ACTIVE TRUNCATED FORM OF THE RNA POLYMERASE OF FLAVIVIRUS

Abstract

ation of two domains from a from a flavivirus is provided. Each domain can function independently. Moreover, one domain codes for a sequence that provide polymerase activity. A process for screening possible modulators of the polymerase activity of an isolated and purified polypeptide from flavivirus is also disclosed.

Description

CROSS REFERENCE TO RELATED APPLICATION

[0001]This application claims the benefit of U.S. Provisional Application No. 60/474,670, filed May 30, 2003, the subject matter of which is fully incorporated herein by reference.

FIELD OF INVENTION

[0002]This invention relates to antiviral molecular biology. More particularly, it relates to the isolation and identification of an active truncated form of the RNA polymerase of flavivirus, capable of being easily reproduced, and serving as a target for high-throughput screening of antiviral drugs.

BACKGROUND

[0003]The genus flavivirus contains approximately 70 positive single-stranded RNA viruses, among which many major human pathogens are found, including Dengue virus ("DV"), West Nile ("WNV"), Yellow Fever virus ("YFV"), Japanese and tick-borne encephalitis viruses. YFV was the first flavivirus to be isolated in 1927, but historically, flavivirus-like diseases have been reported in the medical literature since at least 1780.

[0004]On of the most common and virulent flaviviruses is DV. DV threatens up to 2.5 billion people in 100 endemic countries. Up to 50 million infections occur annually with 500 000 cases of dengue haemorrhagic fever and 22,000 deaths mainly among children. Dengue has been classified by the World Health Organization ("WHO") as a priority as it ranks as the most important mosquito-borne viral disease in the world (http://www.who.int/csr/disease/dengue). In the last 50 years, its incidence has increased 30-fold. Prior to 1970, only 9 countries had experienced cases of dengue haemorrhagic fever ("DHF"); since then the number has increased more than 4-fold and continues to rise.

[0005]WNV also has become much more wide-spread. In 1999, WNV was isolated for the first time in the Americas during an outbreak in New York City. By the end of 2002, WNV activity had been identified in 44 states of the United States and the District of Columbia. The 2002 WNV epidemic resulted in 4,156 reported human cases of WN disease including 2,942 meningoencephalitis cases and 284 deaths (http://www.cdc.gov/ncidod/dvbid/westnile/).

[0006]There have been previous attempts to generate a vaccine. For example, a live, attenuated virus of YFV (strain 17D) was developed in 1936 and has been used as a vaccine for over 400 million people. Unfortunately, the vaccine has not proved 100% successful since there are 200,000 estimated cases of yellow fever (with 30,000 deaths) per year worldwide, 90% of which in Africa. (http://www.who.int/mediacentre/factsheets/fs100/en/). Chimeric live vaccines incorporating genes of either Japanese encephalitis, WNV, or Dengue in a YFV 17D vector are currently in development. However, a number of difficulties are associated with the conception of safe and efficient vaccines, such as vaccine purity, and immunogenic cross responses. That is why antiviral chemotherapy has a major role to play in the control of such diseases.

[0007]Since viral RNA polymerase is critical for replication of the virus and cannot be substituted by any other cellular polymerase, it is an excellent antiviral target. As a result, most of the more than 30 new antiviral agents, which have been developed and approved during the last 5 years, are directed against viral polymerases. They are mainly targeted against human immuno-deficiency virus, but drugs against hepatitis B and C, herpes simplex, varicella-zoster and influenza virus infections have also been made commercially available.

[0008]More than 50% of these antiviral agents are nucleoside analogues, in which the base, the ribose moiety or both have been modified. Nucleoside analogues can act as inhibitory ligands by binding to the template binding site within the polymerase active site and preventing the access of the viral RNA, or by binding to the nucleotide binding site, thus limiting the availability of the natural substrate for complementary strand synthesis. It is generally understood in the art that a nucleoside analogue may be a synthetic molecule that resembles a naturally occurring nucleoside, but lacks a bond site needed to link it to an adjacent nucleotide. Additionally, nucleoside analogues can also act as chain-terminators during DNA or RNA synthesis, by binding themselves as a substrate for the target polymerase, but preventing further chain elongation. Non-nucleoside analogues may bind to allosteric sites thus influencing the local conformation of the active site via long-range conformational changes of the polymerase's structure.

[0009]Another approach whereby many antiviral compounds have been discovered is by using cell cultures infected with the virus of interest. In such cases, addition of an antiviral compound protects the cells from infection, or inhibits virus growth. For this type of experiment, it is useful to identify a large number of antiviral compounds in an efficient manner. As such, another evolving mechanism to identify new antiviral agents through the high-throughput screening ("HTS") of a large number of synthetic or natural compounds. This requires the development of an in vitro assay, which in turn requires large amounts of soluble and active protein.

[0010]When a high number of potentially antiviral compounds are tested by HTS, it is possible to identify antiviral compounds in an efficient manner. This approach has been used successfully for HIV and other viruses. However, in some cases, this approach is difficult due to the absence of a suitable system allowing infection of a cell in vitro.

[0011]In other cases, even if a suitable cell-based assay is available, this procedure may be too cumbersome or expensive. This is the case for certain dangerous viruses--such as those that require BSL-3 and/or BSL-4 facilities. Establishing a screening process for over a large amount of compounds in a BSL-3 or BSL-4 containment facility has not been achieved yet because of this heavy expense and burden. For example, flaviviruses belong to this class of viruses. These viruses require from BSL-2 to BSL-4 facilities (e.g., Dengue, WNV and/or Kyasanur Forest viruses). Thus, in such cases, it is preferable to screen potentially antiviral compounds directly on viral target proteins.

[0012]For efficiency, especially considering the difficulty with certain, more dangerous viruses, the characterization in molecular terms of the target, the viral polymerase, is of prime importance in the screening and selection of antiviral compounds. In the case of the flavivirus RNA polymerase ("NS5" or sometimes referred to herein as "NS5Pol"), this task has proven to be difficult for several reasons. First, polymerase genes have been notoriously difficult to clone in their entirety. When available, recombinant NS5 has been reported to be unstable in bacterial hosts. In addition, the notoriously low yield of soluble purified NS5 is a limiting factor to set up polymerase-activity assays. Another possible reason for the described difficulties is the fact that NS5 does not carry a single enzymatic activity.

[0013]Very recently, we described an N-terminal domain of NS5 (sometimes referred to herein as "NS5 methyltransferase domain") which acts as an S-adenosyl-L-methionine (AdoMet)-utilizing RNA-cap 2'Omethyltransferase, thus participating in mRNA capping, which is generally understood as the process of adding a guanosine nucleotide to the 5' end of mRNA (the methelyated end of guanosine) (Egloff & Benarroch, 2002). Additionally, we showed that the NS5 methyltransferase domain binds GTP analogues.

[0014]Due to the nature and proximity of the NS5 methyltransferase domain to the polymerase domain of the flavivirus, the description and characterization of the NS5 methyltransferase domain clearly shows that some nucleoside analogues and inhibitors of flavivirus replication could potentially be, in fact, mRNA-capping inhibitors without any effect on the polymerase activity. Likewise, it is very possible to mistakenly identify a compound as binding to NS5 and characterizing the binding data as potentially interesting for inhibition of the polymerase, but, in reality, only the RNA-capping has been affected. Therefore, it would be useful to identify and define the "junction" or sequence between the NS5 methyltransferase domain and the polymerase domain.

SUMMARY OF THE INVENTION

[0015]This invention relates to the isolation and purification of a polypeptide from a flavivirus.

[0016]In another aspect of the invention, the polypeptide can be separated into two domains, the N-terminal domain and the C-terminal domain, both of which are separately active.

[0017]In another aspect of the invention, the junction between the N-terminal and C-terminal domains has been identified.

[0018]In yet another aspect of the invention, the results indicated that independent expression of each of the separated domains provided greater expression than the full, unseparated polypeptide.

[0019]In still another aspect of the invention, the C-terminal of the domains in particular is purified and acts as active RNA polymerase.

[0020]In yet another aspect of the invention, the C-terminal domain demonstrates substantial homology with other RNA polymerases of clinical interest.

[0021]In still another aspect of this invention, the polymerase provides a surrogate model and system to screen synthetic and natural compounds against the polymerases of related viruses.

BRIEF DESCRIPTION OF THE FIGURES

[0022]FIG. 1 shows the sequence alignment of NS5 of flaviviruses.

[0023]FIG. 2 shows two Western blots illustrating the expression and purification of NS5_DV and NS5Pol_DV.

[0024]FIG. 3 shows a Western blot (FIG. 3A) and a graph (FIG. 3B) of the purified NS5Pol_DV.

[0025]FIG. 4 shows two graphs (FIG. 4A and FIG. 4B) demonstrating the activity level of NS5Pol_DV on poly(rC).

[0026]FIG. 5 shows a steady-state Km determination of GTP with and poly(rC) (in FIG. 5A) and for monomeric NS5PolDV using poly(rC) without primer (de novo initiation) (in FIG. 5B).

[0027]FIG. 6 shows divalent cation optimum curves for NS5PolDV and NS5PolWNV (in FIG. 6A) on poly(rC) without primer (de novo initiation) (in FIG. 6B).

[0028]FIG. 7 shows two graphs demonstrating the activity level of NS5PolDV on specific heteropolymeric RNA templates.

DETAILED DESCRIPTION

Definitions

[0029]"Structural equivalents" should be understood to mean a protein maintaining its conformational structure as if the protein were the native protein expressed in its natural cell.

[0030]"Substantial homology" or "substantially homologous" means a degree of homology between the isolated and described NS5Pol (as defined herein) and the RNA polymerases of other positive-single-stranded RNA viruses of clinical interest when there is homology at least about 65%, preferably at least about 70%, most preferably in excess of 80%, and even more preferably in excess of 90%, 95% or 99%.

Results and Findings

[0031]In one aspect of this invention, we discovered a way to circumvent the above-described problems associated with viral polymerase. We performed a structural analysis of flaviviruses NS5 genes using biocomputing methods, and isolated and defined two unique domains of NS5. As described in the literature, two distinct domains are generally defined for the large family of flavivirus NS5 genes, and related structural equivalents.

[0032]Specifically, as shown in the experiments, NS5 was separated into the two domains using genetic engineering techniques. We have established the independent folding of these two putative domains using various methods. Moreover, demonstrated in our experiments as set forth below, each domain is separately active, and an appropriate ligand may be mapped to either the N-terminal (capping) domain or the C-terminal (polymerase) domain of NS5. These genetic constructs allow the production of higher quantities of either domain compared to the full-length protein. Thus, simply put, the C-terminal polymerase domain of NS5 (NS5Pol) of DV (subtype 2, Strain New Guinea C), WNV (strain New York 99) and the Kunjin variant of WVN (KV) are easy to purify in large quantities, they are active as a polymerase, and constitute one aspect of our invention.

[0033]As noted, the availability of large quantities of NS5Pol allows its use as a target in HTS. One of the advantages of the isolation of the polymerase domain is that the antiviral compound, which demonstrates the modulating activity of the polymerase domain, is specific to the polymerase activity of the viral protein, without any interference of the other parts of the protein. Indeed, it is possible to detect RNA polymerase activity in a single tube using standard radioactive or nonradioactive methods.

[0034]As described herein, modulation of the polymerase activity of the protein is important in creating antiviral agents for the treatment of the enumerated viral diseases. Since DV, WNV and KV NS5Pol domains are significantly homologous to and demonstrate substantial homology with NS5Pol domains of other flaviviruses, and since DV, WNV and KV NS5Pol are functionally homologous to the RNA polymerases of other positive-single-stranded RNA viruses of clinical interest (such as the related NS5B polymerase of HCV), NS5Pol provides also a surrogate model and system to screen synthetic and natural compounds against such related viruses. Simply put, the invention includes a method of screening antiviral compounds able to modulate the polymerase activity of significantly and functionally homologus NS5 gene encoding viruses (i.e. flaviviruses).

Expression and Purification

[0035]Based on preceding structural and functional studies on a N-terminal methyltransferase or capping domain of protein NS5 of flavivirus (Egloff and Benarroch, 2002) we predicted the limit of a functional and soluble C-terminal polymerase domain of NS5. In particular, FIG. 1 identifies the sequence alignment of various flaviviruses as follows: Dengue 2 (P14340), Dengue 3 (Q99D35), Dengue 1 (Q8VBS3), Dengue 4 (AAA42964), West Nile virus (AAL87234), Japanese encephalitis virus (Q82872), Yellow fever virus (Q89277), Banzi virus (Q67483), Langat virus (Q9IG40), Tick-borne encephalitis virus (Q8VBS4), Louping ill virus (O10383), Modoc virus (CAC82912), Rio Bravo virus (Q9JAD5) were aligned by ClustalW. The secondary-structure elements of the NS5MTaseDV structure as determined by X-ray crystallography and of NS5PolDV as predicted by PredictProtein are displayed in black and red, respectively, above the sequence of NS5 Dengue. NS5Pol starts after the vertical bar just before a predicted alpha-helix. The remaining ca. 550 residues of NS5 are not shown.

[0036]As set forth above, FIG. 1 shows the sequence alignment of the N-terminal part of NS5 of several flaviviruses with the secondary structure elements of the capping domain of Dengue NS5 given above. Essentially, as discussed, the junction between the methyltransferase and polymerase portion of the flaviviruses was isolated, which allows the precise and efficient separation of the domains. The junction (or where the Pol domain starts) is located at amino acid 272.

[0037]Proteins NS5 and their corresponding Pol domains of DV, KV and WNV were expressed as recombinant proteins bearing a His-tag which facilitates subsequent purification. Accordingly, they were purified with immobilized metal-affinity chromatography (IMAC) in a first purification step. As noted above, FIG. 2 illustrates the superiority of NS5PolDV over NS5DV in terms of purification yield after IMAC following the same protocol. The results shown in FIG. 2, the expression and purification of NS5DV and NS5PolDV, were obtained as follows: NS5DV and NS5PolDV were cloned in expression plasmid pQE30, expressed in BL21[pDNAy] overnight at 17° C. after induction with 50 mM IPTG, addition of 2% EtOH and a cold shock (30 min at 4° C.). Sonication was done in 50 mM sodium phosphate lysis buffer, pH 7.5, 300 mM NaCl, 10% glycerol (10 ml of this lysis buffer for 3.6 g cell pellet) in the presence of DNAse, PMSF, protease inhibitors and lysozyme. Recombinant proteins were bound to metal-affinity-chromatography resin talon (Clontech) and eluted with 500 mM imidazole. SDS-PAGE of protein samples from expression and purification: upper panel: NS5DV, lower panel: NS5PolDV, lane 1: total fraction after lysis, lane 2: soluble fraction after lysis and centrifugation, lane 3: flowthrough of metal-affinity column, lane M: molecular mass markers, lanes 4 to 8: eluted fractions from metal-affinity column, F1 to F5, lane 9: metalaffinity resin. The corresponding molecular masses in kD of the markers are given on the left. NS5DV results in low purification yields due to instability resulting in the presence of a protein band the size of which corresponds to the polymerase domain (see arrow in the upper panel).

[0038]The low yield of NS5 is attributed to lower solubility of the recombinant protein and an elevated sensitivity to proteolytic cleavage during purification. This is illustrated in FIG. 2 by the presence of a cleavage product of around 73 kD which could represent the Pol domain. Yields for both proteins are compared in Table 1.

TABLE-US-00001 TABLE 1 Yield after expression and purification Yield (mg per liter expression culture) protein after IMAC after heparin NS5Pol_DV 2 1.2 NS5_DV 0.2 n.d. NS5Pol_KV 10 7 NS5_KV 7 0.6 NS5Pol_WNV 12 8 NS5_WNV n.d. n.d.

[0039]A second purification step consists of heparin affinity chromatography. The results of this purification were illustrated in FIG. 3 and obtained pursuant to the following procedure: NS5PolDV eluates from metal-affinity chromatography were dialyzed against 50 mM sodium phosphate buffer, pH 7.5, 150 mM NaCl, 10% glycerol and submitted to heparin-affinity chromatography applying a salt gradient of 150 mM to 1M NaCl. Pure protein was eluted in two peaks, at 390 mM and 460 mM NaCl.

[0040]A: SDS-PAGE of protein samples from purification steps, lane 1: pooled protein fractions from metal-affinity chromatography after dialysis, lane M: molecular mass markers, lane 2: peak 1 from heparin-affinity chromatography, lane 3: peak 2 from heparin-affinity chromatography, lane 4: flowthrough from heparin-column. The corresponding molecular masses in kD of the markers are given on the left.

[0041]B: Analytical gel filtration (Superdex 200, Pharmacia) of peak 1 and 2 of NS5PolDV from heparin affinity chromatography. The elution volume of peak 1 corresponds to the monomeric form of NS5PolDV whereas peak 2 elutes earlier corresponding to oligomeric NS5PolDV (trimer or tetramer).

[0042]For NS5PolDV it results in two fractions eluting at different salt concentrations both representing NS5PolDV, as shown in FIG. 3A. Analytical gel filtration showed that peak 1 from heparin affinity chromatography represents the monomeric form of NS5PolDV whereas peak 2 represents an oligomeric form as shown in FIG. 3B. Both forms are purified to 98% after heparin purification step, the combined yield of NS5PolDV is 1.2 mg starting from one liter expression culture (Table 1).

[0043]Expression and purification of NS5 KV and NS5PolKV follow a similar tendency compared to Dengue NS5 (sequence identity of NS5 66.4%). Although full-length NS5 KV and NS5PolKV render considerably higher yields compared to the corresponding Dengue proteins, still, full-length NS5 KV shows lower yields after one purification step (Table 1) and, due its instability, dramatically lower yields after a second purification step. In difference to NS5PolDV, NS5PolKV elutes as a single peak after heparin affinity chromatography (data not shown). The same applies to NS5PolWNV (sequence identity to NS5PolKV 94.6%).

[0044]In all cases, the final purification product, for which the purity is adequate for HTS assays, is purified with a >10-fold increase in yield compared to the unengineered polymerase.

Activity Data

[0045]Polymerase activity on NS5Pol was measured on homo- and heteropolymeric templates.

