacattgtg gaggtggatc gtgatacggc 7800 acgcaggcat ttggccgaag ggaaggtgga caccggggtg gcggtctcca gggggaccgc 7860 aaagttaagg tggttccatg agcgtggcta tgtcaagctg gaaggtaggg tgattgacct 7920 ggggtgtggc cgcggaggct ggtgttacta cgctgctgcg caaaaggaag tgagtggggt 7980 caaaggattt actcttggaa gagacggcca tgagaaaccc atgaatgtgc aaagtctggg 8040 atggaacatc atcaccttca aggacaaaac tgatatccac cgcctagaac cagtgaaatg 8100 tgacaccctt ttgtgtgaca ttggagagtc atcatcgtca tcggtcacag agggggaaag 8160 gaccgtgaga gttcttgata ctgtagaaaa atggctggct tgtggggttg acaacttctg 8220 tgtgaaggtg ttagctccat acatgccaga tgttctcgag aaactggaat tgctccaaag 8280 gaggtttggc ggaacagtga tcaggaaccc tctctccagg aattccactc atgaaatgta 8340 ctacgtgtct ggagcccgca gcaatgtcac atttactgtg aaccaaacat cccgcctcct 8400 gatgaggaga atgaggcgtc caactggaaa agtgaccctg gaggctgacg tcatcctccc 8460 aattgggaca cgcagtgttg agacagacaa gggacccctg gacaaagagg ccatagaaga 8520 aagggttgag aggataaaat ctgagtacat gacctcttgg ttttatgaca atgacaaccc 8580 ctacaggacc tggcactact gtggctccta tgtcacaaaa acctcaggaa gtgcggcgag 8640 catggtaaat ggtgttatta aaattctgac atatccatgg gacaggatag aggaggtcac 8700 aagaatggca atgactgaca caaccccttt tggacagcaa agagtgttta aagaaaaagt 8760 tgacaccaga gcaaaggatc caccagcggg aactaggaag atcatgaaag ttgtcaacag 8820 gtggctgttc cgccacctgg ccagagaaaa gaaccccaga ctgtgcacaa aggaagaatt 8880 tattgcaaaa gtccgaagtc atgcagccat tggagcttac ctggaagaac aagaacagtg 8940 gaagactgcc aatgaggctg tccaagaccc aaagttctgg gaactggtgg atgaagaaag 9000 gaagctgcac caacaaggca ggtgtcggac ttgtgtgtac aacatgatgg ggaaaagaga 9060 gaagaagctg tcagagtttg ggaaagcaaa gggaagccgt gccatatggt atatgtggct 9120 gggagcgcgg tatcttgagt ttgaggccct gggattcctg aatgaggacc attgggcttc 9180 cagggaaaac tcaggaggag gagtggaagg cattggctta caatacctag gatatgtgat 9240 cagagacctg gctgcaatgg atggtggtgg attctacgcg gatgacaccg ctggatggga 9300 cacgcgcatc acagaggcag accttgatga tgaacaggag atcttgaact acatgagccc 9360 acatcacaaa aaactggcac aagcagtgat ggaaatgaca tacaagaaca aagtggtgaa 9420 agtgttgaga ccagccccag gagggaaagc ctacatggat gtcataagtc gacgagacca 9480 gagaggatcc gggcaggtag tgacttatgc tctgaacacc atcaccaact tgaaagtcca 9540 attgatcaga atggcagaag cagagatggt gatacatcac caacatgttc aagattgtga 9600 tgaatcagtt ctgaccaggc tggaggcatg gctcactgag cacggatgtg acagactgaa 9660 gaggatggcg gtgagtggag acgactgtgt ggtccggccc atcgatgaca ggttcggcct 9720 ggccctgtcc catctcaacg ccatgtccaa ggttagaaag gacatatctg aatggcagcc 9780 atcaaaaggg tggaatgatt gggagaatgt gcccttctgt tcccaccact tccatgaact 9840 acagctgaag gatggcagga ggattgtggt gccttgccga gaacaggacg agctcattgg 9900 gagaggaagg gtgtctccag gaaacggctg gatgatcaag gaaacagctt gcctcagcaa 9960 agcctatgcc aacatgtggt cactgatgta ttttcacaaa agggacatga ggctactgtc 10020 attggctgtt tcctcagctg ttcccacctc atgggttcca caaggacgca caacatggtc 10080 gattcatggg aaaggggagt ggatgaccac ggaagacatg cttgaggtgt ggaacagagt 10140 atggataacc aacaacccac acatgcagga caagacaatg gtgaaaaaat ggagagatgt 10200 cccttatcta accaagagac aagacaagct gtgcggatca ctgattggaa tgaccaatag 10260 ggccacctgg gcctcccaca tccatttagt catccatcgt atccgaacgc tgattggaca 10320 ggagaaatac actgactacc taacagtcat ggacaggtat tctgtggatg ctgacctgca 10380 actgggtgag cttatctgaa acaccatcta acaggaataa ccgggataca aaccacgggt 10440 ggagaaccgg actccccaca acctgaaacc gggatataaa ccacggctgg agaaccgggc 10500 tccgcactta aaatgaaaca gaaaccggga taaaaactac ggatggagaa ccggactcca 10560 cacattgaga cagaagaagt tgtcagccca gaaccccaca cgagttttgc cactgctaag 10620 ctgtgaggca gtgcaggctg ggacagccga cctccaggtt gcgaaaaacc tggtttctgg 10680 gacctcccac cccagagtaa aaagaacgga gcctccgcta ccaccctccc acgtggtggt 10740 agaaagacgg ggtctagagg ttagaggaga ccctccaggg aacaaatagt gggaccatat 10800 tgacgccagg gaaagaccgg agtggttctc tgcttttcct ccagaggtct gtgagcacag 10860 tttgctcaag aataagcaga cctttggatg acaaacacaa aaccact 10907 <210> 15 <211> 3427 <212> PRT <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct C amino acid sequence <400> 15 Met Lys Asn Pro Lys Lys Lys Ser Gly Gly Phe Arg Ile Val Asn Met   1 5 10 15 Leu Lys Arg Gly Val Ala Arg Val Ser Pro Phe Gly Gly Leu Lys Arg              20 25 30 Leu Pro Ala Gly Leu Leu Leu Gly His Gly Pro Ile Arg Met Val Leu          35 40 45 Ala Ile Leu Ala Phe Leu Arg Phe Thr Ala Ile Lys Pro Ser Leu Gly      50 55 60 Leu Ile Asn Arg Trp Gly Ser Val Gly Lys Lys Glu Ala Met Glu Ile  65 70 75 80 Ile Lys Lys Phe Lys Lys Asp Leu Ala Ala Met Leu Arg Ile Ile Asn                  85 90 95 Ala Arg Lys Glu Lys Lys Arg Arg Gly Ala Asp Thr Ser Val Gly Ile             100 105 110 Val Gly Leu Leu Leu Thr Thr Ala Met Ala Ala Glu Val Thr Arg Arg         115 120 125 Gly Ser Ala Tyr Tyr Met Tyr Leu Asp Arg Asn Asp Ala Gly Glu Ala     130 135 140 Ile Ser Phe Pro Thr Thr Leu Gly Met Asn Lys Cys Tyr Ile Gln Ile 145 150 155 160 Met Asp Leu Gly His Met Cys Asp Ala Thr Met Ser Tyr Glu Cys Pro                 165 170 175 Met Leu Asp Glu Gly Val Glu Pro Asp Asp Val Asp Cys Trp Cys Asn             180 185 190 Thr Thr Ser Thr Trp Val Val Tyr Gly Thr Cys His His Lys Lys Gly         195 200 205 Glu Ala Arg Arg Ser Arg Arg Ala Val Thr Leu Pro Ser His Ser Thr     210 215 220 Arg Lys Leu Gln Thr Arg Ser Gln Thr Trp Leu Glu Ser Arg Glu Tyr 225 230 235 240 Thr Lys His Leu Ile Arg Val Glu Asn Trp Ile Phe Arg Asn Pro Gly                 245 250 255 Phe Ala Leu Ala Ala Ala Ala Ile Ala Trp Leu Leu Gly Ser Ser Thr             260 265 270 Ser Gln Lys Val Ile Tyr Leu Val Met Ile Leu Leu Ile Ala Pro Ala         275 280 285 Tyr Ser Ile Arg Cys Ile Gly Val Ser Asn Arg Asp Phe Val Glu Gly     290 295 300 Met Ser Gly Gly Thr Trp Val Asp Val Val Leu Glu His Gly Gly Cys 305 310 315 320 Val Thr Val Met Ala Gln Asp Lys Pro Thr Val Asp Ile Glu Leu Val                 325 330 335 Thr Thr Thr Val Ser Asn Met Ala Glu Val Arg Ser Tyr Cys Tyr Glu             340 345 350 Ala Ser Ile Ser Asp Met Ala Ser Asp Ser Arg Cys Pro Thr Gln Gly         355 360 365 Glu Ala Tyr Leu Asp Lys Gln Ser Asp Thr Gln Tyr Val Cys Lys Arg     370 375 380 Thr Leu Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys 385 390 395 400 Gly Ser Leu Val Thr Cys Ala Lys Phe Ala Cys Ser Lys Lys Met Thr                 405 410 415 Gly Lys Ser Ile Gln Pro Glu Asn Leu Glu Tyr Arg Ile Met Leu Ser             420 425 430 Val His Gly Ser Gln His Ser Gly Met Ile Val Asn Asp Thr Gly His         435 440 445 Glu Thr Asp Glu Asn Arg Ala Lys Val Glu Ile Thr Pro Asn Ser Pro     450 455 460 Arg Ala Glu Ala Thr Leu Gly Gly Phe Gly Ser Leu Gly Leu Asp Cys 465 470 475 480 Glu Pro Arg Thr Gly Leu Asp Phe Ser Asp Leu Tyr Tyr Leu Thr Met                 485 490 495 Asn Asn Lys His Trp Leu Val His Lys Glu Trp Phe His Asp Ile Pro             500 505 510 Leu Pro Trp His Ala Gly Ala Asp Thr Gly Thr Pro His Trp Asn Asn         515 520 525 Lys Glu Ala Leu Val Glu Phe Lys Asp Ala His Ala Lys Arg Gln Thr     530 535 540 Val Val Val Leu Gly Ser Gln Glu Gly Ala Val His Thr Ala Leu Ala 545 550 555 560 Gly Ala Leu Glu Ala Glu Met Asp Gly Ala Lys Gly Arg Leu Ser Ser                 565 570 575 Gly His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Arg Leu Lys Gly             580 585 590 Val Ser Tyr Ser Leu Cys Thr Ala Ala Phe Thr Phe Thr Lys Ile Pro         595 600 605 Ala Glu Thr Leu His Gly Thr Val Thr Val Glu Val Gln Tyr Ala Gly     610 615 620 Thr Asp Gly Pro Cys Lys Val Pro Ala Gln Met Ala Val Asp Met Gln 625 630 635 640 Thr Leu Thr Pro Val Gly Arg Leu Ile Thr Ala Asn Pro Val Ile Thr                 645 650 655 Glu Ser Thr Glu Asn Ser Lys Met Met Leu Glu Leu Asp Pro Pro Phe             660 665 670 Gly Asp Ser Tyr Ile Val Ile Gly Val Gly Glu Lys Lys Ile Thr His         675 680 685 His Trp His Arg Ser Gly Ser Thr Ile Gly Lys Ala Phe Glu Ala Thr     690 695 700 Val Arg Gly Ala Lys Arg Met Ala Val Leu Gly Asp Thr Ala Trp Asp 705 710 715 720 Phe Gly Ser Val Gly Gly Ala Leu Asn Ser Leu Gly Lys Gly Ile His                 725 730 735 Gln Ile Phe Gly Ala Ala Phe Lys Ser Leu Phe Gly Gly Met Ser Trp             740 745 750 Phe Ser Gln Ile Leu Ile Gly Thr Leu Leu Met Trp Leu Gly Leu Asn         755 760 765 Thr Lys Asn Gly Ser Ile Ser Leu Met Cys Leu Ala Leu Gly Gly Val     770 775 780 Leu Ile Phe Leu Ser Thr Ala Val Ser Ala Asp Gln Gly Cys Ala Ile 785 790 795 800 Asn Phe Gly Lys Arg Glu Leu Lys Cys Gly Asp Gly Ile Phe Ile Phe                 805 810 815 Arg Asp Ser Asp Asp Trp Leu Asn Lys Tyr Ser Tyr Tyr Pro Glu Asp             820 825 830 Pro Val Lys Leu Ala Ser Ile Val Lys Ala Ser Phe Glu Glu Gly Lys         835 840 845 Cys Gly Leu Asn Ser Val Asp Ser Leu Glu His Glu Met Trp Arg Ser     850 855 860 Arg Ala Asp Glu Ile Asn Ala Ile Phe Glu Glu Asn Glu Val Asp Ile 865 870 875 880 Ser Val Val Val Gln Asp Pro Lys Asn Val Tyr Gln Arg Gly Thr His                 885 890 895 Pro Phe Ser Arg Ile Arg Asp Gly Leu Gln Tyr Gly Trp Lys Thr Trp             900 905 910 Gly Lys Asn Leu Val Phe Ser Pro Gly Arg Lys Asn Gly Ser Phe Ile         915 920 925 Ile Asp Gly Lys Ser Arg Lys Glu Cys Pro Phe Ser Asn Arg Val Trp     930 935 940 Asn Ser Phe Gln Ile Glu Glu Phe Gly Thr Gly Val Phe Thr Thr Arg 945 950 955 960 Val Tyr Met Asp Ala Val Phe Glu Tyr Thr Ile Asp Cys Asp Gly Ser                 965 970 975 Ile Leu Gly Ala Ala Val Asn Gly Lys Lys Ser Ala His Gly Ser Pro             980 985 990 Thr Phe Trp Met Gly Ser His Glu Val Asn Gly Thr Trp Met Ile His         995 1000 1005 Thr Leu Glu Ala Leu Asp Tyr Lys Glu Cys Glu Trp Pro Leu Thr His    1010 1015 1020 Thr Ile Gly Thr Ser Val Glu Glu Ser Glu Met Phe Met Pro Arg Ser 1025 1030 1035 1040 Ile Gly Gly Pro Val Ser Ser His Asn His Ile Pro Gly Tyr Lys Val                1045 1050 1055 Gln Thr Asn Gly Pro Trp Met Gln Val Pro Leu Glu Val Lys Arg Glu            1060 1065 1070 Ala Cys Pro Gly Thr Ser Val Ile Asp Gly Asn Cys Asp Gly Arg        1075 1080 1085 Gly Lys Ser Thr Arg Ser Thr Thr Asp Ser Gly Lys Val Ile Pro Glu    1090 1095 1100 Trp Cys Cys Arg Ser Cys Thr Met Pro Pro Val Ser Phe His Gly Ser 1105 1110 1115 1120 Asp Gly Cys Trp Tyr Pro Met Glu Ile Arg Pro Arg Lys Thr His Glu                1125 1130 1135 Ser His Leu Val Arg Ser Trp Val Thr Ala Gly Glu Ile His Ala Val            1140 1145 1150 Pro Phe Gly Leu Val Ser Met Met Ile Ala Met Glu Val Val Leu Arg        1155 1160 1165 Lys Arg Gln Gly Pro Lys Gln Met Leu Val Gly Gly Val Val Leu Leu    1170 1175 1180 Gly Ala Met Leu Val Gly Gln Val Thr Leu Leu Asp Leu Leu Lys Leu 1185 1190 1195 1200 Thr Val Ala Val Gly Leu His Phe His Glu Met Asn Asn Gly Gly Asp                1205 1210 1215 Ala Met Tyr Met