WO2017044507A2 - Sirna/nanoparticle formulations for treatment of middle-east respiratory syndrome coronaviral infection - Google Patents

Abstract

The present invention relates to compositions and methods for siRNA therapeutics for prevention and treatment of Middle East Respiratory Syndrome Corona Virus (MERS-CoV) infections. The compositions include a pharmaceutical composition comprising siRNA cocktails that target viral genes and pharmaceutically acceptable polymeric nanoparticle carriers and liposomal nanoparticle carriers.

Description

s iRN A ANOPARTICLE FORMULATIONS FOR TREATMENT OF

MIDDLE-EAST RESPIRATORY SYNDROME C ORON AVIRAL INFECTION

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent

Application No. 62/215,565, filed September 8, 2015, which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The present invention provides a pharmaceutical product composition of matter comprising siRNA sequences targeting genes or single-stranded viral RNAs of Middle-East Respiratory Syndrome Corona Virus (MERS-CoV), and nanoparticle carrier systems such as Histidine-Lysine co-polymers (HKP), or Spermine-Liposome conjugates (SLiC), or a lung tissue targeted moiety, such as a peptide, a nucleotide, a small molecule, and an antibody. The present invention also involves in methods of use for this pharmaceutical product, including formulations of siRNA/nanoparticle carrier, their process development and specific delivery routes and regimens. This invention presents a novel therapeutic agent for treatment of MERS-CoV infection.

BACKGROUND

MERS-CoV Virus Disease: Biology and Pathology

Middle East respiratory syndrome (MERS) is a highly lethal respiratory disease caused by a novel single-stranded, positive-sense RNA betacoronavirus, MERS-CoV. Dromedary camels, hosts for MERS-CoV, are implicated in direct or indirect transmission to human beings, although the exact mode of transmission is unknown. Recent studies support that camels serve as the primary source of the MERS-CoV infecting humans, while bats may be the ultimate reservoir of the virus. The virus was first isolated from a patient who died from a severe respiratory illness in June, 2012, in Jeddah, Saudi Arabia. As of May 31, 2015, 1180 laboratory-confirmed cases (483 deaths; 40% mortality) have been reported to WHO {Zumbla A. et al. 2015). The Centers for Disease Control and Prevention (CDC) has labelled it as a transmissible disease from human-to-humans. (Jalal S. 2015). Although most cases of MERS have occurred in Saudi Arabia and the United Arab Emirates, cases have been reported in Europe, the USA, and Asia in people who travelled from the Middle East or their contacts. Clinical features of MERS range from asymptomatic or mild disease to acute respiratory distress syndrome and multiorgan failure, resulting in death, especially in individuals with underlying comorbidities. No specific drug treatment exists for MERS and infection prevention, and control measures are crucial to prevent spread in health-care facilities {Zumbla A. Et al 2015). Clinical severity of the disease observed in humans may be explained the ability of MERS-CoV to replicate in the lower respiratory tract (de Wit E, et al. 2013) and is also related to MERS-CoV's ability to infect a broad range of cells with dipeptidyl peptidase 4 receptor (DPP4) expression, evade the host innate immune response, and induce cytokine dysregulation (Chan JF, 2015).

MERS-CoV is an enveloped single-stranded positive sense RNA virus with a genome of 30, 119 nt. The genome structure of MERS-CoV is similar to other coronaviruses, with the 5' two-thirds of the genome encoding the non- structural proteins (NSPs) required for viral genome replication, the remaining 3' third of the genome encoding the structural genes that make up the virion (spike, envelope, membrane, and nucleocapsid proteins), and four accessory genes interspersed within the structural gene region. At the 5' end of the genome, there is a leader sequence (67nt), which is followed by an untranslated region (UTR). At the 3' end of the RNA genome there is another UTR, followed by a poly (A) sequence of variable length. Transcription-regulatory sequences (TRS 5' AACGAA 3') are found at the 3' end of the leader sequence and at different positions upstream of genes in the genomic 3' -proximal domain of MERS-CoV. The MERS-CoV genome contains at least 10 predicted open reading frames (ORFs): ORFla, ORFlb, S, 3, 4a, 4b, 5, E, M and N with sixteen predicted nonstructural proteins being encoded by ORF 1 a/b . Several unique group-specific ORFs that are not essential for virus replication are encoded by MERS-CoV. The functions of these group-specific ORFs are unknown; however, by analogy to other coronaviruses, they may encode structural proteins or interferon antagonist genes (Totura AL, Baric RS, 2012). Open reading frames ORF2, -6, - 7 and -8a are translated from subgenomic mRNAs predicted to encode the four canonical structural genes: a 180/90-kDa spike glycoprotein (S), a ~ 23-kDa membrane glycoprotein (M), a small envelope protein (E) and a ~ 50-kDa nucleocapsidprotein (N), respectively (Abdel- Moneim AS. 2014).

Similar to other RNA viruses, coronavirus replicate in the host cytoplasm. The replication process is initiated by the viral particle after binding with specific cellular receptors, known as S-protein mediated binding. The receptor for MERS-CoV was recently identified as dipeptidyl peptidase 4 (DDP4, also known as CD26), a protein with diverse functions in glucose homeostasis, T-cell activation, neurotransmitter function, and modulation of cardiac signaling. DPP4 is expressed in a variety of cell types, including endothelial cells (kidney, lung, small intestine, spleen) hepatocytes, enterocytes, activated leukocytes, testes, prostate and cells of the renal glomeruli and proximal tubules. DPP4 recognition is mediated by the receptor- binding domain (RBD, amino acids E367- Y606) (PascalK, etal. 2015). Following virus entry, the coronavirus genome, a positive sense, capped and polyadeny!ated RNA strand, is directly translated, resulting in the synthesis of coronavirus replicase gene-encoded NSPs. Coronavirus NSPs are translated as two large polyproteins harboring proteolytic enzymes, namely papain- like and chymotrypsin-iike proteinases that extensively process coronavirus polyproteins to liberate up to 16 NSPs (nsp 1-16) (Lundin A. et al. 2014). After entering into the cell, the virus specially modulates the innate immune response, antigen presentation, and mitogen-activated protein kinase.

Current Prophylaxis and Therapeutics

Although the emergence of highly pathogenic MERS-CoV highlights an urgent need for potent therapeutic and prophylactic agents, no approved antiviral treatments for any human coronavirus infections are currently available. Supportive treatment with extracorporeal membrane oxygenation and dialysis is often required in patients with organ failure. Recently, tremendous efforts have been made in the search for an effective anti-MERS-CoV agent, and a number of antiviral agents have been identified. For example, some compounds with inhibitory activities in the low micromolar range on MERS-CoV replication in cell cultures have been identified from the libraries of FDA-approved drugs, de Wilde AH. and colleges identified four compounds (chloroquine, chlorpromazine, loperamide, and lopinavir) inhibiting MERS-CoV replication in the low-micromolar range (50% effective concentrations [EC(50)s], 3 to 8 LIM ) (de Wilde AH el al. 2014).

Antivirals with potent in vitro activities also include neutralizing monoclonal antibodies, antiviral peptides, interferons, mycophenolic acid. It was reported that rhesus macaques treated with a cocktail of IFN-a2b with ribavirin, a nucleoside analog, exhibited reduced MERS-CoV replication and an improved clinical outcome (Falzarano D, et al. 2013). Lu L. and colleges designed and synthesized a peptide (HR2P) derived from the HR2 domain in the S2 subunit of the spike (S) protein of the MERS-CoV EMC/2012 strain. They found that HR2P could bind with the HRl domain to form a stable six-helix bundle and thus inhibit viral fusion core formation and S protein- mediated cell-cell fusion. HR2P was demonstrated to potently inhibit infection by both pseudotyped and live MERS-CoV in different cell lines. After modification of the HR2P peptide by introducing Glu (E) and Lys (K) residues at the i to i+4 or i to i+3 arrangements, it was found that one of these HR2P analogous peptides, HR2P-M2, exhibited significantly improved stability, solubility and antiviral activity. HR2P-M2 peptide could potently inhibit infection by pseudoviruses expressing MERS-CoV S protein with or without mutation in the HR1 region, suggesting that it could be effective against most currently available MERS-CoV mutants. It was demonstrated that the HR2P-M2 peptide administered via the intranasal route could protect Ad5-hDPP4-transduced mice from challenge by MERS- CoV strains with or without mutations in the HR1 region, indicating that this peptide could be used as a nasal spray to protect high-risk populations, including healthcare workers, MERS patients' family members, and those having close contacts with the patients, from MERS-CoV infection. Intranasal application of the peptide to MERS-CoV-infected patients may suppress viral replication in epithelial cells of the respiratory tract and thus reduce the release of virions, thereby preventing the spreading of MERS-CoV to other people (Lu L. et al. 2015).

Another approach is passive administration of sera from convalescent human MERS patients or other animals to exposed or infected patients. The vast majority of camels in the Middle East have been infected with MERS-CoV, and some contain high titers of antibody to the virus. It was shown that this antibody is protective if delivered either prophylactically or therapeutically to mice infected with MERS-CoV, indicating that this may be a useful intervention in infected patients {Zhao J et al. 2015) .

In April 2014, three studies conducted by separate laboratories around the world reported the development of fully human neutralizing mAbs against MERS-CoV. All these mAbs target the RBD (receptor-binding domain) of the MERS-CoV SI glycoprotein and they were identified from non-immune human antibody libraries. Among these antibodies, three highly potent mAbs (m336, m337, m338) were identified from a very large phage-displayed antibody Fab library that was generated by using B cells from the blood of 40 healthy donors. This library was panned against recombinant MERS-CoV RBD to enrich for high affinity binders. The three identified mAbs, all derived from the VH gene 1-69, which has been the source of many other antiviral antibodies, exhibited exceptionally potent activity and neutralized pseudotyped MERS-CoV with 50% inhibitory concentration (IC50), ranging from 0.005 to 0.017 mg/ml. The most potent mAb, m336, inhibited >90% MERS-CoV pseudovirus infection (IC90) in DPP4- expressing Huh-7 cells at a concentration of 0.039 mg/ml. Similarly, m336 showed the most potent live MERS-CoV neutralizing activity in inhibiting the formation of MERS- CoV-induced CPE during live MERS-CoV infection of permissive Vero E6 cells, with an IC50 of 0.07 mg/ml.

In vivo studies have shown that this mAb is very effective in protecting MERS-CoV- susceptible animals from viral challenge (unpublished data), suggesting that the m336m mAb is a very promising drug candidate for the urgent treatment of MERS-CoV-infected patients (Tianlei Ying et al. 2015). Lu L. et colleges performed in vitro studies demonstrating that the combination of HR2P-M2 peptide with m336 mAb exhibited a strong synergistic effect against MERS-CoV infection (unpublished data). This observation suggests that intranasal administration of HR2P-M2 peptide combined with intravenous administration of m336 mAb may be a powerful strategy for treatment of MERS patients {Lu L. et al. 2015).

Jiang and colleges also identified two potent RBD-specific neutralizing mAbs, MERS-4 and MERS-27, by using a non-immune yeast-displayed scFv library to screen against the recombinant MERS-CoV RBD. The most potent mAb, MERS-4, neutralized the pseudotyped MERS-CoV infection in DPP4- expressing Huh-7 cells with an IC50 of 0.056 mg/ml and inhibited the formation of MERS-CoV-induced CPE during live MERS-CoV infection of permissive Vero E6 cells with an IC50 of 0.5 mg/ml. Tang et colleges identified neutralizing mAbs by using a non-immune phage-displayed scFv library. The panning was performed by sequentially using MERS-CoV spike-containing paramagnetic proteoliposomes and MERS- CoV S glycoprotein-expressing 293T cells as antigens. A panel of 7 anti-Si scFvs was identified and expressed in both scFv-Fc and IgGl formats, and their neutralizing activity against pseudotyped MERS-CoV in DPP4-expressing 293T cells, as well as live MERS-CoV infection in Vero cells, was measured. The most potent antibody, 3B11, neutralized live MERS-CoV in the plaque reduction neutralization tests with an IC50 of 1.83 mg/ml and 3.50 mg/ml in the scFv-Fc and IgG format, respectively {Tianlei Ying et al. 2015).

Fully Human Antibody and Humanized Mouse Model

Pascal K. and colleges used the Veloclmmune platform (a mouse that expresses human antibody-variable heavy chains and κ light chains) to generate a panel of fully human, noncompeting monoclonal antibodies that bind to MERS-CoV S protein and inhibit entry into target cells. It was showed that two of these antibodies (REGN3051 and REGN3048) can potently neutralize pseudoparticles generated with all clinical MERS-CoV S RBD variants isolated to date. Authors demonstrated that the fully human Veloclmmune antibodies neutralize infectious MERS-CoV significantly more efficient than published monoclonals isolated using traditional methods. They also developed a novel humanized model for MERS-CoV infection. They replaced the 79 kb of the mouse Dpp4 gene with 82 kb of its human ortholog. The resulting mice express fully human DPP4 under the control of the mouse regulatory elements, to preserve the proper expression regulation and protein tissue distribution and showed that these antibodies can prevent and treat MERS-CoV infection in vivo (Pascal KE et al. 2015). Coronaviruses

Coronaviruses are enveloped viruses and their positive strand RNA genome, the largest of all RNA viruses, encodes for as many as 16 non-structural proteins (NSPs), 4 major structural proteins, and up to 8 accessory proteins. Many of these proteins provide essential, frequently enzymatic, functions during the viral life cycle, such as coronaviais protease or RNA-dependent RNA polymerase (RdRp) activities. For example, the spike (S) protein mediates binding of different HCoVs to their specific cellular receptors, an event associated with preferential virus tropism for either ciliated or non-ciliated cells of the airway epithelium. The S protein also mediates fusion between lipids of the viral envelope and the host cell plasma membrane or membranes of endocytic vesicles to promote delivery of viral genomic RNA into the cytoplasm . Following virus entry, the coronavirus genome, a positive sense, capped and polyadenylated RNA strand, is directly translated, resulting in the synthesis of coronaviais replicase gene-encoded NSPs. Coronavirus NSPs are translated as two large polyproteins harboring proteolytic enzymes, namely papain-like and chymotrypsin-like proteinases that extensively process coronavirus polyproteins to liberate up to 16 NSPs (nsp 1-16), These proteolytic functions are considered essential for coronavirus replication. Likewise, the coronavirus RdRp activities, which reside in nsp8 and nsp 12, are considered essential for coronaviais replication. Coronaviruses encode an array of RNA -processing enzymes. These include a helicase activity linked to an NTPase activity in nspl3, a 3'-5'-exonuclease activity linked to a ^'N7-methy!transf erase activity in nspl4, an endonuclease activity in nspl 5, and a 2'~ O-methyltransferase activity in nsp 16. Like all positive strand RNA viruses, coronaviruses synthesize viral RNA at organelle- like structures in order to compartmentalize this critical step of the viral life cycle to a specialized environment that is enriched in replicative viral and host-cell factors, and at the same time protected from antiviral host defense mechanisms. There is now a growing body of knowledge concerning the involvement, rearrangement and requirement of cellular membranes for RNA synthesis of a number of positive-stra d RNA viruses, including coronaviruses. Three coronaviral NSPs, i .e., nsp3, nsp4, and nsp6 are thought to participate in formation of these sites for viral RNA synthesis. In particular, these proteins contain multiple trans-membrane domains that are thougfvi to anchor the coronavirus replication complex through recruitment of intracellular membranes to form a reticuiovesicular network (RV ) of modified, frequently paired, membranes that includes convoluted membranes and double membrane vesicles (DVM) interconnected via the outer membrane with the rough ER.

Culture Systems MERS-CoV can replicate in different mammalian cell lines. In humans, it can replicate in the respiratory tract (lung adenocarcinoma cell line A549, embryonic fibroblast cell line HFL and polarized airway epithelium cell line Calu-3), kidney (embryonic kidney cell line; HEK), liver cells (hepatocellular carcinoma cell line; Huh-7), and the intestinal tract (colorectal adenocarcinoma cell line; Caco-2). MERS-CoV can also infect cell lines originating from primates, pigs, bats, civet cats and rabbits (Chan et al. 2013).

Additional Mouse Models

Zhao J and colleges described a novel approach to developing a mouse model for MERS by transducing mice with a recombinant, nonreplicating adenovirus expressing the hDPP4 receptor. After infection with MERS-CoV, mice develop an interstitial pneumonia. Similar to infected patients, Ad5-hDPP4-transduced mice with normal immune systems developed mild disease whereas immunocompromised mice, like patients with underlying diseases, were more profoundly affected. It was shown that these transduced, infected mice can be used to determine antivirus immune responses and to evaluate anti-MERS-CoV vaccines and therapies {Zhao J et al. 2014). Two Mouse Models have been developed Pascal K et al. In the first, a modified adenovirus expressing huDPP4 was administered intranasally to mice leading to huDPP4 expression in all cells of the lung, not just those that natively express DPP4. In this model, mice showed transient huDPP4 expression and mild lung disease. In the second model, a transgenic mouse was produced to expresses huDPP4 in all cells of the body, which in not physiologically relevant. In this model, MERS-CoV infection leads to high levels of viral RNA and inflammation in the lungs, and also significant inflammation and viral RNA in the brains of infected mice. However, no previous reports have documented tropism of MERS-CoV to the brains of an infected host, suggesting that studying pathogenesis of MERS-CoV in this model is limited.

RNAi and siRNA

RNA interference (RNAi) is a sequence-specific RNA degradation process that provides a direct way to knockdown, or silence, theoretically any gene. In naturally occurring RNA interference, a double stranded RNA is cleaved by an RNase Ill/helicase protein, Dicer, into small interfering RNA (siRNA) molecules, a dsRNA of 19-23 nucleotides (nt) with 2-nt overhangs at the 3' ends. These siRNAs are incorporated into a multicomponent-ribonuclease called RNA-induced-silencing-complex (RISC). One strand of siRNA remains associated with RISC, and guides the complex towards a cognate RNA that has sequence complementary to the guider ss-siRNA in RISC. This siRNA-directed endonuclease digests the RNA, thereby inactivating it. Studies have revealed that the use of chemically synthesized 21-25-nt siRNAs exhibit RNAi effects in mammalian cells, and the thermodynamic stability of siRNA hybridization (at terminals or in the middle) plays a central role in determining the molecule's function.

Importantly, it is presently not possible to predict with high degree of confidence which of many possible candidate siRNA sequences potentially targeting an mRNA sequence of a disease gene will, in fact, exhibit effective RNAi activity. Instead, individually specific candidate siRNA polynucleotide or oligonucleotide sequences must be generated and tested to determine whether the intended interference with expression of a targeted gene has occurred. Target Selection

MERS-CoV is enveloped single-stranded positive-sense RNA viruses, belonging to genus Betacoronavirus. The length of the genome is around 30k nt. The genome contains 10 predicted open reading frames (ORFs): ORFla, ORFlb, Spike (S) Protein, 3, 4a, 4b, 5,

Envelope (E) Protein, Membrane (M) Protein and Nucleocapsid (N) Protein, with 5' two third of the genome (ORFla, ORFlb) encoding 16 non-structure proteins (nspl-16), and rest 3' third of the genome encoding 4 structure proteins (S, E, M and N proteins).

The spike (S) protein of MERS-CoV is a glycoprotein with a molecular weight of -180/190 kDa, which is an important determinant of virus virulence and host range. Trimers of S protein form the spikes on the MERS-CoV envelope, which are responsible for the receptor binding and membrane fusion. Similar to the HIV envelope (env) and influenza hemagglutinin (HA), S proteins of MERS-CoV are Class I viral fusion proteins, which requires the protease cleavage between the SI and S2 domains to allow the conformational changes in S2, and initiate the virus entry and syncytia formation. Dipeptidyl peptidase 4

(DDP4, or CD26), a protein with diverse functions in glucose homeostasis, T-cell activation, etc., has been identified as the receptor of MERS-CoV on the host cells. The recognition of DPP4 is mediated by the receptor-binding domain (RBD, aa E367-Y606) of the S protein. DPP4 is expressed in a variety of cell types. It has been discovered on the human cell surface in the airways (such as the lungs) and kidneys recently.

After entry into the cell, two polyproteins, ppla and pplab of MERS-CoV express and undergo cotranslational proteolytic processing into the proteins that form the viral replication complex. During this processing, the activity of nsp-3, papain-like protease (PLpro) and nsp-5, 3C-like proteinase (3CLpro) are critical for the generation of 16 nonstructural proteins from the polyprotein. However, based on the MERS-CoV genome sequences analysis and calculation, we found several siRNA candidates (MPL1-6) match PL^pro as the target, but no good candidate matches 3CL^pro. Meanwhile, the recent studies showed that MERS-CoV PL^pro also has the function to inhibit the innate immune response to viral infection by decreasing the levels of ubiquitinated and ISGylated host cell proteins and down-regulating the cytokines, such as CCL5 and IFN-β in stimulated cells.

MERS-CoV RNA-dependent RNA polymerase (RdRp), encoding by nsp-12, is the most important component of viral replication complex. This complex is responsible for both the transcription of the nested subgenomic mRNAs and the replication of the genomic positive-strand RNA. Both processes take place in the cytoplasm. In the viral mRNA transcription, the negative- strand RNAs generate from genomic RNA at first, and then transcribe a set of 3 '-coterminal nested subgenomic mRNAs by the replication complex, with a common 5' "leader" sequence (67nt) derived from the 5' end of the genome. The newly synthetic genomic RNAs are produced by the taking the negative- strand RNAs as the template.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. The genome structure of MERS-CoV MERS-CoV is enveloped single-stranded positive-sense RNA viruses, belonging to genus Betacoronavirus, with a genome of ~30K nt. The genome contains 10 predicted open reading frames (ORFs): ORFla, ORFlb, Spike (S) Protein, 3, 4a, 4b, 5, Envelope (E) Protein, Membrane (M) Protein and Nucleocapsid (N) Protein with 16 predicted nonstructural proteins being encoded by ORFla/b.

Figure 2. The life cycle of MERS. After binding to the receptor, viral RNA and proteins of MERS-CoV are synthesized entirely in the cytoplasm. Two polyproteins, ppla and pplab undergo cotranslational proteolytic processing into the proteins that form the viral replication complex. This complex is used to produce the negative-strand RNA from genomic RNA, and transcribe a 3 '-coterminal set of nested subgenomic mRNAs from the negative- strand RNA, which have a common 5' "leader" sequence derived from the 5' end of the genome. This viral replication complex is also used to produce the positive-strand genomic RNA taking the negative-strand RNA as the template.

Figure 3. Special design of siRNA sequences targeting critical viral genes: Papain like protein (PL^pro) specific siRNA, total 6 active siRNAs (MPL1-6); RNA dependent RNA protease (RDRP) specific siRNA, total 5 active siRNAs (MRRl-5) and Spike protein specific siRNA, total 8 active siRNAs (MSP 1-8).

Figure 4. Histidine-Lysine co-polymer enhances topical and subcutaneous siRNA deliveries in vivo. The self-assembled HKP/siRNA nanoparticles (average 150nm in diameter) can be dissolved in aqueous solution, can be lyophilized into dry powder, and can be redissovled and mixed with methyl cellulose, or with RNAse free water. HKP/siRNA nanoparticle delivery to mouse respiratory track: upper airway, bronchi, alveoli.

Figure 5. Comparison of target knockdown of lung endogenous gene among HKP, DOTAP and D5W after oral tracheal deliveries of siRNA with three different dosing regimens. HKP demonstrated the efficient siRNA-mediated knockdown of the target gene at the 20μg dose.