Homopolymeric Template

[0046]Activity was tested on three homopolymeric templates: poly(rC), poly(rU) and poly(rA). Only poly(rC) resulted to be a productive template for NS5PolDV. This was illustrated in FIG. 4 based on the following protocol: RNA polymerase activity was tested on a homopolymeric RNA template (polycytidylic acid, Amersham Biosciences) of an average length of 360 nt. A standard assay was carried out in 50 mM HEPES buffer, pH 8.0, 10 mM KCl, 5 mM MgCl2, 5 mM MnCl2, 10 mM DTT containing 1 μM template, 4 mM primer GG, 10 μM GTP, 0.01 mCi [3H]-GTP per μl reaction mixture and the concentration of enzyme given below. Reactions were carried out at 30° C. for given time periods and stopped by spotting a sample on DEAE filter discs (Whatman) presoaked with 50 mM EDTA. Filters were washed 3×10 min with 300 mM (NH4)2SO4 buffer, pH 8.0 and 2×5 min with EtOH and air-dried. Liquid scintillation fluid was added and incorporation in counts per minute (cpm) determined by using a Wallac MicroBeta TriLux Liquid Scintillation Counter.

[0047]A: Influence of specific E. coli RNA polymerase inhibitor rifampicin on NS5PolDV and E. coli polymerase (control). NS5PolDV was tested using the conditions given above at 64-nM enzyme concentration. E. coli RNA polymerase was obtained from USBiochemicals and used in NS5PolDV standard reaction buffer at 37 nM.

[0048]B: Time course of [3H]-GTP incorporation by NS5PolDV peak 1 (monomeric preparation) and peak 2 (oligomeric preparation) from heparin 11 affinity chromatography (see FIG. 3A) tested at 80 nM enzyme concentration using poly(rC) without primer.

[0049]FIG. 4A shows incorporation of radioactively labeled GTP into a nascent poly(rG) polymerization product by NS5PolDV (oligomeric preparation) using primer rGG. In a control reaction E. coli RNA polymerase-inhibitor rifampicin was added and did not show any inhibitory effect. Thus, the observed incorporation of radioactive GTP in a polymerization product is not due to a contamination with E. coli RNA polymerase activity. FIG. 4B shows a parallel test of the oligomeric and monomeric preparation of NS5PolDV. In this experiment no primer was used, thus, polymerization is initiated de novo. Both preparations show identical catalytic efficiency. There are two possible explanations, either the state of oligomerization does not influence polymerase activity or under the conditions of the activity test (80 nM enzyme 6 concentration) both preparations adopt the same oligomerization state. In either way, both preparations can likewise be used for inhibitor screening studies.

[0050]In FIG. 5, the steady-state Km determination of GTP with various ligands was performed as follows: RNA polymerase activity was tested under conditions given in the description set forth above in the experiments related to FIG. 4 without the use of a primer. Initial velocities were determined over a time period of 5 min. GTP concentrations in the range of 2 to 500 μM were tested using poly(rC) at 1 μM. Template poly(rC) was tested in the range of 2 to 1000 nM with GTP fixed at 200 μM.

[0051]A: Plot of apparent intitial velocity (viapp in cpm per min) against GTP concentration. Data were fitted to a Michaelis-Menten hyperbola (viapp=Vmax[S]/(Km+[S]) using Kaleidagraph.

[0052]B: Plot of apparent initial velocity against poly(rC) concentration. Data were fitted as in A.

[0053]The Km of GTP was determined for NS5PolDV (monomeric preparation) as being 27.3±5.1 mM (FIG. 5A). Km values are in a similar range for the oligomeric preparation of NS5PolDV (66.2±6.6 mM) and for full-length NS5DV (13.0±2.9 mM). Thus, the affinity of NS5PolDV from both preparations versus the substrate GTP that binds to a nucleotide-binding site within the active site of the enzyme are close. This again confirms that both preparations can be used likewise. Additionally, the fact that NS5PolDV shows similar substrate affinity as NS5 indicates that the polymerase domain of flavivirus NS5 is a valid model system for the identification of inhibitors of the full-length protein. The Km values of polyC were determined for the monomeric preparation of NS5PolDV as 17.3±2.4 mM, the oligomeric preparation as 14.8±3.8 mM and for NS5DV as 18.0±7.3 mM. As seen above, the corresponding plot for the monomeric preparation of NS5PolDV is shown in FIG. 5B. First, this allows the conclusion that a putatively different oligomerization status of NS5PolDV does not seem to influence the affinity to a long template which could be expected to maintain cooperative interactions with two enzyme molecules at the same time. Secondly, template poly(rC) (around 360 nt long) shows identical affinity to NS5DV and NS5PolDV within the context of its use as a polymerization template. Thus, the capping domain does not seem to be involved in template binding indicating again that the isolated polymerase domain can be used for inhibitor screening experiments of the polymerase activity of full-length NS5.

[0054]FIG. 6A shows the optimum curve for divalent manganese (Mn) and magnesium (Mg) ions of NS5PolDV on poly(rC). Polymerization on poly(rC) works exclusively in the presence of Mn ions. This is also the case for NS5PolWNV as shown in FIG. 6B. The results set forth in FIG. 6 were obtained pursuant to the following protocol:

[0055]A: Tests of NS5PolDV were carried out under standard conditions given in the description set forth above in the experiments related to FIG. 4 at 60 nM enzyme concentration. Influence of Mn2+ was tested either in absence of Mg2+ (Mn) or in presence of 5 mM Mg2+ (Mn (5 mM Mg)). Mg2+ was tested in the absence of Mn2+ (Mg).

[0056]B: Test of NS5PolWNV were carried out under the same reaction conditions as for NS5PolDV with the exception of GTP which was used at 100 μM. NS5PolWNV concentration was 400 nM.

[0057]Clearly, as seen from FIGS. 6A and 6B, the optimum curves for Mn2+ show that NS5PolWNV tolerates higher Mn2+ concentrations for the polymerization on polyC than NS5PolDV. The apparent Km value for substrate GTP is around 2 to 4-fold higher (130 mM) for NS5PolWNV and NS5PolKV in comparison to NS5PolDV (data not shown). These mechanistic differences between flavivirus polymerases can be studied in detail using well expressed and stable independent NS5Pol domains.

Heteropolymeric Template

[0058]NS5PolDV activity was tested on heteropolymeric specific templates comprising 717 nucleotides (225 nt of the 5' and 492 nt of the 3' of the Dengue genome). Specifically, the results are demonstrated in FIG. 7 and were obtained pursuant to the following protocol: Heteropolymeric RNA templates of 717 nt were generated by in vitro transcription using T7 RNA polymerase. The DNA template containing 225 nt of the 5' and 492 nt of the 3' of Dengue genomic RNA was constructed into plasmid pUC18. PCR products with the T7 promoter on the 5' of the positive-sense strand or the 5' of negative-sense strand were generated and used as substrates for in vitro transcription. RNA templates were replicated by NS5PolDV in 50 mM HEPES buffer, pH 8.0 containing 10 mM KCl, 10 mM DTT, 100 nM RNA template, 200 nM NS5PolDV, 500 mM ATP, UTP, GTP, 10 mM CTP and [a-32P]-CTP at 0.1 mCi/ml. Reactions were carried out at 30° C. and stopped by spotting a sample on DEAE 12 filter discs (Whatman) pre-soaked with 50 mM EDTA. Filters were treated as explained above in the experiments related to FIG. 4.

[0059]A: Comparison of incorporation on positive-sense and complementary negative-sense minigenome RNA templates. Reactions were carried out as given above except for the use of 500 mM CTP, 50 mM GTP, 0.1 mCi/ml [a-32P]-GTP and 5 mM Mn2+.

[0060]B: Divalent-cation optimum curves on positive-sense specific RNA template. Mn2+ was used in the absence of Mg2+ and, likewise, Mg2+ in the absence of Mn2+. Reaction were stopped after 60 min. Incorporation of CMP is given in cpm. The axis on the left corresponds to values obtained in presence of Mn2+ and the axis on the right to values obtained in presence of Mg2+.

[0061]This "minigenome" template illustrated in FIG. 7 contains secondary-structure and sequence elements necessary for efficient 7 de novo initiation and replication. Positive-sense and negative-sense RNA was generated by in vitro transcription from PCR products containing the promoter for T7 RNA polymerase in either sense. Specifically, FIG. 7A illustrates that NS5PolDV replicates negative-sense mini-genome RNA with higher efficiency compared to positive-sense RNA. This observation is in accordance with the observation that 10-times more positive-sense genomic RNA is produced in Dengueinfected cells in comparison to negative-sense RNA. Optimum divalent cation concentration (Mn2+ and Mg2+) was determined for replication of the positive-sense template (FIG. 7A). NS5PolDV replicates the template with 20-fold higher efficiency at optimal Mn2+ concentration compared to optimal concentration of Mg2+. The affinity of Mn2+ to active site residues seems to be higher compared to Mg2+ as the optimum concentration is lower.

Selective Inhibition of Dengue Virus Polymerase Activity Using Nucleotide Inhibitors

[0062]GTP analogs were used on NS5PolDV and NS5PolWNV to demonstrate their capacity to inhibit the NS5 polymerase domain using the homopolymeric template poly(rC). IC50 values were determined using GTP concentrations close to the determinded Km values (10 mM for NS5PolDV and 100 mM for NS5PolWNV). Table 2 shows the determined values of three putative chain terminators (3'-deoxy GTP, 3'-dioxolane 3'-deoxy GTP and 2',3'-dideoxy GTP) and 2'-O-methyl-GTP which is expected to be incorporated into the growing RNA chain, thus acting as a competitive inhibitor.

Table 2 IC50 Values for Selected GTP Analogs

[0063]Reactions on poly(rC) were done under conditions given in FIG. 4 without a primer. NS5PolDV was used at 60 nM. Incorporation of [3H]-GTP was measured after 15 min. Inhibitors (TriLink Corp.) were tested in the concentration range of 1 nM to 100 mM.

TABLE-US-00002 IC50 (μM) inhibitor NS5Pol_DV NS5Pol_WNV 3'-deoxy GTP 0.02 0.18 3'-dioxolane 3'-deoxy GTP 1.2 58 2',3'-dideoxy GTP 0.8 30 2'-O-methyl-GTP 5.6 105

[0064]The capacity of 2'-O-methyl-GTP (10 mM) was compared to inhibit NS5PolDV (dimeric preparation) and full-length NS5DV. Initial velocities were determined on poly(rC) at 10 mM GTP and compared to corresponding values without inhibitor. Initial velocities were determined to be 29.3% for NS5PolDV (dimeric preparation) and 19.3% for NS5DV compared to the proteins without inhibitor. This indicates that the NS5PolDV preparation is less inhibited than the full-length polymerase. Since these inhibition results are not identical, questions related to the putative interference of the capping domain remain when using the full-length polymerase.

[0065]It is clear that the removal of the capping domain (methyltransferase domain) which is able to bind GTP and GTP analogs, at the N-terminus of NS5 provides the opportunity to obtain unambiguous data which will show that the present truncated polymerase is the target of these inhibitors. The same applies to non-nucleoside inhibitors.

CONCLUSION

[0066]To summarize our results: the polymerase domain of flavivirus NS5

[0067]1. is purified with higher yields than full-length NS5. The overall increase in yield of the purified product suitable for HTS assays is >10-fold, facilitating mass production for HTS assays.

[0068]2. is much more stable than full-length NS5, as less protein is lost or degraded during the course of purification, giving a cleaner and more homogeneous reagent.

[0069]3. is active on homopolymeric and heteropolymeric specific templates. Thus, the capping domain is not necessary for the polymerase activity defined as the capacity to initiate and incorporate nucleotides into RNA. The polymerase domain defined here is a bonafide polymerase useful to conduct drug-screening assays.

[0070]4. shows similar affinities for template and substrates as the full-length NS5, and is identical in all points tested in terms of polymerase activity.

[0071]5. is unambiguous about being the target of a potential inhibitor. As the capping domain is not present, inhibition of NS5 function by GTP analogues and other molecules cannot be accounted for by inhibition of the capping domain or indirect inhibition of the NS5 polymerase activity by interference of the inhibitor with the capping domain.

[0072]6. is a valid polymerase model to conduct inhibitor screening studies with the aim to identify putative inhibitors of flavivirus RNA polymerases. [0073]Baginski S G, Pevear D C, Seipel M, Sun S C, Benetatos C A, Chunduru S K, Rice C M, Collett M S. "Mechanism of action of a pestivirus antiviral compound." Proc Natl Acad Sci USA 2000, 97:7981-6. [0074]Campiani G, Fabbrini M, Morelli E, Nacci V, Greco G, Novellino E, Maga G, Spadari S, Bergamini A, Faggioli E, Uccella I, Bolacchi F, Marini S, Coletta M, Fracasso C, Caccia S. "Non-nucleoside HIV-1 reverse transcriptase inhibitors: synthesis and biological evaluation of novel quinoxalinylethylpyridylthioureas as potent antiviral agents." Antivir Chem Chemother 2000, 11:141-55. [0075]Carroll S S, Tomassini J E, Bosserman M, Getty K, Stahlhut M W, Eldrup A B, Bhat B, Hall D, Simcoe A L, LaFemina R, Rutkowski C A, Wolanski B, Yang Z, Migliaccio G, De Francesco R, Kuo L C, MacCoss M, Olsen D B. "Inhibition of hepatitis C virus RNA replication by 2'-modified nucleoside analogs." J Biol Chem 2003 278:11979-84. [0076]De Clercq E. "Antiviral drugs: current state of the art." J. Clin. Virol. 2001:73-89. [0077]Egloff M P, Benarroch D, Selisko B, Romette J L, Canard B. "An RNA cap (nucleoside-2'-O--)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization." EMBO J. 2002 21:2757-68. [0078]Khandazhinskaya A L, Shirokova E A, Skoblov Y S, Victorova L S, Goryunova L Y, Beabealashvilli R S, Pronyaeva T R, Fedyuk N V, Zolin V V, Pokrovsky A G, Kukhanova M K. "Carbocyclic dinucleoside polyphosphonates: interaction with HIV reverse transcriptase and antiviral activity." J Med Chem 2002, 45:1284-91. [0079]Lai M M. "RNA polymerase as an antiviral target of hepatitis C virus." Antivir Chem Chemother 2001, 12 Suppl 1:143-7. [0080]Mentel R, Kurek S, Wegner U, Janta-Lipinski M, Gurtler L, Matthes E. "Inhibition of adenovirus DNA polymerase by modified nucleoside triphosphate analogs correlate with their antiviral effects on cellular level." Med Microbiol Immunol (Berl) 2000, 189:91-5. [0081]Mlinaric A, Kreft S, Umek A, Strukelj B. "Screening of selected plant extracts for in vitro inhibitory activity on HIV-1 reverse transcriptase (HIV-1 RT)." Pharmazie 2000, 55:75-7. [0082]Walker M P, Hong Z. "HCV RNA-dependent RNA polymerase as a target for antiviral development." Curr. Opinion Pharmacol. 2002, 2!:1-7. [0083]Wang M, Ng K K, Cherney M M, Chan L, Yannopoulos C G, Bedard J, Morin N, Nguyen-Ba N, Alaoui-Ismaili M H, Bethell R C, James M N. "Non-nucleoside Analogue Inhibitors Bind to an Allosteric Site on HCV NS5B Polymerase." CRYSTAL STRUCTURES AND MECHANISM OF INHIBITION. J Biol Chem 2003 278:9489-95. [0084]Zoulim F. "Therapy of chronic hepatitis B virus infection: inhibition of the viral polymerase and other antiviral strategies." Antiviral Res 1999, 44:1-30.