Ala Leu Ile Ala Ala Phe Ser Ile Arg Pro Gly Leu            1220 1225 1230 Leu Ile Gly Phe Gly Leu Arg Thr Leu Trp Ser Pro Arg Glu Arg Leu        1235 1240 1245 Val Leu Thr Leu Gly Ala Ala Met Val Glu Ile Ala Leu Gly Gly Val    1250 1255 1260 Met Gly Gly Leu Trp Lys Tyr Leu Asn Ala Val Ser Leu Cys Ile Leu 1265 1270 1275 1280 Thr Ile Asn Ala Val Ala Ser Arg Lys Ala Ser Asn Thr Ile Leu Pro                1285 1290 1295 Leu Met Ala Leu Leu Thr Pro Val Thr Met Ala Glu Val Arg Leu Ala            1300 1305 1310 Ala Met Phe Phe Cys Ala Val Val Ile Gly Val Leu His Gln Asn        1315 1320 1325 Phe Lys Asp Thr Ser Met Gln Lys Thr Ile Pro Leu Val Ala Leu Thr    1330 1335 1340 Leu Thr Ser Tyr Leu Gly Leu Thr Gln Pro Phe Leu Gly Leu Cys Ala 1345 1350 1355 1360 Phe Leu Ala Thr Arg Ile Phe Gly Arg Arg Ser Ile Pro Val Asn Glu                1365 1370 1375 Ala Leu Ala Ala Ala Gly Leu Val Gly Val Leu Ala Gly Leu Ala Phe            1380 1385 1390 Gln Glu Met Glu Asn Phe Leu Gly Pro Ile Ala Val Gly Gly Leu Leu        1395 1400 1405 Met Met Leu Val Ser Val Ala Gly Arg Val Asp Gly Leu Glu Leu Lys    1410 1415 1420 Lys Leu Gly Glu Val Ser Trp Glu Glu Glu Ala Glu Ile Ser Gly Ser 1425 1430 1435 1440 Ser Ala Arg Tyr Asp Val Ala Leu Ser Glu Gln Gly Glu Phe Lys Leu                1445 1450 1455 Leu Ser Glu Glu Lys Val Pro Trp Asp Gln Val Val Met Thr Ser Leu            1460 1465 1470 Ala Leu Val Gly Ala Ala Leu His Pro Phe Ala Leu Leu Leu Val Leu        1475 1480 1485 Ala Gly Trp Leu Phe His Val Arg Gly Ala Arg Arg Ser Gly Asp Val    1490 1495 1500 Leu Trp Asp Ile Pro Thr Pro Lys Ile Ile Glu Glu Cys Glu His Leu 1505 1510 1515 1520 Glu Asp Gly Ile Tyr Gly Ile Phe Gln Ser Thr Phe Leu Gly Ala Ser                1525 1530 1535 Gln Arg Gly Val Gly Val Ala Gln Gly Gly Val Phe His Thr Met Trp            1540 1545 1550 His Val Thr Arg Gly Ala Phe Leu Val Arg Asn Gly Lys Lys Leu Ile        1555 1560 1565 Pro Ser Trp Ala Ser Val Lys Glu Asp Leu Val Ala Tyr Gly Gly Ser    1570 1575 1580 Trp Lys Leu Glu Gly Arg Trp Asp Gly Glu Glu Glu Val Gln Leu Ile 1585 1590 1595 1600 Ala Ala Val Pro Gly Lys Asn Val Val Asn Val Gln Thr Lys Pro Ser                1605 1610 1615 Leu Phe Lys Val Arg Asn Gly Gly Glu Ile Gly Ala Val Ala Leu Asp            1620 1625 1630 Tyr Pro Ser Gly Thr Ser Gly Ser Pro Ile Val Asn Arg Asn Gly Glu        1635 1640 1645 Val Ile Gly Leu Tyr Gly Asn Gly Ile Leu Val Gly Asp Asn Ser Phe    1650 1655 1660 Val Ser Ala Ile Ser Gln Thr Glu Val Lys Glu Glu Gly Lys Glu Glu 1665 1670 1675 1680 Leu Gln Glu Ile Pro Thr Met Leu Lys Lys Gly Met Thr Thr Val Leu                1685 1690 1695 Asp Phe His Pro Gly Ala Gly Lys Thr Arg Arg Phe Leu Pro Gln Ile            1700 1705 1710 Leu Ala Glu Cys Ala Arg Arg Arg Leu Arg Thr Leu Val Leu Ala Pro        1715 1720 1725 Thr Arg Val Val Leu Ser Glu Met Lys Glu Ala Phe His Gly Leu Asp    1730 1735 1740 Val Lys Phe His Thr Gln Ala Phe Ser Ala His Gly Ser Gly Arg Glu 1745 1750 1755 1760 Val Ile Asp Ala Met Cys His Ala Thr Leu Thr Tyr Arg Met Leu Glu                1765 1770 1775 Pro Thr Arg Val Val Asn Trp Glu Val Ile Ile Met Asp Glu Ala His            1780 1785 1790 Phe Leu Asp Pro Ala Ser Ile Ala Ala Arg Gly Trp Ala Ala His Arg        1795 1800 1805 Ala Arg Ala Asn Glu Ser Ala Thr Ile Leu Met Thr Ala Thr Pro Pro    1810 1815 1820 Gly Thr Ser Asp Glu Phe Pro His Ser Asn Gly Glu Ile Glu Asp Val 1825 1830 1835 1840 Gln Thr Asp Ile Pro Ser Glu Pro Trp Asn Thr Gly His Asp Trp Ile                1845 1850 1855 Leu Ala Asp Lys Arg Pro Thr Ala Trp Phe Leu Pro Ser Ile Arg Ala            1860 1865 1870 Ala Asn Val Met Ala Ala Ser Leu Arg Lys Ala Gly Lys Ser Val Val        1875 1880 1885 Val Leu Asn Arg Lys Thr Phe Glu Arg Glu Tyr Pro Thr Ile Lys Gln    1890 1895 1900 Lys Lys Pro Asp Phe Ile Leu Ala Thr Asp Ile Ala Glu Met Gly Ala 1905 1910 1915 1920 Asn Leu Cys Val Glu Arg Val Leu Asp Cys Arg Thr Ala Phe Lys Pro                1925 1930 1935 Val Leu Val Asp Glu Gly Arg Lys Val Ala Ile Lys Gly Pro Leu Arg            1940 1945 1950 Ile Ser Ala Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn        1955 1960 1965 Pro Asn Arg Asp Gly Asp Ser Tyr Tyr Tyr Ser Glu Pro Thr Ser Glu    1970 1975 1980 Asn Asn Ala His His Val Cys Trp Leu Glu Ala Ser Met Leu Leu Asp 1985 1990 1995 2000 Asn Met Glu Val Arg Gly Gly Met Val Ala Pro Leu Tyr Gly Val Glu                2005 2010 2015 Gly Thr Lys Thr Pro Val Ser Pro Gly Glu Met Arg Leu Arg Asp Asp            2020 2025 2030 Gln Arg Lys Val Phe Arg Glu Leu Val Arg Asn Cys Asp Leu Pro Val        2035 2040 2045 Trp Leu Ser Trp Gln Val Ala Lys Ala Gly Leu Lys Thr Asn Asp Arg    2050 2055 2060 Lys Trp Cys Phe Glu Gly Pro Glu Glu His Glu Ile Leu Asn Asp Ser 2065 2070 2075 2080 Gly Glu Thr Val Lys Cys Arg Ala Pro Gly Gly Ala Lys Lys Pro Leu                2085 2090 2095 Arg Pro Arg Trp Cys Asp Glu Arg Val Ser Ser Asp Gln Ser Ala Leu            2100 2105 2110 Ser Glu Phe Ile Lys Phe Ala Glu Gly Arg Arg Gly Ala Ala Glu Val        2115 2120 2125 Leu Val Val Leu Ser Glu Leu Pro Asp Phe Leu Ala Lys Lys Gly Gly    2130 2135 2140 Glu Ala Met Asp Thr Ile Ser Val Phe Leu His Ser Glu Glu Gly Ser 2145 2150 2155 2160 Arg Ala Tyr Arg Asn Ala Leu Ser Met Met Pro Glu Ala Met Thr Ile                2165 2170 2175 Val Met Leu Phe Ile Leu Ala Gly Leu Leu Thr Ser Gly Met Val Ile            2180 2185 2190 Phe Phe Met Ser Pro Lys Gly Ile Ser Arg Met Ser Met Ala Met Gly        2195 2200 2205 Thr Met Ala Gly Cys Gly Tyr Leu Met Phe Leu Gly Gly Val Lys Pro    2210 2215 2220 Thr His Ile Ser Tyr Val Met Leu Ile Phe Phe Val Leu Met Val Val 2225 2230 2235 2240 Val Ile Pro Glu Pro Gly Gln Gln Arg Ser Ile Gln Asp Asn Gln Val                2245 2250 2255 Ala Tyr Leu Ile Ile Gly Ile Leu Thr Leu Val Ser Ala Val Ala Ala            2260 2265 2270 Asn Glu Leu Gly Met Leu Glu Lys Thr Lys Glu Asp Leu Phe Gly Lys        2275 2280 2285 Lys Asn Leu Ile Pro Ser Ser Ala Ser Pro Trp Ser Trp Pro Asp Leu    2290 2295 2300 Asp Leu Lys Pro Gly Ala Ala Trp Thr Val Tyr Val Gly Ile Val Thr 2305 2310 2315 2320 Met Leu Ser Pro Met Leu His His Trp Ile Lys Val Glu Tyr Gly Asn                2325 2330 2335 Leu Ser Leu Ser Gly Ile Ala Gln Ser Ala Ser Val Leu Ser Phe Met            2340 2345 2350 Asp Lys Gly Ile Pro Phe Met Lys Met Asn Ile Ser Val Ile Met Leu        2355 2360 2365 Leu Val Ser Gly Trp Asn Ser Ile Thr Val Met Pro Leu Leu Cys Gly    2370 2375 2380 Ile Gly Cys Ala Met Leu His Trp Ser Leu Ile Leu Pro Gly Ile Lys 2385 2390 2395 2400 Ala Gln Gln Ser Lys Leu Ala Gln Arg Arg Val Phe His Gly Val Ala                2405 2410 2415 Glu Asn Pro Val Val Asp Gly Asn Pro Thr Val Asp Ile Glu Glu Ala            2420 2425 2430 Pro Glu Met Pro Ala Leu Tyr Glu Lys Lys Leu Ala Leu Tyr Leu Leu        2435 2440 2445 Leu Ala Leu Ser Leu Ala Ser Val Ala Met Cys Arg Thr Pro Phe Ser    2450 2455 2460 Leu Ala Glu Gly Ile Val Leu Ala Ser Ala Ala Leu Gly Pro Leu Ile 2465 2470 2475 2480 Glu Gly Asn Thr Ser Leu Leu Trp Asn Gly Pro Met Ala Val Ser Met                2485 2490 2495 Thr Gly Val Met Arg Gly Asn His Tyr Ala Phe Val Gly Val Met Tyr            2500 2505 2510 Asn Leu Trp Lys Met Lys Thr Gly Arg Arg Gly Ser Ala Asn Gly Lys        2515 2520 2525 Thr Leu Gly Glu Val Trp Lys Arg Glu Leu Asn Leu Leu Asp Lys Arg    2530 2535 2540 Gln Phe Glu Leu Tyr Lys Arg Thr Asp Ile Val Glu Val Asp Arg Asp 2545 2550 2555 2560 Thr Ala Arg Arg His Leu Ala Glu Gly Lys Val Asp Thr Gly Val Ala                2565 2570 2575 Val Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe His Glu Arg Gly Tyr            2580 2585 2590 Val Lys Leu Glu Gly Arg Val Ile Asp Leu Gly Cys Gly Arg Gly Gly        2595 2600 2605 Trp Cys Tyr Tyr Ala Ala Ala Gln Lys Glu Val Ser Gly Val Lys Gly    2610 2615 2620 Phe Thr Leu Gly Arg Asp Gly His Glu Lys Pro Met Asn Val Gln Ser 2625 2630 2635 2640 Leu Gly Trp Asn Ile Ile Thr Phe Lys Asp Lys Thr Asp Ile His Arg                2645 2650 2655 Leu Glu Pro Val Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser            2660 2665 2670 Ser Ser Ser Ser Val Thr Glu Gly Glu Arg Thr Val Arg Val Leu Asp        2675 2680 2685 Thr Val Glu Lys Trp Leu Ala Cys Gly Val Asp Asn Phe Cys Val Lys    2690 2695 2700 Val Leu Ala Pro Tyr Met Pro Asp Val Leu Glu Lys Leu Glu Leu Leu 2705 2710 2715 2720 Gln Arg Arg Phe Gly Gly Thr Val Ile Arg Asn Pro Leu Ser Arg Asn                2725 2730 2735 Ser Thr His Glu Met Tyr Tyr Val Ser Gly Ala Arg Ser Asn Val Thr            2740 2745 2750 Phe Thr Val Asn Gln Thr Ser Arg Leu Leu Met Arg Arg Met Arg Arg        2755 2760 2765 Pro Thr Gly Lys Val Thr Leu Glu Ala Asp Val Ile Leu Pro Ile Gly    2770 2775 2780 Thr Arg Ser Val Glu Thr Asp Lys Gly Pro Leu Asp Lys Glu Ala Ile 2785 2790 2795 2800 Glu Glu Arg Val Glu Arg Ile Lys Ser Glu Tyr Met Thr Ser Trp Phe                2805 2810 2815 Tyr Asp Asn Asp Asn Pro Tyr Arg Thr Trp His Tyr Cys Gly Ser Tyr            2820 2825 2830 Val Thr Lys Thr Ser Gly Ser Ala Ala Ser Met Val Asn Gly Val Ile        2835 2840 2845 Lys Ile Leu Thr Tyr Pro Trp Asp Arg Ile Glu Glu Val Thr Arg Met    2850 2855 2860 Ala Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu 2865 2870 2875 2880 Lys Val Asp Thr Arg Ala Lys Asp Pro Pro Ala Gly Thr Arg Lys Ile                2885 2890 2895 Met Lys Val Val Asn Arg Trp Leu Phe Arg His Leu Ala Arg Glu Lys            2900 2905 2910 Asn Pro Arg Leu Cys Thr Lys Glu Glu Phe Ile Ala Lys Val Arg Ser        2915 2920 2925 His Ala Ala Ile Gly Ala Tyr Leu Glu Glu Gln Glu Gln Trp Lys Thr    2930 2935 2940 Ala Asn Glu Ala Val Gln Asp Pro Lys Phe Trp Glu Leu Val Asp Glu 2945 2950 2955 2960 Glu Arg Lys Leu His Gln Gln Gly Arg Cys Arg Thr Cys Val Tyr Asn                2965 2970 2975 Met Met Gly Lys Arg Glu Lys Lys Leu Ser Glu Phe Gly Lys Ala Lys            2980 2985 2990 Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Tyr Leu Glu        2995 3000 3005 Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Ala Ser Arg Glu    3010 3015 3020 Asn Ser Gly Gly Gly Val Glu Gly Ile Gly Leu Gln Tyr Leu Gly Tyr 3025 3030 3035 3040 Val Ile Arg Asp Leu Ala Ala Met Asp Gly Gly Gly Phe Tyr Ala Asp                3045 3050 3055 Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Ala Asp Leu Asp Asp            3060 3065 3070 Glu Gln Glu Ile Leu Asn Tyr Met Ser Pro His His Lys Lys Leu Ala        3075 3080 3085 Gln Ala Val Met Glu Met Thr Tyr Lys Asn Lys Val Val Lys Val Leu    3090 3095 3100 Arg Pro Ala Pro Gly Gly Lys Ala