Figure 6. Intraperitoneal delivery of HKP-siRNA nanoparticle formulation demonstrated a prophylactic effect against H1N1 in the viral challenged mice (n =10). The evidence of the anti-influenza efficacy achieved by HKP-siRNA respiratory delivery support our notion that the similar approach can also be applied for anti-MERS siRNA therapeutics. The HKP- siRNA combination (siRNA103-siRNA105 with a 1 : 1 ratio) at a concentration of 40μg/2ml was intraperitoneally administrated on day 1, 2, 3, 4 and 5 (2.5 mg/kg/day, purple arrows). The viral challenges through intranasal administrations of 2x LD50 H1N1 (A/Puerto

Rico/8/1934) were conducted on day 2 (red arrow) for the virus only, Ribavirin and siRNA treatment groups. Ribavirin as a positive control was administered through gavages of 200ul to provide 75mg/kg/day dosing over days 1-5 (orange arrows). The prophylactic efficacy of HKP-siRNA formulation is clearly better than that of Ribavirin.

Figure 7. Intraperitoneal delivery of PAA-siRNA formulation demonstrated a therapeutic efficacy against H1N1 in the viral challenged mice (n = 15). The viral challenges through intranasal administrations of lx LD50 H1N1 (A/California/04/2009) were conducted on day 1 (red arrow) for the virus only, Tamiflu ® and siRNA treatment groups. The H1N1 challenged mice were treated with various dosages of PAA-siRNA combination (siRNA89- siRNA103 with a 1 : 1 ratio), 1 mg/kg, 5 mg/kg and 10 mg/kg, through intraperitoneal administration daily, from day 2 to day 6 (black arrows). Adapting the same route and dosing regimen, 25 mg/kg Tamiflu ® was also administrated daily on the HlNl infected mice. The therapeutic efficacy of 10 mg/kg/day of PAA-siRNA combination resulted in almost equal anti-influenza activity to that of 25 mg/kg/day of Tamiflu ® treatment.

Figure 8. Scheme of the Basic Synthesis Routes and Structure of Spermine-Liposome Conjugates (SLiC) A. The synthesis route for each of the five molecules are listed with the specific liposome chain, such as, Ri, R2, R3, R4 and R5, conjugated at the location of RiH, R2H, R3H, R₄H and R5H respectively. B. The structures of the five SLiC species are illustrated with a spermine head and two lipid legs.

Figure 9. Target Gene Silencing by SLiC Liposome-Mediated siRNA Delivery In Vivo.

TM4-packaged siRNA specific to cyclophilin-B was selected for being tested in a Balb/c mouse model through a respiratory route of delivery. In addition to Blank control and empty TM4 control, a HKP package cyclophilin-B siRNA was used as a positive control. Three different dosage: 25, 40 and 50 μg were tested. Both 40 and 50 μg siRNA dosages achieved significant target gene silencing (N=3, *P<0.05).

Figure 10. Evaluation of the cytokine response in the mouse lung after HKP-siRNA nanoparticles delivery. HKP-siRNA at different dosages were oraltracheally administrated in the mouse lungs. The total lung tissue were harvested for protein isolation and cytokine measurements by ELISA assay.

Figure 11. A. Standard curve to measure protein concentration was prepared according to in- house SOP (Lowry Method); B. Total protein concentration was determined in each sample. Figure 12. A. Standard curve to measure TNF-a concentration was prepared according to in- house SOP (Lowry Method); B. TNF-a concentration in each sample was determined and normalized to total protein.

Figure 13. A. Standard curve to measure IL-6 concentration was prepared according to in- house SOP (Lowry Method); B. IL-6 concentration in each sample was determined and normalized to total protein.

Figure 14. A. Standard curve to measure IFN-a concentration was prepared according to in- house SOP (Lowry Method); B. IFN-a concentration in each sample was determined and normalized to total protein. Figure 15. The HKP siRNA nanoparticle aqueous solution and SLiC siRNA nanoparticle aqueous solution will be administrated through airway, using an ultrasound nebulizer generated aerosol which will have water solution particle size with broad spectrum allowing whole lung distribution.

DESCRIPTION OF THE INVENTION The present invention provides siRNA molecules that inhibit MERS-CoV gene expression, compositions containing the molecules, and methods of using the molecules and compositions to prevent or treat MERS in a subject, such as a human patient.

SiRNA Molecules

As used herein, an "siRNA molecule" or an "siRNA duplex" is a duplex

oligonucleotide, that is a short, double-stranded polynucleotide, that interferes with the expression of a gene in a cell, after the molecule is introduced into the cell, or interferes with the expression of a viral gene. For example, it targets and binds to a complementary nucleotide sequence in a single stranded (ss) target RNA molecule. SiRNA molecules are chemically synthesized or otherwise constructed by techniques known to those skilled in the art. Such techniques are described in U.S. Pat. Nos. 5, 898,031, 6, 107,094, 6,506,559,

7,056,704 and in European Pat. Nos. 1214945 and 1230375, which are incorporated herein by reference in their entireties. By convention in the field, when an siRNA molecule is identified by a particular nucleotide sequence, the sequence refers to the sense strand of the duplex molecule. One or more of the ribonucleotides comprising the molecule can be chemically modified by techniques known in the art. In addition to being modified at the level of one or more of its individual nucleotides, the backbone of the oligonucleotide can be modified. Additional modifications include the use of small molecules (e.g. sugar molecules), amino acids, peptides, cholesterol, and other large molecules for conjugation onto the siRNA molecule. The siRNA molecules of the invention target a conserved region of the genome of a MERS-CoV. As used herein, "target" or "targets" means that the molecule binds to a complementary nucleotide sequence in a MERS-CoV gene, which is an RNA molecule, or it binds to mRNA produced by the gene. This inhibits or silences the expression of the viral gene and/or its replication. As used herein, a "conserved region" of a MERS-CoV gene is a nucleotide sequence that is found in more than one strain of the virus, is identical among the strains, rarely mutates, and is critical for viral infection and/or replication and/or release from the infected cell.

In one embodiment, the siRNA molecule is a double-stranded oligonucleotide with a length of about 17 to about 27 base pairs. In one aspect of this embodiment, the molecule is a double-stranded oligonucleotide with a length of 19 to 25 base pairs. In another aspect of this embodiment, the molecule is a couple- stranded oligonucleotide with a length of 19 to 25 base pairs. In still another aspect of this embodiment, it is a double-stranded oligonucleotide with a length of 25 base pairs. In all of these aspects, the molecule may have blunt ends at both ends, or sticky ends with overhangs at both ends (unpaired bases extending beyond the main strand), or a blunt end at one end and a sticky end at the other. In one particular aspect, it has blunt ends at both ends. In another particular aspect, the molecule has a length of 25 base pairs (25 mer) and has blunt ends at both ends.

In one embodiment, the conserved MERS-CoV genomic regions are the gene sequences coding for the MERS-CoV proteins Papain-like protease (PL^pro), RNA-dependent RNA polymerase (RdRp), and Spike protein. The genomic locations of such genes are shown in Figure 3. In one embodiment, the siRNA molecule targets PL^pro virus gene expression. In another embodiment, the siRNA molecule targets RdRp viral gene expression. In still another embodiment, the siRNA molecule targets Spike viral gene expression.

Particular siRNA sequences that represent some of the siRNA molecules of the invention are disclosed in Tables 1-3. In one embodiment, the siRNA molecules are disclosed in Table 3. In one particular embodiment, the siRNA molecules are the following:

MPL1 CGCAAUACGUAAAGCUAAAGAUUAU,

MPL2 GGGUGUUGAUUAUACUAAGAAGUUU,

MPL3 CGCAUAAUGGUGGUUACAAUUCUU,

MPL4 GGCUUCAUUUUAUUUCAAAGAAUUU, MPL5: GCGCUUUUACAAAUCUAGAUAAGUU,

MPL6: CGCAUUGCAUGCCGUAAGUGUAAUU,

MRR1 : CCCAGUGUUAUUGGUGUUUAUCAUA,

MRR2: GGGAUUUCAUGCUUAAAACAUUGUA,

MRR3 : GGGUGCUAAUGGCAACAAGAUUGUU,

MRR4: CCCCAAAUUUGUUGAUAAAUACUAU,

MRR5: CGGUUGCUUUGUAGAUGAUAUCGUU,

MSP1 : GGCCGUACAUAUUCUAACAUAACUA,

MSP2: GGCCGUACAUAUUCUAACAUAACUA,

MSP3 : CCGAAGAUGAGAUUUUAGAGUGGUU,

MSP4: CCCAGUUUAAUUAUAAACAGUCCUU,

MSP5: GGCUUCACUACAACUAAUGAAGCUU,

MSP6: CCCCUGUUAAUGGCUACUUUAUUAA,

MSP7: CCCUGUUAAUGGCUACUUUAUUAAA, and

MSP8: GCCGCAUAAGGUUCAUGUUCACUAA.

The siRNA molecules of the invention also include ones derived from those listed in Tables 1-3 and otherwise herein. The derived molecules can have less than the 25 base pairs shown for each molecule, down to 17 base pairs, so long as the "core" contiguous base pairs remain. That is, once given the specific sequences shown herein, a person skilled in the art can synthesize molecules that, in effect, "remove" one or more base pairs from either or both ends in any order, leaving the remaining contiguous base pairs, creating shorter molecules that are 24, 23, 22, 21, 20, 19, 18, or 17 base pairs in length, if starting with the 25 base pair molecule. For example, the derived molecules of the 25 mer molecules disclosed in Tables 1-3 include: a) 24 contiguous base pairs of any one or more of the molecules; b) 23 contiguous base pairs of any one or more of the molecules; c) 22 contiguous base pairs of any one or more of the molecules; b) 21 contiguous base pairs of any one or more of the molecules; d) 20 contiguous base pairs of any one or more of the molecules; e) 19 contiguous base pairs of any one or more of the molecules; f) 18 contiguous base pairs of any one or more of the molecules; and g) 17 contiguous base pairs of any one or more of the molecules. It is not expected that molecules shorter than 17 base pairs would have sufficient activity or sufficiently low off-target effects to be pharmaceutically useful; however, if any such constructs did, they would be equivalents within the scope of this invention.

Alternatively, the derived molecules can have more than the 25 base pairs shown for each molecule, so long as the initial 25 contiguous base pairs remain. That is, once given the specific sequences disclosed herein, a person skilled in the art can synthesize molecules that, in effect, "add" one or more base pairs to either or both ends in any order, creating molecules that are 26 or more base pairs in length and containing the original 25 contiguous base pairs.

The siRNA molecule may further comprise an immune stimulatory motif. Such motifs can include specific RNA sequences such as 5'-UGUGU-3' (Judge et al., Nature

Biotechnology 23, 457-462 (1 April 2005)), 5'-GUCCUUCAA-3' (Hornung et al., Nat. Med. 11,263-270(2005). See Kim et al., Mol Cell 24; 247-254 (2007). These articles are incorporated herein by reference in their entireties. These are siRNA sequences that specifically activate immune responses through Toll-like receptor (TLR) activation or through activation of key genes such as RIG-I or PKR. In one embodiment, the motif induces a TH1 pathway immune response. In another embodiment, the motif comprises 5'- UGUGU-3', 5'-GUCCUUCAA-3', 5'-GGGxGG-3' (where x is A, T, G and C), or CpG motifs 5'-GTCGTT-3'.

Pharmaceutical Compositions The invention includes a pharmaceutical composition comprising an siRNA molecule that targets a conserved region of the genome of a MERS-CoV and a pharmaceutically acceptable carrier. In one embodiment, the carrier condenses the molecules to form a nanoparticle. Alternatively, the composition may be formulated into nanoparticles. The compositions may be lyophilized into a dry powder. In one particular embodiment, the pharmaceutically acceptable carrier comprises a polymeric nanoparticle or a liposomal nanoparticle.

In one embodiment, the composition comprises at least two different siRNA molecules that target one or more conserved regions of the genome of a MERS-CoV and a pharmaceutically acceptable carrier. In one aspect of this embodiment, the gene sequences in the conserved regions of the MERS-CoV are critical for the viral infection of a mammal. In one aspect of this embodiment, mammal is a human, mouse, ferret, or monkey. The composition can include one or more additional siRNA molecules that target still other conserved regions of the MERS-CoV genome. In one aspect of this embodiment, a pharmaceutically acceptable carrier comprises a polymeric nanoparticle or a liposomal nanoparticle. In one embodiment, the targeted conserved regions of the genome comprise gene sequences coding for the following MERS-CoV proteins: Papain-like protease (PL^pro), RNA- dependent RNA polymerase (RdRp), and Spike protein. In one aspect of this embodiment, the siRNA molecules target PL^pro viral gene expression. Such siRNA molecules include the following: MPL 1 : CGC AAUACGUAAAGCU AAAGAUUAU,

MPL2: GGGUGUUGAUUAUACUAAGAAGUUU,

MPL3 : CGCAUAAUGGUGGUUACAAUUCUU,

MPL4: GGCUUCAUUUUAUUUCAAAGAAUUU,

MPL5: GCGCUUUUACAAAUCUAGAUAAGUU, and

MPL6 : CGC AUUGC AUGCCGU AAGUGUAAUU.

In another aspect of this embodiment, the siRNA molecules target RdRp viral gene expression. Such siRNA molecules include the following:

MRR1 : CCCAGUGUUAUUGGUGUUUAUCAUA,

MRR2: GGGAUUUCAUGCUUAAAACAUUGUA,

MRR3 : GGGUGCUAAUGGC AAC AAGAUUGUU,

MRR4: CCCCAAAUUUGUUGAUAAAUACUAU, and

MRR5: CGGUUGCUUUGUAGAUGAUAUCGUU.

In still another aspect of this embodiment, the siRNA molecules target Spike viral gene expression. Such siRNA molecules include the following: MSP 1 : GGCCGUAC AUAUUCUAAC AUAACUA,

MSP2: GGCCGUAC AUAUUCUAAC AUAACUA,

MSP3 : CCGAAGAUGAGAUUUUAGAGUGGUU,

MSP4: CCCAGUUUAAUUAUAAACAGUCCUU, MSP5: GGCUUCACUACAACUAAUGAAGCUU,

MSP6: CCCCUGUUAAUGGCUACUUUAUUAA,

MSP7: CCCUGUUAAUGGCUACUUUAUUAAA, and

MSP8: GCCGCAUAAGGUUCAUGUUCACUAA.

In a further aspect of this embodiment, the siRNA molecules are two or more of the following:

MPL1 : CGCAAUACGUAAAGCUAAAGAUUAU,

MPL2: GGGUGUUGAUUAUACUAAGAAGUUU,

MPL3 : CGCAUAAUGGUGGUUACAAUUCUU,

MPL4: GGCUUCAUUUUAUUUCAAAGAAUUU,

MPL5: GCGCUUUUACAAAUCUAGAUAAGUU,

MPL6: CGCAUUGCAUGCCGUAAGUGUAAUU,

MRR1 : CCCAGUGUUAUUGGUGUUUAUCAUA,

MRR2: GGGAUUUCAUGCUUAAAACAUUGUA,

MRR3 : GGGUGCUAAUGGC AAC AAGAUUGUU,

MRR4: CCCCAAAUUUGUUGAUAAAUACUAU,

MRR5: CGGUUGCUUUGUAGAUGAUAUCGUU,

MSP1 : GGCCGUACAUAUUCUAACAUAACUA,

MSP2: GGCCGUACAUAUUCUAACAUAACUA,

MSP3 : CCGAAGAUGAGAUUUUAGAGUGGUU,

MSP4: CCCAGUUUAAUUAUAAACAGUCCUU,

MSP5: GGCUUCACUACAACUAAUGAAGCUU,

MSP6: CCCCUGUUAAUGGCUACUUUAUUAA,

MSP7: CCCUGUUAAUGGCUACUUUAUUAAA, and

MSP8: GCCGCAUAAGGUUCAUGUUCACUAA.

In another embodiment, the composition comprises an siRNA cocktail, MST^PR1, wherein a first siRNA molecule comprises MPL1 : CGCAAUACGUAAAGCUAAAGAUUAU and a second siRNA molecule comprises MRR1 : CCCAGUGUUAUUGGUGUUUAUCAUA.

In another embodiment, the composition comprises an siRNA cocktail, MST^PR2, wherein a first siRNA molecule comprises MPL2:

GGGGUUGAUUAUACUAAGAAGUUU and a second siRNA molecule comprises MRR2: GGGAUUUCAUGCUUAAAACAUUGUA.

In another embodiment, the composition comprises an siRNA cocktail, MST^RS2, wherein a first siRNA molecule comprises MRR2:

GGGAUUUCAUGCUUAAAACAUUGUA and a second siRNA molecule comprises MSP2: GGCCGUACAUAUUCUAACAUAACUA.

In another embodiment, the composition comprises an siRNA cocktail, MST^RS1, wherein a first siRNA molecule comprises MRRl :

CCCAGUGUUAUUGGUGUUUAUCAUA and a second siRNA molecule comprises MSPl : GGCCGUACAUAUUCUAACAUAACUA. In another embodiment, the composition comprises at least three different siRNA molecules that target one or more conserved regions of the genome of a MERS-CoV and a pharmaceutically acceptable carrier. In one aspect of this embodiment, the pharmaceutically acceptable carrier comprises a polymeric nanoparticle or a liposomal nanoparticle.

In another embodiment, the composition comprises an siRNA cocktail, MSTP^RS1, wherein a first siRNA molecule comprises MPL1 :

CGCAAUACGUAAAGCUAAAGAUAU, a second siRNA molecule comprises MRRl : CCCAGUGUUAUUGGUGUUUAUCAUA, and a third siRNA molecule comprises MSPl : GGCCGUACAUAUUCUAACAUAACUA.

In another embodiment, the composition comprises an siRNA cocktail, MST^PRS2, wherein a first siRNA molecule comprises MPL2:

GGGUGUUGAUUAUACUAAGAAGUUU a second siRNA molecule comprises MRR2: GGGAUUUCAUGCUUAAAACAUUGUA, and a third siRNA molecule comprises MSP2: GGCCGUACAUAUUCUAACAUAACUA.

In one aspect of all of these embodiments, the siRNA molecules comprise 25 mer blunt-end siRNA molecules and the carrier comprises a Histidine-Lysine copolymer or Spermine-Lipid Conjugate and cholesterol. Pharmaceutically Acceptable Carriers

Pharmaceutically acceptable carriers include saline, sugars, polypeptides, polymers, lipids, creams, gels, micelle materials, and metal nanoparticles. In one embodiment, the carrier comprises at least one of the following: a glucose solution, a polycationic binding agent, a cationic lipid, a cationic micelle, a cationic polypeptide, a hydrophilic polymer grafted polymer, a non-natural cationic polymer, a cationic polyacetal, a hydrophilic polymer grafted polyacetal, a ligand functionalized cationic polymer, a ligand functionalized- hydrophilic polymer grafted polymer, and a ligand functionalized liposome. In another embodiment, the polymers comprise a biodegradable histidine-lysine polymer, a

biodegradable polyester, such as poly(lactic acid) (PL A), poly(gly colic acid) (PGA), and poly(lactic-co-glycolic acid) (PLGA), a polyamidoamine (PAMAM) dendrimer, a cationic lipid, or a PEGylated PEL Cationic lipids include DOTAP, DOPE, DC-Chol/DOPE, DOTMA, and DOTMA/DOPE. In still another embodiment, the carrier is a histidine-lysine copolymer that forms a nanoparticle with the siRNA molecule, wherein the diameter of the nanoparticle is about lOOnm to about 400 nm.

In one embodiment, the carrier is a polymer. In one aspect of this embodiment, the polymer comprises a histidine-lysine copolymer (HKP). Such copolymers are described in U.S. Pat. Nos. 7,070,807 B2, 7,163,695 B2, and 7,772,201 B2, which are incorporated herein by reference in their entireties. In one aspect of this embodiment, the HKP comprises the structure (R)K(R)-K(R)-(R)K(X), where R =KHHHKHHHKHHHKHHHK, K = lysine, and H = histidine.

In another embodiment, the carrier is a liposome. In one aspect of this embodiment, the liposome comprises a cationic lipid conjugated with cholesterol. In a further aspect, the cationic lipid comprises a spermine head and one or two oleyl alcoholic tails. Examples of such molecules are disclosed in Figure 8. In a further aspect, the liposome comprises Spermine-Liposome-Cholesterol conjugate (SLiC).

Methods of Use

The invention also includes methods of using the siRNA molecules and

pharmaceutical compositions containing them to prevent or treat MERS-CoV disease. A therapeutically effective amount of the composition of the invention is administered to a subject. In one embodiment, the subject is a mammal such as a mouse, ferret, monkey, or human. In one aspect of this embodiment, the mammal is a laboratory animal, such as a rodent. In another aspect of this embodiment, the mammal is a non-human primate, such as a monkey. In still another aspect of this embodiment, the mammal is a human. As used herein, a "therapeutically effective amount" is an amount that prevents, reduces the severity of, or cures MERS disease. Such amounts are determinable by persons skilled in the art, given the teachings contained herein. In one embodiment, a therapeutically effective amount of the pharmaceutical composition administered to a human comprises about 1 mg of the siRNA molecules per kilogram of body weight of the human to about 5 mg of the siRNA molecules per kilogram of body weight of the human. Routes of administration are also determinable by persons skilled in the art, given the teachings contained herein. Such routes include intranasal administration and airway instillation, such as through use of an airway nebulizer. Such routes also include intraperitoneal, intravenous, and subcutaneous administration.

EXAMPLES

We selected Papain-like protease (PL^PR0), RNA-dependent RNA polymerase (RdRp), Spike(S) protein and some of other structure genes (such as M and N protein) and non- structure genes (such as nsp-2, nsp-10 and nsp-15) of MERS-CoV as the targets for an siRNA cocktail-mediated therapeutic approach. The present invention provides siRNA molecules that target a conserved region of MERS-CoV, wherein the siRNA molecules inhibit expression of those genes of MERS-CoV. In a preferred embodiment, the molecule comprises a double-stranded sequence of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In one aspect of this embodiment, the siRNA molecule has blunt ends, or has 3' overhangs of one or more nucleotides on both sides of the double-stranded region. The siRNA cocktail of the invention contains two, three, four, or more sequences targeting those genes of MERS-CoV. Example 1. MERS-CoV Viral Structure and Protein Function

MERS-CoV is enveloped single-stranded positive sense RNA viruses with genomes of 30, 119 nt. The genome structure of MERS-CoV is similar to other coronaviruses, with the 5' two-thirds of the genome encoding the non- structural proteins (NSPs) required for viral genome replication, the remaining 3' third of the genome encoding the structural genes that make up the virion (spike, envelope, membrane, and nucleocapsid proteins), and four accessory genes interspersed within the structural gene region (Figure 1A). At the 5' end of the genome there is a leader sequence (67nt), which is followed by an untranslated region (UTR). At the 3' end of the RNA genome there is another UTR, followed by a poly(A) sequence of variable length. Transcription-regulatory sequences (TRS 5' AACGAA 3' ) are found at the 3' end of the leader sequence and at different positions upstream of genes in the genomic 3' -proximal domain of MERS-CoV. The MERS-CoV genome contains at least 10 predicted open reading frames (ORFs): ORFla, ORFlb, S, 3, 4a, 4b, 5, E, M and N with sixteen predicted nonstructural proteins being encoded by ORFla/b. Several unique group-specific ORFs that are not essential for virus replication are encoded by MERS-CoV. The functions of these group-specific ORFs are unknown; however, by analogy to other coronaviruses, they may encode structural proteins or interferon antagonist genes. Open reading frames ORF2, -6, -7 and -8a are translated from subgenomic mRNAs predicted to encode the four canonical structural genes: a 180/90-kDa spike glycoprotein (S), a ~ 23-kDa membrane glycoprotein(M), a small envelope protein (E) and a ~ 50-kDa nucleocapsidprotein (N), respectively (Figure 1B-C).