Sequence CWU 1

301349PRTFlavivirus sp.MISC_FEATUREDengue2 1Gly Thr Gly Asn Ile Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg1 5 10 15Leu Asn Ala Leu Gly Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly 20 25 30Ile Gln Glu Val Asp Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg Gly 35 40 45Glu Thr Asp His His Ala Val Ser Arg Gly Ser Ala Lys Leu Arg Trp 50 55 60Phe Val Glu Arg Asn Met Val Thr Pro Glu Gly Lys Val Val Asp Leu65 70 75 80Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Gly Gly Leu Lys Asn 85 90 95Val Arg Glu Val Lys Gly Leu Thr Lys Gly Gly Pro Gly His Glu Glu 100 105 110Pro Ile Pro Met Ser Thr Tyr Gly Trp Asn Leu Val Arg Leu Gln Ser 115 120 125Gly Val Asp Val Phe Phe Thr Pro Pro Glu Lys Cys Asp Thr Leu Leu 130 135 140Cys Asp Ile Gly Glu Ser Ser Pro Asn Pro Thr Val Glu Ala Gly Arg145 150 155 160Thr Leu Arg Val Leu Asn Leu Val Glu Asn Trp Leu Asn Asn Asn Thr 165 170 175Gln Phe Cys Ile Lys Val Leu Asn Pro Tyr Met Pro Ser Val Ile Glu 180 185 190Lys Met Glu Ala Leu Gln Arg Lys Tyr Gly Gly Ala Leu Val Arg Asn 195 200 205Pro Leu Ser Arg Asn Ser Thr His Glu Met Tyr Trp Leu Ser Asn Ala 210 215 220Ser Gly Asn Ile Val Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile225 230 235 240Asn Arg Phe Thr Met Arg His Lys Lys Ala Thr Tyr Glu Pro Asp Val 245 250 255Asp Leu Gly Ser Gly Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro 260 265 270Asn Leu Asp Ile Ile Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu His 275 280 285Glu Thr Ser Trp His Tyr Asp Gln Asp His Pro Tyr Lys Thr Trp Ala 290 295 300Tyr His Gly Ser Tyr Glu Thr Lys Gln Thr Gly Ser Ala Ser Ser Met305 310 315 320Gly Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp Asp Val Val Pro 325 330 335Met Val Thr Gln Met Ala Met Thr Asp Thr Thr Pro Phe 340 3452348PRTFlavivirus sp.misc_featureDengue3 2Gly Thr Gly Ser Gln Gly Glu Thr Leu Gly Glu Lys Trp Lys Lys Lys1 5 10 15Leu Asn Gln Leu Ser Arg Lys Glu Phe Asp Leu Tyr Lys Lys Ser Gly 20 25 30Ile Thr Glu Val Asp Arg Thr Glu Ala Lys Glu Gly Leu Lys Arg Gly 35 40 45Glu Ile Thr His His Ala Val Ser Arg Gly Ser Ala Lys Leu Gln Trp 50 55 60Phe Val Glu Arg Asn Met Val Ile Pro Glu Gly Arg Val Ile Asp Leu65 70 75 80Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Ala Gly Leu Lys Lys 85 90 95Val Thr Glu Val Arg Gly Tyr Thr Lys Gly Gly Pro Gly His Glu Glu 100 105 110Pro Val Pro Met Ser Thr Tyr Gly Trp Asn Ile Val Lys Leu Met Ser 115 120 125Gly Lys Asp Val Phe Tyr Leu Pro Pro Glu Lys Cys Asp Thr Leu Leu 130 135 140Cys Asp Ile Gly Glu Ser Ser Pro Ser Pro Thr Val Glu Glu Ser Arg145 150 155 160Thr Ile Arg Val Leu Lys Met Val Glu Pro Trp Leu Lys Asn Asn Gln 165 170 175Phe Cys Ile Lys Val Leu Asn Pro Tyr Met Pro Thr Val Ile Glu His 180 185 190Leu Glu Arg Leu Gln Arg Lys His Gly Gly Met Leu Val Arg Asn Pro 195 200 205Leu Ser Arg Asn Ser Thr His Glu Met Tyr Trp Ile Ser Asn Gly Thr 210 215 220Gly Asn Ile Val Ser Ser Val Asn Met Val Ser Arg Leu Leu Leu Asn225 230 235 240Arg Phe Thr Met Thr His Arg Arg Pro Thr Ile Glu Lys Asp Val Asp 245 250 255Leu Gly Ala Gly Thr Arg His Val Asn Ala Glu Pro Glu Thr Pro Asn 260 265 270Met Asp Val Ile Gly Glu Arg Ile Lys Arg Ile Lys Glu Glu His Ser 275 280 285Ser Thr Trp His Tyr Asp Asp Glu Asn Pro Tyr Lys Thr Trp Ala Tyr 290 295 300His Gly Ser Tyr Glu Val Lys Ala Thr Gly Ser Ala Ser Ser Met Ile305 310 315 320Asn Gly Val Val Lys Leu Leu Thr Lys Pro Trp Asp Val Val Pro Met 325 330 335Val Thr Gln Met Ala Met Thr Asp Thr Thr Pro Phe 340 3453348PRTFlavivirus sp.misc_featureDengue1 3Gly Thr Gly Ala Gln Gly Glu Thr Leu Gly Glu Lys Trp Lys Arg Gln1 5 10 15Leu Asn Gln Leu Ser Lys Ser Glu Phe Asn Thr Tyr Lys Arg Ser Gly 20 25 30Ile Met Glu Val Asp Arg Ser Glu Ala Lys Glu Gly Leu Lys Arg Gly 35 40 45Glu Thr Thr Lys His Ala Val Ser Arg Gly Thr Ala Lys Leu Arg Trp 50 55 60Phe Val Glu Arg Asn Leu Val Lys Pro Glu Gly Lys Val Ile Asp Leu65 70 75 80Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Ala Gly Leu Lys Lys 85 90 95Val Thr Glu Val Lys Gly Tyr Thr Lys Gly Gly Pro Gly His Glu Glu 100 105 110Pro Ile Pro Met Ala Thr Tyr Gly Trp Asn Leu Val Lys Leu His Ser 115 120 125Gly Lys Asp Val Phe Phe Thr Pro Pro Glu Lys Cys Asp Thr Leu Leu 130 135 140Cys Asp Ile Gly Glu Ser Ser Pro Asn Pro Thr Ile Glu Glu Gly Arg145 150 155 160Thr Leu Arg Val Leu Lys Met Val Glu Pro Trp Leu Arg Gly Asn Gln 165 170 175Phe Cys Ile Lys Ile Leu Asn Pro Tyr Met Pro Ser Val Val Glu Thr 180 185 190Leu Glu Gln Met Gln Arg Lys His Gly Gly Met Leu Val Arg Asn Pro 195 200 205Leu Ser Arg Asn Ser Thr His Glu Met Tyr Trp Val Ser Cys Gly Thr 210 215 220Gly Asn Ile Val Ser Ala Val Asn Met Thr Ser Arg Met Leu Leu Asn225 230 235 240Arg Phe Thr Met Ala His Arg Lys Pro Thr Tyr Glu Arg Asp Val Asp 245 250 255Leu Gly Ala Gly Thr Arg His Val Ala Val Glu Pro Glu Val Ala Asn 260 265 270Leu Asp Ile Ile Gly Gln Arg Ile Glu Asn Ile Lys His Glu His Lys 275 280 285Ser Thr Trp His Tyr Asp Glu Asp Asn Pro Tyr Lys Thr Trp Ala Tyr 290 295 300His Gly Ser Tyr Glu Val Lys Pro Ser Gly Ser Ala Ser Ser Met Val305 310 315 320Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp Asp Val Ile Pro Met 325 330 335Val Thr Gln Ile Ala Met Thr Asp Thr Thr Pro Phe 340 3454349PRTFlavivirus spmisc_featureDengue4 4Gly Thr Gly Thr Thr Gly Glu Thr Leu Gly Glu Lys Trp Lys Arg Gln1 5 10 15Leu Asn Ser Leu Asp Arg Lys Glu Phe Glu Glu Tyr Lys Arg Ser Gly 20 25 30Ile Leu Glu Val Asp Arg Thr Glu Ala Lys Ser Ala Leu Lys Asp Gly 35 40 45Ser Lys Ile Lys His Ala Val Ser Arg Gly Ser Ser Lys Ile Arg Trp 50 55 60Ile Val Glu Arg Gly Met Val Lys Pro Lys Gly Lys Val Val Asp Leu65 70 75 80Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Met Ala Thr Leu Lys Asn 85 90 95Val Thr Glu Val Lys Gly Tyr Thr Lys Gly Gly Pro Gly His Glu Glu 100 105 110Pro Ile Pro Met Ala Thr Tyr Gly Trp Asn Leu Val Lys Leu His Ser 115 120 125Gly Val Asp Val Phe Tyr Lys Pro Thr Glu Gln Val Asp Thr Leu Leu 130 135 140Cys Asp Ile Gly Glu Ser Ser Ser Asn Pro Thr Ile Glu Glu Gly Arg145 150 155 160Thr Leu Arg Val Leu Lys Met Val Glu Pro Trp Leu Ser Ser Lys Pro 165 170 175Glu Phe Cys Ile Lys Val Leu Asn Pro Tyr Met Pro Thr Val Ile Glu 180 185 190Glu Leu Glu Lys Leu Gln Arg Lys His Gly Gly Asn Leu Val Arg Cys 195 200 205Pro Leu Ser Arg Asn Ser Thr His Glu Met Tyr Trp Val Ser Gly Ala 210 215 220Ser Gly Asn Ile Val Ser Ser Val Asn Thr Thr Ser Lys Met Leu Leu225 230 235 240Asn Arg Phe Thr Thr Arg His Arg Lys Pro Thr Tyr Glu Lys Asp Val 245 250 255Asp Leu Gly Ala Gly Thr Arg Ser Val Ser Thr Glu Thr Glu Lys Pro 260 265 270Asp Met Thr Ile Ile Gly Arg Arg Leu Gln Arg Leu Gln Glu Glu His 275 280 285Lys Glu Thr Trp His Tyr Asp Gln Glu Asn Pro Tyr Arg Thr Trp Ala 290 295 300Tyr His Gly Ser Tyr Glu Ala Pro Ser Thr Gly Ser Ala Ser Ser Met305 310 315 320Val Asn Gly Val Val Lys Leu Leu Thr Lys Pro Trp Asp Val Ile Pro 325 330 335Met Val Thr Gln Leu Ala Met Thr Asp Thr Thr Pro Phe 340 3455351PRTFlavivirus sp.misc_featureWest Nile 5Gly Gly Ala Lys Gly Arg Thr Leu Gly Glu Val Trp Lys Glu Arg Leu1 5 10 15Asn Gln Met Thr Lys Glu Glu Phe Thr Arg Tyr Arg Lys Glu Ala Ile 20 25 30Ile Glu Val Asp Arg Ser Ala Ala Lys His Ala Arg Lys Glu Gly Asn 35 40 45Val Thr Gly Gly His Ser Val Ser Arg Gly Thr Ala Lys Leu Arg Trp 50 55 60Leu Val Glu Arg Arg Phe Leu Glu Pro Val Gly Lys Val Ile Asp Leu65 70 75 80Gly Cys Gly Arg Gly Gly Trp Cys Tyr Tyr Met Ala Thr Gln Lys Arg 85 90 95Val Gln Glu Val Arg Gly Tyr Thr Lys Gly Gly Pro Gly His Glu Glu 100 105 110Pro Gln Leu Val Gln Ser Tyr Gly Trp Asn Ile Val Thr Met Lys Ser 115 120 125Gly Val Asp Val Phe Tyr Arg Pro Ser Glu Cys Cys Asp Thr Leu Leu 130 135 140Cys Asp Ile Gly Glu Ser Ser Ser Ser Ala Glu Val Glu Glu His Arg145 150 155 160Thr Ile Arg Val Leu Glu Met Val Glu Asp Trp Leu His Arg Gly Pro 165 170 175Arg Glu Phe Cys Val Lys Val Leu Cys Pro Tyr Met Pro Lys Val Ile 180 185 190Glu Lys Met Glu Leu Leu Gln Arg Arg Tyr Gly Gly Gly Leu Val Arg 195 200 205Asn Pro Leu Ser Arg Asn Ser Thr His Glu Met Tyr Trp Val Ser Arg 210 215 220Ala Ser Gly Asn Val Val His Ser Val Asn Met Thr Ser Gln Val Leu225 230 235 240Leu Gly Arg Met Glu Lys Arg Thr Trp Lys Gly Pro Gln Tyr Glu Glu 245 250 255Asp Val Asn Leu Gly Ser Gly Thr Arg Ala Val Gly Lys Pro Leu Leu 260 265 270Asn Ser Asp Thr Ser Lys Ile Asn Asn Arg Ile Glu Arg Leu Arg Arg 275 280 285Glu Tyr Ser Ser Thr Trp His His Asp Glu Asn His Pro Tyr Arg Thr 290 295 300Trp Asn Tyr His Gly Ser Tyr Asp Val Lys Pro Thr Gly Ser Ala Ser305 310 315 320Ser Leu Val Asn Gly Val Val Arg Leu Leu Ser Lys Pro Trp Asp Thr 325 330 335Ile Thr Asn Val Thr Thr Met Ala Met Thr Asp Thr Thr Pro Phe 340 345 3506351PRTFlavivirus sp.misc_featureKunjin 6Gly Gly Ala Lys Gly Arg Thr Leu Gly Glu Val Trp Lys Glu Arg Leu1 5 10 15Asn Gln Met Thr Lys Glu Glu Phe Ile Arg Tyr Arg Lys Glu Ala Ile 20 25 30Thr Glu Val Asp Arg Ser Ala Ala Lys His Ala Arg Lys Glu Arg Asn 35 40 45Ile Thr Gly Gly His Pro Val Ser Arg Gly Thr Ala Lys Leu Arg Trp 50 55 60Leu Val Glu Arg Arg Phe Leu Glu Pro Val Gly Lys Val Ile Asp Leu65 70 75 80Gly Cys Gly Arg Gly Gly Trp Cys Tyr Tyr Met Ala Thr Gln Lys Arg 85 90 95Val Gln Glu Val Arg Gly Tyr Thr Lys Gly Gly Pro Gly His Glu Glu 100 105 110Pro Gln Leu Val Gln Ser Tyr Gly Trp Asn Ile Val Thr Met Lys Ser 115 120 125Gly Val Asp Val Phe Tyr Arg Pro Ser Glu Cys Cys Asp Thr Leu Leu 130 135 140Cys Asp Ile Gly Glu Ser Ser Ser Ser Ala Glu Val Glu Glu His Arg145 150 155 160Thr Leu Arg Val Leu Glu Met Val Glu Asp Trp Leu His Arg Gly Pro 165 170 175Lys Glu Phe Cys Val Lys Val Leu Cys Pro Tyr Met Pro Lys Val Ile 180 185 190Glu Lys Met Glu Leu Leu Gln Arg Arg Tyr Gly Gly Gly Leu Val Arg 195 200 205Asn Pro Leu Ser Arg Asn Ser Thr His Glu Met Tyr Trp Val Ser Arg 210 215 220Ala Ser Gly Asn Val Val His Ser Val Asn Met Thr Ser Gln Val Leu225 230 235 240Leu Gly Arg Met Glu Lys Lys Thr Trp Lys Gly Pro Gln Tyr Glu Glu 245 250 255Asp Val Asn Leu Gly Ser Gly Thr Arg Ala Val Gly Lys Pro Leu Leu 260 265 270Asn Ser Asp Thr Ser Lys Ile Lys Asn Arg Ile Glu Arg Leu Arg Arg 275 280 285Glu Tyr Ser Ser Thr Trp His His Asp Glu Asn His Pro Tyr Arg Thr 290 295 300Trp Asn Tyr His Gly Ser Tyr Glu Val Lys Pro Thr Gly Ser Ala Ser305 310 315 320Ser Leu Val Asn Gly Val Val Arg Leu Leu Ser Lys Pro Trp Asp Thr 325 330 335Ile Thr Asn Val Thr Thr Met Ala Met Thr Asp Thr Thr Pro Phe 340 345 3507351PRTFlavivirus sp.misc_featureJapanese Encephalitis 7Gly Arg Pro Gly Gly Arg Thr Leu Gly Glu Gln Trp Lys Glu Lys Leu1 5 10 15Asn Ala Met Ser Arg Glu Glu Phe Phe Lys Tyr Arg Arg Glu Ala Ile 20 25 30Ile Glu Val Asp Arg Thr Glu Ala Arg Arg Ala Arg Arg Glu Asn Asn 35 40 45Ile Val Gly Gly His Pro Val Ser Arg Gly Ser Ala Lys Leu Arg Trp 50 55 60Leu Val Glu Lys Gly Phe Val Ser Pro Ile Gly Lys Val Ile Asp Leu65 70 75 80Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Ala Ala Thr Leu Lys Lys 85 90 95Val Gln Glu Val Arg Gly Tyr Thr Lys Gly Gly Ala Gly His Glu Glu 100 105 110Pro Met Leu Met Gln Ser Tyr Gly Trp Asn Leu Val Ser Leu Lys Ser 115 120 125Gly Val Asp Val Phe Tyr Lys Pro Ser Glu Pro Ser Asp Thr Leu Phe 130 135 140Cys Asp Ile Gly Glu Ser Ser Pro Ser Pro Glu Val Glu Glu Gln Arg145 150 155 160Thr Leu Arg Val Leu Glu Met Thr Ser Asp Trp Leu His Arg Gly Pro 165 170 175Arg Glu Phe Cys Ile Lys Val Leu Cys Pro Tyr Met Pro Lys Val Ile 180 185 190Glu Lys Met Glu Val Leu Gln Arg Arg Phe Gly Gly Gly Leu Val Arg 195 200 205Leu Pro Leu Ser Arg Asn Ser Asn His Glu Met Tyr Trp Val Ser Gly 210 215 220Ala Ala Gly Asn Val Val His Ala Val Asn Met Thr Ser Gln Val Leu225 230 235 240Leu Gly Arg Met Asp Arg Thr Val Trp Arg Gly Pro Lys Tyr Glu Glu 245 250 255Asp Val Asn Leu Gly Ser Gly Thr Arg Ala Val Gly Lys Gly Glu Val 260 265 270His Ser Asn Gln Glu Lys Ile Lys Lys Arg Ile Gln Lys Leu Lys Glu 275 280 285Glu Phe Ala Thr Thr Trp His Lys Asp Pro Glu His Pro Tyr Arg Thr 290 295 300Trp Thr Tyr His Gly Ser Tyr Glu Val Lys Ala Thr Gly Ser Ala Ser305 310 315 320Ser Leu Val Asn Gly Val Val Lys Leu Met Ser Lys Pro Trp Asp Ala 325