Tyr Met Asp Val Ile Ser Arg Arg 3105 3110 3115 3120 Asp Gln Arg Gly Ser Gly Gln Val Val Thr Tyr Ala Leu Asn Thr Ile                3125 3130 3135 Thr Asn Leu Lys Val Gln Leu Ile Arg Met Ala Glu Ala Glu Met Val            3140 3145 3150 Ile His His Gln His Val Gln Asp Cys Asp Glu Ser Val Leu Thr Arg        3155 3160 3165 Leu Glu Ala Trp Leu Thr Glu His Gly Cys Asp Arg Leu Lys Arg Met    3170 3175 3180 Ala Val Ser Gly Asp Asp Cys Val Val Arg Pro Ile Asp Asp Arg Phe 3185 3190 3195 3200 Gly Leu Ala Leu Ser His Leu Asn Ala Met Ser Lys Val Arg Lys Asp                3205 3210 3215 Ile Ser Glu Trp Gln Pro Ser Lys Gly Trp Asn Asp Trp Glu Asn Val            3220 3225 3230 Pro Phe Cys Ser His His Phe His Glu Leu Gln Leu Lys Asp Gly Arg        3235 3240 3245 Arg Ile Val Val Pro Cys Arg Glu Gln Asp Glu Leu Ile Gly Arg Gly    3250 3255 3260 Arg Val Ser Pro Gly Asn Gly Trp Met Ile Lys Glu Thr Ala Cys Leu 3265 3270 3275 3280 Ser Lys Ala Tyr Ala Asn Met Trp Ser Leu Met Tyr Phe His Lys Arg                3285 3290 3295 Asp Met Arg Leu Leu Ser Leu Ala Val Ser Ser Ala Val Pro Thr Ser            3300 3305 3310 Trp Val Pro Gln Gly Arg Thr Thr Trp Ser Ile His Gly Lys Gly Glu        3315 3320 3325 Trp Met Thr Thr Glu Asp Met Leu Glu Val Trp Asn Arg Val Trp Ile    3330 3335 3340 Thr Asn Asn Pro His Met Gln Asp Lys Thr Met Val Lys Lys Trp Arg 3345 3350 3355 3360 Asp Val Pro Tyr Leu Thr Lys Arg Gln Asp Lys Leu Cys Gly Ser Leu                3365 3370 3375 Ile Gly Met Thr Asn Arg Ala Thr Trp Ala Ser His Ile His Leu Val            3380 3385 3390 Ile His Arg Ile Arg Thr Leu Ile Gly Gln Glu Lys Tyr Thr Asp Tyr        3395 3400 3405 Leu Thr Val Met Asp Arg Tyr Ser Val Asp Ala Asp Leu Gln Leu Gly    3410 3415 3420 Glu leu ile 3425 <210> 16 <211> 10910 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct C nucleic acid sequence <400> 16 agtaaatcct gtgtgctaat tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60 acacatttgg attaatttta atcgttcgtt gagcgattag cagagaactg accagaacat 120 gaaaaaccca aaaaagaaat ccggaggatt ccggattgtc aatatgctaa aacgcggagt 180 agcccgtgtg agcccctttg ggggcttgaa gaggctgcca gccggacttc tgctgggtca 240 tgggcccatc aggatggtct tggcgattct agcctttttg agattcacgg caatcaagcc 300 atcactgggt ctcatcaata gatggggttc agttgggaaa aaagaggcta tggaaataat 360 aaagaagttc aagaaagatc tggctgccat gctgagaata atcaatgcta ggaaggagaa 420 gaagagacga ggcgcagata ctagtgtcgg aattgttggc ctcctgctga ccacagctat 480 ggcagcggag gtcactagac gtgggagtgc atactatatg tacttggaca gaaacgatgc 540 tggggaggcc atatcttttc caaccacatt ggggatgaat aagtgttata tacagatcat 600 ggatcttgga cacatgtgtg atgccaccat gagctatgaa tgccctatgc tggatgaggg 660 ggtggaacca gatgacgtcg attgttggtg caacacgacg tcaacttggg ttgtgtacgg 720 aacctgccat cacaaaaaag gtgaagcacg gagatctaga agagctgtga cgctcccctc 780 ccattccact aggaagctgc aaacgcggtc gcaaacctgg ttggaatcaa gagaatacac 840 aaagcacttg attagagtcg aaaattggat attcaggaac cctggcttcg cgttagcagc 900 agctgccatc gcttggcttt tgggaagctc aacgagccaa aaagtcatat acttggtcat 960 gatactgctg attgccccgg catacagcat caggtgcata ggagtcagca atagggactt 1020 tgtggaaggt atgtcaggtg ggacttgggt tgatgttgtc ttggaacatg gaggttgtgt 1080 caccgtaatg gcacaggaca aaccgactgt cgacatagag ctggttacaa caacagtcag 1140 caacatggcg gaggtaagat cctactgcta tgaggcatca atatcagaca tggcttcgga 1200 cagccgctgc ccaacacaag gtgaagccta ccttgacaag caatcagaca ctcaatatgt 1260 ctgcaaaaga acgttagtgg acagaggctg gggaaatgga tgtggacttt ttggcaaagg 1320 gagcctggtg acatgcgcta agtttgcatg ctccaagaaa atgaccggga agagcatcca 1380 gccagagaat ctggagtacc ggataatgct gtcagttcat ggctcccagc acagtgggat 1440 gatcgttaat gacacaggac atgaaactga tgagaataga gcgaaagttg agataacgcc 1500 caattcacca agagccgaag ccaccctggg gggttttgga agcctaggac ttgattgtga 1560 accgaggaca ggccttgact tttcagattt gtattacttg actatgaata acaagcactg 1620 gttggttcac aaggagtggt tccacgacat tccattacct tggcacgctg gggcagacac 1680 cggaactcca cactggaaca acaaagaagc actggtagag ttcaaggacg cacatgccaa 1740 aaggcaaact gtcgtggttc tagggagtca agaaggagca gttcacacgg cccttgctgg 1800 agctctggag gctgagatgg atggtgcaaa gggaaggctg tcctctggcc acttgaaatg 1860 tcgcctgaaa atggataaac ttagattgaa gggcgtgtca tactccttgt gtactgcagc 1920 gttcacattc accaagatcc cggctgaaac actgcacggg acagtcacag tggaggtaca 1980 gtacgcaggg acagatggac cttgcaaggt tccagctcag atggcggtgg acatgcaaac 2040 tctgacccca gttgggaggt tgataaccgc taaccccgta atcactgaaa gcactgagaa 2100 ctctaagatg atgctggaac ttgatccacc atttggggac tcttacattg tcataggagt 2160 cggggagaag aagatcaccc accactggca caggagtggc agcaccattg gaaaagcatt 2220 tgaagccact gtgagaggtg ccaagagaat ggcagtcttg ggagacacag cctgggactt 2280 tggatcagtt ggaggcgctc tcaactcatt gggcaagggc atccatcaaa tttttggagc 2340 agctttcaaa tcattgtttg gaggaatgtc ctggttctca caaattctca ttggaacgtt 2400 gctgatgtgg ttgggtctga acacaaagaa tggatctatt tcccttatgt gcttggcctt 2460 agggggggtg ttgatcttct tatccacagc cgtctctgct gatcaaggat gcgccatcaa 2520 ctttggcaag agagagctca agtgcggaga tggtatcttc atatttagag actctgatga 2580 ctggctgaac aagtactcat actatccaga agatcctgtg aagcttgcat caatagtgaa 2640 agcctctttt gaagaaggga agtgtggcct aaattcagtt gactcccttg agcatgagat 2700 gtggagaagc agggcagatg agatcaatgc catttttgag gaaaacgagg tggacatttc 2760 tgttgtcgtg caggatccaa agaatgttta ccagagagga actcatccat tttccagaat 2820 tcgggatggt ctgcagtatg gttggaagac ttggggtaag aaccttgtgt tctccccagg 2880 gaggaagaat ggaagcttca tcatagatgg aaagtccagg aaagaatgcc cgttttcaaa 2940 ccgggtctgg aattctttcc agatagagga gtttgggacg ggagtgttca ccacacgcgt 3000 gtacatggac gcagtctttg aatacaccat agactgcgat ggatctatct tgggtgcagc 3060 ggtgaacgga aaaaagagtg cccatggctc tccaacattt tggatgggaa gtcatgaagt 3120 aaatgggaca tggatgatcc acaccttgga ggcattagat tacaaggagt gtgagtggcc 3180 actgacacat acgattggaa catcagttga agagagtgaa atgttcatgc cgagatcaat 3240 cggaggccca gttagctctc acaatcatat ccctggatac aaggttcaga cgaacggacc 3300 ttggatgcag gtaccactag aagtgaagag agaagcttgc ccagggacta gcgtgatcat 3360 tgatggcaac tgtgatggac ggggaaaatc aaccagatcc accacggata gcgggaaagt 3420 tattcctgaa tggtgttgcc gctcctgcac aatgccgcct gtgagcttcc atggtagtga 3480 tgggtgttgg tatcccatgg aaattaggcc aaggaaaacg catgaaagcc atctggtgcg 3540 ctcctgggtt acagctggag aaatacatgc tgtccctttt ggtttggtga gcatgatgat 3600 agcaatggaa gtggtcctaa ggaaaagaca gggaccaaag caaatgttgg ttggaggagt 3660 agtgctcttg ggagcaatgc tggtcgggca agtaactctc cttgatttgc tgaaactcac 3720 agtggctgtg ggattgcatt tccatgagat gaacaatgga ggagacgcca tgtatatggc 3780 gttgattgct gccttttcaa tcagaccagg gctgctcatc ggctttgggc tcaggaccct 3840 atggagccct cgggaacgcc ttgtgctgac cctaggagca gccatggtgg agattgcctt 3900 gggtggcgtg atgggcggcc tgtggaagta tctaaatgca gtttctctct gcatcctgac 3960 aataaatgct gttgcttcta ggaaagcatc aaataccatc ttgcccctca tggctctgtt 4020 gacacctgtc actatggctg aggtgagact tgccgcaatg ttcttttgtg ccgtggttat 4080 cataggggtc cttcaccaga atttcaagga cacctccatg cagaagacta tacctctggt 4140 ggccctcaca ctcacatctt acctgggctt gacacaacct tttttgggcc tgtgtgcatt 4200 tctggcaacc cgcatatttg ggcgaaggag tatcccagtg aatgaggcac tcgcagcagc 4260 tggtctagtg ggagtgctgg caggactggc ttttcaggag atggagaact tccttggtcc 4320 gattgcagtt ggaggactcc tgatgatgct ggttagcgtg gctgggaggg tggatgggct 4380 agagctcaag aagcttggtg aagtttcatg ggaagaggag gcggagatca gcgggagttc 4440 cgcccgctat gatgtggcac tcagtgaaca aggggagttc aagctgcttt ctgaagagaa 4500 agtgccatgg gaccaggttg tgatgacctc gctggccttg gttggggctg ccctccatcc 4560 atttgctctt ctgctggtcc ttgctgggtg gctgtttcat gtcaggggag ctaggagaag 4620 tggggatgtc ttgtgggata ttcccactcc taagatcatc gaggaatgtg aacatctgga 4680 ggatgggatt tatggcatat tccagtcaac cttcttgggg gcctcccagc gaggagtggg 4740 agtggcacag ggaggggtgt tccacacaat gtggcatgtc acaagaggag ctttccttgt 4800 caggaatggc aagaagttga ttccatcttg ggcttcagta aaggaagacc ttgtcgccta 4860 tggtggctca tggaagttgg aaggcagatg ggatggagag gaagaggtcc agttgatcgc 4920 ggctgttcca ggaaagaacg tggtcaacgt ccagacaaaa ccgagcttgt tcaaagtgag 4980 gaatggggga gaaatcgggg ctgtcgctct tgactatccg agtggcactt caggatctcc 5040 tattgttaac aggaacggag aggtgattgg gctgtacggc aatggcatcc ttgtcggtga 5100 caactccttc gtgtccgcca tatcccagac tgaggtgaag gaagaaggaa aggaggagct 5160 ccaagagatc ccgacaatgc taaagaaagg aatgacaact gtccttgatt ttcatcctgg 5220 agctgggaag acaagacgtt tcctcccaca gatcttggcc gagtgcgcac ggagacgctt 5280 gcgcactctt gtgttggccc ccaccagggt tgttctttct gaaatgaagg aggcttttca 5340 cggcctggac gtgaaattcc acacacaggc tttttccgct cacggcagcg ggagagaagt 5400 cattgatgcc atgtgccatg ccaccctaac ttacaggatg ttggaaccaa ctagggttgt 5460 taactgggaa gtgatcatta tggatgaagc ccattttttg gatccagcta gcatagccgc 5520 tagaggttgg gcagcgcaca gagctagggc aaatgaaagt gcaacaatct tgatgacagc 5580 cacaccgcct gggactagtg atgaatttcc acattcaaat ggtgaaatag aagatgttca 5640 aacggacata cccagtgagc cctggaacac agggcatgac tggatcctag ctgacaaaag 5700 gcccacggca tggttccttc catccatcag agctgcaaat gtcatggctg cctctttgcg 5760 taaggctgga aagagtgtgg tggtcctgaa caggaaaacc tttgagagag aataccccac 5820 gataaagcag aagaaacctg actttatatt ggccactgac atagctgaaa tgggagccaa 5880 cctttgcgtg gagcgagtgc tggattgcag gacggctttt aagcctgtgc ttgtggatga 5940 agggaggaag gtggcaataa aagggccact tcgtatctcc gcatcctctg ctgctcaaag 6000 gagggggcgc attgggagaa atcccaacag agatggagac tcatactact attctgagcc 6060 tacaagtgaa aataatgccc accacgtctg ctggttggag gcctcaatgc tcttggacaa 6120 catggaggtg aggggtggaa tggtcgcccc actctatggc gttgaaggaa ctaaaacacc 6180 agtttcccct ggtgaaatga gactgaggga tgaccagagg aaagtcttca gagaactagt 6240 gaggaattgt gacctgcccg tttggctttc gtggcaagtg gccaaggctg gtttgaagac 6300 gaatgatcgt aagtggtgtt ttgaaggccc tgaggaacat gagatcttga atgacagcgg 6360 tgaaacagtg aagtgcaggg ctcctggagg agcaaagaag cctctgcgcc caaggtggtg 6420 tgatgaaagg gtgtcatctg accagagtgc gctgtctgaa tttattaagt ttgctgaagg 6480 taggagggga gctgctgaag tgctagttgt gctgagtgaa ctccctgatt tcctggctaa 6540 aaaaggtgga gaggcaatgg ataccatcag tgtgttcctc cactctgagg aaggctctag 6600 ggcttaccgc aatgcactat caatgatgcc tgaggcaatg acaatagtca tgctgtttat 6660 actggctgga ctactgacat cgggaatggt catctttttc atgtctccca aaggcatcag 6720 tagaatgtct atggcgatgg gcacaatggc cggctgtgga tatctcatgt tccttggagg 6780 cgtcaaaccc actcacatct cctatgtcat gctcatattc tttgtcctga tggtggttgt 6840 gatccccgag ccagggcaac aaaggtccat ccaagacaac caagtggcat acctcattat 6900 tggcatcctg acgctggttt cagcggtggc agccaacgag ctaggcatgc tggagaaaac 6960 caaagaggac ctctttggga agaagaactt aattccatct agtgcttcac cctggagttg 7020 gccggatctt gacctgaagc caggagctgc ctggacagtg tacgttggca ttgttacaat 7080 gctctctcca atgttgcacc actggatcaa agtcgaatat ggcaacctgt ctctgtctgg 7140 aatagcccag tcagcctcag tcctttcttt catggacaag