Attenuated dengue virus

Abstract

To provide attenuated flavivirus, in particular attenuated dengue virus and vaccines.SOLUTION: The invention provides modified flavivirus, e.g., modified dengue virus type 1, 2, 3, and 4, a combination thereof, or tetravalent combinations thereof. The modification in the various aspects of the invention results in the reduced virus protein expression as compared with the parent type virus, and the reduction in the expression results from recoding of one or more regions of the virus. For example, a prM or envelope (E) region may be recoded. In various aspects, one or more regions are recoded by reducing the codon pair bias or codon usage bias of the protein-encoding sequence. These modified flaviviruses are used as vaccine compositions to provide protective immune responses.SELECTED DRAWING: Figure 8B

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 866,477, filed June 25, 2019, the entirety of which is incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present invention provides highly attenuated flaviviruses, and in particular attenuated dengue viruses and vaccines, which provide cross-protection against heterologous viruses in addition to protective immunity from challenge with viruses of the same series. [Background technology]

[0003] 2. Background of the Invention Dengue virus (DENV) is a single-stranded, positive-sense RNA virus that belongs to the Flaviviridae family in the same genus (Flavivirus) that includes Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV). According to the World Health Organization (WHO), approximately 2.5 billion people, nearly half of the world's population, are currently at risk of DENV, and it is estimated that there may be 50 million cases of DENV infection worldwide each year. DENV is phylogenetically closely related to ZIKV and shares a common mosquito vector, Aedes aegypti, with both ZIKV and Chikungunya virus (an alphavirus). There is a special concern regarding the risk of DENV infection for travelers, in addition to concerns about the introduction and spread of the disease to places like the southern United States. The DENV vector, Aedes aegypti, is found throughout the southern United States. The Asian Tiger Mosquito (Aedes albopictus) is also capable of transmitting DENV. If DENV were to enter A. albopictus populations in the United States, it would have a greatly expanded range across the Midwest and Northeast.

[0004] Flavivirus vaccine development is complicated by the phenomenon of antibody-dependent enhancement (ADE). ADE occurs when a previous infection with one flavivirus increases the severity of disease when an individual is reinfected with a different serotype. During ADE, antibodies against the first virus bind to the second virus but do not neutralize it, instead increasing its infectivity. DENV is common in many countries, so any vaccine strategy should consider its impact on populations with established dengue immunity. It is worth noting that people with underlying DENV immunity may experience increased adverse events from a live DENV vaccine, as DENV immunity may enhance the infectivity of the DENV vaccine strain, leading to increased adverse events. These are plausible scenarios that need to be considered when developing a DENV vaccine, whether live, inactivated, or antigen / VLP / backbone carrier. Due to the high possibility of enhancing flavivirus infection due to ADE, all vaccine development strategies need to consider how any given DENV vaccine may interact with pre-existing antibodies to flaviviruses or subsequent flavivirus infections.

[0005] Dengue viruses comprised four serotypes (DENV1-4) of mosquito-borne flaviviruses of the Flaviviridae family. Dengue viruses are enveloped viruses with icosahedral virions containing C (core), M (membrane), and E (envelope) glycoproteins with diameters of 40-65 nanometers. The dengue virus genome is a single positive-stranded RNA molecule of 10,000-11,000 bases in length that encodes structural (C, prM, E) and nonstructural (NS1, NS2, NS3, NS4, and NS5) proteins. Dengue viruses transcribe and replicate their genome in the cytoplasm, where the genome is translated into a single polypeptide that is cleaved and processed by both host and viral proteins. Dengue viruses are emerging pathogens of international concern in tropical regions, and each serotype is a genetically and antigenically distinct virus.

[0006] Thus, there remains a need in the art to develop vaccines to prevent or reduce infectious diseases. Summary of the Invention

[0007] It is described herein that recoded dengue viruses, created by modifying the E region with multiple synonymous nucleotide mutations, are highly effective in providing protective immunity against lethal wild-type challenge and cross-protection against different lineages. Moreover, the viruses have an exceptional safety profile.

[0008] Thus, various embodiments of the present invention provide attenuated dengue viruses in which the expression of viral proteins is reduced through codon pair deoptimization of the E coding region. In certain embodiments, E is the only viral protein coding region targeted. In certain embodiments, when another dengue virus protein coding region other than E is deoptimized, the reduction is smaller compared to the reduction of E. According to the present invention, the reduction in the expression of the viral proteins of the present invention is achieved by changes in the protein coding sequence, for example, by reducing the codon pair bias of the protein coding sequence, substituting rare codons, modifying the G+C content, modifying the CG and / or TA (or UA) dinucleotide content, or a combination. Reduced expression can also be achieved by modifications to the regulatory sequences of the protein.