330 335Ile Ala Asn Val Thr Thr Met Ala Met Thr Asp Thr Thr Pro Phe 340 345 3508350PRTFlavivirus sp.misc_featureYellow Fever 8Gly Thr Ala Asn Gly Lys Thr Leu Gly Glu Val Trp Lys Arg Glu Leu1 5 10 15Asn Leu Leu Asp Lys Gln Gln Phe Glu Leu Tyr Lys Arg Thr Asp Ile 20 25 30Val Glu Val Asp Arg Asp Thr Ala Arg Arg His Leu Ala Glu Gly Lys 35 40 45Val Asp Thr Gly Val Ala Val Ser Arg Gly Thr Ala Lys Leu Arg Trp 50 55 60Phe His Glu Arg Gly Tyr Val Lys Leu Glu Gly Arg Val Ile Asp Leu65 70 75 80Gly Cys Gly Arg Gly Gly Trp Cys Tyr Tyr Ala Ala Ala Gln Lys Glu 85 90 95Val Ser Gly Val Lys Gly Phe Thr Leu Gly Arg Asp Gly His Glu Lys 100 105 110Pro Met Asn Val Gln Ser Leu Gly Trp Asn Ile Ile Thr Phe Lys Asp 115 120 125Lys Thr Asp Ile His Arg Leu Glu Pro Val Lys Cys Asp Thr Leu Leu 130 135 140Cys Asp Ile Gly Glu Ser Ser Ser Ser Ser Val Thr Glu Gly Glu Arg145 150 155 160Thr Val Arg Val Leu Asp Thr Val Glu Lys Trp Leu Ala Cys Gly Val 165 170 175Asp Asn Phe Cys Val Lys Val Leu Ala Pro Tyr Met Pro Asp Val Leu 180 185 190Glu Lys Leu Glu Leu Leu Gln Arg Arg Phe Gly Gly Thr Val Ile Arg 195 200 205Asn Pro Leu Ser Arg Asn Ser Thr His Glu Met Tyr Tyr Val Ser Gly 210 215 220Ala Arg Ser Asn Val Thr Phe Thr Val Asn Gln Thr Ser Arg Leu Leu225 230 235 240Met Arg Arg Met Arg Arg Pro Thr Gly Lys Val Thr Leu Glu Ala Asp 245 250 255Val Thr Leu Pro Ile Gly Thr Arg Ser Val Glu Thr Asp Lys Gly Pro 260 265 270Leu Asp Lys Glu Ala Ile Glu Glu Arg Val Glu Arg Ile Lys Ser Glu 275 280 285Tyr Met Thr Ser Trp Phe Tyr Asp Asn Asp Asn Pro Tyr Arg Thr Trp 290 295 300His Tyr Cys Gly Ser Tyr Val Thr Lys Thr Ser Gly Ser Ala Ala Ser305 310 315 320Met Val Asn Gly Val Ile Lys Ile Leu Thr Tyr Pro Trp Asp Lys Ile 325 330 335Glu Glu Val Thr Arg Met Ala Met Thr Asp Thr Thr Pro Phe 340 345 3509350PRTFlavivirus sp.misc_featureBanzi 9Gly Gly Ser Ser Ala Leu Thr Tyr Gly Glu Val Trp Lys Arg Gln Leu1 5 10 15Asn Leu Leu Gly Lys Gln Glu Phe Met Asn Tyr Lys Val Ser Asp Ile 20 25 30Leu Glu Val Asp Arg Ser His Ala Arg Glu Val Leu Asn Ser Gly Asn 35 40 45Asp Ala Val Gly Val Ala Val Ser Arg Gly Ser Ser Lys Leu Asn Trp 50 55 60Leu Ile Glu Arg Gly Tyr Leu Arg Pro Thr Gly Arg Val Val Asp Leu65 70 75 80Gly Cys Gly Trp Gly Gly Trp Ser Tyr Thr Cys Ala Ala Glu Arg Gln 85 90 95Val Thr Ser Val Lys Ala Tyr Thr Leu Gly Lys Glu Gly His Glu Lys 100 105 110Pro Arg Leu Ile Gln Ser Leu Gly Trp Asn Ile Ile Lys Phe Lys Asp 115 120 125Lys Ser Asp Ile Thr Arg Met Thr Pro His Ala Ser Asp Thr Leu Leu 130 135 140Cys Asp Ile Gly Glu Ser Ser Ser Asn Pro Glu Val Glu Lys Glu Arg145 150 155 160Thr Leu Arg Val Ile Glu Ala Val Glu Lys Trp Met Ser Pro Thr Thr 165 170 175Val Ser Phe Cys Phe Lys Val Leu Ala Pro Tyr Lys Pro Asp Val Ile 180 185 190Glu Ala Leu Glu Arg Phe Gln Leu Lys His Gly Gly Gly Ile Ile Arg 195 200 205Asn Pro Tyr Ser Arg Asn Ser Thr His Glu Met Tyr Tyr Val Ser Gly 210 215 220Val Arg Asn Asn Ile Leu His Met Val Asn Ser Thr Ser Arg Met Leu225 230 235 240Met Arg Arg Met Ser Arg Pro Ser Gly Arg Ser Thr Val Val Pro Asp 245 250 255Leu Ile Tyr Pro Thr Gly Thr Arg Ser Val Ala Ser Glu Ala Gly Pro 260 265 270Leu Asp Leu Glu Lys Val Lys Ala Arg Ile Asn Arg Leu Lys Glu Glu 275 280 285Gln Glu Ser Thr Trp Phe Val Asp Ser Asp His Pro Tyr Arg Thr Trp 290 295 300His Tyr His Gly Ser Tyr Val Ala Lys Gln Ser Gly Thr Ala Ala Ser305 310 315 320Met Ile Asn Gly Val Val Lys Leu Leu Ser Gly Pro Trp Asp Arg Ile 325 330 335Glu Glu Val Thr Asn Met Ala Met Thr Asp Thr Thr Pro Phe 340 345 35010350PRTFlavivirus sp.misc_featureLangat 10Gly Gly Ser Glu Gly Asp Thr Leu Gly Asp Met Trp Lys Ala Arg Leu1 5 10 15Asn Ser Cys Thr Lys Glu Glu Phe Phe Ala Tyr Arg Arg Ala Gly Val 20 25 30Met Glu Thr Asp Arg Glu Lys Ala Arg Glu Leu Leu Lys Arg Gly Glu 35 40 45Thr Asn Met Gly Leu Ala Val Ser Arg Gly Thr Ser Lys Leu Ala Trp 50 55 60Met Glu Glu Arg Gly Tyr Val Thr Leu Lys Gly Glu Val Val Asp Leu65 70 75 80Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Ala Ala Ser Arg Pro Ala 85 90 95Val Met Ser Val Arg Ala Tyr Thr Ile Gly Gly Lys Gly His Glu Ser 100 105 110Pro Arg Met Val Thr Ser Leu Gly Trp Asn Leu Ile Lys Phe Arg Ala 115 120 125Gly Met Asp Val Phe Ser Met Glu Pro His Arg Ala Asp Ala Ile Leu 130 135 140Cys Asp Ile Gly Glu Ser Asn Pro Asp Ala Val Val Glu Gly Glu Arg145 150 155 160Ser Arg Arg Val Ile Leu Leu Met Glu Gln Trp Lys Asn Arg Asn Pro 165 170 175Thr Ala Thr Cys Val Phe Lys Val Leu Ala Pro Tyr Arg Pro Glu Val 180 185 190Ile Glu Ala Leu His Arg Phe Gln Leu Gln Trp Gly Gly Gly Leu Val 195 200 205Arg Thr Pro Phe Ser Arg Asn Ser Thr His Glu Met Tyr Phe Ser Thr 210 215 220Ala Ile Thr Gly Asn Ile Val Asn Ser Val Asn Ile Gln Ser Arg Lys225 230 235 240Leu Leu Ala Arg Phe Gly Asp Gln Arg Gly Pro Thr Arg Val Pro Glu 245 250 255Ile Asp Leu Gly Val Gly Thr Arg Cys Val Val Leu Ala Glu Asp Lys 260 265 270Val Lys Glu Lys Asp Val Met Glu Arg Ile Gln Ala Leu Lys Asp Gln 275 280 285Tyr Cys Asp Thr Trp His Glu Asp His Glu His Pro Tyr Arg Thr Trp 290 295 300Gln Tyr Trp Gly Ser Tyr Lys Thr Ala Ala Thr Gly Ser Ser Ala Ser305 310 315 320Leu Leu Asn Gly Val Val Lys Leu Leu Ser Trp Pro Trp Asn Ala Arg 325 330 335Glu Asp Val Val Arg Met Ala Met Thr Asp Thr Thr Ala Phe 340 345 35011350PRTFlavivirus sp.misc_featurePowassan 11Gly Gly Ala Glu Gly Ser Thr Leu Gly Asp Ile Trp Lys Gln Arg Leu1 5 10 15Asn Ser Cys Thr Lys Glu Glu Phe Phe Ala Tyr Arg Arg Thr Gly Val 20 25 30Met Glu Thr Asn Arg Asp Gln Ala Arg Glu Leu Leu Arg Arg Gly Glu 35 40 45Thr Asn Met Gly Leu Ala Val Ser Arg Gly Cys Ala Lys Leu Ala Trp 50 55 60Leu Glu Glu Arg Gly Tyr Ala Thr Leu Lys Gly Glu Val Val Asp Leu65 70 75 80Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Ala Ala Ser Arg Pro Ser 85 90 95Val Met Ala Val Arg Ala Tyr Thr Ile Gly Gly Lys Gly His Glu Ala 100 105 110Pro Arg Leu Val Thr Ser Leu Gly Trp Asn Leu Ile Lys Phe Arg Ser 115 120 125Gly Met Asp Val Phe Ser Met Ala Thr Thr Arg Ala Asp Thr Ile Leu 130 135 140Cys Asp Ile Gly Glu Ser Ser Pro Asp Pro Glu Lys Glu Gly Ala Arg145 150 155 160Ser Arg Arg Val Ile Leu Leu Met Glu Gln Trp Lys Ala Arg Asn Pro 165 170 175Asp Ala Ala Ala Val Phe Lys Val Leu Ala Pro Tyr Arg Pro Glu Val 180 185 190Leu Glu Ala Leu His Arg Phe Gln Leu Gln Trp Gly Gly Gly Leu Val 195 200 205Arg Val Pro Phe Ser Arg Asn Ser Thr His Glu Met Tyr Tyr Ser Thr 210 215 220Ala Val Thr Gly Asn Leu Val Asn Ser Val Asn Val Leu Ser Arg Lys225 230 235 240Leu Leu Ala Arg Phe Gly Glu Thr Arg Gly Pro Ile Gln Val Pro Glu 245 250 255Ile Asp Leu Gly Thr Gly Thr Arg Cys Val Thr Leu Ala Glu Asp Lys 260 265 270Val Lys Pro Arg Asp Val Ala Glu Arg Ile Gly Ala Leu Arg Glu Gln 275 280 285Tyr Ser Glu Ser Trp His Glu Asp Lys Glu His Pro Tyr Arg Thr Trp 290 295 300Gln Tyr Trp Gly Ser Tyr Arg Thr Pro Ala Thr Gly Ser Ala Ala Ser305 310 315 320Leu Ile Asn Gly Val Val Lys Leu Leu Ser Trp Pro Trp Asn Ala Arg 325 330 335Glu Asp Val Thr Arg Met Ala Met Thr Asp Thr Thr Ala Phe 340 345 35012350PRTFlavivirus sp.misc_featureTick-borne Encephalitis 12Gly Gly Ser Glu Gly Asp Thr Leu Gly Asp Leu Trp Lys Arg Arg Leu1 5 10 15Asn Gly Cys Thr Lys Glu Glu Phe Phe Ala Tyr Arg Arg Thr Gly Ile 20 25 30Leu Glu Thr Glu Arg Asp Lys Ala Arg Glu Leu Leu Arg Arg Gly Glu 35 40 45Thr Asn Met Gly Leu Ala Val Ser Arg Gly Thr Ala Lys Leu Ala Trp 50 55 60Leu Glu Glu Arg Gly Tyr Ala Thr Leu Lys Gly Glu Val Val Asp Leu65 70 75 80Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Ala Ala Ser Arg Pro Ala 85 90 95Val Met Ser Val Lys Ala Tyr Thr Ile Gly Gly Lys Gly His Glu Thr 100 105 110Pro Lys Met Val Thr Ser Leu Gly Trp Asn Leu Ile Lys Phe Arg Ala 115 120 125Gly Val Asp Val Phe Ser Met Gln Pro His Arg Ala Asp Thr Ile Met 130 135 140Cys Asp Ile Gly Glu Ser Asn Pro Asp Ala Val Val Glu Gly Glu Arg145 150 155 160Thr Arg Lys Val Ile Leu Leu Met Glu Gln Trp Lys Asn Arg Asn Pro 165 170 175Thr Ala Thr Cys Val Phe Lys Val Leu Ala Pro Tyr Arg Pro Glu Val 180 185 190Ile Glu Ala Leu His Arg Phe Gln Leu Gln Trp Gly Gly Gly Leu Val 195 200 205Arg Thr Pro Phe Ser Arg Asn Ser Thr His Glu Met Tyr Tyr Ser Thr 210 215 220Ala Val Thr Gly Asn Ile Val Asn Ser Val Asn Ile Gln Ser Arg Lys225 230 235 240Leu Leu Ala Arg Phe Gly Asp Gln Arg Gly Pro Thr Arg Val Pro Glu 245 250 255Leu Asp Leu Gly Val Gly Thr Arg Cys Val Val Leu Ala Glu Asp Lys 260 265 270Val Lys Glu Lys Asp Val Gln Glu Arg Ile Ser Ala Leu Arg Glu Gln 275 280 285Tyr Gly Glu Thr Trp His Met Asp Arg Glu His Pro Tyr Arg Thr Trp 290 295 300Gln Tyr Trp Gly Ser Tyr Arg Thr Ala Pro Thr Gly Ser Ala Ala Ser305 310 315 320Leu Ile Asn Gly Val Val Lys Leu Leu Ser Trp Pro Trp Asn Ala Arg 325 330 335Glu Asp Val Val Arg Met Ala Met Thr Asp Thr Thr Ala Phe 340 345 35013350PRTFlavivirus sp.misc_featureLoupingIll 13Gly Gly Ser Asp Gly Asp Thr Leu Gly Asp Leu Trp Lys Arg Arg Leu1 5 10 15Asn Asn Cys Thr Lys Glu Glu Phe Phe Val Tyr Arg Arg Thr Gly Ile 20 25 30Leu Glu Thr Glu Arg Asp Lys Ala Arg Glu Leu Leu Arg Arg Gly Glu 35 40 45Thr Asn Met Gly Leu Ala Val Ser Arg Gly Thr Ala Lys Leu Ala Trp 50 55 60Leu Glu Glu Arg Gly Tyr Arg Thr Leu Lys Gly Glu Val Val Asp Leu65 70 75 80Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Ala Ala Ser Arg Pro Ala 85 90 95Val Met Ser Val Arg Ala Tyr Thr Ile Gly Gly Arg Gly His Glu Val 100 105 110Pro Lys Met Val Thr Ser Leu Gly Trp Asn Leu Ile Arg Phe Arg Ser 115 120 125Gly Met Asp Val Phe Ser Met Gln Pro His Arg Ala Asp Thr Ile Met 130 135 140Cys Asp Ile Gly Glu Ser Asn Pro Asp Ala Ala Val Glu Gly Glu Arg145 150 155 160Thr Arg Lys Val Ile Ser Leu Met Glu Gln Trp Lys Ile Arg Asn Pro 165 170 175Ala Ala Ala Cys Val Phe Lys Val Leu Ala Pro Tyr Arg Pro Glu Val 180 185 190Ile Glu Ala Leu His Arg Phe Gln Leu Gln Trp Gly Gly Gly Leu Val 195 200 205Arg Thr Pro Phe Ser Arg Asn Ser Thr His Glu Met Tyr Tyr Ser Thr 210 215 220Ala Val Thr Gly Asn Ile Val Asn Ser Val Asn Ile Gln Ser Arg Lys225 230 235 240Leu Leu Ala Arg Phe Gly Asp Gln Arg Gly Pro Thr Lys Val Pro Glu 245 250 255Ala Asp Leu Gly Val Gly Thr Arg Cys Val Val Leu Ala Glu Asp Lys 260 265 270Val Lys Glu Gln Asp Val Gln Glu Arg Ile Arg Ala Leu Arg Lys Gln 275 280 285Tyr Ser Glu Thr Trp His Met Asp Glu Glu His Pro Tyr Arg Thr Trp 290 295 300Gln Tyr Trp Gly Thr Ser Arg Thr Ala Pro Thr Gly Ser Ala Ala Ser305 310 315 320Leu Ile Asn Gly Val Val Lys Leu Leu Ser Trp Pro Trp Asn Ala Arg 325 330 335Glu Asp Val Val Arg Met Ala Met Thr Asp Thr Thr Ala Phe 340 345 35014352PRTFlavivirus sp.misc_featureModoc 14Arg Gly Ile Cys Ser Ser Ala Pro Thr Leu Gly Glu Ile Trp Lys Arg1 5 10 15Lys Leu Asn Gln Leu Asp Ala Lys Glu Phe Met Ala Tyr Arg Arg Arg 20 25 30Phe Val Val Glu Val Asp Arg Asn Glu Ala Arg Glu Ala Leu Ala Lys 35 40 45Gly Lys Thr Asn Thr Gly His Ala Val Ser Arg Gly Thr Ala Lys Leu 50 55 60Ala Trp Ile Asp Glu Arg Gly Gly Val Glu Leu Lys Gly Ser Val Val65 70 75 80Asp Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Ala Ala Ser Gln 85 90 95Pro Asn Val Arg Glu Val Lys Ala Tyr Thr Leu Gly Thr Ser Gly His 100 105 110Glu Lys Pro Arg Leu Val Glu Thr Phe Gly Trp Asn Leu Ile Thr Phe 115 120 125Lys Ser Lys Val Asp Val Arg Lys Met Glu Pro Phe Gln Ala Asp Thr 130 135 140Val Leu Cys Asp Ile Gly Glu Ser Asn Pro Thr Ala Ala Val Glu Ala145 150 155 160Ser Arg Thr Leu Thr Val Leu Asn Val Ile Ser Arg Trp Leu Glu Tyr 165 170 175Asn Gln Gly Cys Gly Phe Cys Val Lys Val Leu Asn Pro Tyr Ser Cys 180 185 190Asp Val Leu Glu Ala Leu Met Lys Met Gln Arg Arg Phe Gly Gly Gly 195 200 205Leu Ile Arg Val Pro Leu Ser Arg Asn Ser Thr His Glu Met Tyr Phe 210 215 220Val Ser Gly Ile Lys Asn Asn Ile Met Gly Asn Val Thr Ala Val Ser225 230 235 240Arg Gln Leu Leu Lys Arg Met Glu Glu Gln Gly Gly Glu Arg Val Val 245 250 255Pro Asp Tyr Lys Phe Ser Thr Gly Thr Arg Ser Asn Leu Thr Gln Lys 260 265 270Ile Glu Val Pro Glu Glu Glu Val Gln Met Arg Val Asp Lys Ile Lys 275 280 285Ala Glu Lys Ser Gly Thr Trp Cys Phe Asp Ser Asn His Pro Tyr Arg 290 295 300Thr Trp Asn