gggataccat tcatgaagat 7200 gaatatctcg gtcataatgc tgctggtcag tggctggaat tcaataacag tgatgcctct 7260 gctctgtggc atagggtgcg ccatgctcca ctggtctctc attttacctg gaatcaaagc 7320 gcagcagtca aagcttgcac agagaagggt gttccatggc gttgccgaga accctgtggt 7380 tgatgggaat ccaacagttg acattgagga agctcctgaa atgcctgccc tttatgagaa 7440 gaaactggct ctatatctcc ttcttgctct cagcctagct tctgttgcca tgtgcagaac 7500 gcccttttca ttggctgaag gcattgtcct agcatcagct gccttagggc cgctcataga 7560 gggaaacacc agccttcttt ggaatggacc catggctgtc tccatgacag gagtcatgag 7620 ggggaatcac tatgcttttg tgggagtcat gtacaatcta tggaagatga aaactggacg 7680 ccgggggagc gcgaatggaa aaactttggg tgaagtctgg aagagggaac tgaatctgtt 7740 ggacaagcga cagtttgagt tgtataaaag gaccgacatt gtggaggtgg atcgtgatac 7800 ggcacgcagg catttggccg aagggaaggt ggacaccggg gtggcggtct ccagggggac 7860 cgcaaagtta aggtggttcc atgagcgtgg ctatgtcaag ctggaaggta gggtgattga 7920 cctggggtgt ggccgcggag gctggtgtta ctacgctgct gcgcaaaagg aagtgagtgg 7980 ggtcaaagga tttactcttg gaagagacgg ccatgagaaa cccatgaatg tgcaaagtct 8040 gggatggaac atcatcacct tcaaggacaa aactgatatc caccgcctag aaccagtgaa 8100 atgtgacacc cttttgtgtg acattggaga gtcatcatcg tcatcggtca cagaggggga 8160 aaggaccgtg agagttcttg atactgtaga aaaatggctg gcttgtgggg ttgacaactt 8220 ctgtgtgaag gtgttagctc catacatgcc agatgttctc gagaaactgg aattgctcca 8280 aaggaggttt ggcggaacag tgatcaggaa ccctctctcc aggaattcca ctcatgaaat 8340 gtactacgtg tctggagccc gcagcaatgt cacatttact gtgaaccaaa catcccgcct 8400 cctgatgagg agaatgaggc gtccaactgg aaaagtgacc ctggaggctg acgtcatcct 8460 cccaattggg acacgcagtg ttgagacaga caagggaccc ctggacaaag aggccataga 8520 agaaagggtt gagaggataa aatctgagta catgacctct tggttttatg acaatgacaa 8580 cccctacagg acctggcact actgtggctc ctatgtcaca aaaacctcag gaagtgcggc 8640 gagcatggta aatggtgtta ttaaaattct gacatatcca tgggacagga tagaggaggt 8700 cacaagaatg gcaatgactg acacaacccc ttttggacag caaagagtgt ttaaagaaaa 8760 agttgacacc agagcaaagg atccaccagc gggaactagg aagatcatga aagttgtcaa 8820 caggtggctg ttccgccacc tggccagaga aaagaacccc agactgtgca caaaggaaga 8880 atttattgca aaagtccgaa gtcatgcagc cattggagct tacctggaag aacaagaaca 8940 gtggaagact gccaatgagg ctgtccaaga cccaaagttc tgggaactgg tggatgaaga 9000 aaggaagctg caccaacaag gcaggtgtcg gacttgtgtg tacaacatga tggggaaaag 9060 agagaagaag ctgtcagagt ttgggaaagc aaagggaagc cgtgccatat ggtatatgtg 9120 gctgggagcg cggtatcttg agtttgaggc cctgggattc ctgaatgagg accattgggc 9180 ttccagggaa aactcaggag gaggagtgga aggcattggc ttacaatacc taggatatgt 9240 gatcagagac ctggctgcaa tggatggtgg tggattctac gcggatgaca ccgctggatg 9300 ggacacgcgc atcacagagg cagaccttga tgatgaacag gagatcttga actacatgag 9360 cccacatcac aaaaaactgg cacaagcagt gatggaaatg acatacaaga acaaagtggt 9420 gaaagtgttg agaccagccc caggagggaa agcctacatg gatgtcataa gtcgacgaga 9480 ccagagagga tccgggcagg tagtgactta tgctctgaac accatcacca acttgaaagt 9540 ccaattgatc agaatggcag aagcagagat ggtgatacat caccaacatg ttcaagattg 9600 tgatgaatca gttctgacca ggctggaggc atggctcact gagcacggat gtgacagact 9660 gaagaggatg gcggtgagtg gagacgactg tgtggtccgg cccatcgatg acaggttcgg 9720 cctggccctg tcccatctca acgccatgtc caaggttaga aaggacatat ctgaatggca 9780 gccatcaaaa gggtggaatg attgggagaa tgtgcccttc tgttcccacc acttccatga 9840 actacagctg aaggatggca ggaggattgt ggtgccttgc cgagaacagg acgagctcat 9900 tgggagagga agggtgtctc caggaaacgg ctggatgatc aaggaaacag cttgcctcag 9960 caaagcctat gccaacatgt ggtcactgat gtattttcac aaaagggaca tgaggctact 10020 gtcattggct gtttcctcag ctgttcccac ctcatgggtt ccacaaggac gcacaacatg 10080 gtcgattcat gggaaagggg agtggatgac cacggaagac atgcttgagg tgtggaacag 10140 agtatggata accaacaacc cacacatgca ggacaagaca atggtgaaaa aatggagaga 10200 tgtcccttat ctaaccaaga gacaagacaa gctgtgcgga tcactgattg gaatgaccaa 10260 tagggccacc tgggcctccc acatccattt agtcatccat cgtatccgaa cgctgattgg 10320 acaggagaaa tacactgact acctaacagt catggacagg tattctgtgg atgctgacct 10380 gcaactgggt gagcttatct gaaacaccat ctaacaggaa taaccgggat acaaaccacg 10440 ggtggagaac cggactcccc acaacctgaa accgggatat aaaccacggc tggagaaccg 10500 ggctccgcac ttaaaatgaa acagaaaccg ggataaaaac tacggatgga gaaccggact 10560 ccacacattg agacagaaga agttgtcagc ccagaacccc acacgagttt tgccactgct 10620 aagctgtgag gcagtgcagg ctgggacagc cgacctccag gttgcgaaaa acctggtttc 10680 tgggacctcc caccccagag taaaaagaac ggagcctccg ctaccaccct cccacgtggt 10740 ggtagaaaga cggggtctag aggttagagg agaccctcca gggaacaaat agtgggacca 10800 tattgacgcc agggaaagac cggagtggtt ctctgctttt cctccagagg tctgtgagca 10860 cagtttgctc aagaataagc agacctttgg atgacaaaca caaaaccact 10910 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> T7 primer Forward <400> 17 taatacgact cactatagga gtaaatcctg 30 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> T7 primer Reverse <400> 18 agtggttttg tgtttgtcat c 21 <210> 19 <211> 2076 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 1_chimeric nucleic acid <400> 19 tcccatgatg ttctgactgt gcaattccta attttgggaa tgctgttgat gacgggtgga 60 gcggaggtca ctagacgtgg gagtgcatac tatatgtact tggacagaaa cgatgctggg 120 gaggccatat cttttccaac cacattgggg atgaataagt gttatataca gatcatggat 180 cttggacaca tgtgtgatgc caccatgagc tatgaatgcc ctatgctgga tgagggggtg 240 gaaccagatg acgtcgattg ttggtgcaac acgacgtcaa cttgggttgt gtacggaacc 300 tgccatcaca aaaaaggtga agcacggaga tctagaagag ctgtgacgct cccctcccat 360 tccactagga agctgcaaac gcggtcgcaa acctggttgg aatcaagaga atacacaaag 420 cacttgatta gagtcgaaaa ttggatattc aggaaccctg gcttcgcgtt agcagcagct 480 gccatcgctt ggcttttggg aagctcaacg agccaaaaag tcatatactt ggtcatgata 540 ctgctgattg ccccggcata cagcatcagg tgcataggag tcagcaatag ggactttgtg 600 gaaggtatgt caggtgggac ttgggttgat gttgtcttgg aacatggagg ttgtgtcacc 660 gtaatggcac aggacaaacc gactgtcgac atagagctgg ttacaacaac agtcagcaac 720 atggcggagg taagatccta ctgctatgag gcatcaatat cagacatggc ttcggacagc 780 cgctgcccaa cacaaggtga agcctacctt gacaagcaat cagacactca atatgtctgc 840 aaaagaacgt tagtggacag aggctgggga aatggatgtg gactttttgg caaagggagc 900 ctggtgacat gcgctaagtt tgcatgctcc aagaaaatga ccgggaagag catccagcca 960 gagaatctgg agtaccggat aatgctgtca gttcatggct cccagcacag tgggatgatc 1020 gttaatgaca caggacatga aactgatgag aatagagcga aagttgagat aacgcccaat 1080 tcaccaagag ccgaagccac cctggggggt tttggaagcc taggacttga ttgtgaaccg 1140 aggacaggcc ttgacttttc agatttgtat tacttgacta tgaataacaa gcactggttg 1200 gttcacaagg agtggttcca cgacattcca ttaccttggc acgctggggc agacaccgga 1260 actccacact ggaacaacaa agaagcactg gtagagttca aggacgcaca tgccaaaagg 1320 caaactgtcg tggttctagg gagtcaagaa ggagcagttc acacggccct tgctggagct 1380 ctggaggctg agatggatgg tgcaaaggga aggctgtcct ctggccactt gaaatgtcgc 1440 ctgaaaatgg ataaacttag attgaagggc gtgtcatact ccttgtgtac tgcagcgttc 1500 acattcacca agatcccggc tgaaacactg cacgggacag tcacagtgga ggtacagtac 1560 gcagggacag atggaccttg caaggttcca gctcagatgg cggtggacat gcaaactctg 1620 accccagttg ggaggttgat aaccgctaac cccgtaatca ctgaaagcac tgagaactct 1680 aagatgatgc tggaacttga tccaccattt ggggactctt acattgtcat aggagtcggg 1740 gagaagaaga tcacccacca ctggcacagg agtggcagca ccattggaaa agcatttgaa 1800 gccactgtga gaggtgccaa gagaatggca gtcttgggag acacagcctg ggactttgga 1860 tcagttggag gcgctctcaa ctcattgggc aagggcatcc atcaaatttt tggagcagct 1920 ttcaaatcat tgtttggagg aatgtcctgg ttctcacaaa ttctcattgg aacgttgctg 1980 atgtggttgg gtctgaacac aaagaatgga tctatttccc ttatgtgctt ggccttaggg 2040 ggggtgttga tcttcttatc cacagccgtc tctgct 2076 <210> 20 <211> 2076 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 2_chimeric nucleic acid <400> 20 tcccatgatg ttctgactgt gcaattccta attttgggaa tgctgttgat gacgggtgga 60 gcggaggtca ctagacgtgg gagtgcatac tatatgtact tggacagaaa cgatgctggg 120 gaggccatat cttttccaac cacattgggg atgaataagt gttatataca gatcatggat 180 cttggacaca tgtgtgatgc caccatgagc tatgaatgcc ctatgctgga tgagggggtg 240 gaaccagatg acgtcgattg ttggtgcaac acgacgtcaa cttgggttgt gtacggaacc 300 tgccatcaca aaaaaggtga agcacggaga tctagaagag ctgtgacgct cccctcccat 360 tccactagga agctgcaaac gcggtcgcaa acctggttgg aatcaagaga atacacaaag 420 cacttgatta gagtcgaaaa ttggatattc aggaaccctg gcttcgcgtt agcagcagct 480 gccatcgctt ggcttttggg aagctcaacg agccaaaaag tcatatactt ggtcatgata 540 ctgctgattg ccccggcata cagcatcagg tgcataggag tcagcaatag ggactttgtg 600 gaaggtatgt caggtgggac ttgggttgat gttgtcttgg aacatggagg ttgtgtcacc 660 gtaatggcac aggacaaacc gactgtcgac atagagctgg ttacaacaac agtcagcaac 720 atggcggagg taagatccta ctgctatgag gcatcaatat cagacatggc ttcggacagc 780 cgctgcccaa cacaaggtga agcctacctt gacaagcaat cagacactca atatgtctgc 840 aaaagaacgt tagtggacag aggctgggga aatggatgtg gactttttgg caaagggagc 900 ctggtgacat gcgctaagtt tgcatgctcc aagaaaatga ccgggaagag catccagcca 960 gagaatctgg agtaccggat aatgctgtca gttcatggct cccagcacag tgggatgatc 1020 gttaatgaca caggacatga aactgatgag aatagagcga aagttgagat aacgcccaat 1080 tcaccaagag ccgaagccac cctggggggt tttggaagcc taggacttga ttgtgaaccg 1140 aggacaggcc ttgacttttc agatttgtat tacttgacta tgaataacaa gcactggttg 1200 gttcacaagg agtggttcca cgacattcca ttaccttggc acgctggggc agacaccgga 1260 actccacact ggaacaacaa agaagcactg gtagagttca aggacgcaca tgccaaaagg 1320 caaactgtcg tggttctagg gagtcaagaa ggagcagttc acacggccct tgctggagct 1380 ctggaggctg agatggatgg tgcaaaggga aggctgtcct ctggccactt gaaatgtcgc 1440 ctgaaaatgg ataaacttag attgaagggc gtgtcatact ccttgtgtac tgcagcgttc 1500 acattcacca agatcccggc tgaaacactg cacgggacag tcacagtgga ggtacagtac 1560 gcagggacag atggaccttg caaggttcca gctcagatgg cggtggacat gcaaactctg 1620 accccagttg ggaggttgat aaccgctaac cccgtaatca ctgaaagcac tgagaactct 1680 aagatgatgc tggaacttga tccaccattt ggggactctt acattgtcat aggagtcggg 1740 gagaagaaga tcacccacca ctggcacagg agtggcagca ccattggaaa agcatttgaa 1800 gccactgtga gaggtgccaa gagaatggca gtcttgggag acacagcctg ggactttgga 1860 tcagttggag gcgctctcaa ctcattgggc aagggcatcc atcaaatttt tggagcagct 1920 ttcaaatcat tgtttggagg aatgtcctgg ttctcacaaa ttctcattgg aacgttgctg 1980 atgtggttgg gtctgaacac aaagaatgga tctatttccc ttatgtgctt ggccttaggg 2040 ggggtgttga tcttcttatc cacagccgtt ggggcg 2076 <210> 21 <211> 2070 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 3_chimeric nucleic acid <400> 21 ggcgcagata ctagtgtcgg aattgttggc ctcctgctga ccacagctat ggcagcggag 60 gtcactagac gtgggagtgc atactatatg tacttggaca gaaacgatgc tggggaggcc 120 atatcttttc caaccacatt ggggatgaat aagtgttata tacagatcat ggatcttgga 180 cacatgtgtg atgccaccat gagctatgaa tgccctatgc tggatgaggg ggtggaacca 240 gatgacgtcg attgttggtg caacacgacg tcaacttggg ttgtgtacgg aacctgccat 300 cacaaaaaag gtgaagcacg gagatctaga agagctgtga cgctcccctc ccattccact 360 aggaagctgc aaacgcggtc gcaaacctgg ttggaatcaa