Example 2. MERS-CoV Viral Genes and RNAs

Similar to other RNA viruses, coronavirus replicate in the host cytoplasm. The replication process is initiated by the viral particle after binding with specific cellular receptors, known as S-protein mediated binding. The receptor for MERS-CoV was recently identified as dipeptidyl peptidase 4 (DDP4, also known as CD26), a protein with diverse functions in glucose homeostasis, T-cell activation, neurotransmitter function, and modulation of cardiac signaling. DPP4 is expressed in a variety of cell types, including endothelial cells (kidney, lung, small intestine, spleen) hepatocytes, enterocytes, activated leukocytes, testes, prostate and cells of the renal glomeruli and proximal tubules. DPP4 recognition is mediated by the receptor- binding domain (RBD, amino acids E367-Y606). Following virus entry, the coronavirus genome, a positive sense, capped and polyadenylated RNA strand, is directly translated, resulting in the synthesis of coronavirus replicase gene-encoded NSPs. Coronavirus NSPs are translated as two large polyproteins harboring proteolytic enzymes, namely papain-like and chym otrypsin-li ke proteinases that extensively process coronavirus polyproteins to liberate up to 16 NSPs (nsp 1 - 16). After entering into the cell the virus specially modulates the innate immune response, antigen presentation, mitogen-activated protein kinase (Figure 2).

Example 3. Design siRNA Targeting Key Genes of MERS-CoV

Using our specific algorithm, we have designed multiple siRNA sequences, including both 25-mer and 23-mer oligos. Table I. siRNA sequences, 25-mer blunt-end oligos and 23- mer sticky-end oligos, targeting various viral RNA Table II. siRNA sequences, 25 -mer blunt-end oligos and 23-mer sticky-end oligos, targeting various viral RNA, where the red labeled siRNAs are the most potent siRNA inhibitors and the gold labeled siRNAs are the second best siRNA inhibitors, based on the prediction of our specific algorithm. Table III. We selected the most potent siRNA oligos, 25-mer blunt-end oligos and 23-mer sticky-end oligos, targeting various viral proteins and genes. As demonstrated in the Figure 3, we are specifically targeting critical viral genes: Papain like protein (PL^pro) specific siRNA, total 6 active siRNAs (MPL1-6); RNA dependent RNA protease (RDRP) specific siRNA, total 5 active siRNAs (MRRl-5) and Spike protein specific siRNA, total 8 active siRNAs (MSP 1-8).

Example 4. Cell Culture Based Screening for Potent Anti-MERS CoV siRNA Oligos Firstly, to identify the most potent siRNA for silencing MERS-CoV genes in Vero cell culture experiments, we used psiCheck plasmid carrying MERS-CoV gene sequences.

Secondly, we used Vero cell infected with real MERS-CoV to test the selected siRNA for their anti- MERS CoV infecting activity.

A. Subc lotting MERS-CoV virus gene fragments as surrogates for siRNA potency

examination in Vero cells In order to investigate the degrading effect of siRNA candidates on targeted MERS-CoV genes, we used a dual luciferase reporter vector, psiCHECK-2, with gene fragments of Papain like viral protein (nsp5), Conoravirus endopeptidase C30 (nsp6), RNA synthesis protein (nsplO), RNA-dependent RNA polymerase (nspl2), and structure proteins S, E, M and N. psiCHECK-2 Vectors are designed to provide a quantitative and rapid approach for initial optimization of RNA interference (RNAi). The vectors enable monitoring of changes in expression of a target gene fused to a reporter gene. The DNA fragments of nsp5, nsp6, nsplO, nspl2 and structure proteins S, E, M and N were amplified by PCR with specific primers to those genes, and then cloned into the multiple cloning sites of psiCHECK-2 Vector. In this vector, Renilla Luciferase is used as a primary reporter gene, and the siRNA targeting genes located downstream of the Renilla translational stop codon.

Vero cells were seeded in 96-well plates and incubated for 12h. The reporter plasmids (recombinant vectors) psi-nsp5, psi-nsp6, psi-nsplO, psi-nspl2, psi-S, psi-E, psi-M and psi-N, and siRNA candidates were co-transfected into Vero cells using Lipofectamine 2000 in the DMEM without FBS. The blank psi vector is taken as a negative control. Six hours post- transfection, the media was replaced with the DMEM supplemented with 10% FBS. 18, 24, 36 and 48 h post-transfection the activity of the firefly luminescence and Renilla Luciferase in each well was detected using the Dual Luciferase Kit. The siRNA candidates dramatically decreased luciferase activity which indicates that siRNA could greatly inhibit the expression of the target genes of MERS-CoV were selected for the assay of infection with MERS-CoV in vitro.

B. Infection of Vero cells with MERS-CoV To investigate whether the real MERS-CoV mRNAs for nsp5, nsp6, nsplO, nspl2 and structure proteins S, E, M and N can be directly degraded by the specific mechanism of RNA interference (RNAi), Vero cells were seeded in 24-well plate and transfected with selected therapeutic single siRNA or siRNA combination candidates using Lipofectamine 2000 in the DMEM without FBS when cell monolayer reached 80% confluency. The transfection efficacy control is

Cy3 labeled siRNA. PBS was taken as a negative control. An siRNA with the sequence unrelated to MERS-CoV was used as another negative control. 24 h post- transfection the media containing the transfection reagent was replaced with fresh media supplemented with 2% FBS , and cells were infected with MERS-CoV. One hour post-infection, the inoculation solution was replaced with DMEM supplemented with 10%FBS. 24 h post-infection, cells were harvested for RNA isolation and 5'- rapid amplification of cDNA ends (5 '-RACE). In the other parallel experiment, at 24, 48 and 72 h post-infection, the cell supernatants were harvested for viral titer determination. All experiments were performed under Biosafety level-2 condition. The viral RNA were extracted from the cell supernatants, and the one-step

quantitative real-time PCR were performed with forward, reverse primers and TaqMan probe specific to the MERS-CoV isolate FRA/UAE spike protein. The total RNA from the harvested cells was extracted, and 5 '-RACE assays were carried out with gene-specific primers for cDNA products of nsp5, nsp6, nsplO, nspl2 and structure proteins S, E, M and N. The single siRNAs or siRNA combinations with high protection efficiency were selected for in vivo studies.

Example 5. HKP/siRNA nanoparticle and pulmonary delivery

Histidine-Lysine co-polymer (FDCP) siRNA nanoparticle formulations can be established by mixing together aqueous solutions of FDCP and siRNA in 4: 1 ratio by a molecular weight (N/P). A typical FDCP/siRNA formulation will provide nanoparticles in average size in 150 nm in diameter (Figure 4A). The self-assembled FDCP/siRNA

nanoparticles can be resuspended in aqueous solution, lyophilized into dry powder, and then resuspended in RNase free water (Figure 4B). After oral-trachial administration of HKP/siRNA (red labeled) nanoparticles to the mouse respiratory track we were able to observe fluorescent siRNA in the upper (bronchi), and lower airway (alveoli) (Figure 4C). We compared the efficacy of RNAi of cyclophiline B in the lung after oraltrachial deliveries of three different doses of siRNA with HKP, DOTAP and D5W . HKP-mediated delivery demonstrated the efficient RNAi of the target gene at the 2C^g dose (Figure 5).

Example 6. HKP/siRNA formulation for intraperitoneal delivery

During evaluation of prophylaxis and therapeutic benefit of siRNA inhibitors against influenza infection, we tested HKP/siRNA formulation through intraperitoneal

administration, using different dosage and regimens. Based on the observations of these treatment results, we found that the prophylactic effect of HKP/siRNA (two siRNAs arespecific to influenza genes) exceed the effect of Ribovirin (Figure 6). Similarly, the therapeutic effect of HKP/siRNA (two siRNAs are specific to influenza genes) is greater than

Tamiflu ® effect (Figure 7). Due to the fact that both influenza and MERS infections occur in the human respiratory system, we are envisioning that the similar therapeutic approach, such as the HKP/siRNA therapeutics, can be applied for treatment of MERS since we observed the positive therapeutic benefit.

Example 7. SLiC/siRNA nanoparticle

SLiC Liposome Preparation. Regular methods were tried at first to prepare liposomes with newly synthesized SLiC molecules, such as thin film method, solvent injection and so on without much success. Norbert Maurer et al reported a method of liposome preparation in which siRNA or oligonucleotide solution was slowly added under vortexing to the 50% ethanol solution (v/v) of liposome and ethanol was later removed by dialysis. The nanoparticles thus derived were small in size and homogeneous. In this method, siRNA was directly wrapped by cationic lipids during formation of liposome, while in most other methods siRNA or nucleic acid molecules are loaded (or entrapped) into preformed liposome, such as Lipofectamine 2000.

Lipids dissolved in ethanol are in so-called metastable state in which liposomes are not very stable and tend to aggregate. We then prepared un-loaded or pre-formed liposomes using modified Norbert Maurer's method. We found that stable liposome solution could be made by simply diluting ethanol to the final concentration of 12.5% (v/v). Liposomes were prepared by addition of lipids (cationic SLiC /cholesterol, 50:50, mol %) dissolved in ethanol to sterile dd-H₂0. The ethanolic lipid solution needs to be added slowly under rapid mixing. Slow addition of ethanol and rapid mixing were critical for the success in making SLiC liposomes, as the process allows formation of small and more homogeneous liposomes. Unlike conventional methods, in which siRNAs are loaded during the process of liposome formulation and ethanol or other solvent is removed at end of manufacturing, our SLiC liposomes were formulated with remaining ethanol still in the solution so that liposomes were thought to be still in metastable state. When siRNA solution was mixed/loaded with liposome solution cationic groups, lipids will interact with anionic siRNA and condense to form core. SLiC liposomes' metastable state helped or facilitated liposome structure transformation to entrap siRNA or nucleic acids more effectively. Because of the entrapment of siRNA, SLiC liposomes become more compact and homogeneous.

Physiochemical Characterization of SLiC Liposome. After the liposome formation, we have developed an array of assays to characterize the physicochemical properties of SLiC liposome, including particle size, surface potential, morphology study, siRNA loading efficiency and biological activity, etc. The particle size and zeta-potentials of SLiC liposomes were measured with Nano ZS Zeta Sizer (Malvern Instruments, UK). Each new SLiC liposome was tested for particle size and zeta-potential when ethanol contents changed from 50% to 25% and to 12.5%. Data were derived from formulations of different ethanol contents. All SLiC liposomes were prepared at lmg/ml in concentration and loaded with siRNA (2: 1, w/w). Each of SLiC Liposomes was composed of cationic SLiC and cholesterol dissolved in ethanol at 12.5%, e.g. TM2 (12.5). The average particle sizes of three sequential measurements and the average zeta-potentials of three sequential measurements were illustrated in Table IV.

Further analysis of the physiochemical perimeters of the SLiC liposome suggested that ethanol concentrations were positively proportional to particle sizes (the lower of ethanol concentration, the smaller of particle sizes), but negatively proportional to zeta-potential (the lower of ethanol concentration, the higher of zeta-potential at the same time). The higher surface potential will render particles more stable in solution. In addition to stability in solution, data shown later also indicated that toxicity was lower with lower ethanol concentration, too. Therefore, to put all factors together, ethanol concentration of 12.5% (v/v) was selected as solvent to suspend cholesterol as well as SLiC into the master working stock solution before they were used to make liposome formulations.

In contrast to bare SLiC liposome formulation, liposomes particle sizes became much smaller when they were loaded with siRNA at 2: 1 (w/w) resulting in particle sizes in the range of 110 to 190nm in diameter and much lower PDI values. Conventional consideration of liposomal structure dictates that siRNA is loaded or interacted with cationic lipids through electrostatic forces and liposomes wraps siRNA to form spherical particles in shape in order to reduce surface tension. As the result, the liposomes particle sizes became much smaller after loaded with siRNA. Liposomes formulated with siRNA also have lower surface charge, which could be explained by neutralizing effect from loaded siRNA.

Example 8. Airway Delivery with Mouse Model

Human host-cell dipeptidyl peptidase 4 (hDPP4) has been shown to be the receptor of MERS-CoV. However, mouse is not a suitable small-animal model for MERS-CoV as it has no receptor being recognized and bound by the virus. In this study, the mice were sensitized to MERS-CoV infection by transduction with Adenoviral or Lentiviral vector expressing hDPP4 in the respiratory tract. This mouse model was used to investigate the efficiency of the siRNA on inhibiting the MERS-CoV infection in vivo. The siRNA combination candidate was delivered by encapsidated with HKP-SLiC nanoparticle system. We performed all mouse studies under Biosafety level-3 conditions.

All BALB/c mice were 18 weeks old and tested as specific pathogen-free at the beginning of this study. To develop the susceptibility to MERS-CoV, 30 mice of Adenoviral vector group and 30 mice of Lentiviral vector group were transduced with Adenoviral and Lentiviral vector expressing hDPP4, respectively. Another 20 mice were transduced with empty Adenoviral or Lentiviral vector as the control. For the Adenoviral vector group, hDDP4 gene was cloned into the Ad5. Then MLE 15 cells were transduced with Ad5-hDDP4 at an MOI of 20. The supernatant were collected at 48 h post-infection. The mice were transduced intranasally with 10⁸ pfu of Ad5-hDDP4. For the Lentiviral vector group, hDDP4 gene was cloned into the plasmid pWPXLd. Then, pWPXLd-hDPP4, along with packaging vector, psPAX2, and envelope vector, pMD2.G, was co-transfected into packaging cell line HEK 293 T using calcium phosphate method. At 48 h post-transfection, the constructed viral vector was harvested and purified, and transducted with CHO cells. The lentivirus was harvested and concentrated. The mice were transduced intranasally with lentivirus expressing hDPP4 at titers of 10⁸ transducing units/ml (TU/ml).

After confirming the hDPP4 was expressed in the respiratory tract of the mice by western blot, the Adenoviral and Lentiviral vector groups were further divided into prophylactic, therapeutic and control subgroup with ten mice in each subgroup. Ten mice from Ad5-hDDP4 or psPAX2-hDDP4 prophylactic subgroup were intranasally inoculated with siRNA combination encapsidated with HKP-SLiC nanoparticle system 24 h before inoculation. 24 h later, all eighty mice including transduced with empty vector were infected intravenously with 10⁵ pfu of MERS-CoV. The prophylactic, therapeutic and control subgroup were intranasally inoculated with siRNA or PBS at 0, 24, 48, 72 and 96 h postinfection.

All mice were weighed and the survivors of each subgroup were counted daily. The nasal washes were collected at 1, 3, 5, 7, 9, and 14 day post-infection for the viral titration. Two infected mice from each group were sacrificed at 3 and 5 day post-infection, respectively. The tissue collection, including lung, trachea, spleen, liver, heart, brain and kidney, were collected for pathological and virological study.

To determine the viral titers, the tissue samples were homogenized in DMEM, and clarified by centrifugation. Both tissue suspensions and nasal washes werelO-fold serially diluted. The dilutions were added to the Vero cells monolayers grown in 96-well plates. The cytopathic effects (CPEs) were observed on day 3 post-infection, and the TCID50 was calculated by the Reed-Muench method.

To investigate the efficiency of siRNA candidates in inhibiting viral gene expression, the total RNAs were extracted from the tissues and the one-step quantitative real-time PCR were performed with forward, reverse primers and TaqMan probe specific to the conserved region of nspl2 (RNA-dependent RNA polymerase) of MERS-CoV.

Example 9. Intraperitoneal siRNA Nanoparticle Solution

In vivo administration of siRNAs. The in vivo experiments were conducted using 6-8 week old female mice. For inoculation, allantoic fluid containing the virus at a dose of 5 ^χ 10⁴ EID5o/mL was used. The infectious activity of the virus in allantoic fluid was determined in vivo by titration of lethality. Titers of the virus were calculated using the Reed and Muench method. Non-infected mice that were kept in the same conditions as the infected animals were used as a negative control. Virus was administered to the animals intranasally under a light ether anesthesia. Each group of animals contained 15 mice. siRNA (1 : 1 ratio of siRNAs 89 and 103) complexed with PAA as described above, was administered to the animals at the dose of 1-10 mg/kg of body weight. siRNA was administered intraperitoneally (200ul per injection). Control animals received PAA without siRNAs. Animals were observed for 14 days post inoculation. The mortality of the animals in control and experimental groups was registered daily. The mortality percentage (M) was calculated in each group as: M=N Nt where: N - the number of animals died withinl4 days after infection; Nt - the total number of animals in the group. The index of protection (IP) was calculated as: IP=((Mc-Me) Mc)xl00%, where: Mc and Me - percentage of mortality in control and experimental groups, correspondingly. The median day of death (MDD) within 14 days was calculated as: MDD=(∑ N D) Nt, where: N - the number of animals surviving D days; Nt - total number of animals in the group Tamiflu ® (oseltamivir phosphate, Roche, Switzerland) was used as a reference compound. It was administered at a dose of 25 mg/kg by the same protocol.

The intraperitoneal administration could be a viable alternative, especially in patients with severe influenza with low gas-exchange volume and/or those on mechanical lung ventilation. Since siRNAs of the same length show similar properties (charge,

hydrophobicity, molecular weight etc) and since siRNAs can be rapidly designed and manufactured, it is feasible that nanoparticle-mediated siRNA delivery may form an intermediate therapeutic strategy in treating rapidly emerging influenza virus strains with high mortality rates that do not respond to existing therapies, while vaccines to protect the general population are under development. The siRNA cocktail demonstrated herein may provide significant value as a prophylactic/ therapeutic with broad anti-influenza strain coverage and this coverage may well extend to as yet unidentified Influenza strains that may emerge in the future. As stated in the Example 6, the therapeutic benefit we observed during the study using siRNA approach against influenza viral infection has provided a good example to follow: the HKP/siRNA nanoparticle delivery through IP route or respiratory route, targeting the conservative regions of the critical viral gene sequences, and siRNA cocktail design, etc.

We demonstrated in this study that polymeric nanoparticle-mediated delivery of a combination of two siRNAs, via IP administration, can result in a potent antiviral effect in the viral-challenged animals. Histidine Lysine Co-Polymer (HKP) nanoparticle-mediated siRNA delivery has been well validated through multiple routes with various animal models (17). We recently completed a full scale safety study for HKP-siRNA nanoparticle formulation via subcutaneous administration into both mouse and monkey models (data not shown). When HKP-siRNA103/105 formulation was IP administrated (10 mg/kg/day), a prophylactic and therapeutic benefit greater than that observed with Ribavirin (75 mg/kg/day) in protecting mice from exposure to a 2xLD50 dose of the virus. (Ribavirin is manufactured by multiple companies in the United States: Copegus produced by Genentech (member of the Roche group), Rebetol by Merck Sharp & Dome, a subsidary of Merck & Co., Inc.,

and Rihasphere by Kadmon Pharmaceuticals (orginally by Three Rivers Pharmaceuticals which was acquired by Kadmon Pharmaceuticals). In addition, several companies, including Sandoz and Teva pharmaceuticals, produce generic ribavirin.) The data obtained suggests that IP injection of peptide nanoparticles containing siRNAs or of a polycationic delivery vehicle carrying siRNAs can both ameliorate the lethality induced by Influenza infection in mice and therefore may suggests the ability to overcome some of these barriers. The amphiphilic poly(allylamine) (PAA) formed polymeric micelles (PM) has been evaluated for siRNA delivery via the GI tract, resulting in efficient siRNA delivery and

endosome/lysosome release. PAA and siRNA can be self-assembled into complexes with nano-sized diameters (150-300 nm) and cationic surface charge (+ 20 to 30 mV). When we IP administered PAA-siRNA89/103 formulation (10 mg/kg) a therapeutic antiviral activity was observed equivalent to that of Tamiflu (25 mg/kg). These results clearly demonstrated that polymeric nanoparticle delivery of siRNA combinations may provide a

prophylactic/therapeutic response against newly emergent strains of influenza virus. A similar approach can be considered for a MERS siRNA therapy.

Example 10. Effects on Innate Immunity in Lung

To evaluate the cytokine response after HKP/siRNA formulation administration to the mouse airway, we collected the lung lavage samples from the Balb/c mice treated with intratracheal instillation of HKP/siRNA aqueous solution (the cohort and dosage described in Table A). The lavage samples were further measured for the TNF-a, IL-6 and IFN-a changes before and after the treatment using commercial available ELISA assay (Figure 10). We first established a standard curve (using Lowry method) of the protein concentration and then measured the protein concentrations of each collected sample, where the STP705 stands for HKP/siRNA groups with different siRNA concentrations (Figure 11). Based on the standard curves for TNF-a, IL-6 and IFN-a we established using the commercial kits (Figure 12A, Figure 13A and Figure 14A), we measured the TNF-a, IL-6 and IFN-a cytokine levels of each collected samples (Figure 12B, Figure 13B and Figure 14B). With comparisons between the normal mouse lungs and LPS treated mouse lungs, and HKP/siRNA treated lungs, we found that (1) HKP/siRNA treatment has little impact on the lung TNF-a level changes (Figure 12B); (2) Various HKP/siRNA formulations with different siRNA concentrations can induce IL-6 level elevation (Figure 13B); (3) There is no significant changes of the IFN-a levels with the HKP/siRNA formulation treatments.

Table A. Effect of siRNA-HKP nano articles on innate immunit at lun

Example 11. Non-human Primate Study

Currently, there is neither an effective vaccine nor drug available for prophylatic or therapeutic strategy. Recently, rhesus macaque has been developed as a model for MERS- CoV using intratracheal inoculation. Similar to human, the infected monkeys showed clinical signs of disease, virus replication, and histological lesions, indicating that rhesus macaque is a good model for evaluation of vaccine and antiviral strategies against MERS-CoV infection.

To investigate the efficiency of the siRNA on protecting and healing from MERS- CoV infection, we plan to perform the non-human primate study in rhesus macaques. The siRNA cocktail candidate will be encapsidated with HKP-SLiC nanoparticle system, and administered intratracheally. This monkey study should be carried out under Biosafety Level- 3 condition.

All rhesus monkeys should be 2-3 years old at the beginning of this study. At the beginning, all monkeys need to be tested negative for MERS-CoV. Twelve monkeys should be divided into three groups—prophylactic, protection, and control group with four animals in each group. Four monkeys of prophylactic group should be intratracheally inoculated with siRNA combination encapsidated in FD P-SLiC nanoparticle system using a nebulizer. 24 h later, all twelve monkeys should be intratracheally inoculated with 6.5 ^χ 10⁷ TCID50 of MERS-CoV in 1 mL. The prophylactic and protection groups should be continuously inoculated with siRNA combination at 0, 24, 48, 72 and 96 h post-infection using the nebulizer. The control group will be inoculated with PBS at the same time points.

All monkeys will be observed twice daily for the symptoms and mortality. Chest X-rays need to be performed 1 day pre-infection and 3, and 5 day post-infection. Oropharyngeal, nasal, and cloacal swabs should be collected at 1, 3, 5, 7, 9, 14, 21, and 28 day post-infection for the viral titration. Two infected monkeys from each group will be sacrificed on the day 3 post-infection. The tissue including lung, trachea, spleen, liver, heart, brain, kidney, and colon tissue will be collected for pathological and virological study.

The viral titers determination in the tissue and swab samples should be performed as described in Example 2. To investigate the efficiency of siRNA candidates on inhibiting viral gene expression, the total RNA will be extracted from the tissues and the one-step

quantitative real-time PCR were performed.

To investigate the efficiency of siRNA candidates on inhibiting viral protein expression, the total RNA will be extracted from the tissues and the one-step quantitative real-time PCR will be performed as described in Example 8.

REFERENCES

1. Zumbla A, et al. (2015) Middle East respiratory syndrome. Lancet SO 140- 6736(15)60454-8.

2. Jalal S. (2015) The emerging threat of MERS. J Pak Med Assoc. 65(3):310-1.

3. de Wit E, et al. (2013) Middle East respiratory syndrome coronavirus (MERS-CoV) causes transient lower respiratory tract infection in rhesus macaques. Proc Natl Acad Sci USA 110: 16598-16603.

4. Chan JF (2015) Middle East Respiratory Syndrome Coronavirus: Another Zoonotic

Betacoronavirus Causing SARS-Like Disease Clin, Microbiol. 28(2): 465-522,

5. Totura AL, Baric RS (2012) SARS coronavirus pathogenesis:Host innate immune

responses and viral antagonism of interferon. Curr Opin Virol 2:264-275.

6. Abdel-Moneim AS (2014) Middle East respiratory syndrome coronavirus (MERS- CoV): evidence and speculations. Arch Virol. 159(7): 1575-84.