Tyr His Gly Ser Tyr Arg Val Arg Asp Val Gly Thr Arg305 310 315 320Ala Ser Ala Val Asn His Val Val Lys Leu Leu Ser Trp Pro Trp Gly 325 330 335Lys Met Glu Lys Val Leu Ala Met Ser Met Thr Asp Thr Thr Ala Phe 340 345 35015352PRTFlavivirus sp.misc_featureRioBravo 15Arg Gly Val Ser Ser Ser Tyr Ile Thr Tyr Gly Glu Gln Trp Lys Arg1 5 10 15Glu Leu Asn Lys Leu Asn Ala Gln Ala Phe Phe Leu Tyr Lys Ser Arg 20 25 30Leu Val His Glu Ile Asp Arg Ala Glu Ala Val Ser Asn Leu Ser Lys 35 40 45Gly Arg Thr Asn Thr Gly His Ala Val Ser Arg Gly Thr Ser Lys Leu 50 55 60Ala Trp Met His Glu Arg Gly Tyr Val Pro Leu Lys Gly Val Val Val65 70 75 80Asp Leu Gly Ser Gly Arg Gly Gly Trp Ser Tyr Tyr Ala Ala Ala Gln 85 90 95Glu Arg Val Arg Lys Val Asn Ala Tyr Thr Leu Ala Thr Thr Lys Gly 100 105 110His Glu Gln Pro Arg Leu Val Gln Ser Tyr Gly Trp Asn Leu Val Thr 115 120 125Phe Lys Lys Ala Asp Val Arg Thr Ile Glu Pro Tyr Pro Val Asp Thr 130 135 140Leu Leu Cys Asp Ile Gly Glu Ser Ser Ser Ser Ser Ser Ile Glu Ala145 150 155 160Lys Arg Thr Leu Gln Ile Met Asp Val Val Gly Lys Trp Leu Glu Ile 165 170 175Ser Pro Gly Ala Ser Phe Cys Ile Lys Ile Leu Cys Pro Tyr Asn Pro 180 185 190Glu Val Ile Glu His Leu Ser Arg Trp Gln His Gln Phe Gly Gly Gly 195 200 205Leu Val Arg Val Pro His Ser Arg Asn Ser Thr His Glu Met Tyr Phe 210 215 220Val Ser Gly Gly Gly Gly Asn Leu Met Ser Ser Ile Thr Ala Val Thr225 230 235 240Thr Gln Leu Met Arg Arg Phe Thr Leu Glu Ala Gly Pro Arg His Val 245 250 255Phe Asp Ile Asn Leu Gly Val Gly Thr Arg Ser Asn Leu Met Glu Lys 260 265 270Ser Glu Ala Asp Lys Ser Leu Ile Ala Asp Arg Ile Thr Ile Ile Gln 275 280 285Asn Glu Asn Lys Ala Ser Trp His Gln Asp Pro Asn Gln Pro Tyr Arg 290 295 300Thr Trp Thr Tyr His Gly Ser Tyr Ser Ile Arg Asp Val Gly Thr Ser305 310 315 320Ala Ser Ala Pro Asn His Val Val Lys Leu Leu Ala Trp Pro Trp Leu 325 330 335Lys Ile Glu Lys Val Val Leu Met Ala Met Thr Asp Thr Thr Ala Phe 340 345 35016629PRTFlavivirus sp.misc_featureDengue2 16Pro Asn Leu Asp Ile Ile Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu1 5 10 15His Glu Thr Ser Trp His Tyr Asp Gln Asp His Pro Tyr Lys Thr Trp 20 25 30Ala Tyr His Gly Ser Tyr Glu Thr Lys Gln Thr Gly Ser Ala Ser Ser 35 40 45Met Gly Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp Asp Val Val 50 55 60Pro Met Val Thr Gln Met Ala Met Thr Asp Thr Thr Pro Phe Gly Gln65 70 75 80Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg Thr Gln Glu Pro Lys 85 90 95Glu Gly Thr Lys Lys Leu Met Lys Ile Thr Ala Glu Trp Leu Trp Lys 100 105 110Glu Leu Gly Lys Lys Lys Thr Pro Arg Met Cys Thr Arg Glu Glu Phe 115 120 125Thr Arg Lys Val Arg Ser Asn Ala Ala Leu Gly Ala Ile Phe Thr Asp 130 135 140Glu Asn Lys Trp Lys Ser Ala Arg Glu Ala Val Glu Asp Ser Arg Phe145 150 155 160Trp Glu Leu Val Asp Lys Glu Arg Asn Leu His Leu Glu Gly Lys Cys 165 170 175Glu Thr Cys Val Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly 180 185 190Glu Phe Gly Lys Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu 195 200 205Gly Ala Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp 210 215 220His Trp Phe Ser Arg Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly225 230 235 240Leu His Lys Leu Gly Tyr Ile Leu Arg Asp Val Ser Lys Lys Glu Gly 245 250 255Gly Ala Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr 260 265 270Leu Glu Asp Leu Lys Asn Glu Glu Met Val Thr Asn His Met Glu Gly 275 280 285Glu His Lys Lys Leu Ala Glu Ala Ile Phe Lys Leu Thr Tyr Gln Asn 290 295 300Lys Val Val Arg Val Gln Arg Pro Thr Pro Arg Gly Thr Val Met Asp305 310 315 320Ile Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly Gln Val Gly Thr Tyr 325 330 335Gly Leu Asn Thr Phe Thr Asn Met Glu Ala Gln Leu Ile Arg Gln Met 340 345 350Glu Gly Glu Gly Val Phe Lys Ser Ile Gln His Leu Thr Val Thr Glu 355 360 365Glu Ile Ala Val Gln Asn Trp Leu Ala Arg Val Gly Arg Glu Arg Leu 370 375 380Ser Arg Met Ala Ile Ser Gly Asp Asp Cys Val Val Lys Pro Leu Asp385 390 395 400Asp Arg Phe Ala Ser Ala Leu Thr Ala Leu Asn Asp Met Gly Lys Val 405 410 415Arg Lys Asp Ile Gln Gln Trp Glu Pro Ser Arg Gly Trp Asn Asp Trp 420 425 430Thr Gln Val Pro Phe Cys Ser His His Phe His Glu Leu Ile Met Lys 435 440 445Asp Gly Arg Val Leu Val Val Pro Cys Arg Asn Gln Asp Glu Leu Ile 450 455 460Gly Arg Ala Arg Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr465 470 475 480Ala Cys Leu Gly Lys Ser Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe 485 490 495His Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala Val 500 505 510Pro Ser His Trp Val Pro Thr Ser Arg Thr Thr Trp Ser Ile His Ala 515 520 525Lys His Glu Trp Met Thr Thr Glu Asp Met Leu Thr Val Trp Asn Arg 530 535 540Val Trp Ile Gln Glu Asn Pro Trp Met Glu Asp Lys Thr Pro Val Glu545 550 555 560Ser Trp Glu Glu Ile Pro Tyr Leu Gly Lys Arg Glu Asp Gln Trp Cys 565 570 575Gly Ser Leu Ile Gly Leu Thr Ser Arg Ala Thr Trp Ala Lys Asn Ile 580 585 590Gln Thr Ala Ile Asn Gln Val Arg Ser Leu Ile Gly Asn Glu Glu Tyr 595 600 605Thr Asp Tyr Met Pro Ser Met Lys Arg Phe Arg Lys Glu Glu Glu Glu 610 615 620Ala Gly Val Leu Trp62517630PRTFlavivirus sp.misc_featureDengue3 17Pro Asn Met Asp Val Ile Gly Glu Arg Ile Lys Arg Ile Lys Glu Glu1 5 10 15His Ser Ser Thr Trp His Tyr Asp Asp Glu Asn Pro Tyr Lys Thr Trp 20 25 30Ala Tyr His Gly Ser Tyr Glu Val Lys Ala Thr Gly Ser Ala Ser Ser 35 40 45Met Ile Asn Gly Val Val Lys Leu Leu Thr Lys Pro Trp Asp Val Val 50 55 60Pro Met Val Thr Gln Met Ala Met Thr Asp Thr Thr Pro Phe Gly Gln65 70 75 80Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg Thr Pro Arg Pro Met 85 90 95Pro Gly Thr Arg Lys Val Met Glu Ile Thr Ala Glu Trp Leu Trp Arg 100 105 110Thr Leu Gly Arg Asn Lys Arg Pro Arg Leu Cys Thr Arg Glu Glu Phe 115 120 125Thr Lys Lys Val Arg Thr Asn Ala Ala Met Gly Ala Val Phe Thr Glu 130 135 140Glu Asn Gln Trp Asp Ser Ala Arg Ala Ala Val Glu Asp Glu Glu Phe145 150 155 160Trp Lys Leu Val Asp Arg Glu Arg Glu Leu His Lys Leu Gly Lys Cys 165 170 175Gly Ser Cys Val Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly 180 185 190Glu Phe Gly Lys Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu 195 200 205Gly Ala Arg Tyr Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp 210 215 220His Trp Phe Ser Arg Glu Asn Ser Tyr Ser Gly Val Glu Gly Glu Gly225 230 235 240Leu His Lys Leu Gly Tyr Ile Leu Arg Asp Ile Ser Lys Ile Pro Gly 245 250 255Gly Ala Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr 260 265 270Glu Asp Asp Leu His Asn Glu Glu Lys Ile Thr Gln Gln Met Asp Pro 275 280 285Glu His Arg Gln Leu Ala Asn Ala Ile Phe Lys Leu Thr Tyr Gln Asn 290 295 300Lys Val Val Lys Val Gln Arg Pro Thr Pro Lys Gly Thr Val Met Asp305 310 315 320Ile Ile Ser Arg Lys Asp Gln Arg Gly Ser Gly Gln Val Gly Thr Tyr 325 330 335Gly Leu Asn Thr Phe Thr Asn Met Glu Ala Gln Leu Ile Arg Gln Met 340 345 350Glu Gly Glu Gly Val Leu Ser Lys Ala Asp Leu Glu Asn Pro His Pro 355 360 365Leu Glu Lys Lys Ile Thr Gln Trp Leu Glu Thr Lys Gly Val Glu Arg 370 375 380Leu Lys Arg Met Ala Ile Ser Gly Asp Asp Cys Val Val Lys Pro Ile385 390 395 400Asp Asp Arg Phe Ala Asn Ala Leu Leu Ala Leu Asn Asp Met Gly Lys 405 410 415Val Arg Lys Asp Ile Pro Gln Trp Gln Pro Ser Lys Gly Trp His Asp 420 425 430Trp Gln Gln Val Pro Phe Cys Ser His His Phe His Glu Leu Ile Met 435 440 445Lys Asp Gly Arg Lys Leu Val Val Pro Cys Arg Pro Gln Asp Glu Leu 450 455 460Ile Gly Arg Ala Arg Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu465 470 475 480Thr Ala Cys Leu Gly Lys Ala Tyr Ala Gln Met Trp Thr Leu Met Tyr 485 490 495Phe His Arg Arg Asp Leu Arg Leu Ala Ser Asn Ala Ile Cys Ser Ala 500 505 510Val Pro Val His Trp Val Pro Thr Ser Arg Thr Thr Trp Ser Ile His 515 520 525Ala His His Gln Trp Met Thr Thr Glu Asp Met Leu Thr Val Trp Asn 530 535 540Arg Val Trp Ile Glu Asp Asn Pro Trp Met Glu Asp Lys Thr Pro Ile545 550 555 560Thr Thr Trp Glu Asp Val Pro Tyr Leu Gly Lys Arg Glu Asp Gln Trp 565 570 575Cys Gly Ser Leu Ile Gly Leu Thr Ser Arg Ala Thr Trp Ala Gln Asn 580 585 590Ile Leu Thr Ala Ile Gln Gln Val Arg Ser Leu Ile Gly Asn Glu Glu 595 600 605Phe Leu Asp Tyr Met Pro Ser Met Lys Arg Phe Arg Lys Glu Glu Glu 610 615 620Ser Glu Gly Ala Ile Trp625 63018629PRTFlavivirus sp.misc_featureDengue1 18Ala Asn Leu Asp Ile Ile Gly Gln Arg Ile Glu Asn Ile Lys Asn Glu1 5 10 15His Lys Ser Thr Trp His Tyr Asp Glu Asp Asn Pro Tyr Lys Thr Trp 20 25 30Ala Tyr His Gly Ser Tyr Glu Val Lys Pro Ser Gly Ser Ala Ser Ser 35 40 45Met Val Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp Asp Val Ile 50 55 60Pro Met Val Thr Gln Ile Ala Met Thr Asp Thr Thr Pro Phe Gly Gln65 70 75 80Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg Thr Pro Lys Ala Lys 85 90 95Arg Gly Thr Ala Gln Ile Met Glu Val Thr Ala Arg Trp Leu Trp Gly 100 105 110Phe Leu Ser Arg Asn Lys Lys Pro Arg Ile Cys Thr Arg Glu Glu Phe 115 120 125Thr Arg Lys Val Arg Ser Asn Ala Ala Ile Gly Ala Val Phe Val Asp 130 135 140Glu Asn Gln Trp Asn Ser Ala Lys Glu Ala Val Ala Asp Glu Arg Phe145 150 155 160Trp Asp Leu Val His Lys Glu Arg Glu Leu His Lys Gln Gly Lys Cys 165 170 175Ala Thr Cys Val Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly 180 185 190Glu Phe Gly Lys Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu 195 200 205Gly Ala Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Met Asn Glu Asp 210 215 220His Trp Phe Ser Arg Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly225 230 235 240Leu His Lys Leu Gly Tyr Ile Leu Arg Asp Ile Ser Lys Ile Pro Gly 245 250 255Gly Asn Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr 260 265 270Glu Asp Asp Leu Gln Asn Glu Ala Lys Ile Thr Asp Ile Met Glu Pro 275 280 285Glu His Ala Leu Leu Ala Thr Ser Ile Phe Lys Leu Thr Tyr Gln Asn 290 295 300Lys Val Val Arg Val Gln Arg Pro Ala Lys Asn Gly Thr Val Met Asp305 310 315 320Val Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly Gln Val Gly Thr Tyr 325 330 335Gly Leu Asn Thr Phe Thr Asn Met Glu Ala Gln Leu Ile Arg Gln Met 340 345 350Glu Ser Glu Gly Ile Phe Ser Pro Ser Glu Leu Glu Thr Pro Asn Leu 355 360 365Ala Glu Arg Val Leu Asp Trp Leu Glu Lys His Gly Val Glu Arg Leu 370 375 380Lys Arg Met Ala Ile Ser Gly Asp Asp Cys Val Val Lys Pro Ile Asp385 390 395 400Asp Arg Phe Ala Thr Ala Leu Thr Ala Leu Asn Asp Met Gly Lys Val 405 410 415Arg Lys Asp Ile Pro Gln Trp Glu Pro Ser Lys Gly Trp Asn Asp Trp 420 425 430Gln Gln Val Pro Phe Cys Ser His His Phe His Gln Leu Ile Met Lys 435 440 445Asp Gly Arg Glu Ile Val Val Pro Cys Arg Asn Gln Asp Glu Leu Val 450 455 460Gly Arg Ala Arg Val Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr465 470 475 480Ala Cys Leu Gly Lys Ser Tyr Ala Gln Met Trp Gln Leu Met Tyr Phe 485 490 495His Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala Val 500 505 510Pro Val Asp Trp Val Pro Thr Ser Arg Thr Thr Trp Ser Ile His Ala 515 520 525His His Gln Trp Met Thr Thr Glu Asp Met Leu Ser Val Trp Asn Arg 530 535 540Val Trp Ile Glu Glu Asn Pro Trp Met Glu Asp Lys Thr His Val Ser545 550 555 560Ser Trp Glu Asp Val Pro Tyr Leu Gly Lys Arg Glu Asp Gln Trp Cys 565 570 575Gly Ser Leu Ile Gly Leu Thr Ala Arg Ala Thr Trp Ala Thr Asn Ile 580 585 590Gln Val Ala Ile Asn Gln Val Arg Arg Leu Ile Gly Asn Glu Asn Tyr 595 600 605Leu Asp Tyr Met Thr Ser Met Lys Arg Phe Lys Asn Glu Ser Asp Pro 610 615 620Glu Gly Ala Leu Trp62519629PRTFlavivirus sp.misc_featureDengue4 19Pro Asp Met Thr Ile Ile Gly Arg Arg Leu Gln Arg Leu Gln Glu Glu1 5 10 15His Lys Glu Thr Trp His Tyr Asp Gln Glu Asn Pro Tyr Arg Thr Trp 20 25 30Ala Tyr His Gly Ser Tyr Glu Ala Pro Ser Thr Gly Ser Ala Ser Ser 35 40 45Met Val Asn Gly Val Val Lys Leu Leu Thr Lys Pro Trp Asp Val Ile 50 55 60Pro Met Val Thr Gln Leu Ala Met Thr Asp Thr Thr Pro Phe Gly Gln65 70 75 80Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg Thr Pro Gln Pro Lys 85 90 95Pro Gly Thr Arg Met Val Met Thr Thr Thr Ala Asn Trp Leu Trp Ala 100 105 110Leu Leu Gly Lys Lys Lys Asn Pro Arg Leu Cys Thr Arg Glu Glu Phe 115 120 125Ile Ser Lys Val Arg Ser Asn Ala Ala Ile Gly Ala Val Phe Gln Glu 130 135 140Glu Gln Gly Trp Thr Ser Ala Ser Glu Ala Val Asn Asp Ser Arg Phe145 150 155 160Trp Glu Leu Val Asp Lys Glu Arg Ala Leu His Gln Glu Gly Lys Cys 165