gagaatacac aaagcacttg 420 attagagtcg aaaattggat attcaggaac cctggcttcg cgttagcagc agctgccatc 480 gcttggcttt tgggaagctc aacgagccaa aaagtcatat acttggtcat gatactgctg 540 attgccccgg catacagcat caggtgcata ggagtcagca atagggactt tgtggaaggt 600 atgtcaggtg ggacttgggt tgatgttgtc ttggaacatg gaggttgtgt caccgtaatg 660 gcacaggaca aaccgactgt cgacatagag ctggttacaa caacagtcag caacatggcg 720 gaggtaagat cctactgcta tgaggcatca atatcagaca tggcttcgga cagccgctgc 780 ccaacacaag gtgaagccta ccttgacaag caatcagaca ctcaatatgt ctgcaaaaga 840 acgttagtgg acagaggctg gggaaatgga tgtggacttt ttggcaaagg gagcctggtg 900 acatgcgcta agtttgcatg ctccaagaaa atgaccggga agagcatcca gccagagaat 960 ctggagtacc ggataatgct gtcagttcat ggctcccagc acagtgggat gatcgttaat 1020 gacacaggac atgaaactga tgagaataga gcgaaagttg agataacgcc caattcacca 1080 agagccgaag ccaccctggg gggttttgga agcctaggac ttgattgtga accgaggaca 1140 ggccttgact tttcagattt gtattacttg actatgaata acaagcactg gttggttcac 1200 aaggagtggt tccacgacat tccattacct tggcacgctg gggcagacac cggaactcca 1260 cactggaaca acaaagaagc actggtagag ttcaaggacg cacatgccaa aaggcaaact 1320 gtcgtggttc tagggagtca agaaggagca gttcacacgg cccttgctgg agctctggag 1380 gctgagatgg atggtgcaaa gggaaggctg tcctctggcc acttgaaatg tcgcctgaaa 1440 atggataaac ttagattgaa gggcgtgtca tactccttgt gtactgcagc gttcacattc 1500 accaagatcc cggctgaaac actgcacggg acagtcacag tggaggtaca gtacgcaggg 1560 acagatggac cttgcaaggt tccagctcag atggcggtgg acatgcaaac tctgacccca 1620 gttgggaggt tgataaccgc taaccccgta atcactgaaa gcactgagaa ctctaagatg 1680 atgctggaac ttgatccacc atttggggac tcttacattg tcataggagt cggggagaag 1740 aagatcaccc accactggca caggagtggc agcaccattg gaaaagcatt tgaagccact 1800 gtgagaggtg ccaagagaat ggcagtcttg ggagacacag cctgggactt tggatcagtt 1860 ggaggcgctc tcaactcatt gggcaagggc atccatcaaa tttttggagc agctttcaaa 1920 tcattgtttg gaggaatgtc ctggttctca caaattctca ttggaacgtt gctgatgtgg 1980 ttgggtctga acacaaagaa tggatctatt tcccttatgt gcttggcctt agggggggtg 2040 ttgatcttct tatccacagc cgtctctgct 2070 <210> 22 <211> 2076 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 4_chimeric nucleic acid <400> 22 tcccatggcg cagatactag tgtcggaatt gttggcctcc tgctgaccac agctatggca 60 gcggaggtca ctagacgtgg gagtgcatac tatatgtact tggacagaaa cgatgctggg 120 gaggccatat cttttccaac cacattgggg atgaataagt gttatataca gatcatggat 180 cttggacaca tgtgtgatgc caccatgagc tatgaatgcc ctatgctgga tgagggggtg 240 gaaccagatg acgtcgattg ttggtgcaac acgacgtcaa cttgggttgt gtacggaacc 300 tgccatcaca aaaaaggtga agcacggaga tctagaagag ctgtgacgct cccctcccat 360 tccactagga agctgcaaac gcggtcgcaa acctggttgg aatcaagaga atacacaaag 420 cacttgatta gagtcgaaaa ttggatattc aggaaccctg gcttcgcgtt agcagcagct 480 gccatcgctt ggcttttggg aagctcaacg agccaaaaag tcatatactt ggtcatgata 540 ctgctgattg ccccggcata cagcatcagg tgcataggag tcagcaatag ggactttgtg 600 gaaggtatgt caggtgggac ttgggttgat gttgtcttgg aacatggagg ttgtgtcacc 660 gtaatggcac aggacaaacc gactgtcgac atagagctgg ttacaacaac agtcagcaac 720 atggcggagg taagatccta ctgctatgag gcatcaatat cagacatggc ttcggacagc 780 cgctgcccaa cacaaggtga agcctacctt gacaagcaat cagacactca atatgtctgc 840 aaaagaacgt tagtggacag aggctgggga aatggatgtg gactttttgg caaagggagc 900 ctggtgacat gcgctaagtt tgcatgctcc aagaaaatga ccgggaagag catccagcca 960 gagaatctgg agtaccggat aatgctgtca gttcatggct cccagcacag tgggatgatc 1020 gttaatgaca caggacatga aactgatgag aatagagcga aagttgagat aacgcccaat 1080 tcaccaagag ccgaagccac cctggggggt tttggaagcc taggacttga ttgtgaaccg 1140 aggacaggcc ttgacttttc agatttgtat tacttgacta tgaataacaa gcactggttg 1200 gttcacaagg agtggttcca cgacattcca ttaccttggc acgctggggc agacaccgga 1260 actccacact ggaacaacaa agaagcactg gtagagttca aggacgcaca tgccaaaagg 1320 caaactgtcg tggttctagg gagtcaagaa ggagcagttc acacggccct tgctggagct 1380 ctggaggctg agatggatgg tgcaaaggga aggctgtcct ctggccactt gaaatgtcgc 1440 ctgaaaatgg ataaacttag attgaagggc gtgtcatact ccttgtgtac tgcagcgttc 1500 acattcacca agatcccggc tgaaacactg cacgggacag tcacagtgga ggtacagtac 1560 gcagggacag atggaccttg caaggttcca gctcagatgg cggtggacat gcaaactctg 1620 accccagttg ggaggttgat aaccgctaac cccgtaatca ctgaaagcac tgagaactct 1680 aagatgatgc tggaacttga tccaccattt ggggactctt acattgtcat aggagtcggg 1740 gagaagaaga tcacccacca ctggcacagg agtggcagca ccattggaaa agcatttgaa 1800 gccactgtga gaggtgccaa gagaatggca gtcttgggag acacagcctg ggactttgga 1860 tcagttggag gcgctctcaa ctcattgggc aagggcatcc atcaaatttt tggagcagct 1920 ttcaaatcat tgtttggagg aatgtcctgg ttctcacaaa ttctcattgg aacgttgctg 1980 atgtggttgg gtctgaacac aaagaatgga tctatttccc ttatgtgctt ggccttaggg 2040 ggggtgttga tcttcttatc cacagccgtc tctgct 2076 <210> 23 <211> 2080 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 5_chimeric nucleic acid <400> 23 ttcccatgat gttctgactg tgcaattcct aattttggga atgctgttga tgacgggtgg 60 agcagcggag gtcactagac gtgggagtgc atactatatg tacttggaca gaaacgatgc 120 tggggaggcc atatcttttc caaccacatt ggggatgaat aagtgttata tacagatcat 180 ggatcttgga cacatgtgtg atgccaccat gagctatgaa tgccctatgc tggatgaggg 240 ggtggaacca gatgacgtcg attgttggtg caacacgacg tcaacttggg ttgtgtacgg 300 aacctgccat cacaaaaaag gtgaagcacg gagatctaga agagctgtga cgctcccctc 360 ccattccact aggaagctgc aaacgcggtc gcaaacctgg ttggaatcaa gagaatacac 420 aaagcacttg attagagtcg aaaattggat attcaggaac cctggcttcg cgttagcagc 480 agctgccatc gcttggcttt tgggaagctc aacgagccaa aaagtcatat acttggtcat 540 gatactgctg attgccccgg catacagcat caggtgcata ggagtcagca atagggactt 600 tgtggaaggt atgtcaggtg ggacttgggt tgatgttgtc ttggaacatg gaggttgtgt 660 caccgtaatg gcacaggaca aaccgactgt cgacatagag ctggttacaa caacagtcag 720 caacatggcg gaggtaagat cctactgcta tgaggcatca atatcagaca tggcttcgga 780 cagccgctgc ccaacacaag gtgaagccta ccttgacaag caatcagaca ctcaatatgt 840 ctgcaaaaga acgttagtgg acagaggctg gggaaatgga tgtggacttt ttggcaaagg 900 gagcctggtg acatgcgcta agtttgcatg ctccaagaaa atgaccggga agagcatcca 960 gccagagaat ctggagtacc ggataatgct gtcagttcat ggctcccagc acagtgggat 1020 gatcgttaat gacacaggac atgaaactga tgagaataga gcgaaagttg agataacgcc 1080 caattcacca agagccgaag ccaccctggg gggttttgga agcctaggac ttgattgtga 1140 accgaggaca ggccttgact tttcagattt gtattacttg actatgaata acaagcactg 1200 gttggttcac aaggagtggt tccacgacat tccattacct tggcacgctg gggcagacac 1260 cggaactcca cactggaaca acaaagaagc actggtagag ttcaaggacg cacatgccaa 1320 aaggcaaact gtcgtggttc tagggagtca agaaggagca gttcacacgg cccttgctgg 1380 agctctggag gctgagatgg atggtgcaaa gggaaggctg tcctctggcc acttgaaatg 1440 tcgcctgaaa atggataaac ttagattgaa gggcgtgtca tactccttgt gtactgcagc 1500 gttcacattc accaagatcc cggctgaaac actgcacggg acagtcacag tggaggtaca 1560 gtacgcaggg acagatggac cttgcaaggt tccagctcag atggcggtgg acatgcaaac 1620 tctgacccca gttgggaggt tgataaccgc taaccccgta atcactgaaa gcactgagaa 1680 ctctaagatg atgctggaac ttgatccacc atttggggac tcttacattg tcataggagt 1740 cggggagaag aagatcaccc accactggca caggagtggc agcaccattg gaaaagcatt 1800 tgaagccact gtgagaggtg ccaagagaat ggcagtcttg ggagacacag cctgggactt 1860 tggatcagtt ggaggcgctc tcaactcatt gggcaagggc atccatcaaa tttttggagc 1920 agctttcaaa tcattgtttg gaggaatgtc ctggttctca caaattctca ttggaacgtt 1980 gctgatgtgg ttgggtctga acacaaagaa tggatctatt tcccttatgt gcttggcctt 2040 agggggggtg ttgatcttct tatccacagc cgtctctgct 2080 <210> 24 <211> 2076 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 6_chimeric nucleic acid <400> 24 tcccatgatg ttctgactgt gcaattccta attttgggaa tgctgttgat ggctatggca 60 gcggaggtca ctagacgtgg gagtgcatac tatatgtact tggacagaaa cgatgctggg 120 gaggccatat cttttccaac cacattgggg atgaataagt gttatataca gatcatggat 180 cttggacaca tgtgtgatgc caccatgagc tatgaatgcc ctatgctgga tgagggggtg 240 gaaccagatg acgtcgattg ttggtgcaac acgacgtcaa cttgggttgt gtacggaacc 300 tgccatcaca aaaaaggtga agcacggaga tctagaagag ctgtgacgct cccctcccat 360 tccactagga agctgcaaac gcggtcgcaa acctggttgg aatcaagaga atacacaaag 420 cacttgatta gagtcgaaaa ttggatattc aggaaccctg gcttcgcgtt agcagcagct 480 gccatcgctt ggcttttggg aagctcaacg agccaaaaag tcatatactt ggtcatgata 540 ctgctgattg ccccggcata cagcatcagg tgcataggag tcagcaatag ggactttgtg 600 gaaggtatgt caggtgggac ttgggttgat gttgtcttgg aacatggagg ttgtgtcacc 660 gtaatggcac aggacaaacc gactgtcgac atagagctgg ttacaacaac agtcagcaac 720 atggcggagg taagatccta ctgctatgag gcatcaatat cagacatggc ttcggacagc 780 cgctgcccaa cacaaggtga agcctacctt gacaagcaat cagacactca atatgtctgc 840 aaaagaacgt tagtggacag aggctgggga aatggatgtg gactttttgg caaagggagc 900 ctggtgacat gcgctaagtt tgcatgctcc aagaaaatga ccgggaagag catccagcca 960 gagaatctgg agtaccggat aatgctgtca gttcatggct cccagcacag tgggatgatc 1020 gttaatgaca caggacatga aactgatgag aatagagcga aagttgagat aacgcccaat 1080 tcaccaagag ccgaagccac cctggggggt tttggaagcc taggacttga ttgtgaaccg 1140 aggacaggcc ttgacttttc agatttgtat tacttgacta tgaataacaa gcactggttg 1200 gttcacaagg agtggttcca cgacattcca ttaccttggc acgctggggc agacaccgga 1260 actccacact ggaacaacaa agaagcactg gtagagttca aggacgcaca tgccaaaagg 1320 caaactgtcg tggttctagg gagtcaagaa ggagcagttc acacggccct tgctggagct 1380 ctggaggctg agatggatgg tgcaaaggga aggctgtcct ctggccactt gaaatgtcgc 1440 ctgaaaatgg ataaacttag attgaagggc gtgtcatact ccttgtgtac tgcagcgttc 1500 acattcacca agatcccggc tgaaacactg cacgggacag tcacagtgga ggtacagtac 1560 gcagggacag atggaccttg caaggttcca gctcagatgg cggtggacat gcaaactctg 1620 accccagttg ggaggttgat aaccgctaac cccgtaatca ctgaaagcac tgagaactct 1680 aagatgatgc tggaacttga tccaccattt ggggactctt acattgtcat aggagtcggg 1740 gagaagaaga tcacccacca ctggcacagg agtggcagca ccattggaaa agcatttgaa 1800 gccactgtga gaggtgccaa gagaatggca gtcttgggag acacagcctg ggactttgga 1860 tcagttggag gcgctctcaa ctcattgggc aagggcatcc atcaaatttt tggagcagct 1920 ttcaaatcat tgtttggagg aatgtcctgg ttctcacaaa ttctcattgg aacgttgctg 1980 atgtggttgg gtctgaacac aaagaatgga tctatttccc ttatgtgctt ggccttaggg 2040 ggggtgttga tcttcttatc cacagccgtc tctgct 2076 <210> 25 <211> 2070 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 3-B_chimeric nucleic acid <400> 25 ggcgcagata ctagtgtcgg aattgttggc ctcctgctga ccacagctat ggcagcggag 60 gtcactagac gtgggagtgc atactatatg tacttggaca gaaacgatgc tggggaggcc 120 atatcttttc caaccacatt ggggatgaat aagtgttata tacagatcat ggatcttgga 180 cacatgtgtg atgccaccat gagctatgaa tgccctatgc tggatgaggg ggtggaacca 240 gatgacgtcg attgttggtg caacacgacg tcaacttggg ttgtgtacgg aacctgccat 300 cacaaaaaag gtgaagcacg gagatctaga agagctgtga cgctcccctc ccattccact 360 aggaagctgc aaacgcggtc gcaaacctgg ttggaatcaa gagaatacac aaagcacttg 420 attagagtcg aaaattggat attcaggaac cccttttttg cagtgacggc tctgaccatt 480 gcctaccttg tgggaagcaa catgacgcaa cgagtcgtga