7. Pascal K, et al. (2015) Pre- and postexposure efficacy of fully human antibodies against Spike protein in a novel humanized mouse model of MERS-CoV infection. Proc Natl Acad Sci U S A. pii: 201510830. 8. de Wilde AH et al. (20 14 ) Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture. Chemother. 58(8):4875-84. doi: 10.1 128/AAC.0301 1-14.

9. Falzarano D, et al.,( 2013) Treatment with interferon-a2b and ribavirin improves

outcome in MERS-CoV-infected rhesus macaques. Nat Med 19(10): 1313-1317.

10. Lu L. et al.(2015) Urgent development of effective therapeutic and prophylactic agents to control the emerging threat of Middle East respiratory Syndrome (MERS). Emerging Microbes & Infections (2015) 4, e37; doi: 10.1038/emi.2015.37.

1 1. Zhao J, et al (2015) Passive immunotherapy with dromedary immune serum in an

experimental animal model for middle East respiratory syndrome coronavirus infection.

Virol. 89(1 1):61 17-20. doi: 10.1 128/JVI.00446-15.

12. Tianlei Ying et al (2015) Development of human neutralizing monoclonal antibodies for prevention and therapy of MERS-CoV infections. Microbes and Infection 17 (2015) 142-148.

13. Needle D. et al. (2015) Structures of the Middle East respiratory syndrome coronavirus 3C-like protease reveal insights into substrate specificity Acta Cryst. (2015). D71, 1 102- 1 1 1 1.

14. Chan et al. (2013)Differential cell line susceptibility to the emerging novel

humanbetacoronavirus 2c EMC/2012: implications for disease pathogenesisand clinical manifestation. J Infect Dis 207: 1743-1752

15. Lundin A et al .( 2014) Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus. PLoS Pathogens, vol. 10, no. 5, Article ID el004166, 2014.

16. Zhao J, et al. (2014) Rapid generation of a mouse model for Middle East respiratory syndrome. Proc Natl Acad Sci USA 1 1 1(13):4970-4975.

17. Leng, Q and Mixson J. et al. Systemic delivery of HK Raf-l siRNA Polyplexes Inhibits MDA-MB-435 Xenografts. Cancer Gene Therapy.1-11(2008).

The disclosures of all publications identified herein, including issued patents and published patent applications, and all database entries identified herein by url addresses or accession numbers are incorporated herein by reference in their entirety.

Although this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention. Table 1

GGGAGAGUUCGUUGUCAACGAUGUU 1 times at 1738 NSP2

GCCGGCCCAUUCAUGGAUAAUGCUA 1 times at 1880 NSP2

CCGGCCCAUUCAUGGAUAAUGCUAU 1 times at 1881 NSP2

CGGCCCAUUCAUGGAUAAUGCUAUU 1 times at 1882 NSP2

GGCCCAUUCAUGGAUAAUGCUAUUA 1 times at 1883 NSP2

GCCCAUUCAUGGAUAAUGCUAUUAA 1 times at 1884 NSP2 GCCCAUUCAUGGAUAAUGCUAUU

CCCAUUCAUGGAUAAUGCUAUUAAU 1 times at 1885 NSP2 CCCAUUCAUGGAUAAUGCUAUUA

GCUAUUAAUGUUGGUGGUACAGGAU 1 times at 1901 NSP2

CGCCAUUACUGCACCUUAUGUAGUU 1 times at 1936 NSP2 CGCCAUUACUGCACCUUAUGUAG

GCUCACAGCGUGUUGUACAGAGUUU 1 times at 2048 NSP2

GCGUGUUGUACAGAGUUUUUCCUUA 1 times at 2055 NSP2

CGUGUUGUACAGAGUUUUUCCUUAU 1 times at 2056 NSP2 GUGUUGUACAGAGUUUUUCCUUA

GGCGACUUUAUGUCUACAAUUAUUA 1 times at 2186 NSP2 GGCGACUUUAUGUCUACAAUUAU

CCAAACUGCUGUUAGUAAGCUUCUA 1 times at 2218 NSP2

GCUGUUAGUAAGCUUCUAGAUACAU 1 times at 2225 NSP2 CUGUUAGUAAGCUUCUAGAUACA

GCAACAU U U AACU UCUUGUUAGAUU 1 times at 2267 NSP2 AACAUUUAACUUCUUGUUAGAUU

CCUAUGUGUACACUUCACAAGGGUU 1 times at 2325 NSP2

GGAACCUAUUACUGUGUCACCACUA 1 times at 2504 NSP2

GGUUGAAACUGUUGUGGGUCAACUU 1 times at 2653 NSP2

GCAAACUAAUAUGCAUAGUCCUGAU 1 times at 2680 NSP2

GGUGACUAUGUCAUUAUUAGUGAAA 1 times at 2714 NSP2

GGGAGGUGCACCUGUAAAAAAAGUA 1 times at 2830 NSP2

CG AG U ACAACAU UCAUGCUGUAUUA 1 times at 2908 NSP3

GCUGUAUUAGACACACUACUUGCUU 1 times at 2924 NSP3

GGAGUUUGCUGACGUAGUAAAGGAA 1 times at 2995 NSP3

GCGUGGAAUGCCGAUUCCAGAUUUU 1 times at 3049 NSP3

GGAAUGCCGAUUCCAGAUUUUGAUU 1 times at 3053 NSP3

CCAG AU U U U G AU U U AG ACG AU U U U A 1 times at 3065 NSP3

CGAUUUUAUUGACGCACCAUGCUAU 1 times at 3082 NSP3

CCCGUCGAGUGUGACGAGGAGUGUU 1 times at 3164 NSP3

CGAGUGUGACGAGGAGUGUUCUGAA 1 times at 3169 NSP3

GGCUUCAGAUUUAGAAGAAGGUGAA 1 times at 3199 NSP3

GCUUCAGAUUUAGAAGAAGGUGAAU 1 times at 3200 NSP3

CGACGAGUGGGCUGCUGCAGUUGAU 1 times at 3283 NSP3

CGAGUGGGCUGCUGCAGUUGAUGAA 1 times at 3286 NSP3

GGGCUGCUGCAGUUGAUGAAGCGUU 1 times at 3291 NSP3

GCAAGAAGAAGCACAACCAGUAGAA 1 times at 3352 NSP3

CCAGUAGAAGUACCUGUUGAAGAUA 1 times at 3368 NSP3

GCAGGUUGUCAUAGCUGACACCUUA 1 times at 3397 NSP3

GGUUAUUACAGAGUGCGUUACCAUA 1 times at 3628 NSP3

GGCGGUGGUAUCGCUGGUGCUAUUA 1 times at 3734 NSP3

GCGGUGGUAUCGCUGGUGCUAUUAA 1 times at 3735 NSP3

CGGUGGUAUCGCUGGUGCUAUUAAU 1 times at 3736 NSP3

GCUGGUGCUAUUAAUGCGGCUUCAA 1 times at 3746 NSP3

GCGGCUUCAAAAGGGGCUGUCCAAA 1 times at 3761 NSP3 CGGCUUCAAAAGGGGCUGUCCAAAA 1 times at 3762 NSP3

GGCUUCAAAAGGGGCUGUCCAAAAA 1 times at 3763 NSP3

GCCGUUACAAGUAGGAGAUUCAGUU 1 times at 3817 NSP3

CGUAGGCCCAGAUGCCCGCGCUAAA 1 times at 3883 NSP3

CCCAGAUGCCCGCGCUAAACAGGAU 1 times at 3889 NSP3

GGCUAUGAAUGCAUAUCCUCUUGUA 1 times at 3940 NSP3

CCAGCUGUGUCUUUUGAUUAUCUUA 1 times at 4004 NSP3

GCUGUGUCUUUUGAUUAUCUUAUUA 1 times at 4007 NSP3 CUGUGUCUUUUGAUUAUCUUAUU

CGUCGUUAAUU CCCAAG AU G U CU AU 1 times at 4057 NSP3

GGCGCAAUACGUAAAGCUAAAGAUU 1 times at 4142 NSP3

GCGCAAUACGU AAAG CU AAAG AU U A 1 times at 4143 NSP3

CGCAAUACGUAAAGCUAAAGAUUAU 1 times at 4144 NSP3 CGCAAUACGUAAAGCUAAAGAUU

CGUAAAGCUAAAGAUUAUGGUUUUA 1 times at 4151 NSP3

GCUAAAGAUUAUGGUUUUACUGUUU 1 times at 4157 NSP3

GCACAGACAACUCUGCUAACACUAA 1 times at 4188 NSP3

GGAACAAGGGUGUUGAUUAUACUAA 1 times at 4221 NSP3

GGGUGUUGAUUAUACUAAGAAGUUU 1 times at 4228 NSP3 GGGUGUUGAUUAUACUAAGAAGU

CGUCUAAGGACACUUUAGAUGAUAU 1 times at 4287 NSP3

GGACACUUUAGAUGAUAUCUUACAA 1 times at 4294 NSP3 GACACUUUAGAUGAUAUCUUACA

GCUAAUAAGUCUGUUGGUAUUAUAU 1 times at 4322 NSP3

GGUAUUAUAUCUAUGCCUUUGGGAU 1 times at 4337 NSP3

CCUUUGGGAUAUGUGUCUCAUGGUU 1 times at 4352 NSP3

GCCCUACGUGUGUCUCCUAGCUAAU 1 times at 4420 NSP3

CCCUACGUGUGUCUCCUAGCUAAUA 1 times at 4421 NSP3

CCUACGUGUGUCUCCUAGCUAAUAA 1 times at 4422 NSP3

GCUAAUAAAGAGCAAGAAGCUAUUU 1 times at 4439 NSP3

GCAAGAAGCUAUUUUGAUGUCUGAA 1 times at 4450 NSP3

GCUAUUUUGAUGUCUGAAGACGUUA 1 times at 4457 NSP3

CG U U AAG U U AAACCC U U C AG AAG AU 1 times at 4477 NSP3

CGUCCGCACUAAUGGUGGUUACAAU 1 times at 4513 NSP3

CGCACUAAUGGUGGUUACAAUUCUU 1 times at 4517 NSP3 CGCACUAAUGGUGGUUACAAUUC

CCUGCAUUGGUCUGAUCAAACCAUA 1 times at 4594 NSP3

GGAUUCACGCACGACACAGCAGUUA 1 times at 4702 NSP3

GCGUUUUCUUUAAUGGUGCUGAUAU 1 times at 4815 NSP3

CGUUUUCUUUAAUGGUGCUGAUAUU 1 times at 4816 NSP3

GCAGACAAUUUGACUGCUGAUGAAA 1 times at 4889 NSP3

CCUACUUUCUUACACAGAUUCUAUU 1 times at 4949 NSP3 UACU UUCU U ACACAGAU UCUAU U

CGGUUACUUCAUACCGUGCUUGCAA 1 times at 5222 NSP3

GGUUACUUCAUACCGUGCUUGCAAA 1 times at 5223 NSP3

GCAUGGUUUGGAGAGAGUGGUGCAA 1 times at 5271 NSP3

GCUUGUUGUUACGUGGGUGUGCAAA 1 times at 5336 NSP3

CGUGGGUGUGCAAACUGUUGAAGAU 1 times at 5347 NSP3

GGUUGCUGCUCUCAGGCACACCAAA 1 times at 5448 NSP3

GCUGCUCUCAGGCACACCAAAUGAA 1 times at 5452 NSP3

GCU CU CAG GCACACCAAAU G AAAAA 1 times at 5455 NSP3 GGUGACAACCUCCACGGCGCCUGAU 1 times at 5482 NSP3

GGGCAUUGAAACGGCUGUUGGCCAU 1 times at 5530 NSP3

GGCAUUGAAACGGCUGUUGGCCAUU 1 times at 5531 NSP3

GCAUUGAAACGGCUGUUGGCCAUUA 1 times at 5532 NSP3

CCG U U AG CAAGACU U CAG ACU GG AA 1 times at 5607 NSP3

GCAAGACUUCAGACUGGAAGUGCAA 1 times at 5613 NSP3

G G CC AAAAAU ACAG U AG CG AU U G U A 1 times at 5660 NSP3

G CC AAAAAU ACAG UAGCGAUUGUAA 1 times at 5661 NSP3

CCAAAAAUACAGUAGCGAUUGUAAU 1 times at 5662 NSP3

CGUACGGUAUUCUUUGGACGGUAAU 1 times at 5689 NSP3

GGACGGUAAUUUCAGAACAGAGGUU 1 times at 5704 NSP3

CGGUAAUUUCAGAACAGAGGUUGAU 1 times at 5707 NSP3

CCCGACCUAUCUGCUUUCUAUGUUA 1 times at 5732 NSP3

CCGACCUAUCUGCUUUCUAUGUUAA 1 times at 5733 NSP3 GACCUAUCUGCUUUCUAUGUUAA

CCUAUCUGCUUUCUAUGUUAAGGAU 1 times at 5737 NSP3

GCUUUCUAUGUUAAGGAUGGUAAAU 1 times at 5744 NSP3

GGAUGGUAAAUACUUUACAAGUGAA 1 times at 5758 NSP3

CCACCCGUAACAUAUUCACCAGCUA 1 times at 5783 NSP3

CCCGUAACAUAUUCACCAGCUACAA 1 times at 5786 NSP3

CCG UAACAUAU UCACCAGCU ACAAU 1 times at 5787 NSP3

CGUAACAUAUUCACCAGCUACAAUU 1 times at 5788 NSP3

GGACAACCUGGCGGUGAUGCUAUUA 1 times at 5858 NSP3

GGCGGUGAUGCUAUUAGUUUGAGUU 1 times at 5867 NSP3

GCGGUGAUGCUAUUAGUUUGAGUUU 1 times at 5868 NSP3

CGGUGAUGCUAUUAGUUUGAGUUUU 1 times at 5869 NSP3

GGUGAUGCUAUUAGUUUGAGUUUUA 1 times at 5870 NSP3 GUGAUGCUAUUAGUUUGAGUUUU

CGGCGAUGUGUUGUUGGCUGAGUUU 1 times at 5968 NSP3

GCUGAGUUUGACACUUAUGACCCUA 1 times at 5984 NSP3

GGUGCCAUGUAU AAAG G CAAACC AA 1 times at 6020 NSP3

GCAUCUUAUGAUACUAAUCUUAAUA 1 times at 6062 NSP3 AUCUUAUGAUACUAAUCUUAAUA

CGUAGCCCCCAUUGAACUCGAAAAU 1 times at 6121 NSP3

GCCCCCAUUGAACUCGAAAAUAAAU 1 times at 6125 NSP3 CCCCAUUGAACUCGAAAAUAAAU

CCCCCAU U G AACU CG AAAAU AAAU U 1 times at 6126 NSP3 CCCAUUGAACUCGAAAAUAAAUU

CCUUUCGUGAAGGACAAUGUCAGUU 1 times at 6254 NSP3

CGUGAAGGACAAUGUCAGUUUCGUU 1 times at 6259 NSP3

GGACAAUGUCAGUUUCGUUGCUGAU 1 times at 6265 NSP3

CCCUAAGUAUCAAGUCAUUGUCUUA 1 times at 6352 NSP3

CCUAAGUAUCAAGUCAUUGUCUUAA 1 times at 6353 NSP3 CCCUAAGUAUCAAGUCAUUGUCU

GCACACCGUUGAGUCAGGUGAUAUU 1 times at 6409 NSP3

CGUUGAGUCAGGUGAUAUUAACGUU 1 times at 6415 NSP3

GGUGAUAUUAACGUUGUUGCAGCUU 1 times at 6425 NSP3

GGGCUUCAUUUUAUUUCAAAGAAUU 1 times at 6486 NSP3

GGCUUCAUUUUAUUUCAAAGAAUUU 1 times at 6487 NSP3 GGCUUCAUUUUAUUUCAAAGAAU

GCUACCACUGCUGUAGGUAGUUGUA 1 times at 6530 NSP3

CCACUGCUGUAGGUAGUUGUAUAAA 1 times at 6534 NSP3 GGCAUAUUGACAGGCUGUUUUAGUU 1 times at 6590 NSP3

GCAUAUUGACAGGCUGUUUUAGUUU 1 times at 6591 NSP3

GCUUCCACUAGCUUACUUUAGUGAU 1 times at 6634 NSP3

CCACUAGCUUACUUUAGUGAUUCAA 1 times at 6638 NSP3 CACUAGCUUACUUUAGUGAUUCA

CCACAG AG G UUAAAG UGAGUGCUUU 1 times at 6672 NSP3

GGCGUUGUGACAGGUAAUGUUGUAA 1 times at 6707 NSP3

GCGUUGUGACAGGUAAUGUUGUAAA 1 times at 6708 NSP3

CGUUGUGACAGGUAAUGUUGUAAAA 1 times at 6709 NSP3

GCACUGCUGCUGUUGAUUUAAGUAU 1 times at 6741 NSP3

GCUGCUGUUGAUUUAAGUAUGGAUA 1 times at 6746 NSP3

CCGUGUGGAUUGGAAAUCAACCCUA 1 times at 6778 NSP3

CGGUUGUUACUUAUGUUAUGCACAA 1 times at 6803 NSP3

CCCAAGGU U UGAAAAAG UUCUACAA 1 times at 6906 NSP3 CCCAAG G U U UGAAAAAG U U CU AC

CCAAGGUUUG A AAAAG U U C U AC AAA 1 times at 6907 NSP3 AAG G U U U G AAAAAG UUCUACAAA

GCUUGUGACGGUCUUGCUUCAGCUU 1 times at 6962 NSP3

GCGCAAACCGUUCUGCAAUGUGUAA 1 times at 7020 NSP3

CGCAAACCGUUCUGCAAUGUGUAAU 1 times at 7021 NSP3

GCAAACCGUUCUGCAAUGUGUAAUU 1 times at 7022 NSP3

GCAAUGUGUAAUUGGUGCUUGAUUA 1 times at 7034 NSP3

GGUGCUUGAUUAGCCAAGAUUCCAU 1 times at 7047 NSP3

CCAUAACUCACUACCCAGCUCUUAA 1 times at 7068 NSP3

GGUUCAAACACAUCUUAGCCACUAU 1 times at 7096 NSP3

GGCAGGUACAUUGCAUUAUUUCUUU 1 times at 7207 NSP3 CAGGUACAUUGCAUUAUUUCUUU

CCAUAUUUGUAGACUGGCGGUCAUA 1 times at 7242 NSP3

CGGUCAUACAAUUAUGCUGUGUCUA 1 times at 7259 NSP3

GCUGUGUCUAGUGCCUUCUGGUUAU 1 times at 7274 NSP3

GCUUUUACGCAAGUUUUAUCAGCAU 1 times at 7357 NSP3

GCAAGUUUUAUCAGCAUGUAAUCAA 1 times at 7365 NSP3

GCAUGUAAUCAAUGGUUGCAAAGAU 1 times at 7378 NSP3

GCUCUGCUAUAAGAGGAACCGACUU 1 times at 7414 NSP3

CGACUUACUAGAGUUGAAGCUUCUA 1 times at 7433 NSP3

GCUUCUACCGUUGUCUGUGGUGGAA 1 times at 7451 NSP3

CGGUAUUUCAUUCUGUCGUAGGCAU 1 times at 7504 NSP3

GGUAUUUCAUUCUGUCGUAGGCAUA 1 times at 7505 NSP3

GGGGAAUACCUUCAUCUGUGAAGAA 1 times at 7564 NSP3

CCUUCAUCUGUGAAGAAGUCGCAAA 1 times at 7572 NSP3

GCCCUACGCAGGCCUAUUAACGCUA 1 times at 7610 NSP3

CGCAGGCCUAUUAACGCUACGGAUA 1 times at 7616 NSP3

CGCUACGGAUAGAUCACAUUAUUAU 1 times at 7630 NSP3

GGAUAGAUCACAUUAUUAUGUGGAU 1 times at 7636 NSP3

CGUUACAGUUAAAGAGACUGUUGUU 1 times at 7663 NSP3

CCUCUGCGCUUUUACAAAUCUAGAU 1 times at 7735 NSP3

GCGCUUUUACAAAUCUAGAUAAGUU 1 times at 7740 NSP3 GCGCUUUUACAAAUCUAGAUAAG

GGUCUGUAAAACUACUACUGGUAUA 1 times at 7777 NSP3

GCUAGGUCUGCAUGUGUUUAUUAUU 1 times at 7856 NSP3 GGUGAUUCUAGUGAAAUCGCCACUA 1 times at 7937 NSP3