[0009] In certain embodiments, reducing codon pair bias may include identifying codon pairs in parent protein coding sequences that have a codon pair score that can be reduced, and reducing the codon pair bias by replacing the codon pair with a codon pair with a lower codon pair score. In other embodiments, reducing codon pair bias includes rearranging the codons of the parent protein coding sequence. In certain embodiments, the E protein coding sequence has a codon pair bias of less than -0.1, or less than -0.2, or less than -0.3, or less than -0.4. The codon pair bias of the protein coding sequence (i.e., open reading frame) is calculated as described in Coleman et al., 2000 and herein.

[0010] In an embodiment of the invention, expression of the E protein coding sequence is reduced by replacing one or more codons with a synonymous codon that is less frequent in the host.

[0011] In an embodiment of the invention, the E protein coding sequence DENV serotype is present in a background from a different strain of the same DENV serotype or from a homologous strain.

[0012] The present invention also provides a dengue vaccine composition for inducing a protective immune response in a subject, comprising a virus having reduced viral translation while maintaining at least 90% antigenic identity with the wt virus. In some embodiments, viral translation is reduced while maintaining at least 95, 96, 97, 98 or 99% antigenic identity with the wt virus. In some embodiments, viral translation is reduced while maintaining 100% antigenic identity with the wt virus.

[0013] The present invention also provides a method of inducing a protective immune response in a subject, comprising administering to the subject a prophylactically or therapeutically effective dose of a vaccine composition comprising an attenuated dengue virus, wherein the expression of viral proteins is reduced by at least 10%. In various embodiments, the expression of viral proteins is reduced by at least 15%, at least 20%, or at least 25%. In an embodiment of the present invention, an effective immune response is induced against a dengue virus of the same series as the attenuated virus of the vaccine. In another embodiment, an effective immune response is induced against a heterologous dengue virus.

[0014] The present invention also provides a method for making attenuated dengue virus genome, comprising: (a) obtaining genomic nucleotides; (b) recoding the envelope-encoding nucleotide sequence to reduce expression; and recoding the nonstructural protein 3-encoding nucleotide sequence to reduce expression; and substituting the recoded nucleotide sequence into the dengue virus genome to make attenuated dengue virus genome.In certain embodiments, only the E region is targeted.In some embodiments, the expression of other viral protein coding regions is also reduced.

[0015] The present invention also provides a method for constructing template dengue virus DNA sequences for transcription of infectious viral RNA genomes by T7 polymerase using overlapping PCR. All dengue genomes with a homogenous backbone were divided into three fragments starting from the 5' end (fragment 1: nt 1-3596; fragment 2: nt 3030-6959, and fragment 3: nt 6851-end) and chemically / biochemically synthesized. Instead of constructing infectious cDNA clones, a long overlap extension PCR strategy was used to obtain full-length dengue genomes (or syn-wt and min dengue genomes simultaneously). To construct heterologous dengue genomes in the DENV2 16681 backbone, a first 2.5Kb fragment was synthesized, containing the DENV2 16681 5'UTR, C domains and prM / M, E (wild-type or deoptimized) domains from individual DENV serotypes and a small proportion of the DENV2 16681 NS1 domain. An 8Kb DENV2 16681 backbone containing all NS domains and 3'UTR was obtained from infectious clones encoding wild-type or deoptimized DENV2 16681. The 2.5Kb and 8Kb fragments were fused using asymmetric fusion PCR.

[0016] Various embodiments of the present invention provide modified dengue viruses comprising a recoded prM protein, a recoded envelope (E) protein, or both, wherein the recoded prM protein has reduced codon pair bias compared to its parent prM protein coding sequence, or has at least five codons replaced with less frequently used synonymous codons, or has an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence, and the recoded E protein has reduced codon pair bias compared to its parent E protein coding sequence, or has at least five codons replaced with less frequently used synonymous codons, or has an increased number of CpG or UpA dinucleotides compared to its parent E protein coding sequence.

[0017] In various embodiments, expression of the prM protein or the E protein, or both, can be reduced compared to its parent dengue virus.