ttgccctact ggtcttggct 540 gttggtccgg cctactcaat caggtgcata ggagtcagca atagggactt tgtggaaggt 600 atgtcaggtg ggacttgggt tgatgttgtc ttggaacatg gaggttgtgt caccgtaatg 660 gcacaggaca aaccgactgt cgacatagag ctggttacaa caacagtcag caacatggcg 720 gaggtaagat cctactgcta tgaggcatca atatcagaca tggcttcgga cagccgctgc 780 ccaacacaag gtgaagccta ccttgacaag caatcagaca ctcaatatgt ctgcaaaaga 840 acgttagtgg acagaggctg gggaaatgga tgtggacttt ttggcaaagg gagcctggtg 900 acatgcgcta agtttgcatg ctccaagaaa atgaccggga agagcatcca gccagagaat 960 ctggagtacc ggataatgct gtcagttcat ggctcccagc acagtgggat gatcgttaat 1020 gacacaggac atgaaactga tgagaataga gcgaaagttg agataacgcc caattcacca 1080 agagccgaag ccaccctggg gggttttgga agcctaggac ttgattgtga accgaggaca 1140 ggccttgact tttcagattt gtattacttg actatgaata acaagcactg gttggttcac 1200 aaggagtggt tccacgacat tccattacct tggcacgctg gggcagacac cggaactcca 1260 cactggaaca acaaagaagc actggtagag ttcaaggacg cacatgccaa aaggcaaact 1320 gtcgtggttc tagggagtca agaaggagca gttcacacgg cccttgctgg agctctggag 1380 gctgagatgg atggtgcaaa gggaaggctg tcctctggcc acttgaaatg tcgcctgaaa 1440 atggataaac ttagattgaa gggcgtgtca tactccttgt gtactgcagc gttcacattc 1500 accaagatcc cggctgaaac actgcacggg acagtcacag tggaggtaca gtacgcaggg 1560 acagatggac cttgcaaggt tccagctcag atggcggtgg acatgcaaac tctgacccca 1620 gttgggaggt tgataaccgc taaccccgta atcactgaaa gcactgagaa ctctaagatg 1680 atgctggaac ttgatccacc atttggggac tcttacattg tcataggagt cggggagaag 1740 aagatcaccc accactggca caggagtggc agcaccattg gaaaagcatt tgaagccact 1800 gtgagaggtg ccaagagaat ggcagtcttg ggagacacag cctgggactt tggatcagtt 1860 ggaggcgctc tcaactcatt gggcaagggc atccatcaaa tttttggagc agctttcaaa 1920 tcattgtttg gaggattgaa ctggataaca aaggtcatca tgggggcggt acttatatgg 1980 gttggcatca acacaagaaa catgacaatg tccatgagca tgatcttggt aggagtgatc 2040 atgatgtttt tgtctctagg agttggggcg 2070 <210> 26 <211> 2076 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 4-B_chimeric nucleic acid <400> 26 tcccatggcg cagatactag tgtcggaatt gttggcctcc tgctgaccac agctatggca 60 gcggaggtca ctagacgtgg gagtgcatac tatatgtact tggacagaaa cgatgctggg 120 gaggccatat cttttccaac cacattgggg atgaataagt gttatataca gatcatggat 180 cttggacaca tgtgtgatgc caccatgagc tatgaatgcc ctatgctgga tgagggggtg 240 gaaccagatg acgtcgattg ttggtgcaac acgacgtcaa cttgggttgt gtacggaacc 300 tgccatcaca aaaaaggtga agcacggaga tctagaagag ctgtgacgct cccctcccat 360 tccactagga agctgcaaac gcggtcgcaa acctggttgg aatcaagaga atacacaaag 420 cacttgatta gagtcgaaaa ttggatattc aggaacccct tttttgcagt gacggctctg 480 accattgcct accttgtggg aagcaacatg acgcaacgag tcgtgattgc cctactggtc 540 ttggctgttg gtccggccta ctcaatcagg tgcataggag tcagcaatag ggactttgtg 600 gaaggtatgt caggtgggac ttgggttgat gttgtcttgg aacatggagg ttgtgtcacc 660 gtaatggcac aggacaaacc gactgtcgac atagagctgg ttacaacaac agtcagcaac 720 atggcggagg taagatccta ctgctatgag gcatcaatat cagacatggc ttcggacagc 780 cgctgcccaa cacaaggtga agcctacctt gacaagcaat cagacactca atatgtctgc 840 aaaagaacgt tagtggacag aggctgggga aatggatgtg gactttttgg caaagggagc 900 ctggtgacat gcgctaagtt tgcatgctcc aagaaaatga ccgggaagag catccagcca 960 gagaatctgg agtaccggat aatgctgtca gttcatggct cccagcacag tgggatgatc 1020 gttaatgaca caggacatga aactgatgag aatagagcga aagttgagat aacgcccaat 1080 tcaccaagag ccgaagccac cctggggggt tttggaagcc taggacttga ttgtgaaccg 1140 aggacaggcc ttgacttttc agatttgtat tacttgacta tgaataacaa gcactggttg 1200 gttcacaagg agtggttcca cgacattcca ttaccttggc acgctggggc agacaccgga 1260 actccacact ggaacaacaa agaagcactg gtagagttca aggacgcaca tgccaaaagg 1320 caaactgtcg tggttctagg gagtcaagaa ggagcagttc acacggccct tgctggagct 1380 ctggaggctg agatggatgg tgcaaaggga aggctgtcct ctggccactt gaaatgtcgc 1440 ctgaaaatgg ataaacttag attgaagggc gtgtcatact ccttgtgtac tgcagcgttc 1500 acattcacca agatcccggc tgaaacactg cacgggacag tcacagtgga ggtacagtac 1560 gcagggacag atggaccttg caaggttcca gctcagatgg cggtggacat gcaaactctg 1620 accccagttg ggaggttgat aaccgctaac cccgtaatca ctgaaagcac tgagaactct 1680 aagatgatgc tggaacttga tccaccattt ggggactctt acattgtcat aggagtcggg 1740 gagaagaaga tcacccacca ctggcacagg agtggcagca ccattggaaa agcatttgaa 1800 gccactgtga gaggtgccaa gagaatggca gtcttgggag acacagcctg ggactttgga 1860 tcagttggag gcgctctcaa ctcattgggc aagggcatcc atcaaatttt tggagcagct 1920 ttcaaatcat tgtttggagg attgaactgg ataacaaagg tcatcatggg ggcggtactt 1980 atatgggttg gcatcaacac aagaaacatg acaatgtcca tgagcatgat cttggtagga 2040 gtgatcatga tgtttttgtc tctaggagtt ggggcg 2076 <210> 27 <211> 2080 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 5-B_chimeric nucleic acid <400> 27 ttcccatgat gttctgactg tgcaattcct aattttggga atgctgttga tgacgggtgg 60 agcagcggag gtcactagac gtgggagtgc atactatatg tacttggaca gaaacgatgc 120 tggggaggcc atatcttttc caaccacatt ggggatgaat aagtgttata tacagatcat 180 ggatcttgga cacatgtgtg atgccaccat gagctatgaa tgccctatgc tggatgaggg 240 ggtggaacca gatgacgtcg attgttggtg caacacgacg tcaacttggg ttgtgtacgg 300 aacctgccat cacaaaaaag gtgaagcacg gagatctaga agagctgtga cgctcccctc 360 ccattccact aggaagctgc aaacgcggtc gcaaacctgg ttggaatcaa gagaatacac 420 aaagcacttg attagagtcg aaaattggat attcaggaac cctggcttcg cgttagcagc 480 agctgccatc gcttggcttt tgggaagctc aacgagccaa aaagtcatat acttggtcat 540 gatactgctg attgccccgg catacagcat caggtgcata ggagtcagca atagggactt 600 tgtggaaggt atgtcaggtg ggacttgggt tgatgttgtc ttggaacatg gaggttgtgt 660 caccgtaatg gcacaggaca aaccgactgt cgacatagag ctggttacaa caacagtcag 720 caacatggcg gaggtaagat cctactgcta tgaggcatca atatcagaca tggcttcgga 780 cagccgctgc ccaacacaag gtgaagccta ccttgacaag caatcagaca ctcaatatgt 840 ctgcaaaaga acgttagtgg acagaggctg gggaaatgga tgtggacttt ttggcaaagg 900 gagcctggtg acatgcgcta agtttgcatg ctccaagaaa atgaccggga agagcatcca 960 gccagagaat ctggagtacc ggataatgct gtcagttcat ggctcccagc acagtgggat 1020 gatcgttaat gacacaggac atgaaactga tgagaataga gcgaaagttg agataacgcc 1080 caattcacca agagccgaag ccaccctggg gggttttgga agcctaggac ttgattgtga 1140 accgaggaca ggccttgact tttcagattt gtattacttg actatgaata acaagcactg 1200 gttggttcac aaggagtggt tccacgacat tccattacct tggcacgctg gggcagacac 1260 cggaactcca cactggaaca acaaagaagc actggtagag ttcaaggacg cacatgccaa 1320 aaggcaaact gtcgtggttc tagggagtca agaaggagca gttcacacgg cccttgctgg 1380 agctctggag gctgagatgg atggtgcaaa gggaaggctg tcctctggcc acttgaaatg 1440 tcgcctgaaa atggataaac ttagattgaa gggcgtgtca tactccttgt gtactgcagc 1500 gttcacattc accaagatcc cggctgaaac actgcacggg acagtcacag tggaggtaca 1560 gtacgcaggg acagatggac cttgcaaggt tccagctcag atggcggtgg acatgcaaac 1620 tctgacccca gttgggaggt tgataaccgc taaccccgta atcactgaaa gcactgagaa 1680 ctctaagatg atgctggaac ttgatccacc atttggggac tcttacattg tcataggagt 1740 cggggagaag aagatcaccc accactggca caggagtggc agcaccattg gaaaagcatt 1800 tgaagccact gtgagaggtg ccaagagaat ggcagtcttg ggagacacag cctgggactt 1860 tggatcagtt ggaggcgctc tcaactcatt gggcaagggc atccatcaaa tttttggagc 1920 agctttcaaa tcattgtttg gaggattgaa ctggataaca aaggtcatca tgggggcggt 1980 acttatatgg gttggcatca acacaagaaa catgacaatg tccatgagca tgatcttggt 2040 aggagtgatc atgatgtttt tgtctctagg agttggggcg 2080 <210> 28 <211> 2076 <212> DNA <213> Artificial Sequence <220> <223> Chimeric ZIKV- construct 6-B_chimeric nucleic acid <400> 28 tcccatgatg ttctgactgt gcaattccta attttgggaa tgctgttgat ggctatggca 60 gcggaggtca ctagacgtgg gagtgcatac tatatgtact tggacagaaa cgatgctggg 120 gaggccatat cttttccaac cacattgggg atgaataagt gttatataca gatcatggat 180 cttggacaca tgtgtgatgc caccatgagc tatgaatgcc ctatgctgga tgagggggtg 240 gaaccagatg acgtcgattg ttggtgcaac acgacgtcaa cttgggttgt gtacggaacc 300 tgccatcaca aaaaaggtga agcacggaga tctagaagag ctgtgacgct cccctcccat 360 tccactagga agctgcaaac gcggtcgcaa acctggttgg aatcaagaga atacacaaag 420 cacttgatta gagtcgaaaa ttggatattc aggaacccct tttttgcagt gacggctctg 480 accattgcct accttgtggg aagcaacatg acgcaacgag tcgtgattgc cctactggtc 540 ttggctgttg gtccggccta ctcaatcagg tgcataggag tcagcaatag ggactttgtg 600 gaaggtatgt caggtgggac ttgggttgat gttgtcttgg aacatggagg ttgtgtcacc 660 gtaatggcac aggacaaacc gactgtcgac atagagctgg ttacaacaac agtcagcaac 720 atggcggagg taagatccta ctgctatgag gcatcaatat cagacatggc ttcggacagc 780 cgctgcccaa cacaaggtga agcctacctt gacaagcaat cagacactca atatgtctgc 840 aaaagaacgt tagtggacag aggctgggga aatggatgtg gactttttgg caaagggagc 900 ctggtgacat gcgctaagtt tgcatgctcc aagaaaatga ccgggaagag catccagcca 960 gagaatctgg agtaccggat aatgctgtca gttcatggct cccagcacag tgggatgatc 1020 gttaatgaca caggacatga aactgatgag aatagagcga aagttgagat aacgcccaat 1080 tcaccaagag ccgaagccac cctggggggt tttggaagcc taggacttga ttgtgaaccg 1140 aggacaggcc ttgacttttc agatttgtat tacttgacta tgaataacaa gcactggttg 1200 gttcacaagg agtggttcca cgacattcca ttaccttggc acgctggggc agacaccgga 1260 actccacact ggaacaacaa agaagcactg gtagagttca aggacgcaca tgccaaaagg 1320 caaactgtcg tggttctagg gagtcaagaa ggagcagttc acacggccct tgctggagct 1380 ctggaggctg agatggatgg tgcaaaggga aggctgtcct ctggccactt gaaatgtcgc 1440 ctgaaaatgg ataaacttag attgaagggc gtgtcatact ccttgtgtac tgcagcgttc 1500 acattcacca agatcccggc tgaaacactg cacgggacag tcacagtgga ggtacagtac 1560 gcagggacag atggaccttg caaggttcca gctcagatgg cggtggacat gcaaactctg 1620 accccagttg ggaggttgat aaccgctaac cccgtaatca ctgaaagcac tgagaactct 1680 aagatgatgc tggaacttga tccaccattt ggggactctt acattgtcat aggagtcggg 1740 gagaagaaga tcacccacca ctggcacagg agtggcagca ccattggaaa agcatttgaa 1800 gccactgtga gaggtgccaa gagaatggca gtcttgggag acacagcctg ggactttgga 1860 tcagttggag gcgctctcaa ctcattgggc aagggcatcc atcaaatttt tggagcagct 1920 ttcaaatcat tgtttggagg attgaactgg ataacaaagg tcatcatggg ggcggtactt 1980 atatgggttg gcatcaacac aagaaacatg acaatgtcca tgagcatgat cttggtagga 2040 gtgatcatga tgtttttgtc tctaggagtt ggggcg 2076 <210> 29 <211> 10862 <212> DNA <213> Yellow fever virus <400> 29 agtaaatcct gtgtgctaat tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60 acacatttgg attaatttta atcgttcgtt gagcgattag cagagaactg accagaacat 120 gtctggtcgt aaagctcagg gaaaaaccct gggcgtcaat atggtacgac gaggagttcg 180 ctccttgtca aacaaaataa aacaaaaaac aaaacaaatt ggaaacagac ctggaccttc 240 aagaggtgtt caaggattta tctttttctt tttgttcaac attttgactg gaaaaaagat 300 cacagcccac ctaaagaggt tgtggaaaat gctggaccca agacaaggct tggctgttct 360 aaggaaagtc aagagagtgg tggccagttt gatgagagga ttgtcctcaa ggaaacgccg 420 ttcccatgat gttctgactg tgcaattcct aattttggga atgctgttga tgacgggtgg 480 agtgaccttg gtgcggaaaa acagatggtt gctcctaaat gtgacatctg aggacctcgg 540 gaaaacattc tctgtgggca caggcaactg cacaacaaac attttggaag ccaagtactg 600 gtgcccagac tcaatggaat acaactgtcc caatctcagt