CGCCACUAAAAUGUUUGAUUCCUUU 1 times at 7954 NSP3

CGCUGUAUAAUGUCACACGCGAUAA 1 times at 7995 NSP3

CGUGAUGGCGUAAGGCGAGGCGAUA 1 times at 8045 NSP3

CGUAAGGCGAGGCGAUAACUUCCAU 1 times at 8053 NSP3

GGCGAUAACUUCCAUAGUGUCUUAA 1 times at 8063 NSP3

CCAUAGUGUCUUAACAACAUUCAUU 1 times at 8074 NSP3

CGGCUUCAGUUAACCAAAUUGUCUU 1 times at 8286 NSP3 GGCUUCAGUUAACCAAAUUGUCU

CCAAAUUGUCUUGCGUAAUUCUAAU 1 times at 8299 NSP3

CGACAGAUUCGCAUUGCAUGCCGUA 1 times at 8378 NSP3

CGCAUUGCAUGCCGUAAGUGUAAUU 1 times at 8387 NSP3 CGCAUUGCAUGCCGUAAGUGUAA

GCAUUGCAUGCCGUAAGUGUAAUUU 1 times at 8388 NSP3

GCAUGCCGUAAGUGUAAUUUAGCUU 1 times at 8393 NSP3

CCUCAAAGCUACGCGCUAAUGAUAA 1 times at 8430 NSP3 CUCAAAGCUACGCGCUAAUGAUA

GCUACGCGCUAAUGAUAAUAUCUUA 1 times at 8437 NSP3

CGCUAAUGAUAAUAUCUUAUCAGUU 1 times at 8443 NSP3

GCUAAUGAUAAUAUCUUAUCAGUUA 1 times at 8444 NSP3

CCGCAUCUUGGACUUUAAAGUUCUU 1 times at 8638 NSP4 CCGCAUCUUGGACUUUAAAGUUC

CCUGAUGAUAAGUGCUUUGCUAAUA 1 times at 8690 NSP4

GCUUUGCUAAUAAGCACCGGUCCUU 1 times at 8703 NSP4

GCACCGGUCCUUCACACAAUGGUAU 1 times at 8716 NSP4

CCG G U CCU UCACACAAU G G U AUCAU 1 times at 8719 NSP4

GGUGCUCGCAUUCCAGACGUACCUA 1 times at 8816 NSP4

GCUCGCAUUCCAGACGUACCUACUA 1 times at 8819 NSP4

CGCAUUCCAGACGUACCUACUACAU 1 times at 8822 NSP4

GCAUUCCAGACGUACCUACUACAUU 1 times at 8823 NSP4

CCAGACGUACCUACUACAUUGGCUU 1 times at 8828 NSP4

GCAUUCUUCCAUCUGAGUGCACUAU 1 times at 8964 NSP4

GGGCCGUAUGACACCAUACUGCCAU 1 times at 9004 NSP4

CCGUAUGACACCAUACUGCCAUGAU 1 times at 9007 NSP4

CCAUACUGCCAUGAUCCUACUGUUU 1 times at 9017 NSP4

GGCCUCAUGUUCGUUACGACUUGUA 1 times at 9072 NSP4

GCCUCAUGUUCGUUACGACUUGUAU 1 times at 9073 NSP4

CGACUUGUAUGAUGGUAACAUGUUU 1 times at 9088 NSP4

CCACAAAUGGCUCGUGGGCCAUUUU 1 times at 9225 NSP4

GGCCAU U U U U AAU G ACCACCAU CU U 1 times at 9241 NSP4

GCCAUUUUUAAUGACCACCAUCUUA 1 times at 9242 NSP4

CCAU U U U U AAU G ACCACCAUCU U AA 1 times at 9243 NSP4

CCAUCUUAAUAGACCUGGUGUCUAU 1 times at 9259 NSP4

CCUGGUGUCUAUUGUGGCUCUGAUU 1 times at 9272 NSP4

GGUGUCUAUUGUGGCUCUGAUUUUA 1 times at 9275 NSP4

GCAGUAUCACUGUUCCAGCCUAUUA 1 times at 9320 NSP4

CCU AU U ACU U AU U U CCAAU U G ACU A 1 times at 9338 NSP4

CCUCAUUGGUCUUGGGUAUAGGUUU 1 times at 9363 NSP4

CCUGACUUUGCUCUUCUAUUAUAUU 1 times at 9397 NSP4 GCUCUUCUAUUAUAUUAAUAAAGUA 1 times at 9406 NSP4 CUCUUCUAUUAUAUUAAUAAAGU

GCUGUUGUUGCUGCUGUUCUUAAUA 1 times at 9470 NSP4

CCUGCAUUUAUUAUGCAUGUUUCUU 1 times at 9587 NSP4

CCAGGACGCUGCCUCUAAUAUCUUU 1 times at 9760 NSP4

GGACGCUGCCUCUAAUAUCUUUGUU 1 times at 9763 NSP4

CGCUGCCUCUAAUAUCUUUGUUAUU 1 times at 9766 NSP4

GCUGCCUCUAAUAUCUUUGUUAUUA 1 times at 9767 NSP4 UGCCUCUAAUAUCUUUGUUAUUA

CCUCUAAUAUCUUUGUUAUUAACAA 1 times at 9771 NSP4 CUCUAAUAUCUUUGUUAUUAACA

GCAGCUCUUAGAAACUCUUUAACUA 1 times at 9806 NSP4 CAGCUCUUAGAAACUCUUUAACU

CCUAUUCACGAUUUUUGGGGUUGUU 1 times at 9837 NSP4

GGUUGUUUAACAAGUAUAAGUACUU 1 times at 9855 NSP4

GCCGCUUAUCGUGAAGCUGCAGCAU 1 times at 9899 NSP4

GCGAGACUGGUAGUGAUCUUCUUUA 1 times at 9954 NSP4

CCUCUGGCGUGUUGCAAAGCGGUUU 1 times at 10002 NSP4

GCGUGUUGCAAAGCGGUUUGGUGAA 1 times at 10008 NSP4

CGUGUUGCAAAGCGGUUUGGUGAAA 1 times at 10009 NSP4

GGUUACCUGCGGUAGCAUGACUCUU 1 times at 10075 NSP5

CGGUAGCAUGACUCUUAAUGGUCUU 1 times at 10084 NSP5

GGUAGCAUGACUCUUAAUGGUCUUU 1 times at 10085 NSP5

CCUAAUUAUGAUGCCUUGUUGAUUU 1 times at 10172 NSP5

CGCUCCAGCAAACUUGCGUGUUGUU 1 times at 10237 NSP5

GGUCAUGCCAUGCAAGGCACUCUUU 1 times at 10262 NSP5

GGCGCAGCAUUUAGUGUGUUAGCAU 1 times at 10352 NSP5

GCAUUUAGUGUGUUAGCAUGCUAUA 1 times at 10358 NSP5

CCGACUGGUACAUUCACUGUUGUAA 1 times at 10391 NSP5

CGACUGGUACAUUCACUGUUGUAAU 1 times at 10392 NSP5

CGCCCUAACUACACAAUUAAGGGUU 1 times at 10418 NSP5

CCGGUUCAGCAUUUGAUGGUACUAU 1 times at 10545 NSP5

G CACCAAG U U CAG U U AACAG ACAAA 1 times at 10597 NSP5

GCUUGGCUUUACGCAGCAAUACUUA 1 times at 10643 NSP5

GCAGCAAUACUUAAUGGUUGCGCUU 1 times at 10655 NSP5

GGCGUUGCUAUUGAACAGCUGCUUU 1 times at 10793 NSP5

GCGUUGCUAUUGAACAGCUGCUUUA 1 times at 10794 NSP5

CGUUGCUAUUGAACAGCUGCUUUAU 1 times at 10795 NSP5

GGAAGAUGAAUUCACACCUGAGGAU 1 times at 10879 NSP5

CCUGAGGAUGUUAAUAUGCAGAUUA 1 times at 10895 NSP5

GGUUAUGCAGAGUGGUGUGAGAAAA 1 times at 10927 NSP5

GGUGUGAGAAAAGUUACAUAUGGUA 1 times at 10940 NSP6

CGACCCUUGUCUCAACCUAUGUGAU 1 times at 10983 NSP6

CCCUUGUCUCAACCUAUGUGAUAAU 1 times at 10986 NSP6

CCACUAAAUUUACUUUGUGGAACUA 1 times at 11019 NSP6

CCCACACAGUUGUUCCCACUCUUAU 1 times at 11060 NSP6

CCACACAGUUGUUCCCACUCUUAUU 1 times at 11061 NSP6

GGCCUUCGUUAUGUUGUUGGUUAAA 1 times at 11095 NSP6

CGUUAUGUUGUUGGUUAAACACAAA 1 times at 11101 NSP6 GCCUGUGGCUAUUUGUUUGACUUAU 1 times at 11152 NSP6

GCAAACAUAGUCUACGAGCCCACUA 1 times at 11177 NSP6

CGUCAGCGCUGAUUGCAGUUGCAAA 1 times at 11211 NSP6

GCUGAUUGCAGUUGCAAAUUGGCUU 1 times at 11218 NSP6

GGCUUGCCCCCACUAAUGCUUAUAU 1 times at 11238 NSP6

CCCACUAAUGCUUAUAUGCGCACUA 1 times at 11246 NSP6

GGUGUAAUGUGGUUGUACACUUAUA 1 times at 11378 NSP6

GCAUUGGAGAAGCCUCAAGCCCCAU 1 times at 11403 NSP6

CCGGAAGUGAAGAUGAUACUUUUAU 1 times at 11555 NSP6

CGGAAGUGAAGAUGAUACUUUUAUU 1 times at 11556 NSP6 CGGAAGUGAAGAUGAUACUUUUA

GGAAGUGAAGAUGAUACUUUUAUUA 1 times at 11557 NSP6 AAGUGAAGAUGAUACUUUUAUUA

GCUUAGAGCACCUAUGGGUGUCUAU 1 times at 11644 NSP6

GCACCUAUGGGUGUCUAUGACUUUA 1 times at 11651 NSP6

GCUAACAAUCUAACUGCACCUAGAA 1 times at 11708 NSP6

GCACCUAGAAAUUCUUGGGAGGCUA 1 times at 11723 NSP6

GGGAGGCUAUGGCUCUGAACUUUAA 1 times at 11739 NSP6

GGUUGCUGCUAUGCAGUCUAAACUU 1 times at 11797 NSP6

G C AG U C U AAAC UUACAGAUCUU AAA 1 times at 11809 NSP6

CCAACAGUUACACUUAGAGGCUAAU 1 times at 11863 NSP7

GGGCUUUCUGUGUUAAAUGCCAUAA 1 times at 11898 NSP7

GGCUUUCUGUGUUAAAUGCCAUAAU 1 times at 11899 NSP7

GCAGCAACAGACCCCAGUGAGGCUU 1 times at 11933 NSP7

GCUAGUGAUAUUUUUGACACUCCUA 1 times at 12026 NSP7

CCUAGCGUACUUCAAGCUACUCUUU 1 times at 12047 NSP7

GCGCAGAAAGCCUAUCAGGAAGCUA 1 times at 12113 NSP8

CGCAGAAAGCCUAUCAGGAAGCUAU 1 times at 12114 NSP8

GGACUCUGGUGACACCUCACCACAA 1 times at 12139 NSP8

GG U G ACACCU CACCACAAG U U CU U A 1 times at 12146 NSP8

CCUCACCACAAGUUCUUAAGGCUUU 1 times at 12153 NSP8

GGCUUUGCAGAAGGCUGUUAAUAUA 1 times at 12172 NSP8

GCAGAAGGCUGUUAAUAUAGCUAAA 1 times at 12178 NSP8

GCUAAAAACGCCUAUGAGAAGGAUA 1 times at 12197 NSP8

GGAUAAGGCAGUGGCCCGUAAGUUA 1 times at 12217 NSP8

GCAGUGGCCCGUAAGUUAGAACGUA 1 times at 12224 NSP8

GGCUAUGACUUCUAUGUAUAAGCAA 1 times at 12259 NSP8 GGCUAUGACUUCUAUGUAUAAGC

G CAAAAAU UGUCAGUGCUAUG CAAA 1 times at 12305 NSP8

GCUAUGCAAACUAUGUUGUUUGGUA 1 times at 12320 NSP8

GCAAACUAUGUUGUUUGGUAUGAUU 1 times at 12325 NSP8

GCUUCAAAUAAACUUCGCGUUGUAA 1 times at 12434 NSP8

CCGUCUGGAAU CAG G U AG U CACAU A 1 times at 12471 NSP8

CGUCUGGAAUCAGGUAGUCACAUAU 1 times at 12472 NSP8

CCCUCGCUUAACUACGCUGGGGCUU 1 times at 12497 NSP8

CCUCGCUUAACUACGCUGGGGCUUU 1 times at 12498 NSP8

GGGGCUUUGUGGGACAUUACAGUUA 1 times at 12515 NSP8

GGGCUUUGUGGGACAUUACAGUUAU 1 times at 12516 NSP8 GGCUUUGUGGGACAUUACAGUUAUA 1 times at 12517 NSP8

GCUUUGUGGGACAUUACAGUUAUAA 1 times at 12518 NSP8

GGGCAUCCACUUCUGCCGUUAAGUU 1 times at 12630 NSP8

CCACUUCUGCCGUUAAGUUGCAAAA 1 times at 12636 NSP8

CCG U U AAG U U G C AAAAU AAU G AG AU 1 times at 12645 NSP8

GGUCAAGAGCAAACUAACUGUAAUA 1 times at 12707 NSP9

GGGUCGUAAAAUGCUGAUGGCUCUU 1 times at 12763 NSP9

CGUAAAAUGCUGAUGGCUCUUCUUU 1 times at 12767 NSP9

GCUGAUGGCUCUUCUUUCUGAUAAU 1 times at 12775 NSP9

GGCUCUUCUUUCUGAUAAUGCCUAU 1 times at 12781 NSP9

GCGCGUGUUGAAGGUAAGGACGGAU 1 times at 12815 NSP9

CGCGUGUUGAAGGUAAGGACGGAUU 1 times at 12816 NSP9

GCGUGUUGAAGGUAAGGACGGAUUU 1 times at 12817 NSP9

GCAAAUUCUUGAUUGCGGGACCAAA 1 times at 12867 NSP9

GGACCAAAAGGACCUGAAAUCCGAU 1 times at 12884 NSP9

GGGCACAUUGCUGCGACUGUUAGAU 1 times at 12959 NSP9

GGCACAUUGCUGCGACUGUUAGAUU 1 times at 12960 NSP9

GCGACUGUUAGAUUGCAAGCUGGUU 1 times at 12971 NSP9

GCAAGCUGGUUCUAACACCGAGUUU 1 times at 12985 NSP9

GGUUCUAACACCGAGUUUGCCUCUA 1 times at 12992 NSP10

CCUAAAACUGGUACAGGUAUAGCUA 1 times at 13127 NSP10

GGUACAGGUAUAGCUAUAUCUGUUA 1 times at 13136 NSP10 UACAGGUAUAGCUAUAUCUGUUA

GCUAUAUCUGUUAAACCAGAGAGUA 1 times at 13148 NSP10

CCGUGCGCAUAUAGAACAUCCUGAU 1 times at 13219 NSP10

CCUGUAAUGUCUGUCAAUAUUGGAU 1 times at 13335 NSP10

GCCCCAAUCUAAAGAUUCCAAUUUU 1 times at 13402 NSP10

CCCCAAU CU AAAG AU U CCAAU U U U U 1 times at 13403 NSP10 CCCCAAU CU AAAG AU U CCAAU U U

CCCAAUCUAAAGAUUCCAAUUUUUU 1 times at 13404 NSP10 CCCAAUCU AAAG AU U CCAAU U U U

CCAAUCUAAAGAUUCCAAUUUUUUA 1 times at 13405 NSP10

CGGGGUUCUAUUGUAAAUGCCCGAA 1 times at 13438 NSP12

GGGGUUCUAUUGUAAAUGCCCGAAU 1 times at 13439 NSP12

GGGUUCUAUUGUAAAUGCCCGAAUA 1 times at 13440 NSP12

CG AAU AG AACCCU G U U CAAGUGG U U 1 times at 13459 NSP12

GGGCAUUUGACAUCUGCAACUAUAA 1 times at 13505 NSP12

GGCUAAGGUUGCUGGUAUUGGAAAA 1 times at 13530 NSP12

GCUAAGGUUGCUGGUAUUGG AAAAU 1 times at 13531 NSP12

GGUAUUGGAAAAUACUACAAGACUA 1 times at 13543 NSP12

GGAAAAUACUACAAGACUAAUACUU 1 times at 13549 NSP12

CCAAGGGCAUCAUUUAGACUCCUAU 1 times at 13596 NSP12

CGUUAAGAGGCAUACUAUGGAGAAU 1 times at 13626 NSP12

GCAUACUAUGGAGAAUUAUGAACUA 1 times at 13635 NSP12

CCAUGAUUUCUUCAUCUUUGAUGUA 1 times at 13707 NSP12

CCUCAUAUUGUACGUCAGCGUUUAA 1 times at 13747 NSP12

CGUCAGCGUUUAACUGAGUACACUA 1 times at 13759 NSP12

GCCCUGAGGCACUUUGAUCAAAAUA 1 times at 13801 NSP12 GCUUAAGGCUAUCUUAGUGAAGUAU 1 times at 13833 NSP12

GCUGUGAUGUUACCUACUUUGAAAA 1 times at 13862 NSP12 CUGUGAUGUUACCUACUUUGAAA

CCUACUUUGAAAAUAAACUCUGGUU 1 times at 13874 NSP12

CCCAGUGUUAUUGGUGUUUAUCAUA 1 times at 13915 NSP12 CCCAGUGUUAUUGGUGUUUAUCA

CCAGUGUUAUUGGUGUUUAUCAUAA 1 times at 13916 NSP12 CAGUGUUAUUGGUGUUUAUCAUA

CGCCAAGCUAUCUUAAACACUGUUA 1 times at 13957 NSP12

GCCAAGCUAUCUUAAACACUGUUAA 1 times at 13958 NSP12

CCAAGCUAUCUUAAACACUGUUAAA 1 times at 13959 NSP12

GCUAUCUUAAACACUGUUAAAUUUU 1 times at 13963 NSP12

GCUCACACUAGACAACCAGGACCUU 1 times at 14022 NSP12

CCAGGACCUUAAUGGCAAGUGGUAU 1 times at 14037 NSP12

GGACCUUAAUGGCAAGUGGUAUGAU 1 times at 14040 NSP12

CCUUAAUGGCAAGUGGUAUGAUUUU 1 times at 14043 NSP12

GCAAGUGGUAUGAUUUUGGUGACUU 1 times at 14051 NSP12

GGUAUGAUUUUGGUGACUUCGUAAU 1 times at 14057 NSP12

GGUUCAGGAGUAGCUAUAGUUGAUA 1 times at 14092 NSP12

GCUAUAGUUGAUAGCUACUAUUCUU 1 times at 14104 NSP12

CGAUUGUCUGGCCGCUGAGACACAU 1 times at 14154 NSP12

CGCUGAGACACAUAGGGAUUGUGAU 1 times at 14166 NSP12

GCUGAGACACAUAGGGAUUGUGAUU 1 times at 14167 NSP12

GGUACAACUCUUUGAGAAGUACUUU 1 times at 14247 NSP12 UACAACUCUUUGAGAAGUACUUU

CGCAAAUUGCGUUAAUUGUACUGAU 1 times at 14295 NSP12

CCGUUGUGUGUUACAUUGUGCUAAU 1 times at 14322 NSP12

CGUUGUGUGUUACAUUGUGCUAAUU 1 times at 14323 NSP12

GCUAAUUUCAAUGUAUUGUUUGCUA 1 times at 14341 NSP12

GCCUAAGACUUGUUUCGGACCCAUA 1 times at 14373 NSP12

CGGACCCAUAGUCCGAAAGAUCUUU 1 times at 14388 NSP12

GCCAUUUGUAGUAUCUUGUGGUUAU 1 times at 14424 NSP12

GGUUAUCACUACAAAGAAUUAGGUU 1 times at 14443 NSP12

GGUUUAGUCAUGAAUAUGGAUGUUA 1 times at 14464 NSP12

CCAGCCAUGCACAUUGCCUCCUCUA 1 times at 14542 NSP12

GCACAUUGCCUCCUCUAACGCUUUU 1 times at 14550 NSP12

GCCUCCUCUAACGCUUUUCUUGAUU 1 times at 14557 NSP12

CCUCCUCUAACGCUUUUCUUGAUUU 1 times at 14558 NSP12 CUCCUCUAACGCUUUUCUUGAUU

GCUUUUCUUGAUUUGAGGACAUCAU 1 times at 14569 NSP12

GCUGCACUUACAACUGGUUUGACUU 1 times at 14605 NSP12

GGCCUGGCAAUUUUAACCAAGACUU 1 times at 14642 NSP12

CCAAGACUUCUAUGAUUUCGUGGUA 1 times at 14658 NSP12

GCUCAAACAUUUUUUCUUUGCUCAA 1 times at 14718 NSP12

GCUCAAGAUGGUAAUGCUGCUAUUA 1 times at 14737 NSP12

GGUAAUGCUGCUAUUACAGAUUAUA 1 times at 14746 NSP12

GCUAUUACAGAUUAUAAUUACUAUU 1 times at 14755 NSP12

GCCUACUAUGUGUGACAUCAAACAA 1 times at 14790 NSP12

CCUACUAUGUGUGACAU C A AAC AAA 1 times at 14791 NSP12 UACUAUGUGUG ACAU CAAAC AAA

GCAUGGAAGUUGUAAACAAGUACUU 1 times at 14825 NSP12 GGAAGUUGUAAACAAGUACUUCGAA 1 times at 14829 NSP12

CGAAAUCUAUGACGGUGGUUGUCUU 1 times at 14850 NSP12

CGGUGGUUGUCUUAAUGCUUCUGAA 1 times at 14862 NSP12

GCUUCUGAAGUGGUUGUUAAUAAUU 1 times at 14878 NSP12

GCCAUCCUUUUAAUAAGUUUGGCAA 1 times at 14918 NSP12

CCAUCCUUUUAAUAAGUUUGGCAAA 1 times at 14919 NSP12

CGUGUCUAUUAUGAGAGCAUGUCUU 1 times at 14947 NSP12

GCAGGCGUGUCCAUACUUAG CACAA 1 times at 15082 NSP12

CGCCAGUACCAUCAGAAAAUGCUUA 1 times at 15115 NSP12

G CC AG U ACCAU CAG AAAAU G CU U AA 1 times at 15116 NSP12

CGUGGAGCGACUUGCGUCAUUGGUA 1 times at 15157 NSP12

GGAGCGACUUGCGUCAUUGGUACUA 1 times at 15160 NSP12

GCGACUUGCGUCAUUGGUACUACAA 1 times at 15163 NSP12

CGACUUGCGUCAUUGGUACUACAAA 1 times at 15164 NSP12

GCGUCAUUGGUACUACAAAGUUCUA 1 times at 15170 NSP12

GGUGGCUGGGAUUUCAUGCUUAAAA 1 times at 15196 NSP12

GGCUGGGAUUUCAUGCUUAAAACAU 1 times at 15199 NSP12

GCUGGGAUUUCAUGCUUAAAACAUU 1 times at 15200 NSP12 UGGGAUUUCAUGCUUAAAACAUU

GGGAUUUCAUGCUUAAAACAUUGUA 1 times at 15203 NSP12 GGGAUUUCAUGCUUAAAACAUUG

GGGUUGGGAUUACCCUAAGUGUGAU 1 times at 15255 NSP12

GGUUGGGAUUACCCUAAGUGUGAUA 1 times at 15256 NSP12

CCUAAGUGUGAUAGAGCUAUGCCUA 1 times at 15268 NSP12

CCUAAUAUGUGUAGAAUCUUCGCUU 1 times at 15289 NSP12

CGCUUCACUCAUAUUAGCUCGUAAA 1 times at 15309 NSP12

GGGACAGAUUUUAUCGCUUGGCAAA 1 times at 15356 NSP12

GGACAGAUUUUAUCGCUUGGCAAAU 1 times at 15357 NSP12

GGCAAAUGAGUGUGCUCAGGUGCUA 1 times at 15375 NSP12

GCAAAUGAGUGUGCUCAGGUGCUAA 1 times at 15376 NSP12

GGUUACUACGUCAAACCUGGAGGUA 1 times at 15424 NSP12

CCACUGCAUAUGCCAAUAGUGUCUU 1 times at 15467 NSP12 CACUGCAUAUGCCAAUAGUGUCU

GGGUGCUAAUGGCAACAAGAUUGUU 1 times at 15534 NSP12 GGGUGCUAAUGGCAACAAGAUUG

GGAGCACUAGCCCAGACCCCAAAUU 1 times at 15608 NSP12

GCCCAGACCCCAAAUUUGUUGAUAA 1 times at 15617 NSP12

CCCAG ACCCCAAAU U UG U U G AU AAA 1 times at 15618 NSP12

CCAGACCCCAAAUUUGUUGAUAAAU 1 times at 15619 NSP12

CCCCAAAUUUGUUGAUAAAUACUAU 1 times at 15624 NSP12 CCCCAAAUUUGUUGAUAAAUACU

GCUUUUCUUAAUAAGCACUUUUCUA 1 times at 15649 NSP12

CGGUGUCGUUUGCUAUAAUAGUGAU 1 times at 15693 NSP12

GGUGUCGUUUGCUAUAAUAGUGAUU 1 times at 15694 NSP12

GCUAUAAUAGUGAUUAUGCAGCUAA 1 times at 15704 NSP12

GCAGCUAAGGGUUACAUUGCUGGAA 1 times at 15721 NSP12

GGGUUACAUUGCUGGAAUACAGAAU 1 times at 15729 NSP12

GGUUACAUUGCUGGAAUACAGAAUU 1 times at 15730 NSP12

GGAAACGCUGUAUUAUCAGAACAAU 1 times at 15759 NSP12

CGCUGUAUUAUCAGAACAAUGUCUU 1 times at 15764 NSP12 GCUGUAUUAUCAGAACAAUGUCUUU 1 times at 15765 NSP12