170 175Glu Ser Cys Val Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly 180 185 190Glu Phe Gly Arg Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu 195 200 205Gly Ala Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp 210 215 220His Trp Phe Gly Arg Glu Asn Ser Trp Ser Gly Val Glu Gly Glu Gly225 230 235 240Leu His Arg Leu Gly Tyr Ile Leu Glu Glu Ile Asp Lys Lys Asp Gly 245 250 255Asp Leu Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr 260 265 270Glu Asp Asp Leu Gln Asn Glu Glu Leu Ile Thr Glu Gln Met Ala Pro 275 280 285His His Lys Ile Leu Ala Lys Ala Ile Phe Lys Leu Thr Tyr Gln Asn 290 295 300Lys Val Val Lys Val Leu Arg Pro Thr Pro Arg Gly Ala Val Met Asp305 310 315 320Ile Ile Ser Arg Lys Asp Gln Arg Gly Ser Gly Gln Val Gly Thr Tyr 325 330 335Gly Leu Asn Thr Phe Thr Asn Met Glu Val Gln Leu Ile Arg Gln Met 340 345 350Glu Ala Glu Gly Val Ile Thr Gln Asp Asp Met Gln Asn Pro Lys Gly 355 360 365Leu Lys Glu Arg Val Glu Lys Trp Leu Lys Glu Cys Gly Val Asp Arg 370 375 380Leu Lys Arg Met Ala Ile Ser Gly Asp Asp Cys Val Val Lys Pro Leu385 390 395 400Asp Glu Arg Phe Gly Thr Ser Leu Leu Phe Leu Asn Asp Met Gly Lys 405 410 415Val Arg Lys Asp Ile Pro Gln Trp Glu Pro Ser Lys Gly Trp Lys Asn 420 425 430Trp Gln Glu Val Pro Phe Cys Ser His His Phe His Lys Ile Phe Met 435 440 445Lys Asp Gly Arg Ser Leu Val Val Pro Cys Arg Asn Gln Asp Glu Leu 450 455 460Ile Gly Arg Ala Arg Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu465 470 475 480Thr Ala Cys Leu Gly Lys Ala Tyr Ala Gln Met Trp Ser Leu Met Tyr 485 490 495Phe His Arg Arg Asp Leu Arg Leu Ala Ser Met Ala Ile Cys Ser Ala 500 505 510Val Pro Thr Glu Trp Phe Pro Thr Ser Arg Thr Thr Trp Ser Ile His 515 520 525Ala His His Gln Trp Met Thr Thr Glu Asp Met Leu Lys Val Trp Asn 530 535 540Arg Val Trp Ile Glu Asp Asn Pro Asn Met Thr Asp Lys Thr Pro Val545 550 555 560His Ser Trp Glu Asp Ile Pro Tyr Leu Gly Lys Arg Glu Asp Leu Trp 565 570 575Cys Gly Ser Leu Ile Gly Leu Ser Ser Arg Ala Thr Trp Ala Lys Asn 580 585 590Ile His Thr Ala Ile Thr Gln Val Arg Asn Leu Ile Gly Lys Glu Glu 595 600 605Tyr Val Asp Tyr Met Pro Val Met Lys Arg Tyr Ser Ala Pro Ser Glu 610 615 620Ser Glu Gly Val Leu62520632PRTFlavivirus sp.misc_featureWest Nile 20Ser Asp Thr Ser Lys Ile Asn Asn Arg Ile Glu Arg Leu Arg Arg Glu1 5 10 15Tyr Ser Ser Thr Trp His His Asp Glu Asn His Pro Tyr Arg Thr Trp 20 25 30Asn Tyr His Gly Ser Tyr Asp Val Lys Pro Thr Gly Ser Ala Ser Ser 35 40 45Leu Val Asn Gly Val Val Arg Leu Leu Ser Lys Pro Trp Asp Thr Ile 50 55 60Thr Asn Val Thr Thr Met Ala Met Thr Asp Thr Thr Pro Phe Gly Gln65 70 75 80Gln Arg Val Phe Lys Glu Lys Val Asp Thr Lys Ala Pro Glu Pro Pro 85 90 95Glu Gly Ala Lys Tyr Val Leu Asn Glu Thr Thr Asn Trp Leu Trp Ala 100 105 110Phe Leu Ala Arg Glu Lys Arg Pro Arg Met Cys Ser Arg Glu Glu Phe 115 120 125Ile Arg Lys Val Asn Ser Asn Ala Ala Leu Gly Ala Met Phe Glu Glu 130 135 140Gln Asn Gln Trp Arg Ser Ala Arg Glu Ala Val Glu Asp Pro Lys Phe145 150 155 160Trp Glu Met Val Asp Glu Glu Arg Glu Ala His Leu Arg Gly Glu Cys 165 170 175His Thr Cys Ile Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Pro Gly 180 185 190Glu Phe Gly Lys Ala Lys Gly Ser Arg Ala Ile Trp Phe Met Trp Leu 195 200 205Gly Ala Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp 210 215 220His Trp Leu Gly Arg Lys Asn Ser Gly Gly Gly Val Glu Gly Leu Gly225 230 235 240Leu Gln Lys Leu Gly Tyr Ile Leu Arg Glu Val Gly Thr Arg Pro Gly 245 250 255Gly Lys Ile Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr 260 265 270Arg Ala Asp Leu Glu Asn Glu Ala Lys Val Leu Glu Leu Leu Asp Gly 275 280 285Glu His Arg Arg Leu Ala Arg Ala Ile Ile Glu Leu Thr Tyr Arg His 290 295 300Lys Val Val Lys Val Met Arg Pro Ala Ala Asp Gly Arg Thr Val Met305 310 315 320Asp Val Ile Ser Arg Glu Asp Gln Arg Gly Ser Gly Gln Val Val Thr 325 330 335Tyr Ala Leu Asn Thr Phe Thr Asn Leu Ala Val Gln Leu Val Arg Met 340 345 350Met Glu Gly Glu Gly Val Ile Gly Pro Asp Asp Val Glu Lys Leu Thr 355 360 365Lys Gly Lys Gly Pro Lys Val Arg Thr Trp Leu Phe Glu Asn Gly Glu 370 375 380Glu Arg Leu Ser Arg Met Ala Val Ser Gly Asp Asp Cys Val Val Lys385 390 395 400Pro Leu Asp Asp Arg Phe Ala Thr Ser Leu His Phe Leu Asn Ala Met 405 410 415Ser Lys Val Arg Lys Asp Ile Gln Glu Trp Lys Pro Ser Thr Gly Trp 420 425 430Tyr Asp Trp Gln Gln Val Pro Phe Cys Ser Asn His Phe Thr Glu Leu 435 440 445Ile Met Lys Asp Gly Arg Thr Leu Val Val Pro Cys Arg Gly Gln Asp 450 455 460Glu Leu Val Gly Arg Ala Arg Ile Ser Pro Gly Ala Gly Trp Asn Val465 470 475 480Arg Asp Thr Ala Cys Leu Ala Lys Ser Tyr Ala Gln Met Trp Leu Leu 485 490 495Leu Tyr Phe His Arg Arg Asp Leu Arg Leu Met Ala Asn Ala Ile Cys 500 505 510Ser Ala Val Pro Val Asn Trp Val Pro Thr Gly Arg Thr Thr Trp Ser 515 520 525Ile His Ala Gly Gly Glu Trp Met Thr Thr Glu Asp Met Leu Glu Val 530 535 540Trp Asn Arg Val Trp Ile Glu Glu Asn Glu Trp Met Glu Asp Lys Thr545 550 555 560Pro Val Glu Lys Trp Ser Asp Val Pro Tyr Ser Gly Lys Arg Glu Asp 565 570 575Ile Trp Cys Gly Ser Leu Ile Gly Thr Arg Ala Arg Ala Thr Trp Ala 580 585 590Glu Asn Ile Gln Val Ala Ile Asn Gln Val Arg Ala Ile Ile Gly Asp 595 600 605Glu Lys Tyr Val Asp Tyr Met Ser Ser Leu Lys Arg Tyr Glu Asp Thr 610 615 620Thr Leu Val Glu Asp Thr Val Leu625 63021632PRTFlavivirus sp.misc_featureKunjin 21Ser Asp Thr Ser Lys Ile Lys Asn Arg Ile Glu Arg Leu Arg Arg Glu1 5 10 15Tyr Ser Ser Thr Trp His His Asp Glu Asn His Pro Tyr Arg Thr Trp 20 25 30Asn Tyr His Gly Ser Tyr Glu Val Lys Pro Thr Gly Ser Ala Ser Ser 35 40 45Leu Val Asn Gly Val Val Arg Leu Leu Ser Lys Pro Trp Asp Thr Ile 50 55 60Thr Asn Val Thr Thr Met Ala Met Thr Asp Thr Thr Pro Phe Gly Gln65 70 75 80Gln Arg Val Phe Lys Glu Lys Val Asp Thr Lys Ala Pro Glu Pro Pro 85 90 95Glu Gly Val Lys Tyr Val Leu Asn Glu Thr Thr Asn Trp Leu Trp Ala 100 105 110Phe Leu Ala Arg Glu Lys Arg Pro Arg Met Cys Ser Arg Glu Glu Phe 115 120 125Ile Arg Lys Val Asn Ser Asn Ala Ala Leu Gly Ala Met Phe Glu Glu 130 135 140Gln Asn Gln Trp Arg Ser Ala Arg Glu Ala Val Glu Asp Pro Lys Phe145 150 155 160Trp Glu Met Val Asp Glu Glu Arg Glu Ala His Leu Arg Gly Glu Cys 165 170 175His Thr Cys Ile Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Pro Gly 180 185 190Glu Phe Gly Lys Ala Lys Gly Ser Arg Ala Ile Trp Phe Met Trp Leu 195 200 205Gly Ala Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp 210 215 220His Trp Leu Gly Arg Lys Asn Ser Gly Gly Gly Val Glu Gly Leu Gly225 230 235 240Leu Gln Lys Leu Gly Tyr Ile Leu Arg Glu Val Gly Thr Arg Pro Gly 245 250 255Gly Arg Ile Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr 260 265 270Arg Ala Asp Leu Glu Asn Glu Ala Lys Val Leu Glu Leu Leu Asp Gly 275 280 285Glu His Arg Arg Leu Ala Arg Ala Ile Ile Glu Leu Thr Tyr Arg His 290 295 300Lys Val Val Lys Val Met Arg Pro Ala Ala Asp Gly Arg Thr Val Met305 310 315 320Asp Val Ile Ser Arg Glu Asp Gln Arg Gly Ser Gly Gln Val Val Thr 325 330 335Tyr Ala Leu Asn Thr Phe Thr Asn Leu Ala Val Gln Leu Val Arg Met 340 345 350Met Glu Gly Glu Gly Val Ile Gly Pro Asp Asp Val Glu Lys Leu Thr 355 360 365Lys Gly Lys Gly Pro Lys Val Arg Thr Trp Leu Ser Glu Asn Gly Glu 370 375 380Glu Arg Leu Ser Arg Met Ala Val Ser Gly Asp Asp Cys Val Val Lys385 390 395 400Pro Leu Asp Asp Arg Phe Ala Thr Ser Leu His Phe Leu Asn Ala Met 405 410 415Ser Lys Val Arg Lys Asp Ile Gln Glu Trp Lys Pro Ser Thr Gly Trp 420 425 430Tyr Asp Trp Gln Gln Val Pro Phe Cys Ser Asn His Phe Thr Glu Leu 435 440 445Ile Met Lys Asp Gly Arg Thr Leu Val Thr Pro Cys Arg Gly Gln Asp 450 455 460Glu Leu Val Gly Arg Ala Arg Ile Ser Pro Gly Ala Gly Trp Asn Val465 470 475 480Arg Asp Thr Ala Cys Leu Ala Lys Ser Tyr Ala Gln Met Trp Leu Leu 485 490 495Leu Tyr Phe His Arg Arg Asp Leu Arg Leu Met Ala Asn Ala Ile Cys 500 505 510Ser Ala Val Pro Val Asn Trp Val Pro Thr Gly Arg Thr Thr Trp Ser 515 520 525Ile His Ala Gly Gly Glu Trp Met Thr Thr Glu Asp Met Leu Glu Val 530 535 540Trp Asn Arg Val Trp Ile Glu Glu Asn Glu Trp Met Glu Asp Lys Thr545 550 555 560Pro Val Glu Lys Trp Ser Asp Val Pro Tyr Ser Gly Lys Arg Glu Asp 565 570 575Ile Trp Cys Gly Ser Leu Ile Gly Thr Arg Ala Arg Ala Thr Trp Ala 580 585 590Glu Asn Ile Gln Val Ala Ile Asn Gln Val Arg Ser Ile Ile Gly Asp 595 600 605Glu Lys Tyr Val Asp Tyr Met Ser Ser Leu Lys Arg Tyr Glu Asp Thr 610 615 620Thr Leu Val Glu Asp Thr Val Leu625 63022632PRTFlavivirus sp.misc_featureJapanese Encephalitis 22Ser Asn Gln Glu Lys Ile Lys Lys Arg Ile Gln Lys Leu Lys Glu Glu1 5 10 15Phe Ala Thr Thr Trp His Lys Asp Pro Glu His Pro Tyr Arg Thr Trp 20 25 30Thr Tyr His Gly Ser Tyr Glu Val Lys Ala Thr Gly Ser Ala Ser Ser 35 40 45Leu Val Asn Gly Val Val Lys Leu Met Ser Lys Pro Trp Asp Ala Ile 50 55 60Ala Asn Val Thr Thr Met Ala Met Thr Asp Thr Thr Pro Phe Gly Gln65 70 75 80Gln Arg Val Phe Lys Glu Lys Val Asp Thr Lys Ala Pro Glu Pro Pro 85 90 95Ala Gly Ala Lys Glu Val Leu Asn Glu Thr Thr Asn Trp Leu Trp Ala 100 105 110His Leu Ser Arg Glu Lys Arg Pro Arg Leu Cys Thr Lys Glu Glu Phe 115 120 125Ile Lys Lys Val Asn Ser Asn Ala Ala Leu Gly Ala Val Phe Ala Glu 130 135 140Gln Asn Gln Trp Ser Thr Ala Arg Glu Ala Val Asp Asp Pro Arg Phe145 150 155 160Trp Glu Met Val Asp Glu Glu Arg Glu Asn His Leu Arg Gly Glu Cys 165 170 175His Thr Cys Ile Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Pro Gly 180 185 190Glu Phe Gly Lys Ala Lys Gly Ser Arg Ala Ile Trp Phe Met Trp Leu 195 200 205Gly Ala Arg Tyr Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp 210 215 220His Trp Leu Ser Arg Glu Asn Ser Gly Gly Gly Val Glu Gly Ser Gly225 230 235 240Val Gln Lys Leu Gly Tyr Ile Leu Arg Asp Ile Ala Gly Lys Gln Gly 245 250 255Gly Lys Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr 260 265 270Arg Thr Asp Leu Glu Asn Glu Ala Lys Val Leu Glu Leu Leu Asp Gly 275 280 285Glu His Arg Met Leu Ala Arg Ala Ile Ile Glu Leu Thr Tyr Arg His 290 295 300Lys Val Val Lys Val Met Arg Pro Ala Ala Glu Gly Lys Thr Val Met305 310 315 320Asp Val Ile Ser Arg Glu Asp Gln Arg Gly Ser Gly Gln Val Val Thr 325 330 335Tyr Ala Leu Asn Thr Phe Thr Asn Ile Ala Val Gln Leu Val Arg Leu 340 345 350Met Glu Ala Glu Gly Val Ile Gly Pro Gln His Leu Glu Gln Leu Pro 355 360 365Arg Lys Asn Lys Ile Ala Val Arg Thr Trp Leu Phe Glu Asn Gly Glu 370 375 380Glu Arg Val Thr Arg Met Ala Ile Ser Gly Asp Asp Cys Val Val Lys385 390 395 400Pro Leu Asp Asp Arg Phe Ala Thr Ala Leu His Phe Leu Asn Ala Met 405 410 415Ser Lys Val Arg Lys Asp Ile Gln Glu Trp Lys Pro Ser His Gly Trp 420 425 430His Asp Trp Gln Gln Val Pro Phe Cys Ser Asn His Phe Gln Glu Ile 435 440 445Val Met Lys Asp Gly Arg Ser Ile Val Val Pro Cys Arg Gly Gln Asp 450 455 460Glu Leu Ile Gly Arg Ala Arg Ile Ser Pro Gly Ala Gly Trp Asn Val465 470 475 480Lys Asp Thr Ala Cys Leu Ala Lys Ala Tyr Ala Gln Met Trp Leu Leu 485 490 495Leu Tyr Phe His Arg Arg Asp Leu Arg Leu Met Ala Asn Ala Ile Cys 500 505 510Ser Ala Val Pro Val Asp Trp Val Pro Thr Gly Arg Thr Ser Trp Ser 515 520 525Ile His Ser Lys Gly Glu Trp Met Thr Thr Glu Asp Met Leu Gln Val 530 535 540Trp Asn Arg Val Trp Ile Glu Glu Asn Glu Trp Met Met Asp Lys Thr545 550 555 560Pro Ile Thr Ser Trp Thr Asp Val Pro Tyr Val Gly Lys Arg Glu Asp 565 570 575Ile Trp Cys Gly Ser Leu Ile Gly Thr Arg Ser Arg Ala Thr Trp Ala 580 585 590Glu Asn Ile Tyr Ala Ala Ile Asn Gln Val Arg Ala Val Ile Gly Lys 595 600 605Glu Asn Tyr Val Asp Tyr Met Thr Ser Leu Arg Arg Tyr Glu Asp Val 610 615 620Leu Ile Gln Glu Asp Arg Val Ile625 63023633PRTFlavivirus sp.misc_featureYellow Fever 23Leu Asp Lys Glu Ala Ile Glu Glu Arg Val Glu Arg Ile Lys Ser Glu1 5 10 15Tyr Met Thr Ser Trp Phe Tyr Asp Asn Asp Asn Pro Tyr Arg Thr Trp 20 25 30His Tyr Cys Gly Ser Tyr Val Thr Lys Thr Ser Gly Ser Ala Ala Ser 35 40 45Met Val Asn Gly Val Ile Lys Ile Leu Thr Tyr Pro Trp Asp Lys Ile 50 55 60Glu Glu Val Thr Arg Met Ala Met Thr Asp Thr Thr Pro Phe Gly Gln65 70 75 80Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg Ala Lys Asp Pro Pro 85 90 95Ala Gly Thr Arg Lys Ile Met Lys Val Val Asn Arg Trp Leu Phe Arg 100 105 110His