ccaagagagg agccagatga 660 cattgattgc tggtgctatg gggtggaaaa cgttagagtc gcatatggta agtgtgactc 720 agcaggcagg tctaggaggt caagaagggc cattgacttg cctacgcatg aaaaccatgg 780 tttgaagacc cggcaagaaa aatggatgac tggaagaatg ggtgaaaggc aactccaaaa 840 gattgagaga tggttcgtga ggaacccctt ttttgcagtg acggctctga ccattgccta 900 ccttgtggga agcaacatga cgcaacgagt cgtgattgcc ctactggtct tggctgttgg 960 tccggcctac tcagctcact gcattggaat tactgacagg gatttcattg agggggtgca 1020 tggaggaact tgggtttcag ctaccctgga gcaagacaag tgtgtcactg ttatggcccc 1080 tgacaagcct tcattggaca tctcactaga gacagtagcc attgatagac ctgctgaggt 1140 gaggaaagtg tgttacaatg cagttctcac tcatgtgaag attaatgaca agtgccccag 1200 cactggagag gcccacctag ctgaagagaa cgaaggggac aatgcgtgca agcgcactta 1260 ttctgataga ggctggggca atggctgtgg cctatttggg aaagggagca ttgtggcatg 1320 cgccaaattc acttgtgcca aatccatgag tttgtttgag gttgatcaga ccaaaattca 1380 gtatgtcatc agagcacaat tgcatgtagg ggccaagcag gaaaattgga ataccgacat 1440 taagactctc aagtttgatg ccctgtcagg ctcccaggaa gtcgagttca ttgggtatgg 1500 aaaagctaca ctggaatgcc aggtgcaaac tgcggtggac tttggtaaca gttacatcgc 1560 tgagatggaa acagagagct ggatagtgga cagacagtgg gcccaggact tgaccctgcc 1620 atggcagagt ggaagtggcg gggtgtggag agagatgcat catcttgtcg aatttgaacc 1680 tccgcatgcc gccactatca gagtactggc cctgggaaac caggaaggct ccttgaaaac 1740 agctcttact ggcgcaatga gggttacaaa ggacacaaat gacaacaacc tttacaaact 1800 acatggtgga catgtttctt gcagagtgaa attgtcagct ttgacactca aggggacatc 1860 ctacaaaata tgcactgaca aaatgttttt tgtcaagaac ccaactgaca ctggccatgg 1920 cactgttgtg atgcaggtga aagtgtcaaa aggagccccc tgcaggattc cagtgatagt 1980 agctgatgat cttacagcgg caatcaataa aggcattttg gttacagtta accccatcgc 2040 ctcaaccaat gatgatgaag tgctgattga ggtgaaccca ccttttggag acagctacat 2100 tatcgttggg agaggagatt cacgtctcac ttaccagtgg cacaaagagg gaagctcaat 2160 aggaaagttg ttcactcaga ccatgaaagg cgtggaacgc ctggccgtca tgggagacac 2220 cgcctgggat ttcagctccg ctggagggtt cttcacttcg gttgggaaag gaattcatac 2280 ggtgtttggc tctgcctttc aggggctatt tggcggcttg aactggataa caaaggtcat 2340 catgggggcg gtacttatat gggttggcat caacacaaga aacatgacaa tgtccatgag 2400 catgatcttg gtaggagtga tcatgatgtt tttgtctcta ggagttgggg cggatcaagg 2460 atgcgccatc aactttggca agagagagct caagtgcgga gatggtatct tcatatttag 2520 agactctgat gactggctga acaagtactc atactatcca gaagatcctg tgaagcttgc 2580 atcaatagtg aaagcctctt ttgaagaagg gaagtgtggc ctaaattcag ttgactccct 2640 tgagcatgag atgtggagaa gcagggcaga tgagatcaat gccatttttg aggaaaacga 2700 ggtggacatt tctgttgtcg tgcaggatcc aaagaatgtt taccagagag gaactcatcc 2760 attttccaga attcgggatg gtctgcagta tggttggaag acttggggta agaaccttgt 2820 gttctcccca gggaggaaga atggaagctt catcatagat ggaaagtcca ggaaagaatg 2880 cccgttttca aaccgggtct ggaattcttt ccagatagag gagtttggga cgggagtgtt 2940 caccacacgc gtgtacatgg acgcagtctt tgaatacacc atagactgcg atggatctat 3000 cttgggtgca gcggtgaacg gaaaaaagag tgcccatggc tctccaacat tttggatggg 3060 aagtcatgaa gtaaatggga catggatgat ccacaccttg gaggcattag attacaagga 3120 gtgtgagtgg ccactgacac atacgattgg aacatcagtt gaagagagtg aaatgttcat 3180 gccgagatca atcggaggcc cagttagctc tcacaatcat atccctggat acaaggttca 3240 gacgaacgga ccttggatgc aggtaccact agaagtgaag agagaagctt gcccagggac 3300 tagcgtgatc attgatggca actgtgatgg acggggaaaa tcaaccagat ccaccacgga 3360 tagcgggaaa gttattcctg aatggtgttg ccgctcctgc acaatgccgc ctgtgagctt 3420 ccatggtagt gatgggtgtt ggtatcccat ggaaattagg ccaaggaaaa cgcatgaaag 3480 ccatctggtg cgctcctggg ttacagctgg agaaatacat gctgtccctt ttggtttggt 3540 gagcatgatg atagcaatgg aagtggtcct aaggaaaaga cagggaccaa agcaaatgtt 3600 ggttggagga gtagtgctct tgggagcaat gctggtcggg caagtaactc tccttgattt 3660 gctgaaactc acagtggctg tgggattgca tttccatgag atgaacaatg gaggagacgc 3720 catgtatatg gcgttgattg ctgccttttc aatcagacca gggctgctca tcggctttgg 3780 gctcaggacc ctatggagcc ctcgggaacg ccttgtgctg accctaggag cagccatggt 3840 ggagattgcc ttgggtggcg tgatgggcgg cctgtggaag tatctaaatg cagtttctct 3900 ctgcatcctg acaataaatg ctgttgcttc taggaaagca tcaaatacca tcttgcccct 3960 catggctctg ttgacacctg tcactatggc tgaggtgaga cttgccgcaa tgttcttttg 4020 tgccgtggtt atcatagggg tccttcacca gaatttcaag gacacctcca tgcagaagac 4080 tatacctctg gtggccctca cactcacatc ttacctgggc ttgacacaac cttttttggg 4140 cctgtgtgca tttctggcaa cccgcatatt tgggcgaagg agtatcccag tgaatgaggc 4200 actcgcagca gctggtctag tgggagtgct ggcaggactg gcttttcagg agatggagaa 4260 cttccttggt ccgattgcag ttggaggact cctgatgatg ctggttagcg tggctgggag 4320 ggtggatggg ctagagctca agaagcttgg tgaagtttca tgggaagagg aggcggagat 4380 cagcgggagt tccgcccgct atgatgtggc actcagtgaa caaggggagt tcaagctgct 4440 ttctgaagag aaagtgccat gggaccaggt tgtgatgacc tcgctggcct tggttggggc 4500 tgccctccat ccatttgctc ttctgctggt ccttgctggg tggctgtttc atgtcagggg 4560 agctaggaga agtggggatg tcttgtggga tattcccact cctaagatca tcgaggaatg 4620 tgaacatctg gaggatggga tttatggcat attccagtca accttcttgg gggcctccca 4680 gcgaggagtg ggagtggcac agggaggggt gttccacaca atgtggcatg tcacaagagg 4740 agctttcctt gtcaggaatg gcaagaagtt gattccatct tgggcttcag taaaggaaga 4800 ccttgtcgcc tatggtggct catggaagtt ggaaggcaga tgggatggag aggaagaggt 4860 ccagttgatc gcggctgttc caggaaagaa cgtggtcaac gtccagacaa aaccgagctt 4920 gttcaaagtg aggaatgggg gagaaatcgg ggctgtcgct cttgactatc cgagtggcac 4980 ttcaggatct cctattgtta acaggaacgg agaggtgatt gggctgtacg gcaatggcat 5040 ccttgtcggt gacaactcct tcgtgtccgc catatcccag actgaggtga aggaagaagg 5100 aaaggaggag ctccaagaga tcccgacaat gctaaagaaa ggaatgacaa ctgtccttga 5160 ttttcatcct ggagctggga agacaagacg tttcctccca cagatcttgg ccgagtgcgc 5220 acggagacgc ttgcgcactc ttgtgttggc ccccaccagg gttgttcttt ctgaaatgaa 5280 ggaggctttt cacggcctgg acgtgaaatt ccacacacag gctttttccg ctcacggcag 5340 cgggagagaa gtcattgatg ccatgtgcca tgccacccta acttacagga tgttggaacc 5400 aactagggtt gttaactggg aagtgatcat tatggatgaa gcccattttt tggatccagc 5460 tagcatagcc gctagaggtt gggcagcgca cagagctagg gcaaatgaaa gtgcaacaat 5520 cttgatgaca gccacaccgc ctgggactag tgatgaattt ccacattcaa atggtgaaat 5580 agaagatgtt caaacggaca tacccagtga gccctggaac acagggcatg actggatcct 5640 agctgacaaa aggcccacgg catggttcct tccatccatc agagctgcaa atgtcatggc 5700 tgcctctttg cgtaaggctg gaaagagtgt ggtggtcctg aacaggaaaa cctttgagag 5760 agaatacccc acgataaagc agaagaaacc tgactttata ttggccactg acatagctga 5820 aatgggagcc aacctttgcg tggagcgagt gctggattgc aggacggctt ttaagcctgt 5880 gcttgtggat gaagggagga aggtggcaat aaaagggcca cttcgtatct ccgcatcctc 5940 tgctgctcaa aggagggggc gcattgggag aaatcccaac agagatggag actcatacta 6000 ctattctgag cctacaagtg aaaataatgc ccaccacgtc tgctggttgg aggcctcaat 6060 gctcttggac aacatggagg tgaggggtgg aatggtcgcc ccactctatg gcgttgaagg 6120 aactaaaaca ccagtttccc ctggtgaaat gagactgagg gatgaccaga ggaaagtctt 6180 cagagaacta gtgaggaatt gtgacctgcc cgtttggctt tcgtggcaag tggccaaggc 6240 tggtttgaag acgaatgatc gtaagtggtg ttttgaaggc cctgaggaac atgagatctt 6300 gaatgacagc ggtgaaacag tgaagtgcag ggctcctgga ggagcaaaga agcctctgcg 6360 cccaaggtgg tgtgatgaaa gggtgtcatc tgaccagagt gcgctgtctg aatttattaa 6420 gtttgctgaa ggtaggaggg gagctgctga agtgctagtt gtgctgagtg aactccctga 6480 tttcctggct aaaaaaggtg gagaggcaat ggataccatc agtgtgttcc tccactctga 6540 ggaaggctct agggcttacc gcaatgcact atcaatgatg cctgaggcaa tgacaatagt 6600 catgctgttt atactggctg gactactgac atcgggaatg gtcatctttt tcatgtctcc 6660 caaaggcatc agtagaatgt ctatggcgat gggcacaatg gccggctgtg gatatctcat 6720 gttccttgga ggcgtcaaac ccactcacat ctcctatgtc atgctcatat tctttgtcct 6780 gatggtggtt gtgatccccg agccagggca acaaaggtcc atccaagaca accaagtggc 6840 atacctcatt attggcatcc tgacgctggt ttcagcggtg gcagccaacg agctaggcat 6900 gctggagaaa accaaagagg acctctttgg gaagaagaac ttaattccat ctagtgcttc 6960 accctggagt tggccggatc ttgacctgaa gccaggagct gcctggacag tgtacgttgg 7020 cattgttaca atgctctctc caatgttgca ccactggatc aaagtcgaat atggcaacct 7080 gtctctgtct ggaatagccc agtcagcctc agtcctttct ttcatggaca aggggatacc 7140 attcatgaag atgaatatct cggtcataat gctgctggtc agtggctgga attcaataac 7200 agtgatgcct ctgctctgtg gcatagggtg cgccatgctc cactggtctc tcattttacc 7260 tggaatcaaa gcgcagcagt caaagcttgc acagagaagg gtgttccatg gcgttgccga 7320 gaaccctgtg gttgatggga atccaacagt tgacattgag gaagctcctg aaatgcctgc 7380 cctttatgag aagaaactgg ctctatatct ccttcttgct ctcagcctag cttctgttgc 7440 catgtgcaga acgccctttt cattggctga aggcattgtc ctagcatcag ctgccttagg 7500 gccgctcata gagggaaaca ccagccttct ttggaatgga cccatggctg tctccatgac 7560 aggagtcatg agggggaatc actatgcttt tgtgggagtc atgtacaatc tatggaagat 7620 gaaaactgga cgccggggga gcgcgaatgg aaaaactttg ggtgaagtct ggaagaggga 7680 actgaatctg ttggacaagc gacagtttga gttgtataaa aggaccgaca ttgtggaggt 7740 ggatcgtgat acggcacgca ggcatttggc cgaagggaag gtggacaccg gggtggcggt 7800 ctccaggggg accgcaaagt taaggtggtt ccatgagcgt ggctatgtca agctggaagg 7860 tagggtgatt gacctggggt gtggccgcgg aggctggtgt tactacgctg ctgcgcaaaa 7920 ggaagtgagt ggggtcaaag gatttactct tggaagagac ggccatgaga aacccatgaa 7980 tgtgcaaagt ctgggatgga acatcatcac cttcaaggac aaaactgata tccaccgcct 8040 agaaccagtg aaatgtgaca cccttttgtg tgacattgga gagtcatcat cgtcatcggt 8100 cacagagggg gaaaggaccg tgagagttct tgatactgta gaaaaatggc tggcttgtgg 8160 ggttgacaac ttctgtgtga aggtgttagc tccatacatg ccagatgttc tcgagaaact 8220 ggaattgctc caaaggaggt ttggcggaac agtgatcagg aaccctctct ccaggaattc 8280 cactcatgaa atgtactacg tgtctggagc ccgcagcaat gtcacattta ctgtgaacca 8340 aacatcccgc ctcctgatga ggagaatgag gcgtccaact ggaaaagtga ccctggaggc 8400 tgacgtcatc ctcccaattg ggacacgcag tgttgagaca gacaagggac ccctggacaa 8460 agaggccata gaagaaaggg ttgagaggat aaaatctgag tacatgacct cttggtttta 8520 tgacaatgac aacccctaca ggacctggca ctactgtggc tcctatgtca caaaaacctc 8580 aggaagtgcg gcgagcatgg taaatggtgt tattaaaatt ctgacatatc catgggacag 8640 gatagaggag gtcacaagaa tggcaatgac tgacacaacc ccttttggac agcaaagagt 8700 gtttaaagaa aaagttgaca ccagagcaaa ggatccacca gcgggaacta ggaagatcat 8760 gaaagttgtc aacaggtggc tgttccgcca cctggccaga gaaaagaacc ccagactgtg 8820 cacaaaggaa gaatttattg caaaagtccg aagtcatgca gccattggag cttacctgga 8880 agaacaagaa cagtggaaga ctgccaatga ggctgtccaa gacccaaagt tctgggaact 8940 ggtggatgaa gaaaggaagc tgcaccaaca aggcaggtgt cggacttgtg tgtacaacat 9000 gatggggaaa agagagaaga agctgtcaga gtttgggaaa gcaaagggaa gccgtgccat 9060 atggtatatg tggctgggag cgcggtatct tgagtttgag gccctgggat tcctgaatga 9120 ggaccattgg gcttccaggg aaaactcagg aggaggagtg gaaggcattg gcttacaata 9180 cctaggatat gtgatcagag acctggctgc aatggatggt ggtggattct acgcggatga 9240 caccgctgga tgggacacgc gcatcacaga ggcagacctt gatgatgaac aggagatctt 9300 gaactacatg agcccacatc acaaaaaact ggcacaagca gtgatggaaa tgacatacaa 9360 gaacaaagtg gtgaaagtgt