GCUGGGUGGAAACCGAUCUGAAGAA 1 times at 15806 NSP12

CGAUCUGAAGAAAGGGCCACAUGAA 1 times at 15819 NSP12

G CC ACAU G AAU U CU G U U CACAG C AU 1 times at 15834 NSP12

CCACAUGAAUUCUGUUCACAGCAUA 1 times at 15835 NSP12

GCUUUAUAUUAAGGAUGGCGACGAU 1 times at 15861 NSP12

GGAUGGCGACGAUGGUUACUUCCUU 1 times at 15873 NSP12

GGCGACGAUGGUUACUUCCUUCCUU 1 times at 15877 NSP12

GCGACGAUGGUUACUUCCUUCCUUA 1 times at 15878 NSP12

CGACGAUGGUUACUUCCUUCCUUAU 1 times at 15879 NSP12

CCU U AU CCAG ACCCU U CAAG AAU U U 1 times at 15898 NSP12

CCUUCAAGAAUUUUGUCUGCCGGUU 1 times at 15910 NSP12

CGGUUGCUUUGUAGAUGAUAUCGUU 1 times at 15930 NSP12 CGGUUGCUUUGUAGAUGAUAUCG

GGUUGCUUUGUAGAUGAUAUCGUUA 1 times at 15931 NSP12 UUGCUUUGUAGAUGAUAUCGUUA

GCGGUUUGUGUCUUUGGCUAUAGAU 1 times at 15981 NSP12

GCUAUAGAUGCUUACCCUCUCACAA 1 times at 15997 NSP12

CCC U C U C AC AAAG CAU G AAG AU AU A 1 times at 16011 NSP12 CUCUCACAAAGCAUGAAGAUAUA

GCAUGAAGAUAUAGAAUACCAGAAU 1 times at 16023 NSP12

CCAGAAUGUAUUCUGGGUCUACUUA 1 times at 16041 NSP12

GGGUCUACUUACAGUAUAUAGAAAA 1 times at 16055 NSP12

GGUCUACUUACAGUAUAUAGAAAAA 1 times at 16056 NSP12 GUCUACUUACAGUAUAUAGAAAA

GCUUGACAGUUAUUCUGUCAUGCUA 1 times at 16107 NSP12

CCUACCACUUUGCAGGCUGUCGGUU 1 times at 16192 NSP12

GCAGGCUGUCGGUUCAUGCGUUGUA 1 times at 16203 NSP12

CCACAUAAGAUGGUUUUGUCUGUUU 1 times at 16318 NSP13

CCACUUUGCGCUAAUGGUCUUGUAU 1 times at 16450 NSP13

GCGCUAAUGGUCUUGUAUUCGGCUU 1 times at 16457 NSP13

CGCUAAUGGUCUUGUAUUCGGCUUA 1 times at 16458 NSP13

GCUAAUGGUCUUGUAUUCGGCUUAU 1 times at 16459 NSP13

GGUGAUUACACCCUUGCCAAUACUA 1 times at 16558 NSP13

CCAAUACUACAACAGAACCACUCAA 1 times at 16574 NSP13

CCACCACU CAAU CG U AAU U AU G U U U 1 times at 16726 NSP13 ACCACUCAAUCGUAAUUAUGUUU

CCACUCAAUCGUAAUUAUGUUUUUA 1 times at 16729 NSP13

GGUUAUCAU AU AACCAAAAAU AG U A 1 times at 16756 NSP13

GCGCAUUGAUUAUAGUGAUGCUGUA 1 times at 16809 NSP13

CGCAUUGAUUAUAGUGAUGCUGUAU 1 times at 16810 NSP13

GCUGUAUCCUACAAGUCUAGUACAA 1 times at 16828 NSP13

CCUACAAGUCUAGUACAACGUAUAA 1 times at 16835 NSP13 U ACAAG U CU AG U ACAACG U AU AA

CGUAUAAACUGACUGUAGGUGACAU 1 times at 16853 NSP13

GGCUACCUUGACGGCGCCCACAAUU 1 times at 16902 NSP13

GGUAUGUUAAAAUUACUGGGUUGUA 1 times at 16940 NSP13

G CC AACU U CCAAAAAU CAG G U U AU A 1 times at 17005 NSP13

CCAAAAAU CAG G U U AU AG U AAAU AU 1 times at 17013 NSP13

GCACGUGUUGUUUAUACAGCAUGUU 1 times at 17110 NSP13

CGCAGCUGUUGAUGCUUUGUGUGAA 1 times at 17139 NSP13 GCAGCUGUUGAUGCUUUGUGUGAAA 1 times at 17140 NSP13

GCUUUGUGUGAAAAAGCUUUUAAAU 1 times at 17152 NSP13

GCUUUUAAAUAUUUGAACAUUGCUA 1 times at 17167 NSP13

CGUGUUGAGUGCUAUGACAGGUUUA 1 times at 17221 NSP13

GGUUAGUAUGUGCACUAAUUAUGAU 1 times at 17331 NSP13

GCACUAAUUAUGAUCUUUCAAUUAU 1 times at 17342 NSP13 CACUAAUUAUGAUCUUUCAAUUA

GCACAGUUGCCAGCUCCUAGGACUU 1 times at 17413 NSP13

CCAGCUCCUAGGACUUUGUUGACUA 1 times at 17422 NSP13

GGACUUUGUUGACUAGAGGCACAUU 1 times at 17432 NSP13

GCACUGUGAGCGCUCUUGUCUACAA 1 times at 17555 NSP13

GCGCUCUUGUCUACAAUAAUAAAUU 1 times at 17564 NSP13

GCUUUAAAAUACUCUAUAAGGGCAA 1 times at 17618 NSP13

CGCAUGAUGCUAGCUCUGCCAUUAA 1 times at 17648 NSP13

GCAUGAUGCUAGCUCUGCCAUUAAU 1 times at 17649 NSP13

GCCAUUAAUAGACCACAACUCACAU 1 times at 17665 NSP13

CCAU U AAU AGACCACAACUCACAU U 1 times at 17666 NSP13

CCACAACU CACAU U U G U G AAG AAU U 1 times at 17677 NSP13

CCGGCAUGGAGUAAGGCAGUCUUUA 1 times at 17716 NSP13

CGGCAUGGAGUAAGGCAGUCUUUAU 1 times at 17717 NSP13

GGCAUGGAGUAAGGCAGUCUUUAUU 1 times at 17718 NSP13

GCAUGGAGUAAGGCAGUCUUUAUUU 1 times at 17719 NSP13 AUGGAGUAAGGCAGUCUUUAUUU

CCU CACAG G G U U CAG AAU ACCAG U A 1 times at 17810 NSP13

GCACAUGCUAACAACAUUAACAGAU 1 times at 17863 NSP13 CACAUGCUAACAACAUUAACAGA

G CAAU CACU CG U G CCCAAAAAG G U A 1 times at 17896 NSP13

GCCCAAAAAGGUAUUCUUUGUGUUA 1 times at 17908 NSP13 GCCCAAAAAGGUAUUCUUUGUGU

CCCAAAAAGGUAUUCUUUGUGUUAU 1 times at 17909 NSP13

GGCACUCUUUGAGUCCUUAGAGUUU 1 times at 17943 NSP13

GCACUCUUUGAGUCCUUAGAGUUUA 1 times at 17944 NSP13 CACUCUUUGAGUCCUUAGAGUUU

CCUUAGAGUUUACUGAAUUGUCUUU 1 times at 17957 NSP13

CCUUUUUAAAGAUUGCUCUAGAGAA 1 times at 18018 NSP14

GGCCUCUCACCUGCUUAUGCACCAA 1 times at 18049 NSP14

GCGUGAAUCUUAAUUUACCCGCAAA 1 times at 18119 NSP14

CGUGAAUCUUAAUUUACCCGCAAAU 1 times at 18120 NSP14

CGCAAAUGUCCCAUACUCUCGUGUU 1 times at 18138 NSP14

GCAAAUGUCCCAUACUCUCGUGUUA 1 times at 18139 NSP14

CGUGUUAUUUCCAGGAUGGGCUUUA 1 times at 18157 NSP14

GGGCUUUAAACUCGAUGCAACAGUU 1 times at 18174 NSP14

GGCAAGUUCGAAGCUGGAUAGGCUU 1 times at 18242 NSP14

GGUGCUCAUGCUUCCCGUAAUGCAU 1 times at 18277 NSP14

CCAAUGUGCCUCUACAAUUAGGAUU 1 times at 18308 NSP14

GGUGUUGUAGACACUGAGUGGGGUA 1 times at 18367 NSP14

CG U CCU CCACCAG G UGAACAG U U U A 1 times at 18415 NSP14

CGUUUGUUUGUUGGGCUCAUGGCUU 1 times at 18545 NSP14

GGCUUUGAAUUAACGUCUGCAUCAU 1 times at 18565 NSP14

GCUUUGAAUUAACGUCUGCAUCAUA 1 times at 18566 NSP14 CGUCUGCAUCAUACUUUUGCAAGAU 1 times at 18578 NSP14

GCAUCAUACUUUUGCAAGAUAGGUA 1 times at 18583 NSP14

GCAGCGUACUCUUCACCUCUGCAAU 1 times at 18643 NSP14

GCGUACUCUUCACCUCUGCAAUCUU 1 times at 18646 NSP14

CGUACUCUUCACCUCUGCAAUCUUA 1 times at 18647 NSP14

GCAAUCUUAUGCCUGCUGGACUCAU 1 times at 18663 NSP14

GCCUGCUGGACUCAUUCCUGCGGUU 1 times at 18673 NSP14

CCUGCUGGACUCAUUCCUGCGGUUA 1 times at 18674 NSP14

GGACUCAUUCCUGCGGUUAUGAUUA 1 times at 18680 NSP14

CCUGCGGUUAUGAUUAUGUCUACAA 1 times at 18689 NSP14 CUGCGGUUAUGAUUAUGUCUACA

GGUUAUGAUUAUGUCUACAACCCUU 1 times at 18694 NSP14

CGAUGUUCAACAGUGGGGUUAUGUA 1 times at 18726 NSP14

CGAUCGUUAUUGCUCUGUCCAUCAA 1 times at 18771 NSP14

GCUCAUGUGGCUUCUAAUGAUGCAA 1 times at 18799 NSP14

GCAAUAAUGACUCGUUGUUUAGCUA 1 times at 18820 NSP14

CGUUGUUUAGCUAUUCAUUCUUGUU 1 times at 18832 NSP14

CCUUAUAUCUCACAUGAAAAGAAAU 1 times at 18889 NSP14

GCGCAACGUCGUACGUGCUGCUCUU 1 times at 18939 NSP14

CGGUUCAUUUGACAAAGUCUAUGAU 1 times at 18969 NSP14 CGGUUCAUUUGACAAAGUCUAUG

GGUUCAUUUGACAAAGUCUAUGAUA 1 times at 18970 NSP14 UUCAUUUGACAAAGUCUAUGAUA

GGCAUUAUUUUGAUGCACAGCCCUU 1 times at 19043 NSP14

GGACAUGGCCUCAAGAUUUGCUGAU 1 times at 19101 NSP14

GCACGCUUUUCAUACACCAGCAUAU 1 times at 19257 NSP14

CGCUUUUCAUACACCAGCAUAUGAU 1 times at 19260 NSP14

CCUUUACCAUUCUUUUAUUAUUCUA 1 times at 19309 NSP14

GGUAAUGGUAGUAUGAUAGAGGAUA 1 times at 19354 NSP14

GGUAGUAUGAUAGAGGAUAUUGAUU 1 times at 19360 NSP14 UAGUAUGAUAGAGGAUAUUGAUU

GGAUAUUGAUUAUGUACCCCUAAAA 1 times at 19374 NSP14

CCCCUAAAAUCUGCAGUCUGUAUUA 1 times at 19390 NSP14

GGUGUUAUAAGACCUUUGAUAUUUA 1 times at 19517 NSP14 GUGUUAUAAGACCUUUGAUAUUU

CCAUUUUAUUGGUGUUGAGGGUGAA 1 times at 19611 NSP15

CCACUUUGCCUACUAAUAUAGCUUU 1 times at 19712 NSP15

GCGUGCUGUACGCUCGCAUCCCGAU 1 times at 19752 NSP15

CGUGCUGUACGCUCGCAUCCCGAUU 1 times at 19753 NSP15

CCCGAUUUCAAAUUGCUACACAAUU 1 times at 19771 NSP15

CCGAUUUCAAAUUGCUACACAAUUU 1 times at 19772 NSP15

CGAUUUCAAAUUGCUACACAAUUUA 1 times at 19773 NSP15

G CU ACACAAU U U ACAAG CAG ACAU U 1 times at 19785 NSP15

GCUACAAGUUCGUCCUUUGGGAUUA 1 times at 19811 NSP15

CCUUUGGGAUUAUGAACGUAGCAAU 1 times at 19824 NSP15

GGGAUUAUGAACGUAGCAAUAUUUA 1 times at 19829 NSP15 GGGAUUAUGAACGUAGCAAUAUU

GGAUUAUGAACGUAGCAAUAUUUAU 1 times at 19830 NSP15

CGUAGCAAUAUUUAUGGUACUGCUA 1 times at 19840 NSP15

GCAAUAUUUAUGGUACUGCUACUAU 1 times at 19844 NSP15

CCCAAUGCCAUCUUUAUUUCUGAUA 1 times at 19966 NSP15 GCCAUCUUUAUUUCUGAUAGAAAAA 1 times at 19972 NSP15 GCCAUCUUUAUUUCUGAUAGAAA

CCAU CU U U AU U U CU G AU AG AAAAAU 1 times at 19973 NSP15 AUCUUUAUUUCUGAUAGAAAAAU

CCCUUGUAUGGUAGGUCCUGAUUAU 1 times at 20007 NSP15

CCGUGAUAGUGAUGUUGUUAAACAA 1 times at 20055 NSP15 CCGUGAUAGUGAUGUUGUUAAAC

GGAAAACUAUGCUUUUGAGCACGUA 1 times at 20244 NSP15

CGUUAGGCGGUCUUCACUUGCUUAU 1 times at 20294 NSP15

GGCGGUCUUCACUUGCUUAUUGGUU 1 times at 20299 NSP15

GCGGUCUUCACUUGCUUAUUGGUUU 1 times at 20300 NSP15

CGGUCUUCACUUGCUUAUUGGUUUA 1 times at 20301 NSP15

GGUCUUCACUUGCUUAUUGGUUUAU 1 times at 20302 NSP15 GUCUUCACUUGCUUAUUGGUUUA

GCUUAUUGGUUUAUACAAGAAGCAA 1 times at 20313 NSP15

GGAAGGUCAUAUUAUUAUGGAAGAA 1 times at 20340 NSP15

GCUAAAAGGUAGCUCAACUAUUCAU 1 times at 20367 NSP15

GGUAGCUCAACUAUUCAUAACUAUU 1 times at 20374 NSP15

GCUCAACUAUUCAUAACUAUUUUAU 1 times at 20378 NSP15 CUCAACUAUUCAUAACUAUUUUA

GGCUUUUAAGGCGGUGUGUUCUGUU 1 times at 20421 NSP15

GCUUUUAAGGCGGUGUGUUCUGUUA 1 times at 20422 NSP15

GGCGGUGUGUUCUGUUAUAGAUUUA 1 times at 20430 NSP15

GCGGUGUGUUCUGUUAUAGAUUUAA 1 times at 20431 NSP15

CGGUGUGUUCUGUUAUAGAUUUAAA 1 times at 20432 NSP15

GCUUGACGACUUUGUUAUGAUUUUA 1 times at 20457 NSP15

CGUAGUAUCCAAGGUUGUCAAGGUU 1 times at 20499 NSP15

GGUUGUCAAGGUUCCUAUUGACUUA 1 times at 20511 NSP15

GGUUCCUAUUGACUUAACAAUGAUU 1 times at 20520 NSP15 UUCCUAUUGACUUAACAAUGAUU

CCCUCGACUCCAGGCUUCUGCAGAU 1 times at 20589 NSP15

CCUCGACUCCAGGCUUCUGCAGAUU 1 times at 20590 NSP15

GCCAUCCCUCUUUAAAGUUCAAAAU 1 times at 20634 NSP16

CCCUCUUUAAAGUUCAAAAUGUAAA 1 times at 20639 NSP16 CU CU UUAAAG U U CAAAAU G U AAA

CGCGGUGUGCACAUGAACAUCGCUA 1 times at 20713 NSP16

GCGGUGUGCACAUGAACAUCGCUAA 1 times at 20714 NSP16

CGGUGUGCACAUGAACAUCGCUAAA 1 times at 20715 NSP16

GGUGUGCACAUGAACAUCGCUAAAU 1 times at 20716 NSP16

GCCAGU AU U UAAAUACU UGCACAU U 1 times at 20753 NSP16

CCAGUAUUUAAAUACUUGCACAUUA 1 times at 20754 NSP16

GCCUGCCAAUAUGCGUGUUAUACAU 1 times at 20784 NSP16

CCUGCCAAUAUGCGUGUUAUACAUU 1 times at 20785 NSP16 UGCCAAUAUGCGUGUUAUACAUU

CGUGUUAUACAUUUUGGCGCUGGUU 1 times at 20797 NSP16

GCCAUUAUUAUAGAUAAUGAUUUAA 1 times at 20878 NSP16

CCAU U AU U AUAG AUAAUGAUU U AAA 1 times at 20879 NSP16

CGUGUCAGAUGCUGACAUAACUUUA 1 times at 20910 NSP16

GCUGACAUAACUUUAUUUGGAGAUU 1 times at 20920 NSP16

CCGACAUGUAUGAUCCUACUACUAA 1 times at 20987 NSP16 GACAUGUAUGAUCCUACUACUAA

CCUACUACUAAGAAUGUAACAGGUA 1 times at 21001 NSP16

GGUAGUAAUGAGUCAAAGGCUUUAU 1 times at 21022 NSP16

GCUUUAUUCUUUACUUACCUGUGUA 1 times at 21040 NSP16 CCUGUGUAACCUCAUUAAUAAUAAU 1 times at 21057 NSP16

GGUGGGUCUGUUGCUAUUAAAAUAA 1 times at 21091 NSP16

G C U AU U AAAAU AACAGAACACUCUU 1 times at 21103 NSP16

GGAGCGUUGAACUUUAUGAACUUAU 1 times at 21128 NSP16 GAGCGUUGAACUUUAUGAACUUA

GGGAAAAUUUGCUUGGUGGACUGUU 1 times at 21153 NSP16

GGAAAAUUUGCUUGGUGGACUGUUU 1 times at 21154 NSP16

GCAAAUGCAUCCUCAUCUGAAGGAU 1 times at 21190 NSP16

GGUAUUAAUUACUUGGGUACUAUUA 1 times at 21223 NSP16

GGGUACUAUUAAAG AAAAU AUAGAU 1 times at 21237 NSP16 GGGUACUAUUAAAG AAAAU AU AG

GGUGGUGCUAUGCACGCCAACUAUA 1 times at 21262 NSP16

GGUGCUAUGCACGCCAACUAUAUAU 1 times at 21265 NSP16

GCUAUGCACGCCAACUAUAUAUUUU 1 times at 21268 NSP16

CGCCAACUAUAUAUUUUGGAGAAAU 1 times at 21276 NSP16

GCCAACUAUAUAUUUUGGAGAAAUU 1 times at 21277 NSP16

CCACUCCUAUGAAUCUGAGUACUUA 1 times at 21302 NSP16

G G AG AG U CAAAU U AACG AACU CG U A 1 times at 21390 NSP16

GGGUAAGUUACUUAUCCGUGACAAU 1 times at 21432 NSP16

CCGUGACAAUGAUACACUCAGUGUU 1 times at 21447 NSP16

CG U G ACAAU G AU ACACU CAG U G U U U 1 times at 21448 NSP16

GGCUGACGGUAUUAUAUACCCUCAA 1 times at 21610 S protein

GGUAUUAUAUACCCUCAAGGCCGUA 1 times at 21617 S protein

GGCCGUACAUAUUCUAACAUAACUA 1 times at 21635 S protein GGCCGUACAUAUUCUAACAUAAC

GCCGUACAUAUUCUAACAUAACUAU 1 times at 21636 S protein GCCGUACAUAUUCUAACAUAACU

CCCUAUCAGGGAGACCAUGGUGAUA 1 times at 21680 S protein

CCUAUCAGGGAGACCAUGGUGAUAU 1 times at 21681 S protein

GGGAGACCAUGGUGAUAUGUAUGUU 1 times at 21688 S protein CACUUUACUUAGAGCUUUUUAUU

GGAGACCAUGGUGAUAUGUAUGUUU 1 times at 21689 S protein

CCAUCUACCAGCGCUACUAUACGAA 1 times at 21854 S protein

CCAGCGCUACUAUACGAAAAAUUUA 1 times at 21861 S protein

GGGCCGCUUCUUCAAUCAUACUCUA 1 times at 21937 S protein

GCCCGAUGGAUGUGGCACUUUACUU 1 times at 21970 S protein

CCCGAUGGAUGUGGCACUUUACUUA 1 times at 21971 S protein

GGAUGUGGCACUUUACUUAGAGCUU 1 times at 21977 S protein

GGCACUUUACUUAGAGCUUUUUAUU 1 times at 21983 S protein

CCUGCUGGCAAUUCCUAUACUUCUU 1 times at 22040 S protein

GCAACAGAUUGUUCUGAUGGCAAUU 1 times at 22085 S protein

CGUAAUGCCAGUCUGAACUCUUUUA 1 times at 22115 S protein

CCAGUCUGAACUCUUUUAAGGAGUA 1 times at 22122 S protein

CGUAACUGCACCUUUAUGUACACUU 1 times at 22157 S protein

GCACCUUUAUGUACACUUAUAACAU 1 times at 22164 S protein CACCUUUAUGUACACUUAUAACA

CCGAAGAUGAGAUUUUAGAGUGGUU 1 times at 22191 S protein CCGAAGAUGAGAUUUUAGAGUGG

CGAAGAUGAGAUUUUAGAGUGGUUU 1 times at 22192 S protein

GCUCAAGGUGUUCACCUCUUCUCAU 1 times at 22232 S protein

CCUCUUCUCAUCUCGGUAUGUUGAU 1 times at 22246 S protein

GGUAUGUUGAUUUGUACGGCGGCAA 1 times at 22260 S protein CCGUUAACUUUCCUGUUGGAUUUUU 1 times at 22412 S protein

GGAUUUUUCUGUUGAUGGUUAUAUA 1 times at 22429 S protein

CGCAGAGCUAUAGACUGUGGUUUUA 1 times at 22454 S protein

GCAGAGCUAUAGACUGUGGUUUUAA 1 times at 22455 S protein

GCUAUAGACUGUGGUUUUAAUGAUU 1 times at 22460 S protein

CCACUGCUCAUAUGAAUCCUUCGAU 1 times at 22495 S protein

CCUUCGAUGUUGAAUCUGGAGUUUA 1 times at 22512 S protein

CGAAGCAAAACCUUCUGGCUCAGUU 1 times at 22552 S protein

GGCUGAAGGUGUUGAAUGUGAUUUU 1 times at 22585 S protein

GCUGAAGGUGUUGAAUGUGAUUUUU 1 times at 22586 S protein

GGCACACCUCCUCAGGUUUAUAAUU 1 times at 22625 S protein

GCACACCU CCU CAG G U U U AU AAU U U 1 times at 22626 S protein

CCUCAGGUUUAUAAUUUCAAGCGUU 1 times at 22634 S protein

GGUUUAUAAUUUCAAGCGUUUGGUU 1 times at 22639 S protein

GCGUUUGGUUUUUACCAAUUGCAAU 1 times at 22654 S protein

CGUUUGGUUUUUACCAAUUGCAAUU 1 times at 22655 S protein

GGUUUUUACCAAUUGCAAUUAUAAU 1 times at 22660 S protein

GCUUUCACUUUUUUCUGUGAAUGAU 1 times at 22696 S protein

GCUGGUCCAAUAUCCCAGUUUAAUU 1 times at 22835 S protein

GGUCCAAUAUCCCAGUUUAAUUAUA 1 times at 22838 S protein UG G U CCAAU AU CCCAG U U U AAU U