Leu Ala Arg Glu Lys Asn Pro Arg Leu Cys Thr Lys Glu Glu Phe 115 120 125Ile Ala Lys Val Arg Ser His Ala Ala Ile Gly Ala Tyr Leu Glu Glu 130 135 140Gln Glu Gln Trp Lys Thr Ala Asn Glu Ala Val Gln Asp Pro Lys Phe145 150 155 160Trp Glu Leu Val Asp Glu Glu Arg Lys Leu His Gln Gln Gly Arg Cys 165 170 175Arg Thr Cys Val Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Leu Ser 180 185 190Glu Phe Gly Lys Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu 195 200 205Gly Ala Arg Tyr Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp 210 215 220His Trp Ala Ser Arg Glu Asn Ser Gly Gly Gly Val Glu Gly Ile Gly225 230 235 240Leu Gln Tyr Leu Gly Tyr Val Ile Arg Asp Leu Ala Ala Met Asp Gly 245 250 255Gly Gly Phe Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr 260 265 270Glu Ala Asp Leu Asp Asp Glu Gln Glu Ile Leu Asn Tyr Met Ser Pro 275 280 285His His Lys Lys Leu Ala Gln Ala Val Met Glu Met Thr Tyr Lys Asn 290 295 300Lys Val Val Lys Val Leu Arg Pro Ala Pro Gly Gly Lys Ala Tyr Met305 310 315 320Asp Val Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly Gln Val Val Thr 325 330 335Tyr Ala Leu Asn Thr Ile Thr Asn Leu Lys Val Gln Leu Ile Arg Met 340 345 350Ala Glu Ala Glu Met Val Ile His His Gln His Val Gln Asp Cys Asp 355 360 365Glu Ser Val Leu Thr Arg Leu Glu Ala Trp Leu Thr Glu His Gly Cys 370 375 380Asn Arg Leu Arg Arg Met Ala Val Ser Gly Asp Asp Cys Val Val Arg385 390 395 400Pro Ile Asp Asp Arg Phe Gly Leu Ala Leu Ser His Leu Asn Ala Met 405 410 415Ser Lys Val Arg Lys Asp Ile Ser Glu Trp Gln Pro Ser Lys Gly Trp 420 425 430Asn Asp Trp Glu Asn Val Pro Phe Cys Ser His His Phe His Glu Leu 435 440 445Gln Leu Lys Asp Gly Arg Arg Ile Val Val Pro Cys Arg Glu Gln Asp 450 455 460Glu Leu Ile Gly Arg Gly Arg Val Ser Pro Gly Asn Gly Trp Met Ile465 470 475 480Lys Glu Thr Ala Cys Leu Ser Lys Ala Tyr Ala Asn Met Trp Ser Leu 485 490 495Met Tyr Phe His Lys Arg Asp Met Arg Leu Leu Ser Leu Ala Val Ser 500 505 510Ser Ala Val Pro Thr Ser Trp Val Pro Gln Gly Arg Thr Thr Trp Ser 515 520 525Ile His Gly Lys Gly Glu Trp Met Thr Thr Glu Asp Met Leu Glu Val 530 535 540Trp Asn Arg Val Trp Ile Thr Asn Asn Pro His Met Gln Asp Lys Thr545 550 555 560Met Val Lys Glu Trp Arg Asp Val Pro Tyr Leu Thr Lys Arg Gln Asp 565 570 575Lys Leu Cys Gly Ser Leu Ile Gly Met Thr Asn Arg Ala Thr Trp Ala 580 585 590Ser His Ile His Leu Val Ile His Arg Ile Arg Thr Leu Val Gly Gln 595 600 605Glu Lys Tyr Thr Asp Tyr Leu Thr Val Met Asp Arg Tyr Ser Val Asp 610 615 620Ala Asp Leu Gln Pro Gly Glu Leu Ile625 63024633PRTFlavivirus sp.misc_featureBanzi 24Leu Asp Leu Glu Lys Val Lys Ala Arg Ile Asn Arg Leu Lys Glu Glu1 5 10 15Gln Glu Ser Thr Trp Phe Val Asp Ser Asp His Pro Tyr Arg Thr Trp 20 25 30His Tyr His Gly Ser Tyr Val Ala Lys Gln Ser Gly Thr Ala Ala Ser 35 40 45Met Ile Asn Gly Val Val Lys Leu Leu Ser Gly Pro Trp Asp Arg Ile 50 55 60Glu Glu Val Thr Asn Met Ala Met Thr Asp Thr Thr Pro Phe Gly Gln65 70 75 80Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg Ala Pro Glu Pro Pro 85 90 95Gln Gly Thr Arg Glu Ile Met Lys Val Val Asn Gln Trp Leu Phe Asp 100 105 110Tyr Leu Gly Arg Thr Lys Gln Pro Arg Ile Cys Thr Lys Glu Glu Phe 115 120 125Ile Asn Lys Val Arg Ser His Ala Ala Leu Gly Gly Ile Leu Thr Glu 130 135 140Gln Glu Gly Trp Ser Ser Ala Ala Glu Ala Val Ala Asp Pro Arg Phe145 150 155 160Trp Ser Leu Val Asp Lys Glu Arg Gln Ala His Leu Glu Gly Arg Cys 165 170 175Glu Thr Cys Ile Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Pro Ser 180 185 190Glu Phe Gly Arg Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu 195 200 205Gly Ala Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp 210 215 220His Trp Leu Gly Arg Glu Asn Ser Lys Ala Gly Val Glu Gly Ile Gly225 230 235 240Leu Gln Tyr Leu Gly Tyr Val Val Glu Glu Val Ala Arg Lys Gly Asn 245 250 255Gly Leu Val Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr 260 265 270Glu Ala Asp Leu Glu Asp Glu Gln Tyr Ile Met Lys Arg Met Ser Ala 275 280 285Glu His Arg Gln Leu Ala Trp Ala Val Met Glu Leu Thr Tyr Arg Asn 290 295 300Lys Val Val Lys Val Pro Arg Pro Gly Pro Gly Gly Lys Ile Leu Met305 310 315 320Asp Val Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly Gln Val Val Thr 325 330 335Tyr Pro Leu Asn Thr Ala Thr Asn Met Lys Val Gln Leu Ile Arg Met 340 345 350Ala Glu Ala Glu Asn Val Ile Thr Arg Asn Asp Val Glu Lys Val Ser 355 360 365Leu Ile Thr Leu Lys Glu Leu Gln Leu Trp Leu Glu Val Asn Gly Val 370 375 380Asn Arg Leu Glu Arg Met Ala Val Ser Gly Asp Asp Cys Ile Val Ala385 390 395 400Pro Val Asp Glu Ser Phe Ala Gly Ala Leu His His Leu Asn Ala Met 405 410 415Ser Lys Thr Arg Lys Asp Ile Ser Glu Trp Glu Asn Ser Arg Gly Trp 420 425 430Thr Asp Trp Glu Ser Val Pro Phe Cys Ser His His Phe His Thr Leu 435 440 445Tyr Leu Lys Asp Gly Arg Thr Ile Ile Ala Pro Cys Arg Cys Gln Asp 450 455 460Glu Leu Ile Gly Arg Ala Arg Ile Ser Pro Gly Asn Gly Trp Met Ile465 470 475 480Lys Glu Thr Ala Gly Leu Ser Lys Ala Tyr Ala Gln Met Trp Thr Leu 485 490 495Met Tyr Phe His Arg Arg Asp Leu Arg Leu Met Ala Asn Ala Ile Cys 500 505 510Ser Ala Val Pro Ile Asp Trp Val Pro Thr Gly Arg Thr Thr Trp Ser 515 520 525Ile His Ala Thr Gly Glu Trp Met Ser Ser Asp Asp Met Leu Glu Val 530 535 540Trp Asn Lys Val Trp Ile Gln Asp Asn Pro His Val Lys Asp Lys Thr545 550 555 560Pro Ile Phe Ala Trp Arg Asp Val Pro Tyr Ile Gln Lys Gly Gln Asp 565 570 575Arg Ala Cys Gly Ser Leu Val Gly Thr Ser Leu Arg Ala Ser Trp Ala 580 585 590Glu Ser Ile Met Thr Ser Val His Arg Val Arg Met Leu Ile Gly Asn 595 600 605Glu Arg Tyr Val Asn Tyr Met Glu Ser Met Asp Arg Tyr Ala Thr Gln 610 615 620Arg Cys Ser Ala Tyr Gly Glu Leu Leu625 63025631PRTFlavivirus sp.misc_featureLangat 25Val Lys Glu Lys Asp Val Met Glu Arg Ile Gln Ala Leu Lys Asp Gln1 5 10 15Tyr Cys Asp Thr Trp His Glu Asp His Glu His Pro Tyr Arg Thr Trp 20 25 30Gln Tyr Trp Gly Ser Tyr Lys Thr Ala Ala Thr Gly Ser Ser Ala Ser 35 40 45Leu Leu Asn Gly Val Val Lys Leu Leu Ser Trp Pro Trp Asn Ala Arg 50 55 60Glu Asp Val Val Arg Met Ala Met Thr Asp Thr Thr Ala Phe Gly Gln65 70 75 80Gln Arg Val Phe Lys Asp Lys Val Asp Thr Lys Ala Gln Glu Pro Gln 85 90 95Pro Gly Thr Lys Ile Ile Met Arg Ala Val Asn Asp Trp Leu Leu Glu 100 105 110Arg Leu Val Lys Lys Ser Arg Pro Arg Met Cys Ser Arg Glu Glu Phe 115 120 125Ile Ala Lys Val Arg Ser Asn Ala Ala Leu Gly Ala Trp Ser Asp Glu 130 135 140Gln Asn Lys Trp Lys Ser Ala Arg Glu Ala Val Glu Asp Pro Glu Phe145 150 155 160Trp Ser Leu Val Glu Ala Glu Arg Glu Arg His Leu Gln Gly Arg Cys 165 170 175Ala His Cys Val Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly 180 185 190Glu Phe Gly Val Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu 195 200 205Gly Ser Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp 210 215 220His Trp Ala Ser Arg Ala Ser Ser Gly Ala Gly Val Glu Gly Ile Ser225 230 235 240Leu Asn Tyr Leu Gly Trp His Leu Lys Lys Leu Ala Ser Leu Ser Gly 245 250 255Gly Leu Phe Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr 260 265 270Asn Ala Asp Leu Asp Asp Glu Glu Gln Ile Leu Arg Tyr Met Asp Gly 275 280 285Asp His Lys Lys Leu Ala Ala Thr Val Leu Arg Lys Ala Tyr His Ala 290 295 300Lys Val Val Arg Val Ala Arg Pro Ser Arg Glu Gly Gly Cys Val Met305 310 315 320Asp Ile Ile Thr Arg Arg Asp Gln Arg Gly Ser Gly Gln Val Val Thr 325 330 335Tyr Ala Leu Asn Thr Ile Thr Asn Ile Lys Val Gln Leu Val Arg Met 340 345 350Met Glu Gly Glu Gly Val Ile Glu Val Ala Asp Ser His Asn Pro Arg 355 360 365Leu Leu Arg Val Glu Lys Trp Leu Glu Glu His Gly Glu Glu Arg Leu 370 375 380Ser Arg Met Leu Val Ser Gly Asp Asp Cys Val Val Arg Pro Val Asp385 390 395 400Asp Arg Phe Ser Lys Ala Leu Tyr Phe Leu Asn Asp Met Ala Lys Thr 405 410 415Arg Lys Asp Thr Gly Glu Trp Glu Pro Ser Thr Gly Phe Ala Ser Trp 420 425 430Glu Glu Val Pro Phe Cys Ser His His Phe His Glu Leu Val Met Lys 435 440 445Asp Gly Arg Ala Leu Val Val Pro Cys Arg Asp Gln Asp Glu Leu Val 450 455 460Gly Arg Ala Arg Val Ser Pro Gly Cys Gly Trp Ser Val Arg Glu Thr465 470 475 480Ala Cys Leu Ser Lys Ala Tyr Gly Gln Met Trp Leu Leu Ser Tyr Phe 485 490 495His Arg Arg Asp Leu Arg Thr Leu Gly Phe Ala Ile Cys Ser Ala Val 500 505 510Pro Val Asp Trp Val Pro Thr Gly Arg Thr Thr Trp Ser Ile His Ala 515 520 525Ser Gly Ala Trp Met Thr Thr Glu Asp Met Leu Glu Val Trp Asn Arg 530 535 540Val Trp Ile Tyr Asp Asn Pro Phe Met Glu Asp Lys Thr Arg Val Asp545 550 555 560Glu Trp Arg Asp Thr Pro Tyr Leu Pro Lys Ser Gln Asp Ile Leu Cys 565 570 575Ser Ser Leu Val Gly Arg Gly Glu Arg Ala Glu Trp Ala Lys Asn Ile 580 585 590Trp Gly Ala Val Glu Lys Val Arg Arg Met Ile Gly Pro Glu His Tyr 595 600 605Arg Asp Tyr Leu Ser Ser Met Asp Arg His Asp Leu His Trp Glu Leu 610 615 620Lys Leu Glu Ser Ser Ile Phe625 63026631PRTFlavivirus sp.misc_featurePowassan 26Val Lys Pro Arg Asp Val Ala Glu Arg Ile Gly Ala Leu Arg Glu Gln1 5 10 15Tyr Ser Glu Ser Trp His Glu Asp Lys Glu His Pro Tyr Arg Thr Trp 20 25 30Gln Tyr Trp Gly Ser Tyr Arg Thr Pro Ala Thr Gly Ser Ala Ala Ser 35 40 45Leu Ile Asn Gly Val Val Lys Leu Leu Ser Trp Pro Trp Asn Ala Arg 50 55 60Glu Asp Val Thr Arg Met Ala Met Thr Asp Thr Thr Ala Phe Gly Gln65 70 75 80Gln Arg Val Phe Lys Glu Lys Val Asp Thr Lys Ala Gln Glu Pro Gln 85 90 95Pro Gly Thr Arg Val Ile Met Arg Ala Val Ser Asp Trp Leu Leu Glu 100 105 110His Leu Ser Arg Arg Ala Lys Val Arg Met Cys Thr Lys Asp Glu Phe 115 120 125Ile Ala Lys Val Arg Ser Asn Ala Ala Leu Gly Ala Trp Ser Asp Glu 130 135 140Gln Asn Lys Trp Ser Ser Ala Lys Glu Ala Val Glu Asp Pro Glu Phe145 150 155 160Trp Lys Leu Val Asp Glu Glu Arg Ser Arg His Leu Lys Gly Gln Cys 165 170 175Arg His Cys Val Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly 180 185 190Glu Phe Gly Val Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu 195 200 205Gly Ser Arg Phe Leu Glu Phe Glu Val Leu Gly Phe Leu Asn Glu Glu 210 215 220His Trp Ala Ser Arg Glu Val Ser Gly Ala Gly Val Glu Gly Thr Ser225 230 235 240Leu Asn Tyr Leu Gly Trp Leu Leu Arg Glu Leu Gly Met Lys Asp Gly 245 250 255Gly Lys Leu Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr 260 265 270Asn Ala Asp Leu Glu Asp Glu Glu Gln Ile Leu Arg Tyr Met Glu Gly 275 280 285Glu His His Val Leu Ala Lys Thr Ile Leu Glu Lys Ala Tyr His Ala 290 295 300Lys Val Val Lys Val Ala Arg Pro Ser Pro Gln Gly Gly Cys Val Met305 310 315 320Asp Val Ile Thr Arg Arg Asp Gln Arg Gly Ser Gly Gln Val Val Thr 325 330 335Tyr Ala Leu Asn Thr Ile Thr Asn Met Lys Val Gln Leu Ile Arg Met 340 345 350Met Glu Gly Glu Gly Val Ile Gly Pro Ala Asp Ser Gln Asp Pro Arg 355 360 365Leu Lys Arg Val Glu Thr Trp Leu Lys Glu Tyr Gly Val Glu Arg Leu 370 375 380Gly Arg Met Leu Val Ser Gly Asp Asp Cys Val Val Lys Pro Ile Asp385 390 395 400Asp Arg Phe Gly Lys Ala Leu Tyr Phe Leu Asn Asp Met Asp Lys Val 405 410 415Arg Lys Asp Val Gly Glu Trp Glu Pro Ser Met Gly Leu Thr Glu Trp 420 425 430Glu Glu Val Pro Phe Cys Ser His His Phe His Glu Leu Val Met Lys 435 440 445Asp Gly Arg Ser Leu Ile Val Pro Cys Arg Asp Gln Asp Glu Leu Val 450 455 460Gly Arg Ala Arg Val Ser Pro Gly Cys Gly Trp Ser Val Arg Glu Thr465 470 475 480Ala Cys Leu Ser Lys Ala Tyr Gly His Met Trp Leu Leu Asn Tyr Phe 485 490 495His Arg Arg Asp Leu Arg Thr Leu Gly Phe Ala Ile Cys Ser Ala Val 500 505 510Pro Val Ser Trp Val Pro Met Gly Arg Thr Thr Trp Ser Ile His Ala 515 520 525Ser Gly Glu Trp Met Thr Thr Glu Asp Met Leu Arg Ile Trp Asn Lys 530 535 540Val Trp Ile Leu Asp Asn Pro His Met Glu Asp Lys Gln Thr Val Asp545 550 555 560Glu Trp Arg Asp Ile Pro Tyr Leu Pro Lys Thr Gln Asp Leu Val Cys 565 570 575Ser Ser Leu Val Gly Arg Lys Glu Arg Ala Glu Trp Ala Lys Asn Ile 580 585 590Trp Gly Ser Val Glu Lys Val Arg Lys Leu Ile Gly Pro Glu Asp Tyr 595 600 605Arg Asp Tyr Leu Ser Ser Met Asp Arg His Asp Leu His Trp Glu Leu 610 615 620Lys Leu Glu Ser Ser Ile Ile625 63027631PRTFlavivirus sp.misc_featureTick-borne Encephalitis 27Val Lys Glu Lys Asp Val Gln Glu Arg Ile Ser Ala Leu Arg Glu Gln1 5 10 15Tyr Gly Glu Thr Trp His Met Asp Arg Glu His Pro Tyr Arg Thr Trp 20 25 30Gln Tyr Trp Gly Ser Tyr Arg Thr Ala Pro Thr Gly Ser Ala Ala Ser 35 40 45Leu Ile Asn