tgagaccagc cccaggaggg aaagcctaca tggatgtcat 9420 aagtcgacga gaccagagag gatccgggca ggtagtgact tatgctctga acaccatcac 9480 caacttgaaa gtccaattga tcagaatggc agaagcagag atggtgatac atcaccaaca 9540 tgttcaagat tgtgatgaat cagttctgac caggctggag gcatggctca ctgagcacgg 9600 atgtgacaga ctgaagagga tggcggtgag tggagacgac tgtgtggtcc ggcccatcga 9660 tgacaggttc ggcctggccc tgtcccatct caacgccatg tccaaggtta gaaaggacat 9720 atctgaatgg cagccatcaa aagggtggaa tgattgggag aatgtgccct tctgttccca 9780 ccacttccat gaactacagc tgaaggatgg caggaggatt gtggtgcctt gccgagaaca 9840 ggacgagctc attgggagag gaagggtgtc tccaggaaac ggctggatga tcaaggaaac 9900 agcttgcctc agcaaagcct atgccaacat gtggtcactg atgtattttc acaaaaggga 9960 catgaggcta ctgtcattgg ctgtttcctc agctgttccc acctcatggg ttccacaagg 10020 acgcacaaca tggtcgattc atgggaaagg ggagtggatg accacggaag acatgcttga 10080 ggtgtggaac agagtatgga taaccaacaa cccacacatg caggacaaga caatggtgaa 10140 aaaatggaga gatgtccctt atctaaccaa gagacaagac aagctgtgcg gatcactgat 10200 tggaatgacc aatagggcca cctgggcctc ccacatccat ttagtcatcc atcgtatccg 10260 aacgctgatt ggacaggaga aatacactga ctacctaaca gtcatggaca ggtattctgt 10320 ggatgctgac ctgcaactgg gtgagcttat ctgaaacacc atctaacagg aataaccggg 10380 atacaaacca cgggtggaga accggactcc ccacaacctg aaaccgggat ataaaccacg 10440 gctggagaac cgggctccgc acttaaaatg aaacagaaac cgggataaaa actacggatg 10500 gagaaccgga ctccacacat tgagacagaa gaagttgtca gcccagaacc ccacacgagt 10560 tttgccactg ctaagctgtg aggcagtgca ggctgggaca gccgacctcc aggttgcgaa 10620 aaacctggtt tctgggacct cccaccccag agtaaaaaga acggagcctc cgctaccacc 10680 ctcccacgtg gtggtagaaa gacggggtct agaggttaga ggagaccctc cagggaacaa 10740 atagtgggac catattgacg ccagggaaag accggagtgg ttctctgctt ttcctccaga 10800 ggtctgtgag cacagtttgc tcaagaataa gcagaccttt ggatgacaaa cacaaaacca 10860 ct 10862

Claims (30)

Induces an immune response to Zika virus infection,
I) a gene encoding a signal peptide existing upstream of a gene encoding a pre-membrane (prM) protein of a yellow fever virus 17D vaccine strain nucleic acid molecule; ii) genes encoding pre-membrane (prM) and its transmembrane proteins; And iii) inserted into the gene region encoding envelope (E) and its transmembrane protein in place of the yellow fever virus 17D vaccine strain gene,
I) a gene encoding a signal peptide existing upstream of a gene encoding a pre-membrane (prM) protein of Zika virus; ii) genes encoding pre-membrane (prM) and its transmembrane proteins; And iii) a gene encoding the envelope (E) and its transmembrane protein, wherein some or all of the nucleic acids constituting the genes i), ii), and iii) are substituted, deleted, or inserted, Nucleic Acid Molecules. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is any one of the following sequences, and includes both a nucleic acid derived from a yellow fever virus 17D vaccine strain and a Zika virus derived nucleic acid.
a) the nucleic acid molecule of SEQ ID NO: 19;
b) the nucleic acid molecule of SEQ ID NO: 20;
c) the nucleic acid molecule of SEQ ID NO: 21;
d) the nucleic acid molecule of SEQ ID NO: 22;
e) the nucleic acid molecule of SEQ ID NO: 23; or
f) the nucleic acid molecule of SEQ ID NO: 24. The chimeric nucleic acid molecule of claim 1, wherein the chimeric nucleic acid molecule is any one of the following sequences.
a) the nucleic acid molecule of SEQ ID NO: 25;
b) the nucleic acid molecule of SEQ ID NO: 26;
c) the nucleic acid molecule of SEQ ID NO: 27; or
d) the nucleic acid molecule of SEQ ID NO: 28. An immunogenic composition comprising a chimeric nucleic acid molecule consisting of any one of SEQ ID NOs: 19 to 25. Chimeric Zika virus formed by introducing a Zika virus antigen gene into the gene sequence of the attenuated yellow fever virus of SEQ ID NO: 29. The method of claim 5, wherein the Zika virus antigen gene is i) pre-membrane (prM) protein and its transmembrane protein gene, ii) envelope (E) protein and its transmembrane protein gene, and iii) capsid (C) protein Chimeric Zika virus, characterized in that consisting of the gene. The method according to claim 5, wherein the chimeric Zika virus is chimeric Zika virus for inducing an immune response to Zika virus,
I) pre-membrane (prM) protein and its transmembrane protein region of attenuated yellow fever virus, and ii) the envelope (E) protein and its transmembrane protein region
A chimeric zika virus, characterized in that it is substituted from the i) pre-membrane (prM) protein and its transmembrane protein region of wild-type Zika virus, and ii) the envelope (E) protein and its transmembrane protein region. The method of claim 5,
The chimeric Zika virus is chimeric Zika virus for inducing an immune response to Zika virus,
The i) pre-membrane (prM) protein region of the attenuated yellow fever virus is replaced with an amino acid sequence derived from the pre-membrane (prM) protein region of the wild-type Zika virus, and ii) the envelope (E) of the attenuated yellow fever virus. The protein region is substituted with an amino acid sequence derived from the envelope (E) protein region of wild-type Zika virus,
Wherein the i) transmembrane protein region of the pre-membrane (prM) protein region and ii) the transmembrane protein region of the envelope (E) protein are each derived from an attenuated yellow fever virus. The method of claim 7, wherein
The chimeric Zika virus chimeric Zika virus, characterized in that containing the capsid (C) protein of the wild type Zika virus. The method of claim 7, wherein
The chimeric Zika virus, characterized in that consisting of the amino acid sequence of SEQ ID NO: 1, chimeric Zika virus. The method of claim 8,
The chimeric Zika virus, characterized in that consisting of the amino acid sequence of SEQ ID NO: 13, chimeric Zika virus. The chimeric Zika virus according to claim 10, wherein the chimeric Zika virus is located at position 792 in the amino acid sequence of SEQ ID NO: 1 from S to G. The chimeric Zika virus of claim 10, wherein the chimeric Zika virus is substituted with GADTSVGIVGLLLTTAMA at SHDVLTVQFLILGMLLMTGG at positions 102-121 in the amino acid sequence of SEQ ID NO: 1. The chimeric Zika virus according to claim 10, wherein the chimeric Zika virus has a sequence of 104-121 amino acid positions in the amino acid sequence of SEQ ID NO: 1 replaced by GADTSVGIVGLLLTTAMA in DVLTVQFLILGMLLMTGG. The chimeric Zika virus according to claim 10, wherein the chimeric Zika virus is caused by the insertion of one or more amino acids after the G amino acid which is the amino acid at position 121 in the amino acid sequence of SEQ ID NO: 1. 16. The chimeric zika virus according to claim 15, wherein the inserted amino acid is one and amino acid A. The chimeric Zika virus according to claim 10, wherein the chimeric Zika virus has a substitution induced in TGG which is an amino acid at positions 119-121 in the amino acid sequence of SEQ ID NO: 1. 18. The chimeric Zika virus according to claim 17, wherein the chimeric Zika virus is TGG which is an amino acid at positions 119-121 in the amino acid sequence of SEQ ID NO: 1. A chimeric Zika virus of any one of claims 5 to 17, which is administered to the animal to induce immunity to Zika virus in the body of the animal; And
Zikavirus immunogenic composition comprising a pharmaceutically acceptable excipient. 20. The zikavirus immunogenicity of claim 19, wherein the pharmaceutically acceptable excipient is at least one selected from the group consisting of water, PBS buffer solution, glycine solution, D-trehalose dehydrate, lactose, and sorbitol. Composition. 20. The Zikavirus immunogenic composition according to claim 19, wherein said animal is a human. 20. The Zikavirus immunogenic composition of claim 19, wherein the Zikavirus immunogenic composition is administered via an intramuscular route, an intradermal, mucosal or subcutaneous route. 20. The method of claim 19, wherein said immunogenic composition is used to prevent Zika virus infection or Guillain-Barr.

Zikavirus immunogenic composition for use in the prevention of syndrome, microcephaly, neuropathy, congenital malformations. In a method for producing an immunogenic composition comprising a chimeric Zika virus,
Preparing the attenuated yellow fever virus 17D vaccine strain;
Preparing a 17D-Zika chimeric genome by replacing the nucleotide sequence encoding the structural protein of the yellow fever virus 17D vaccine strain with the nucleotide sequence encoding the structural protein of the wild-type Zika virus;
Cloning the dielectric into a vector;
Introducing the cloned vector into competent cells to transform the cloned vector; And
Recovering viral DNA from the competent cells. The method of claim 24, wherein the cloning into the vector comprises forming a pCC1-chimeric zikavirus vector using the pCC1 vector as a vector. 25. The method of claim 24, wherein said 17D-Zika chimeric genome has any one base selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. . 25. The method of claim 24, wherein the pCC1-chimeric zikavirus vector comprises a restriction enzyme AscI coding sequence (5'-GGCGCGCC) at the 5 'end in sequence; T7 promoter sequence (TAATACGACTCACTATA); transcription start codon (GG); Nucleic acids of any one chimeric zika virus selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16; Method 3, characterized in that the restriction enzyme SmaI-NotI coding sequence (3'-GGGCCCCGCCGGCG) is included at the end. A method for preparing chimeric zika virus having immunogenicity against Zika virus infection, comprising the following steps.
(a) preparing a YF 17D genome which is an attenuated yellow fever virus vaccine strain having SEQ ID NO: 29; And
(b) replacing the nucleic acid at the positions 482 to 2452 of the nucleotide sequence of the YF 17D genome of SEQ ID NO: 29 with the nucleotide sequence of any one of SEQ ID NOs: 19 to 28 to prepare a chimeric Zika virus gene. The method of claim 28, wherein
(c) adding a T7 promoter sequence (TAATACGACTCACTATA) and a transcription start codon (GG) to the 5 'end of the chimeric Zika virus gene prepared in step (b), and ascI (5`-GGCGCGCC) restriction enzymes at both ends; And SmaI-NotI (3'-GGGCCCCGCCGGCG), respectively, further comprising preparing for cloning into a vector. The method of claim 29, wherein the vector is a pCC1 vector.

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