CCCAGUUUAAUUAUAAACAGUCCUU 1 times at 22848 S protein CCCAGUUUAAUUAUAAACAGUCC

CCAGUUUAAUUAUAAACAGUCCUUU 1 times at 22849 S protein

CCUUUUCUAAUCCCACAUGUUUGAU 1 times at 22869 S protein

CCUUACUACUAUUACUAAGCCUCUU 1 times at 22915 S protein

CCUCAGUUAGUGAACGCUAAUCAAU 1 times at 22997 S protein

CGCUAAUCAAUACUCACCCUGUGUA 1 times at 23011 S protein

GCUAAUCAAUACUCACCCUGUGUAU 1 times at 23012 S protein

GGGAAGACGGUGAUUAUUAUAGGAA 1 times at 23058 S protein

GGAAGACGGUGAUUAUUAUAGGAAA 1 times at 23059 S protein

CGGUGAUUAUUAUAGGAAACAACUA 1 times at 23065 S protein

GGUGAUUAUUAUAGGAAACAACUAU 1 times at 23066 S protein

GGCUGGCUUGUUGCUAGUGGCUCAA 1 times at 23108 S protein

GCUUGUUGCUAGUGGCUCAACUGUU 1 times at 23113 S protein

GCAAUUACAGAUGGGCUUUGGUAUU 1 times at 23149 S protein

GGGCUUUGGUAUUACAGUUCAAUAU 1 times at 23161 S protein

GCUUGAAUUUGCUAAUGACACAAAA 1 times at 23215 S protein

GCAAUUGCGUGGAAUAUUCCCUCUA 1 times at 23256 S protein

CGUGGAAUAUUCCCUCUAUGGUGUU 1 times at 23263 S protein

GGUGUUCGACAGCAGCGCUUUGUUU 1 times at 23324 S protein

GCUAUUAUUCUGAUGAUGGCAACUA 1 times at 23373 S protein

CCCGUUCUACGCGAUCAAUGCUUAA 1 times at 23523 S protein

GGUUGUGUCCUAGGACUUGUUAAUU 1 times at 23588 S protein

CCUCUUUGUUCGUAGAGGACUGCAA 1 times at 23613 S protein

GCGCUUGGCAUCCAUUGCUUUUAAU 1 times at 23725 S protein

GGUUGAUCAACUUAAUAGUAGUUAU 1 times at 23761 S protein UUGAUCAACUUAAUAGUAGUUAU CCUUUGGUGUGACUCAGGAGUACAU 1 times at 23814 S protein

CCAUGGUGCCAAUUUACGCCAGGAU 1 times at 23959 S protein

GGUGCCAAUUUACGCCAGGAUGAUU 1 times at 23963 S protein

CGCCAGGAUGAUUCUGUACGUAAUU 1 times at 23975 S protein

GCCAGGAUGAUUCUGUACGUAAUUU 1 times at 23976 S protein CAGGAUGAUUCUGUACGUAAUUU

GGAUGAUUCUGUACGUAAUUUGUUU 1 times at 23980 S protein AUGAUUCUGUACGUAAUUUGUUU

CGUAAUUUGUUUGCGAGCGUGAAAA 1 times at 23993 S protein

GCGAGCGUGAAAAGCUCUCAAUCAU 1 times at 24005 S protein

CCAGGUUUUGGAGGUGACUUUAAUU 1 times at 24041 S protein CAGGUUUUGGAGGUGACUUUAAU

GGCAGUCGUAGUGCACGUAGUGCUA 1 times at 24098 S protein

GCAGUCGUAGUGCACGUAGUGCUAU 1 times at 24099 S protein

CGUAGUGCUAUUGAGGAUUUGCUAU 1 times at 24113 S protein

GCUGAUCCUGGUUAUAUGCAAGGUU 1 times at 24155 S protein

GGUUAUAUGCAAGGUUACGAUGAUU 1 times at 24164 S protein

GGUCCAGCAUCAGCUCGUGAUCUUA 1 times at 24200 S protein

CCAGCAUCAGCUCGUGAUCUUAUUU 1 times at 24203 S protein

GCUCGUGAUCUUAUUUGUGCUCAAU 1 times at 24212 S protein

GGAUGUUAAUAUGGAAGCCGCGUAU 1 times at 24271 S protein

GGUGUUGGCUGGACUGCUGGCUUAU 1 times at 24323 S protein

GCUGGACUGCUGGCUUAUCCUCCUU 1 times at 24330 S protein

GCUGGCUUAUCCUCCUUUGCUGCUA 1 times at 24338 S protein

GCUGCUAUUCCAUUUGCACAGAGUA 1 times at 24356 S protein

CGGUGUUGGCAUUACUCAACAGGUU 1 times at 24397 S protein

GGUUCUUUCAGAGAACCAAAAGCUU 1 times at 24418 S protein

CCAAAAGCU U AU U G CCAAUAAG U U U 1 times at 24433 S protein

GGAGCUAUGCAAACAGGCUUCACUA 1 times at 24470 S protein

GCUAUGCAAACAGGCUUCACUACAA 1 times at 24473 S protein

GCAAACAGGCUUCACUACAACUAAU 1 times at 24478 S protein

GGCUUCACUACAACUAAUGAAGCUU 1 times at 24485 S protein GGCUUCACUACAACUAAUGAAGC

GCUUCACUACAACUAAUGAAGCUUU 1 times at 24486 S protein

GCUAUCUAAUACUUUUGGUGCUAUU 1 times at 24571 S protein

GGCACAAUCCAAGCGUUCUGGAUUU 1 times at 24778 S protein

GCACAAUCCAAGCGUUCUGGAUUUU 1 times at 24779 S protein

CCCUAGCAACCACAUUGAGGUUGUU 1 times at 24880 S protein

CCUAGCAACCACAUUGAGGUUGUUU 1 times at 24881 S protein

CCACAUUGAGGUUGUUUCUGCUUAU 1 times at 24889 S protein

CCCUACUAAUUGUAUAGCCCCUGUU 1 times at 24934 S protein

CCUACUAAUUGUAUAGCCCCUGUUA 1 times at 24935 S protein

GCCCCUGUUAAUGGCUACUUUAUUA 1 times at 24950 S protein

CCCCUGUUAAUGGCUACUUUAUUAA 1 times at 24951 S protein CCCCUGUUAAUGGCUACUUUAUU

CCCUGUUAAUGGCUACUUUAUUAAA 1 times at 24952 S protein CCCUGUUAAUGGCUACUUUAUUA

CCUGUUAAUGGCUACUUUAUUAAAA 1 times at 24953 S protein

GGUCAUAUACUGGCUCGUCCUUCUA 1 times at 25005 S protein

CCUUAAUGAGUCUUACAUAGACCUU 1 times at 25279 S protein

GGCAAUUAUACUUAUUACAACAAAU 1 times at 25313 S protein GGCCGUGGUACAUUUGGCUUGGUUU 1 times at 25338 S protein

GCUGGGCUUGUUGCCUUAGCUCUAU 1 times at 25367 S protein

GCACUGGUUGUGGCACAAACUGUAU 1 times at 25413 S protein

GGUUGUGGCACAAACUGUAUGGGAA 1 times at 25418 S protein

GGCACAAACUGUAUGGGAAAACUUA 1 times at 25424 S protein

G C AC AAAC UGUAUGGG AAAAC U U AA 1 times at 25425 S protein CACAAACUGUAUGGGAAAACUUA

GGAAAACUUAAGUGUAAUCGUUGUU 1 times at 25439 S protein

CGUUGUUGUGAUAGAUACGAGGAAU 1 times at 25457 S protein

GCCGCAUAAGGUUCAUGUUCACUAA 1 times at 25492 S protein GCCGCAUAAGGUUCAUGUUCACU

CCGCAUAAGGUUCAUGUUCACUAAU 1 times at 25493 S protein

CGCAUAAGGUUCAUGUUCACUAAUU 1 times at 25494 S protein

GCAUAAGGUUCAUGUUCACUAAUUA 1 times at 25495 S protein

GGUUGCAUGCUUAGGGCUUGUAUUA 1 times at 25639 orf 3

CCAAGCUGAUACAGCUGGUCUUUAU 1 times at 25671 orf 3

GCUGAUACAGCUGGUCUUUAUACAA 1 times at 25675 orf 3

CGAAUUGACGUCCCAUCUGCAGAAU 1 times at 25705 orf 3

CCCUGUGCUGUGGAACUGUCAGCUA 1 times at 25973 orf 4a

CCUGUGCUGUGGAACUGUCAGCUAU 1 times at 25974 orf 4a

GCUGUGGAACUGUCAGCUAUCCUUU 1 times at 25979 orf 4a

GCUAUCCUUUGCUGGUUAUACUGAA 1 times at 25994 orf 4a

GCUGGUUAUACUGAAUCUGCUGUUA 1 times at 26004 orf 4a

GGUUAUACUGAAUCUGCUGUUAAUU 1 times at 26007 orf 4a

G CC AAACAG G ACG C AG C U CAG CG AA 1 times at 26046 orf 4a

CCAAACAG G ACG CAG CU CAG CG AAU 1 times at 26047 orf 4a

GGUUGCUACAUAAGGAUGGAGGAAU 1 times at 26077 orf 4a

CGGCACUCAAGUUUAUUCGCGCAAA 1 times at 26127 orf 4a

CCAACACACUAUGUCAGGGUUACAU 1 times at 26248 orf4b

GGGUUACAUUUUCAGACCCCAACAU 1 times at 26264 orf4b

GGUAUCUACGUUCGGGUCAUCAUUU 1 times at 26291 orf4b

GCCAACCUGUUUCUGAGUACCAUAU 1 times at 26351 orf4b

CCAACCU G U U U CU G AG U ACCAU AU U 1 times at 26352 orf4b

CCAUAUUACUCUAGCUUUGCUAAAU 1 times at 26370 orf4b

GCUAAAUCUCACUGAUGAAGAUUUA 1 times at 26388 orf4b

CGCCUUGCUGCGCAAAACUCUUGUU 1 times at 26475 orf4b

GCUGCGCAAAACUCUUGUUCUUAAU 1 times at 26481 orf4b CUGCGCAAAACUCUUGUUCUUAA

CGCAAAACUCUUGUUCUUAAUGCAU 1 times at 26485 orf4b CGCAAAACUCUUGUUCUUAAUGC

GGAUUGGCUUCUCGUUCAGGGAUUU 1 times at 26583 orf4b

GCUUCUCGUUCAGGGAUUUUCCCUU 1 times at 26589 orf4b

CG U U CAG GG AU U U U CCCU U U ACCAU 1 times at 26595 orf4b

CCCUUUACCAUAGUGGCCUCCCUUU 1 times at 26609 orf4b

CCUUUACCAUAGUGGCCUCCCUUUA 1 times at 26610 orf4b

CGCAAU UACAUCAU UACAAUGCCAU 1 times at 26677 orf4b

CCUCAACAAAUGUUUGUUACUCCUU 1 times at 26716 orf4b

CCAUACGGUCUUCCAAUCAGGGUAA 1 times at 26759 orf4b

GGUAAUAAACAAAUUGUUCAUUCUU 1 times at 26779 orf4b GGCUUUCUCGGCGUCUUUAUUUAAA 1 times at 26841 orf5 GGCUUUCUCGGCGUCUUUAUUUA

CCUAUUAUUACUGCUACGUCAAGAU 1 times at 26991 orf5

CCUUGUUCUGUAUAACUUUUUAUUA 1 times at 27057 orf5 UUGUUCUGUAUAACUUUUUAUUA

GGUGUACAUUAUCCAACUGGAAGUU 1 times at 27100 orf5

CCUCAUAAUACUUUGGUUUGUAGAU 1 times at 27147 orf5

CCAAACCAU U AU U U AU U AG AAACU U 1 times at 27284 orf5

GCGUUGCAGCUGUUCUCGUUGUUUU 1 times at 27315 orf5

CGUUGCAGCUGUUCUCGUUGUUUUU 1 times at 27316 orf5

GCAGCUGUUCUCGUUGUUUUUAUUU 1 times at 27320 orf5

CCACUUAUAUAGAGUGCACUUAUAU 1 times at 27353 orf5

GCACUUAUAUUAGCCGUUUUAGUAA 1 times at 27368 orf5

CCG UU U UAGUAAGAU U AGCCU AG U U 1 times at 27381 orf5

CG U U U U AG U AAG AU U AGCCU AG U U U 1 times at 27382 orf5

CGCGCGAUUCAGUUCCUCUUCACAU 1 times at 27461 orf5

GCGCGAUUCAGUUCCUCUUCACAUA 1 times at 27462 orf5

CGCG AU U CAG U U CCU CU U CACAU AA 1 times at 27463 orf5

GCGAUUCAGUUCCUCUUCACAUAAU 1 times at 27464 orf5

CGCCCCGAGCUCGCUUAUCGUUUAA 1 times at 27489 orf5

CGUUUAAGCAGCUCUGCGCUACUAU 1 times at 27507 orf5

GGGUCCCGUGUAGAGGCUAAUCCAU 1 times at 27532

GGUCCCGUGUAGAGGCUAAUCCAUU 1 times at 27533

GGACAUAUGGAAAACGAACUAUGUU 1 times at 27569 GACAUAUGGAAAACGAACUAUGU

CCGUAGUAUGUGCUAUAACACUCUU 1 times at 27647 E

GGCUUUCCUUACGGCUACUAGAUUA 1 times at 27681 E

GCUUUCCUUACGGCUACUAGAUUAU 1 times at 27682 E

GCUACUAGAUUAUGUGUGCAAUGUA 1 times at 27694 E

CCCUGUUAGUUCAGCCCGCAUUAUA 1 times at 27734 E

CCCAUCCCGUAGUAUGACUGUCUAU 1 times at 27965 M

GGCCAUCUUCCAUGGCGCUAUCAAU 1 times at 28021 M

GCCAUCUUCCAUGGCGCUAUCAAUA 1 times at 28022 M

CCAUCUUCCAUGGCGCUAUCAAUAU 1 times at 28023 M

CCAAUUGAUCUAGCUUCCCAGAUAA 1 times at 28062 M

GGCAUUGUAGCAGCUGUUUCAGCUA 1 times at 28092 M GGCAUUGUAGCAGCUGUUUCAGC

GCAUUGUAGCAGCUGUUUCAGCUAU 1 times at 28093 M

GCUGUUUCAGCUAUGAUGUGGAUUU 1 times at 28104 M

GGAUUUCCUACUUUGUGCAGAGUAU 1 times at 28123 M

CGGCUGUUUAUGAGAACUGGAUCAU 1 times at 28149 M

CCAGUGUAACUGCUGUUGUAACCAA 1 times at 28261 M

CCACCUCAAAAUGGCUGGCAUGCAU 1 times at 28289 M

GCAUGCAUUUCGGUGCUUGUGACUA 1 times at 28306 M

CGGUGCUUGUGACUACGACAGACUU 1 times at 28316 M

GCUUGUGACUACGACAGACUUCCUA 1 times at 28320 M

G CU U U AAAAAU GGUGAAGCGG CAAA 1 times at 28380 M

GGAACUAAUUCCGGCGUUGCCAUUU 1 times at 28410 M

CCGGCGUUGCCAUUUACCAUAGAUA 1 times at 28420 M CGGCGUUGCCAUUUACCAUAGAUAU 1 times at 28421 M

GGCGUUGCCAUUUACCAUAGAUAUA 1 times at 28422 M

GCGUUGCCAUUUACCAUAGAUAUAA 1 times at 28423 M

GCAGGUAAUUACAGGAGUCCGCCUA 1 times at 28449 M

GGUAAUUACAGGAGUCCGCCUAUUA 1 times at 28452 M

GGAGUCCGCCUAUUACGGCGGAUAU 1 times at 28462 M

GCCUAUUACGGCGGAUAUUGAACUU 1 times at 28469 M GCCUAUUACGGCGGAUAUUGAAC

GGCGGAUAUUGAACUUGCAUUGCUU 1 times at 28478 M

GCAUUGCUUCGAGCUUAGGCUCUUU 1 times at 28494 M

GCUUCGAGCUUAGGCUCUUUAGUAA 1 times at 28499 M

GGCAGGGUGUACCUCUUAAUGCCAA 1 times at 28743 N

GCAGGGUGUACCUCUUAAUGCCAAU 1 times at 28744 N

GGGUAUUGGCGGAGACAGGACAGAA 1 times at 28790 N

GGUAUUGGCGGAGACAGGACAGAAA 1 times at 28791 N

GGCGGAGACAGGACAGAAAAAUUAA 1 times at 28797 N

GCGGAGACAGGACAGAAAAAUUAAU 1 times at 28798 N

CGGAGACAGGACAGAAAAAUUAAUA 1 times at 28799 N

GGACAGAAAAAUUAAUACCGGGAAU 1 times at 28807 N

GCAGCACUCCCAUUCCGGGCUGUUA 1 times at 28889 N

CCGGGCUGUUAAGGAUGGCAUCGUU 1 times at 28903 N

CGGGCUGUUAAGGAUGGCAUCGUUU 1 times at 28904 N

GGAUGGCAUCGUUUGGGUCCAUGAA 1 times at 28915 N

GGCGCCACUGAUGCUCCUUCAACUU 1 times at 28943 N

GCGCCACUGAUGCUCCUUCAACUUU 1 times at 28944 N

CGCCACUGAUGCUCCUUCAACUUUU 1 times at 28945 N

GGGACGCGGAACCCUAACAAUGAUU 1 times at 28970 N

CCGGUACUAAGCUUCCUAAAAACUU 1 times at 29019 N CCGGUACUAAGCUUCCUAAAAAC

CCACAUUGAGGGGACUGGAGGCAAU 1 times at 29044 N

GGGACUGGAGGCAAUAGUCAAUCAU 1 times at 29054 N

GGAGGCAAUAGUCAAUCAUCUUCAA 1 times at 29060 N GAGGCAAUAGUCAAUCAUCUUCA

CGGAGCAGUAGGAGGUGAUCUACUU 1 times at 29182 N

GGAGCAGUAGGAGGUGAUCUACUUU 1 times at 29183 N

CCUUGAUCUUCUGAACAGACUACAA 1 times at 29209 N

G G CAAAG U AAAG C AAU CG C AG CCAA 1 times at 29246 N

G CAAAG U AAAG CAAU CG CAG CCAAA 1 times at 29247 N

CG C AG CC AAAAG U AAU CACU AAG AA 1 times at 29262 N

GCGCCACAAGCGCACUUCCACCAAA 1 times at 29314 N

CGCCACAAGCGCACUUCCACCAAAA 1 times at 29315 N

G CACU U CCACCAAAAG U U U CAACAU 1 times at 29325 N

CGCGGACCAGGAGACCUCCAGGGAA 1 times at 29369 N

GCGGACCAGGAGACCUCCAGGGAAA 1 times at 29370 N

CCUCCAGGGAAACUUUGGUGAUCUU 1 times at 29383 N

CCAGGGAAACUUUGGUGAUCUUCAA 1 times at 29386 N

CCCCAAAUUGCUGAGCUUGCUCCUA 1 times at 29444 N

GCUUGCUCCUACAGCCAGUGCUUUU 1 times at 29458 N CCUACAGCCAGUGCUUUUAUGGGUA 1 times at 29465 N

GCUUUUAUGGGUAUGUCGCAAUUUA 1 times at 29477 N

CGCAAUUUAAACUUACCCAUCAGAA 1 times at 29493 N

GCAACCCUGUGUACUUCCUUCGGUA 1 times at 29532 N

CCUUCGGUACAGUGGAGCCAUUAAA 1 times at 29548 N

GGUUGGAGCUUCUUGAGCAAAAUAU 1 times at 29604 N

GGAGCUUCUUGAGCAAAAUAUUGAU 1 times at 29608 N GAGCUUCUUGAGCAAAAUAUUGA

G G AA AAG AAAC A AAAG G C ACC AA AA 1 times at 29656 N

CGUCCAAGUGUUCAGCCUGGUCCAA 1 times at 29759 N

CCAAU GAU U G AU G U U AACACU G AU U 1 times at 29780 N

Table 2

CGGCUUCAGUUAACCAAAUUGUCUU 1 times at 8286 NSP3 GGCUUCAGUUAACCAAAUUGUCU

CGCAUUGCAUGCCGUAAGUGUAAUU 1 times at 8387 NSP3 CGCAUUGCAUGCCGUAAGUGUAA

CCUCAAAGCUACGCGCUAAUGAUAA 1 times at 8430 NSP3 CUCAAAGCUACGCGCUAAUGAUA

CCGCAUCUUGGACUUUAAAGUUCUU 1 times at 8638 NSP4 CCGCAUCUUGGACUUUAAAGUUC

GCUCUUCUAUUAUAUUAAUAAAGUA 1 times at 9406 NSP4 CUCUUCUAUUAUAUUAAUAAAGU

GCUGCCUCUAAUAUCUUUGUUAUUA 1 times at 9767 NSP4 UGCCUCUAAUAUCUUUGUUAUUA

CCUCUAAUAUCUUUGUUAUUAACAA 1 times at 9771 NSP4 CUCUAAUAUCUUUGUUAUUAACA

GCAGCUCUUAGAAACUCUUUAACUA 1 times at 9806 NSP4 CAGCUCUUAGAAACUCUUUAACU

CGGAAGUGAAGAUGAUACUUUUAUU 1 times at 11556 NSP6 CGGAAGUGAAGAUGAUACUUUUA

GGAAGUGAAGAUGAUACUUUUAUUA 1 times at 11557 NSP6 AAGUGAAGAUGAUACUUUUAUUA

GGCUAUGACUUCUAUGUAUAAGCAA 1 times at 12259 NSP8 GGCUAUGACUUCUAUGUAUAAGC

CCCCAAU CU AAAG AU U CCAAU U U U U 1 times at 13403 NSP10 CCCCAAU CU AAAG AU U CCAAU U U

CCCAAUCUAAAGAUUCCAAUUUUUU 1 times at 13404 NSP10 CCCAAU CU AAAG AU U CCAAU U U U

GCUGUGAUGUUACCUACUUUGAAAA 1 times at 13862 NSP12 CUGUGAUGUUACCUACUUUGAAA

CCCAGUGUUAUUGGUGUUUAUCAUA 1 times at 13915 NSP12 CCCAGUGUUAUUGGUGUUUAUCA

CCAGUGUUAUUGGUGUUUAUCAUAA 1 times at 13916 NSP12 CAGUGUUAUUGGUGUUUAUCAUA

GGUACAACUCUUUGAGAAGUACUUU 1 times at 14247 NSP12 UACAACUCUUUGAGAAGUACUUU

CCUCCUCUAACGCUUUUCUUGAUUU 1 times at 14558 NSP12 CUCCUCUAACGCUUUUCUUGAUU

CCUACUAUGUGUGACAUCAAACAAA 1 times at 14791 NSP12 UACUAUGUGUGACAU C AAAC AAA

GCUGGGAUUUCAUGCUUAAAACAUU 1 times at 15200 NSP12 UGGGAUUUCAUGCUUAAAACAUU

GGGAUUUCAUGCUUAAAACAUUGUA 1 times at 15203 NSP12 GGGAUUUCAUGCUUAAAACAUUG

CCACUGCAUAUGCCAAUAGUGUCUU 1 times at 15467 NSP12 CACUGCAUAUGCCAAUAGUGUCU

GGGUGCUAAUGGCAACAAGAUUGUU 1 times at 15534 NSP12 GGGUGCUAAUGGCAACAAGAUUG

CCCCAAAUUUGUUGAUAAAUACUAU 1 times at 15624 NSP12 CCCCAAAUUUGUUGAUAAAUACU

CGGUUGCUUUGUAGAUGAUAUCGUU 1 times at 15930 NSP12 CGGUUGCUUUGUAGAUGAUAUCG

GGUUGCUUUGUAGAUGAUAUCGUUA 1 times at 15931 NSP12 UUGCUUUGUAGAUGAUAUCGUUA

CCCUCUCACAAAGCAUGAAGAUAUA 1 times at 16011 NSP12 CUCUCACAAAGCAUGAAGAUAUA

GGUCUACUUACAGUAUAUAGAAAAA 1 times at 16056 NSP12 GUCUACUUACAGUAUAUAGAAAA

CCACCACUCAAUCGUAAUUAUGUUU 1 times at 16726 NSP13 ACCACUCAAUCGUAAUUAUGUUU

CCUACAAGUCUAGUACAACGUAUAA 1 times at 16835 NSP13 U ACAAG U C U AG U ACAACG U AU AA

GCACUAAUUAUGAUCUUUCAAUUAU 1 times at 17342 NSP13 CACUAAUUAUGAUCUUUCAAUUA

GCAUGGAGUAAGGCAGUCUUUAUUU 1 times at 17719 NSP13 AUGGAGUAAGGCAGUCUUUAUUU

G CACAU G CU AACAACAU U AACAG AU 1 times at 17863 NSP13 CACAU G CU AACAACAU U AACAG A

GCCCAAAAAGGUAUUCUUUGUGUUA 1 times at 17908 NSP13 GCCCAAAAAGGUAUUCUUUGUGU

GCACUCUUUGAGUCCUUAGAGUUUA 1 times at 17944 NSP13 CACUCUUUGAGUCCUUAGAGUUU

CCUGCGGUUAUGAUUAUGUCUACAA 1 times at 18689 NSP14 CUGCGGUUAUGAUUAUGUCUACA

CGGUUCAUUUGACAAAGUCUAUGAU 1 times at 18969 NSP14 CGGUUCAUUUGACAAAGUCUAUG

GGUUCAUUUGACAAAGUCUAUGAUA 1 times at 18970 NSP14 UUCAUUUGACAAAGUCUAUGAUA

GGUAGUAUGAUAGAGGAUAUUGAUU 1 times at 19360 NSP14 UAGUAUGAUAGAGGAUAUUGAUU

GGUGUUAUAAGACCUUUGAUAUUUA 1 times at 19517 NSP14 GUGUUAUAAGACCUUUGAUAUUU

GGGAUUAUGAACGUAGCAAUAUUUA 1 times at 19829 NSP15 GGGAUUAUGAACGUAGCAAUAUU

GCCAUCUUUAUUUCUGAUAGAAAAA 1 times at 19972 NSP15 GCCAUCUUUAUUUCUGAUAGAAA

CCAUCUUUAUUUCUGAUAGAAAAAU 1 times at 19973 NSP15 AUCUUUAUUUCUGAUAGAAAAAU

CCGUGAUAGUGAUGUUGUUAAACAA 1 times at 20055 NSP15 CCGUGAUAGUGAUGUUGUUAAAC

GGUCUUCACUUGCUUAUUGGUUUAU 1 times at 20302 NSP15 GUCUUCACUUGCUUAUUGGUUUA GCUCAACUAUUCAUAACUAUUUUAU 1 times at 20378 NSP15 CUCAACUAUUCAUAACUAUUUUA