Gly Val Val Lys Leu Leu Ser Trp Pro Trp Asn Ala Arg 50 55 60Glu Asp Val Val Arg Met Ala Met Thr Asp Thr Thr Ala Phe Gly Gln65 70 75 80Gln Arg Val Phe Lys Glu Lys Val Asp Thr Lys Ala Gln Glu Pro Gln 85 90 95Pro Gly Thr Lys Val Ile Met Arg Ala Val Asn Asp Trp Ile Leu Glu 100 105 110Arg Leu Ala Arg Lys Ser Lys Pro Arg Met Cys Ser Arg Glu Glu Phe 115 120 125Ile Ala Lys Val Lys Ser Asn Ala Ala Leu Gly Ala Trp Ser Asp Glu 130 135 140Gln Asn Arg Trp Ser Ser Ala Lys Glu Ala Val Glu Asp Pro Ala Phe145 150 155 160Trp Gln Leu Val Asp Glu Glu Arg Glu Arg His Leu Ala Gly Arg Cys 165 170 175Ala His Cys Val Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly 180 185 190Glu Phe Gly Val Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu 195 200 205Gly Ser Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp 210 215 220His Trp Ala Ser Arg Gly Ser Ser Gly Ser Gly Val Glu Gly Ile Ser225 230 235 240Leu Asn Tyr Leu Gly Trp His Leu Lys Gly Leu Ser Thr Leu Glu Gly 245 250 255Gly Leu Phe Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Lys Val Thr 260 265 270Asn Ala Asp Leu Glu Asp Glu Glu Gln Leu Leu Arg Tyr Met Glu Gly 275 280 285Glu His Lys Gln Leu Ala Ala Thr Ile Met Gln Lys Ala Tyr His Ala 290 295 300Lys Val Val Lys Val Ala Arg Pro Ser Arg Asp Gly Gly Cys Ile Met305 310 315 320Asp Val Ile Thr Arg Arg Asp Gln Arg Gly Ser Gly Gln Val Val Thr 325 330 335Tyr Ala Leu Asn Thr Leu Thr Asn Ile Lys Val Gln Leu Ile Arg Met 340 345 350Met Glu Gly Glu Gly Val Ile Glu Ala Ser Asp Ala His Asn Pro Arg 355 360 365Leu Leu Arg Val Glu Arg Trp Leu Arg Asp His Gly Glu Glu Arg Leu 370 375 380Gly Arg Met Leu Val Ser Gly Asp Asp Cys Val Val Arg Pro Val Asp385 390 395 400Asp Arg Phe Ser Gly Ala Leu Tyr Phe Leu Asn Asp Met Ala Lys Thr 405 410 415Arg Lys Asp Ile Gly Glu Trp Glu His Ser Val Gly Phe Ser Asn Trp 420 425 430Glu Glu Val Pro Phe Cys Ser His His Phe His Glu Leu Val Met Lys 435 440 445Asp Gly Arg Ala Leu Ile Val Pro Cys Arg Asp Gln Asp Glu Leu Val 450 455 460Gly Arg Ala Arg Val Ser Pro Gly Cys Gly Trp Ser Val Arg Glu Thr465 470 475 480Ala Cys Leu Ser Lys Ala Tyr Gly Gln Met Trp Leu Leu Ser Tyr Phe 485 490 495His Arg Arg Asp Leu Arg Thr Leu Gly Leu Ala Ile Cys Ser Ala Val 500 505 510Pro Val Asp Trp Val Pro Thr Gly Arg Thr Thr Trp Ser Ile His Ala 515 520 525Ser Gly Ala Trp Met Thr Thr Glu Asp Met Leu Asp Val Trp Asn Arg 530 535 540Val Trp Ile Leu Asp Asn Pro Phe Met His Ser Lys Glu Lys Ile Ala545 550 555 560Glu Trp Arg Asp Val Pro Tyr Leu Pro Lys Ser His Asp Met Leu Cys 565 570 575Ser Ser Leu Val Gly Arg Lys Glu Arg Ala Glu Trp Ala Lys Asn Ile 580 585 590Trp Gly Ala Val Glu Lys Val Arg Lys Met Ile Gly Gln Glu Lys Phe 595 600 605Lys Asp Tyr Leu Ser Cys Met Asp Arg His Asp Leu His Trp Glu Leu 610 615 620Lys Leu Glu Ser Ser Ile Ile625 63028631PRTFlavivirus sp.misc_featureLoupingIll 28Val Lys Glu Gln Asp Val Gln Glu Arg Ile Arg Ala Leu Arg Lys Gln1 5 10 15Tyr Ser Glu Thr Trp His Met Asp Glu Glu His Pro Tyr Arg Thr Trp 20 25 30Gln Tyr Trp Gly Thr Ser Arg Thr Ala Pro Thr Gly Ser Ala Ala Ser 35 40 45Leu Ile Asn Gly Val Val Lys Leu Leu Ser Trp Pro Trp Asn Ala Arg 50 55 60Glu Asp Val Val Arg Met Ala Met Thr Asp Thr Thr Ala Phe Gly Gln65 70 75 80Gln Arg Val Phe Lys Glu Lys Val Asp Thr Lys Ala Gln Glu Pro Gln 85 90 95Pro Gly Thr Arg Val Ile Thr Arg Ala Val Asn Asp Trp Ile Leu Glu 100 105 110Arg Leu Ala Gln Lys Ser Lys Pro Arg Met Cys Ser Arg Glu Glu Phe 115 120 125Ile Ala Lys Val Arg Ser Asn Ala Ala Leu Gly Ala Trp Ser Asp Glu 130 135 140Gln Asn Arg Trp Ala Ser Ala Arg Glu Ala Val Val Val Pro Ala Phe145 150 155 160Trp Ala Leu Val Asp Glu Val Arg Glu Arg His Leu Val Gly Trp Cys 165 170 175Ala His Cys Val Tyr Ile Met Met Gly Met Arg Glu Lys Lys Leu Gly 180 185 190Glu Phe Gly Val Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu 195 200 205Gly Ser Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Lys Asp 210 215 220His Trp Ala Ser Arg Glu Ser Ser Gly Gly Gly Val Glu Gly Ile Ser225 230 235 240Leu Asn Tyr Leu Gly Trp His Leu Lys Lys Leu Thr Thr Leu Asn Gly 245 250 255Gly Leu Phe Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Lys Gly Thr 260 265 270Asn Ser Asp Pro Glu Asp Glu Glu Gln Ile Leu Arg Tyr Met Glu Gly 275 280 285Glu His Lys Gln Leu Ala Thr Thr Ile Met Gln Lys Ala Tyr His Ala 290 295 300Lys Val Val Lys Val Ala Arg Pro Ser Arg Asp Gly Gly Cys Ile Met305 310 315 320Asp Val Ile Thr Arg Arg Asp Gln Arg Gly Ser Gly Gln Val Val Thr 325 330 335Tyr Ala Leu Asn Thr Leu Thr Asn Ile Lys Val Gln Ser Thr Arg Met 340 345 350Met Glu Gly Glu Gly Val Ile Glu Ala Glu Asp Ala His Asn Pro Arg 355 360 365Leu Leu Arg Val Glu Arg Trp Leu Lys Glu His Gly Glu Glu Arg Leu 370 375 380Gly Arg Met Leu Val Ser Gly Asp Asp Cys Val Val Arg Pro Ile Asp385 390 395 400Asp Arg Phe Gly Lys Ala Leu Tyr Phe Leu Asn Asp Met Ala Lys Thr 405 410 415Arg Lys Asp Met Gly Glu Trp Glu Pro Ser Ala Gly Phe Ser Ser Trp 420 425 430Glu Glu Val Pro Phe Cys Ser His His Phe His Glu Leu Val Met Lys 435 440 445Asp Gly Arg Thr Leu Val Val Pro Cys Arg Asp Gln Asp Glu Leu Val 450 455 460Gly Arg Ala Arg Val Ser Pro Gly Cys Gly Trp Ser Val Arg Glu Thr465 470 475 480Ala Cys Leu Ser Lys Ala Tyr Gly Gln Met Trp Leu Leu Ser Tyr Phe 485 490 495His Arg Arg Asp Leu Arg Thr Leu Gly Phe Ala Ile Ser Pro Ala Val 500 505 510Pro Val Asp Trp Val Pro Thr Gly Arg Thr Thr Trp Ser Ile His Ala 515 520 525Ser Gly Ala Trp Met Thr Thr Glu Asp Met Leu Asp Val Trp Asn Arg 530 535 540Val Trp Ile Leu Asp Asn Pro Phe Met Gln Asn Lys Glu Arg Ile Met545 550 555 560Glu Trp Arg Asp Val Pro Tyr Leu Pro Lys Thr Gln Asp Met Ile Cys 565 570 575Ser Ser Leu Val Gly Arg Lys Glu Arg Ala Glu Trp Ala Lys Asn Ile 580 585 590Trp Gly Ala Val Glu Lys Val Arg Lys Met Ile Gly Pro Glu Arg Phe 595 600 605Lys Asp Tyr Leu Ser Cys Met Asp Arg His Asp Leu His Trp Glu Leu 610 615 620Lys Leu Glu Ser Ser Ile Ile625 63029625PRTFlavivirus sp.misc_featureModoc 29Val Pro Glu Glu Glu Val Gln Met Arg Val Asp Lys Ile Lys Ala Glu1 5 10 15Lys Ser Gly Thr Trp Cys Phe Asp Ser Asn His Pro Tyr Arg Thr Trp 20 25 30Asn Tyr His Gly Ser Tyr Arg Val Arg Asp Val Gly Thr Arg Ala Ser 35 40 45Ala Val Asn His Val Val Lys Leu Leu Ser Trp Pro Trp Gly Lys Met 50 55 60Glu Lys Val Leu Ala Met Ser Met Thr Asp Thr Thr Ala Phe Gly Gln65 70 75 80Gln Arg Val Phe Lys Gln Lys Val Asp Thr Lys Ala Pro Glu Pro Asn 85 90 95Ile Gln Val Lys Lys Val Met Arg Lys Val Phe Lys Trp Leu Ile Glu 100 105 110Arg Ile Lys Thr Lys Gly Gly Lys Val Arg Thr Cys Thr Lys Glu Glu 115 120 125Phe Ile Gln Lys Val Arg Ser His Ala Ala Ile Gly Ala Trp Ser Ser 130 135 140Asp Met Glu Gly Trp Ser Ser Ala Val Glu Ala Val Asp Asp Pro Arg145 150 155 160Phe Trp Asn Met Val Gln Lys Glu Arg Asp Leu His Leu Gln Gly Lys 165 170 175Cys Glu Met Cys Val Tyr Asn Leu Met Gly Lys Ser Glu Lys Lys Pro 180 185 190Gly Asp Phe Gly Val Ala Lys Gly Ser Arg Thr Ile Trp Tyr Met Trp 195 200 205Leu Gly Ser Arg Phe Leu Glu Phe Glu Ser Phe Gly Phe Leu Asn Glu 210 215 220Glu His Trp Ala Ser Arg Glu Leu Ser Gly Gly Gly Val Glu Gly Ile225 230 235 240Pro Leu Asn Tyr Leu Gly Tyr His Leu Arg Glu Met Ala Gln Lys Pro 245 250 255Gly Val Leu Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr 260 265 270Met Ala Asp Leu Glu Asp Glu Gly Met Leu Leu Asp Met Met Ser Gly 275 280 285Glu His Lys Lys Leu Ala Ser Ala Leu Phe Ser Lys Ala Tyr Lys Val 290 295 300Lys Val Ala Leu Cys Pro Arg Pro Gly Pro Lys Gly Gly Thr Leu Met305 310 315 320Asp Val Ile Ser Arg Thr Asp Gln Arg Gly Ser Gly Gln Val Val Thr 325 330 335Tyr Ala Leu Asn Thr Leu Thr Asn Ile Lys Val Gln Leu Ile Arg Met 340 345 350Ala Glu Ala Glu Gly Val Leu Gly Ala Thr Phe Glu Asp Phe Gly Ile 355 360 365Asp Arg Trp Leu Gln Glu His Gly Glu Asp Arg Val Glu Arg Met Leu 370 375 380Val Ser Gly Asp Asp Cys Val Val Asn Ala Ile Asp Glu Arg Phe Gly385 390 395 400Ser Ser Leu Asn Trp Leu Asn Ala Met Glu Lys Val Arg Lys Asp Ile 405 410 415Asp Leu Trp Lys Pro Ser Pro Ser Phe Arg Asn Trp Glu Arg Val Glu 420 425 430Phe Cys Ser Asn His Phe His Glu Met Thr Met Lys Asp Gly Arg Val 435 440 445Ile Val Ala Pro Cys Arg Gly Gln Thr Glu Leu Ile Ala Arg Gly Thr 450 455 460Val Asn Gln Gly Gly Cys Val Gly Val Glu Ser Thr Gly Cys Leu Ala465 470 475 480Lys Ala Tyr Ala Gln Met Trp Leu Leu Leu Tyr Phe His Arg Arg Asp 485 490 495Leu Arg Thr Leu Ala Leu Ala Val Met Ser Ala Val Pro Ser Asn Trp 500 505 510Ile Pro Thr Gly Arg Thr Thr Trp Ser Leu Met Val Lys Gly Glu Trp 515 520 525Met Thr Asp Glu Asp Met Leu Ala Val Trp Asn Arg Val Trp Ile Glu 530 535 540Asp Asn Pro Phe Met Glu Asp Lys Arg Glu Val Glu Arg Trp Ser Glu545 550 555 560Val Pro Tyr Leu Pro Arg Asn Gln Asp Lys Ser Cys Gly Ser Leu Ile 565 570 575Gly Thr Thr Ala Arg Ala Glu Trp Ala Lys Leu Leu Pro Gly Ala Val 580 585 590Glu Lys Val Arg Asn Ile Phe Gly Lys Gln Arg Phe Arg Asn Tyr Leu 595 600 605Arg Asn Met Gly Arg Tyr Glu Ser Gln Glu Glu Ala Pro Phe Ser Met 610 615 620Tyr62530624PRTFlavivirus sp.misc_featureRio Bravo 30Ala Asp Lys Ser Leu Ile Ala Asp Arg Ile Thr Ile Ile Gln Asn Glu1 5 10 15Asn Lys Ala Ser Trp His Gln Asp Pro Asn Gln Pro Tyr Arg Thr Trp 20 25 30Thr Tyr His Gly Ser Tyr Ser Ile Arg Asp Val Gly Thr Ser Ala Ser 35 40 45Ala Pro Asn His Val Val Lys Leu Leu Ala Trp Pro Trp Leu Lys Ile 50 55 60Glu Lys Val Val Leu Met Ala Met Thr Asp Thr Thr Ala Phe Gly Gln65 70 75 80Gln Arg Val Phe Lys Glu Lys Val Asp Thr Lys Ala Pro Glu Pro Pro 85 90 95Lys Glu Val Lys Lys Val Met Arg Leu Val Phe Arg Trp Leu Leu Asn 100 105 110His Ile Lys Ser Lys Gly Ala Val Val Arg Arg Cys Thr Lys Glu Glu 115 120 125Phe Ile Asn Lys Val Asn Ser Asn Ala Ser Ile Gly Ala Tyr Leu Arg 130 135 140Glu Met Gly Glu Trp Ser Ser Ala Lys Glu Ala Val Ser Asp Pro Lys145 150 155 160Phe Trp Asn Met Val Asp Lys Glu Arg Gln Leu His Leu Lys Gly Lys 165 170 175Cys His Asn Cys Val Tyr Asn Leu Met Gly Lys Arg Glu Lys Lys Pro 180 185 190Gly Glu Phe Gly Val Ala Lys Gly Ser Arg Thr Ile Trp Tyr Met Trp 195 200 205Leu Gly Ser Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu 210 215 220Glu Arg Trp Ala Ser Arg Asp Ile Ser Gly Gly Gly Val Glu Gly Ile225 230 235 240Gly Ile Asn Tyr Leu Gly Tyr His Leu Glu Lys Met Ala Arg Lys Val 245 250 255Gly Val Leu Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Val Thr 260 265 270Gln Ala Asp Leu Glu Asp Glu Arg Glu Leu Leu His Phe Met Glu Gly 275 280 285Glu His Lys Arg Leu Ala Glu Ala Ile Phe Lys Leu Thr Tyr Glu Asn 290 295 300Lys Val Ala Leu Cys Pro Arg Pro Gly Ser Lys Gly Gly Thr Val Met305 310 315 320Asp Val Ile Ser Arg Lys Asp Gln Arg Gly Ser Gly Gln Val Val Thr 325 330 335Tyr Ala Leu Asn Thr Leu Thr Asn Ile Lys Val Gln Leu Val Arg Met 340 345 350Ala Glu Ser Glu Gly Ile Leu Thr Pro Glu Leu Glu Asp Leu Gly Ile 355 360 365Glu Gln Trp Leu Lys Gln Asn Gly Glu Asp Arg Leu Ser Arg Leu Leu 370 375 380Val Ser Gly Asp Asp Cys Val Val Asn Ala Leu Asp Glu Arg Phe Gly385 390 395 400Lys Ala Leu Thr Trp Leu Asn Thr Met Glu Lys Thr Arg Lys Asp Ile 405 410 415Glu Ala Trp Lys Pro Ser Arg Ala Tyr Arg Thr Trp Gln Glu Val Glu 420 425 430Phe Cys Ser His His Phe His Glu Leu Phe Leu Arg Asp Gly Arg Lys 435 440 445Leu Ile Val Pro Cys Arg Asp Gln His Glu Leu Val Gly Arg Ser Met 450 455 460Val Ser Gln Gly Gly Ser Ser Gly Val Ala Gly Thr Ala Cys Leu Ala465 470 475 480Lys Ala Tyr Ala Gln Met Trp Leu Met Ser Tyr Phe His Arg Arg Asp 485 490 495Leu Arg Thr Leu Gly Phe Ala Ile Met Ser Ser Val Pro Lys Asp Trp 500 505 510Phe Pro Thr Gly Arg Thr Thr Trp Ser Val His Ala Asn His Glu Trp 515 520 525Leu Ser Asn Glu Asn Ile Leu Glu Ile Trp Asn Arg Val Trp Ile Glu 530 535 540Glu Asn Pro Phe Met Glu Asp Lys Thr Thr Val Ala Glu Trp Lys Asn545 550 555 560Ile Pro Tyr Leu Gln Arg Asn Gln Asp Leu Ser Cys Ser Ser Leu Ile 565 570 575Asp Asn Pro Thr Arg Ala Gln Trp Ala Lys Leu Leu Lys Gly Ala Val 580 585 590Met Lys Val Arg Glu Met Ile Gly Arg Glu His Tyr Ser Asp Tyr Leu 595 600 605Ser Asn Met Gly Arg Tyr Gln Glu Gly Thr Glu Glu Phe His Met Trp 610 615 620

Claims

1-20. (canceled)

21. An isolated and purified polypeptide from a flavivirus, wherein the full-length amino acid sequence of said polypeptide has at least 53% of identity with the SEQ ID NO: 19.

22. The isolated and purified polypeptide of claim 21, which is from Dengue 4 virus.

23. The isolated and purified polypeptide of claim 21, which has polymerase activity.

24. An isolated and purified polypeptide comprising the polymerase domain of the C-terminus of the NS5 polypeptide of a flavivirus, wherein said polypeptide has polymerase activity and wherein the full-length amino acid sequence of said polypeptide has at least 53% of identity with the SEQ ID NO: 19.

25. The isolated and purified polypeptide of claim 24, wherein the flavivirus is a Dengue 4 virus.

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