GGUUCCUAUUGACUUAACAAUGAUU 1 times at 20520 NSP15 UUCCUAUUGACUUAACAAUGAUU

CCCUCUUUAAAGUUCAAAAUGUAAA 1 times at 20639 NSP16 CU CU U UAAAG U UCAAAAU G U AAA

CCUGCCAAUAUGCGUGUUAUACAUU 1 times at 20785 NSP16 UGCCAAUAUGCGUGUUAUACAUU

CCGACAUGUAUGAUCCUACUACUAA 1 times at 20987 NSP16 GACAUGUAUGAUCCUACUACUAA

GGAGCGUUGAACUUUAUGAACUUAU 1 times at 21128 NSP16 GAGCGUUGAACUUUAUGAACUUA

GGGUACUAUUAAAGAAAAUAUAGAU 1 times at 21237 NSP16 GGGUACUAUU AAAG AAAAU AU AG

GGCCGUACAUAUUCUAACAUAACUA 1 times at 21635 S protein GGCCGUACAUAUUCUAACAUAAC

GCCGUACAUAUUCUAACAUAACUAU 1 times at 21636 S protein GCCGUACAUAUUCUAACAUAACU

GGGAGACCAUGGUGAUAUGUAUGUU 1 times at 21688 S protein CACUUUACUUAGAGCUUUUUAUU

GCACCUU U AUG UACACU UAUAACAU 1 times at 22164 S protein CACCUUUAUGUACACUUAUAACA

CCGAAGAUGAGAUUUUAGAGUGGUU 1 times at 22191 S protein CCGAAGAUGAGAUUUUAGAGUGG

GGUCCAAUAUCCCAGUUUAAUUAUA 1 times at 22838 S protein U GG U CCAAU AU CCCAG U U UAAU U

CCCAGUUUAAUUAUAAACAGUCCUU 1 times at 22848 S protein CCCAGUUUAAUUAUAAACAGUCC

GGUUGAUCAACUUAAUAGUAGUUAU 1 times at 23761 S protein UUGAUCAACUUAAUAGUAGUUAU

GCCAGGAUGAUUCUGUACGUAAUUU 1 times at 23976 S protein CAGGAUGAUUCUGUACGUAAUUU

GGAUGAUUCUGUACGUAAUUUGUUU 1 times at 23980 S protein AUGAUUCUGUACGUAAUUUGUUU

CCAGGUUUUGGAGGUGACUUUAAUU 1 times at 24041 S protein CAGGUUUUGGAGGUGACUUUAAU

GGCUUCACUACAACUAAUGAAGCUU 1 times at 24485 S protein GGCUUCACUACAACUAAUGAAGC

CCCCUGUUAAUGGCUACUUUAUUAA 1 times at 24951 S protein CCCCUGUUAAUGGCUACUUUAUU

CCCUGUUAAUGGCUACUUUAUUAAA 1 times at 24952 S protein CCCUGUUAAUGGCUACUUUAUUA

G C AC AAAC U G U A U G G G AAAAC U U AA 1 times at 25425 S protein C AC AAAC UGUAUGGG AAAAC U U A

GCCGCAUAAGGUUCAUGUUCACUAA 1 times at 25492 S protein GCCGCAUAAGGUUCAUGUUCACU

GCUGCGCAAAACUCUUGUUCUUAAU 1 times at 26481 orf4b CUGCGCAAAACUCUUGUUCUUAA

CGCAAAACUCUUGUUCUUAAUGCAU 1 times at 26485 orf4b CGCAAAACUCUUGUUCUUAAUGC

GGCUUUCUCGGCGUCUUUAUUUAAA 1 times at 26841 orf5 GGCUUUCUCGGCGUCUUUAUUUA

CCUUGUUCUGUAUAACUUUUUAUUA 1 times at 27057 orf5 UUGUUCUGUAUAACUUUUUAUUA

GGACAUAUGGAAAACGAACUAUGUU 1 times at 27569 GACAUAUGGAAAACGAACUAUGU

GGCAUUGUAGCAGCUGUUUCAGCUA 1 times at 28092 M GGCAUUGUAGCAGCUGUUUCAGC

GCCUAUUACGGCGGAUAUUGAACUU 1 times at 28469 M GCCUAUUACGGCGGAUAUUGAAC

CCGGUACUAAGCUUCCUAAAAACUU 1 times at 29019 N CCGGUACUAAGCUUCCUAAAAAC

Table 3

Predicted 25 mer siRNA targeting

25mer blunt ended sequences

SiRNA sequence Start Protein 23 mer Sequences passing all

Base Name metrics and BLAST search

CCCAGAAUCUGCUUAAGAAGUUGAU 1 times at 825 NSP1 CCCAGAAUCUGCUUAAGAAGUUG

GCCCAUUCAUGGAUAAUGCUAUUAA 1 times at 1884 NSP2 GCCCAUUCAUGGAUAAUGCUAUU

CCCAUUCAUGGAUAAUGCUAUUAAU 1 times at 1885 NSP2 CCCAUUCAUGGAUAAUGCUAUUA

CGCCAUUACUGCACCUUAUGUAGUU 1 times at 1936 NSP2 CGCCAUUACUGCACCUUAUGUAG

GGCGACUUUAUGUCUACAAUUAUUA 1 times at 2186 NSP2 GGCGACUUUAUGUCUACAAUUAU

CGCAAUACGUAAAGCUAAAGAUUAU 1 times at 4144 NSP3 CGCAAUACGUAAAGCUAAAGAUU

GGGUGUUGAUUAUACUAAGAAGUUU 1 times at 4228 NSP3 GGGUGUUGAUUAUACUAAGAAGU

CGCACUAAUGGUGGUUACAAUUCUU 1 times at 4517 NSP3 CGCACUAAUGGUGGUUACAAUUC

GGCUUCAUUUUAUUUCAAAGAAUUU 1 times at 6487 NSP3 GGCUUCAUUUUAUUUCAAAGAAU

GCGCUUUUACAAAUCUAGAUAAGUU 1 times at 7740 NSP3 GCGCUUUUACAAAUCUAGAUAAG

CGCAUUGCAUGCCGUAAGUGUAAUU 1 times at 8387 NSP3 CGCAUUGCAUGCCGUAAGUGUAA

CCGCAUCUUGGACUUUAAAGUUCUU 1 times at 8638 NSP4 CCGCAUCUUGGACUUUAAAGUUC

CGGAAGUGAAGAUGAUACUUUUAUU 1 times at 11556 NSP6 CGGAAGUGAAGAUGAUACUUUUA

GGCUAUGACUUCUAUGUAUAAGCAA 1 times at 12259 NSP8 GGCUAUGACUUCUAUGUAUAAGC

CCCCAAUCUAAAGAUUCCAAUUUUU 1 times at 13403 NSP10 CCCCAAUCUAAAGAUUCCAAUUU

CCCAAUCUAAAGAUUCCAAUUUUUU 1 times at 13404 NSP10 CCCAAUCUAAAGAUUCCAAUUUU

CCCAGUGUUAUUGGUGUUUAUCAUA 1 times at 13915 NSP12 CCCAGUGUUAUUGGUGUUUAUCA

GGGAUUUCAUGCUUAAAACAUUGUA 1 times at 15203 NSP12 GGGAUUUCAUGCUUAAAACAUUG

GGGUGCUAAUGGCAACAAGAUUGUU 1 times at 15534 NSP12 GGGUGCUAAUGGCAACAAGAUUG

CCCCAAAU U UG U U G AU AAAU ACU AU 1 times at 15624 NSP12 CCCCAAAU U U G U U GAU AAAU ACU

CGGUUGCUUUGUAGAUGAUAUCGUU 1 times at 15930 NSP12 CGGUUGCUUUGUAGAUGAUAUCG

GCCCAAAAAGGUAUUCUUUGUGUUA 1 times at 17908 NSP13 GCCCAAAAAGGUAUUCUUUGUGU

CG G U UCAU U UGACAAAG U CU AU G AU 1 times at 18969 NSP14 CGGUUCAUUUGACAAAGUCUAUG

GGGAUUAUGAACGUAGCAAUAUUUA 1 times at 19829 NSP15 GGGAUUAUGAACGUAGCAAUAUU

GCCAUCUUUAUUUCUGAUAGAAAAA 1 times at 19972 NSP15 GCCAUCUUUAUUUCUGAUAGAAA

CCGUGAUAGUGAUGUUGUUAAACAA 1 times at 20055 NSP15 CCGUGAUAGUGAUGUUGUUAAAC

GGGUACUAUUAAAGAAAAUAUAGAU 1 times at 21237 NSP16 GGGUACUAUUAAAGAAAAUAUAG

GGCCGUACAUAUUCUAACAUAACUA 1 times at 21635 S protein GGCCGUACAUAUUCUAACAUAAC

GCCGUACAUAUUCUAACAUAACUAU 1 times at 21636 S protein GCCGUACAUAUUCUAACAUAACU

CCGAAGAUGAGAUUUUAGAGUGGUU 1 times at 22191 S protein CCGAAGAUGAGAUUUUAGAGUGG

CCCAGUUUAAUUAUAAACAGUCCUU 1 times at 22848 S protein CCCAG U UUAAU U AU AAACAG U CC

GGCUUCACUACAACUAAUGAAGCUU 1 times at 24485 S protein GGCUUCACUACAACUAAUGAAGC

CCCCUGUUAAUGGCUACUUUAUUAA 1 times at 24951 S protein CCCCUGUUAAUGGCUACUUUAUU

CCCUGUUAAUGGCUACUUUAUUAAA 1 times at 24952 S protein CCCUGUUAAUGGCUACUUUAUUA

GCCGCAUAAGGUUCAUGUUCACUAA 1 times at 25492 S protein GCCGCAUAAGGUUCAUGUUCACU

CGCAAAACUCUUGUUCUUAAUGCAU 1 times at 26485 orf4b CGCAAAACUCUUGUUCUUAAUGC

GGCUUUCUCGGCGUCUUUAUUUAAA 1 times at 26841 orf5 GGCUUUCUCGGCGUCUUUAUUUA

GGCAUUGUAGCAGCUGUUUCAGCUA 1 times at 28092 M GGCAUUGUAGCAGCUGUUUCAGC

GCCUAUUACGGCGGAUAUUGAACUU 1 times at 28469 M GCCUAUUACGGCGGAUAUUGAAC

CCGGUACUAAGCUUCCUAAAAACUU 1 times at 29019 N CCGGUACUAAGCUUCCUAAAAAC Table 4 Characterization indexes of five SLiC species and five SLiC-siRNA nanoparticles, including particle sizes, poly-dispersity index (PDI) and Zeta-potential.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising at least two different siRNA molecules that target one or more conserved regions of the genome of a Middle-East Respiratory Syndrome Corona Virus (MERS-CoV) and a pharmaceutically acceptable carrier comprising a polymeric nanoparticle or a liposomal nanoparticle.

2. The composition of claim 1, wherein the gene sequences in the conserved regions of the MERS-CoV are critical for the viral infection of a mammal.

3. The composition of claim 2, wherein the mammal is a human, mouse, ferret, or

monkey.

4. The composition of claim 1, wherein the targeted conserved regions of the genome comprise gene sequences coding for MERS-CoV proteins selected from the group consisting of Papain-like protease (PL^pro), RNA-dependent RNA polymerase (RdRp), and Spike protein.

5. The composition of claim 4, wherein the siRNA molecules target PL^pro viral gene

expression.

6. The composition of claim 4, wherein the siRNA molecules target RdRp viral gene expression.

7. The composition of claim 4, wherein the siRNA molecules target Spike viral gene expression.

8. The composition of claim 4, wherein the siRNA molecules are selected from the group consisting of:

MPL1 : CGCAAUACGUAAAGCUAAAGAUUAU,

MPL2: GGGUGUUGAUUAUACUAAGAAGUUU,

MPL3 : CGCAUAAUGGUGGUUACAAUUCUU,

MPL4: GGCUUCAUUUUAUUUCAAAGAAUUU,

MPL5: GCGCUUUUACAAAUCUAGAUAAGUU,

MPL6: CGCAUUGCAUGCCGUAAGUGUAAUU,

MRR1 : CCCAGUGUUAUUGGUGUUUAUCAUA,

MRR2: GGGAUUUCAUGCUUAAAACAUUGUA,

MRR3 : GGGUGCUAAUGGCAACAAGAUUGUU,

MRR4: CCCCAAAUUUGUUGAUAAAUACUAU, MRR5: CGGUUGCUUUGUAGAUGAUAUCGUU,

MSP1 : GGCCGUACAUAUUCUAACAUAACUA,

MSP2: GGCCGUACAUAUUCUAACAUAACUA,

MSP3 : CCGAAGAUGAGAUUUUAGAGUGGUU,

MSP4: CCCAGUUUAAUUAUAAACAGUCCUU,

MSP5: GGCUUCACUACAACUAAUGAAGCUU,

MSP6: CCCCUGUUAAUGGCUACUUUAUUAA,

MSP7: CCCUGUUAAUGGCUACUUUAUUAAA, and

MSP8: GCCGCAUAAGGUUCAUGUUCACUAA.

9. The composition of claim 5, wherein the siRNA molecules that target the PL^pro gene are selected from the group consisting of:

MPL1 : CGCAAUACGUAAAGCUAAAGAUUAU,

MPL2: GGGUGUUGAUUAUACUAAGAAGUUU,

MPL3 : CGCAUAAUGGUGGUUACAAUUCUU,

MPL4: GGCUUCAUUUUAUUUCAAAGAAUUU,

MPL5: GCGCUUUUACAAAUCUAGAUAAGUU, and

MPL6: CGCAUUGCAUGCCGUAAGUGUAAUU.

10. The composition of claim 6, wherein the siRNA molecules that target the RdRp gene are selected from the group consisting of:

MRR1 : CCCAGUGUUAUUGGUGUUUAUCAUA,

MRR2: GGGAUUUCAUGCUUAAAACAUUGUA,

MRR3 : GGGUGCUAAUGGCAACAAGAUUGUU,

MRR4: CCCCAAAUUUGUUGAUAAAUACUAU, and

MRR5: CGGUUGCUUUGUAGAUGAUAUCGUU.

11. The composition of claim 7, wherein the siRNA molecules that target the Spike gene are selected from the group consisting of:

MSP1 : GGCCGUACAUAUUCUAACAUAACUA, MSP2: GGCCGUACAUAUUCUAACAUAACUA,

MSP3 : CCGAAGAUGAGAUUUUAGAGUGGUU,

MSP4: CCCAGUUUAAUUAUAAACAGUCCUU,

MSP5: GGCUUCACUACAACUAAUGAAGCUU,

MSP6: CCCCUGUUAAUGGCUACUUUAUUAA,

MSP7: CCCUGUUAAUGGCUACUUUAUUAAA, and

MSP8: GCCGCAUAAGGUUCAUGUUCACUAA.

12. The composition of claim 1, comprising a siRNA cocktail, MST^PR1, wherein a first siRNA molecule comprises MPL1 : C GC A AU AC GU A A AGCU A A AGAUU AU and a second siRNA molecule comprises MRRl :

CCCAGUGUUAUUGGUGUUUAUCAUA.

13. The composition of claim 1, comprising a siRNA cocktail, MST^PR2, wherein a first siRNA molecule comprises MPL2: GGGGUUGAUUAUACUAAGAAGUUU and a second siRNA molecule comprises MRR2:

GGGAUUUCAUGCUUAAAACAUUGUA

14. The composition of claim 1, comprising a siRNA cocktail, MST^RS2, wherein a first siRNA molecule comprises MRR2: GGGAUUUCAUGCUUAAAACAUUGUA and a second siRNA molecule comprises MSP2: GGCCGUACAUAUUCUAACAUAACUA.

15. The composition of claim 1, comprising a siRNA cocktail, MST^RS1, wherein a first siRNA molecule compri ses MRRl : CCCAGUGUUAUUGGUGUUUAUCAUA and a second siRNA molecule comprises MSPl : GGCCGUACAUAUUCUAACAUAACUA.

16. The composition of claim 1, comprising a siRNA cocktail, MST^PRS1, wherein a first siRNA molecule comprises MPL1 : C GC A AU AC GU A A AGCU A A AGAU AU, a second siRNA molecule comprises MRRl :

CCCAGUGUUAUUGGUGUUUAUCAUA, and a third siRNA molecule comprises

MSPl : GGCCGUACAUAUUCUAACAUAACUA.

17. The composition of claim 1, comprising a siRNA cocktail, MST^PRS2, wherein a first siRNA molecule comprises MPL2: GGGUGUUGAUUAUACUAAGAAGUUU a second siRNA molecule comprises MRR2:

GGGAUUUCAUGCUUAAAACAUUGUA, and a third siRNA molecule comprises

MSP2: GGCCGUACAUAUUCUAACAUAACUA.

18. The composition of any one of claims 1-17, wherein the polymeric nanoparticle carrier comprises a Histidine-Lysine co-polymer (HKP).

19. The composition of claim 18, wherein the HKP comprises the structure (R)K(R)-K(R)- (R)K(X), where R =KHHHKHHHKHHHKHHHK, K = lysine, and H = histidine.

20. The composition of any one of claims 1-17, wherein the liposomal nanoparticle carrier comprises a Spermine-Lipid Conjugate (SLiC) and cholesterol.

21. The composition of claim 20, wherein the SLiC comprises one of the structures shown in Figure 8.

22. The composition of any one of claims 1-17, wherein the HKP and siRNA molecules are formulated into nanoparticles.

23. The composition of any one of claims 1-17, wherein the SLiC and the siRNA molecules are formulated into nanoparticles.

24. The composition of claim 18, wherein the HKP and the siRNA molecules self-assemble into nanoparticles.

25. The composition of claim 20, wherein the SLiC and the siRNA self-assemble into

nanoparticles in formulation with cholesterol.

26. The composition of any one of claims 1-25, wherein the siRNA molecules comprise oligonucleotides with a length of 19-25 base pairs.

27. The composition of any one of claims 1-25, wherein the siRNA molecules comprise oligonucleotides with a length of 21-25 base pairs.

28. The composition of any one of claims 1-25, wherein the siRNA molecules comprise oligonucleotides with a length of 25 base pairs.

29. A method of treating a subject with a MERS infection comprising administering to said subject a pharmaceutically effective amount of the composition of claim 1.

30. A method of treating a subject with a MERS infection comprising administering to said subject a pharmaceutically effective amount of the composition of any one of claims 2- 28.

31. The method of claims 29 or 30, wherein the composition is administered to the subject through airway instillation.

32. The method of claims 29 or 30, wherein the composition is administered to the subject through intraperitoneal administration.

33. The method of claims 29 or 30, wherein the composition is administered to the subject through an airway nebulizer.

34. The method of claims 29 or 30, wherein the composition is administered to the subject through subcutaneous administration.

35. The method of any one of claims 26-34, wherein the subject is a mammal.

36. The method of claim 35, wherein the mammal is a human, mouse, ferret, or monkey.

37. The method of claim 35, wherein the mammal is a human.

38. An siRNA molecule that targets a conserved region of the genome of a MERS-CoV.

39. The siRNA molecule of claim 38, wherein the targeted conserved region of the genome comprises gene sequences coding for MERS-CoV proteins selected from the group consisting of Papain-like protease (PL^pro), RNA-dependent RNA polymerase (RdRp), and Spike protein.

40. The siRNA molecule of claim 39, wherein the siRNA molecule targets PL^pro virus gene expression.

41. The siRNA molecule of claim 39, wherein the siRNA molecule targets RdRp viral gene expression.

42. The siRNA molecule of claim 39, wherein the siRNA molecule targets Spike viral gene expression.

43. The siRNA molecule of claim 38, wherein the molecule is selected from the group consisting of the molecules identified in Table 3.

44. The siRNA molecule of claim 38, wherein the wherein the siRNA molecules are

selected from the group consisting of:

MPL1 : CGCAAUACGUAAAGCUAAAGAUUAU,

MPL2: GGGUGUUGAUUAUACUAAGAAGUUU,

MPL3 : CGCAUAAUGGUGGUUACAAUUCUU,

MPL4: GGCUUCAUUUUAUUUCAAAGAAUUU,

MPL5: GCGCUUUUACAAAUCUAGAUAAGUU,

MPL6: CGCAUUGCAUGCCGUAAGUGUAAUU,

MRR1 : CCCAGUGUUAUUGGUGUUUAUCAUA,

MRR2: GGGAUUUCAUGCUUAAAACAUUGUA,

MRR3 : GGGUGCUAAUGGCAACAAGAUUGUU,

MRR4: CCCCAAAUUUGUUGAUAAAUACUAU,

MRR5: CGGUUGCUUUGUAGAUGAUAUCGUU,

MSP1 : GGCCGUACAUAUUCUAACAUAACUA, MSP2 GGCCGUACAUAUUCUAACAUAACUA,

MSP3 CCGAAGAUGAGAUUUUAGAGUGGUU,

MSP4 CCCAGUUUAAUUAUAAACAGUCCUU,

MSP5 GGCUUCACUACAACUAAUGAAGCUU,

MSP6 CCCCUGUUAAUGGCUACUUUAUUAA,

MSP7: CCCUGUUAAUGGCUACUUUAUUAAA, and

MSP8: GCCGCAUAAGGUUCAUGUUCACUAA.

45. The siRNA molecule of any one of claims 38-44, wherein the siRNA molecule

comprises oligonucleotides with a length of 19-25 base pairs.

46. The siRNA molecule of any one of claims 38-44, wherein the siRNA molecule

comprises oligonucleotides with a length of 21-25 base pairs.

47. The siRNA molecule of any one of claims 38-44, wherein the siRNA molecule

comprises oligonucleotides with a length of 25 base pairs.

48. A composition comprising the siRNA molecule of any one of claims 38-47 and a

pharmaceutically acceptable carrier comprising a polymeric nanoparticle or a liposomal nanoparticle.

49. A method of treating a subject with a MERS infection comprising administering to said subject a pharmaceutically effective amount of the composition of claim 48.

50. The method of claim 49, wherein the subject is a human.

51. The composition of claim 44, wherein the siRNA molecules comprise derivatives of the identified siRNA molecules, the derivatives having 17-24 contiguous base pairs of original 25 contiguous base pairs of the identified molecules or one or more base pairs in addition to the original 25 contiguous base pairs of the identified molecules.