[0018] In various embodiments, the recoded prM protein has a reduced codon pair bias compared to its parent prM protein coding sequence. In various embodiments, the recoded prM protein may have at least five codons replaced with less frequently used synonymous codons. In various embodiments, the recoded prM protein may have an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence. In various embodiments, the recoded E protein may have a reduced codon pair bias compared to its parent E protein coding sequence. In various embodiments, the recoded E protein may have at least five codons replaced with less frequently used synonymous codons. In various embodiments, the recoded E protein may have an increased number of CpG or UpA dinucleotides compared to its parent E protein coding sequence. In various embodiments, each of the recoded prM or E protein coding sequences may have a codon pair bias of less than -0.05. In various embodiments, the codon pair bias of each of the recoded prM or E protein coding sequences may be reduced by at least 0.05.

[0019] In various embodiments, the modified dengue virus may be selected from type 1, type 2, type 3, type 4, or a combination thereof. In various embodiments, the modified dengue virus is a modified tetravalent dengue virus.

[0020] Various aspects of the present invention provide a dengue vaccine composition for inducing a protective immune response in a subject comprising a modified dengue virus as described herein above and a pharma- ceutically acceptable excipient or carrier.Various aspects of the present invention provide a method of inducing an immune response in a subject comprising administering to the subject an effective dose of a composition comprising a modified dengue virus as described herein above and a pharma- ceutically acceptable excipient or carrier.

[0021] In various embodiments, the immune response can be a protective immune response and a prophylactically or therapeutically effective dose of the claimed vaccine composition can be administered. In various embodiments, the immune response can be cross-protective against a heterologous dengue virus.

[0022] In various embodiments, the methods may further comprise administering to the subject at least one adjuvant.

[0023] Various aspects of the invention relate to a method of eliciting an immune response in a subject in need thereof, comprising administering to a subject a prime dose of (i) an attenuated dengue virus produced by methods other than codon pair deoptimization or codon deoptimization or an increase in CpG or UpA dinucleotides, or (ii) a modified dengue virus comprising a recoded prM protein, a recoded envelope (E) protein, or both, wherein the recoded prM protein has reduced codon pair bias compared to its parent prM protein coding sequence, or has at least five codons replaced with less frequently used synonymous codons, or has an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence; and administering to a subject in need thereof a modified dengue virus, wherein the recoded E protein has reduced codon pair bias compared to its parent E protein coding sequence, or has at least five codons replaced with less frequent synonymous codons, or has an increased number of CpG or UpA dinucleotides compared to its parent E protein coding sequence; and administering one or more boost doses of (i) an attenuated dengue virus made by a method other than codon pair deoptimization or codon deoptimization, or an increase in CpG or UpA dinucleotides, or (ii) the modified dengue virus, wherein at least the prime dose or the one or more boost doses is a modified dengue virus.

[0024] In various embodiments, the first of the one or more boost doses may be administered about two weeks after the prime dose.

[0025] In various embodiments, expression of the prM protein or the E protein, or both, can be reduced compared to its parent dengue virus.

[0026] In various embodiments, the recoded prM protein may have a reduced codon pair bias compared to its parent prM protein coding sequence. In various embodiments, the recoded prM protein may have at least five codons replaced with less frequently used synonymous codons. In various embodiments, the recoded prM protein may have an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence. In various embodiments, the recoded E protein may have a reduced codon pair bias compared to its parent E protein coding sequence. In various embodiments, the recoded E protein may have at least five codons replaced with less frequently used synonymous codons. In various embodiments, the recoded E protein may have an increased number of CpG or UpA dinucleotides compared to its parent E protein coding sequence. In various embodiments, each of the recoded prM or E protein coding sequences has a codon pair bias of less than -0.05. In various embodiments, the codon pair bias of each of the recoded prM or E protein coding sequences is reduced by at least 0.05.

[0027] In various embodiments, the modified dengue virus is selected from type 1, type 2, type 3, type 4, or a combination thereof. In various embodiments, the modified dengue virus is a modified tetravalent dengue virus.

[0028] Various aspects of the present invention provide a method of making a modified dengue virus genome, comprising the steps of obtaining a nucleotide sequence encoding an envelope protein of a dengue virus; recoding the envelope-coding nucleotide sequence to reduce protein expression, and substituting a nucleic acid having the recoded envelope-coding nucleotide sequence into a parent dengue virus genome to make a modified dengue virus genome, wherein expression of the recoded envelope-coding nucleotide sequence is reduced compared to the parent virus. [The present invention 1001] A modified dengue virus comprising: a recoded prM protein, a recoded envelope (E) protein, or both; the recoded prM protein has a reduced codon pair bias compared to its parent prM protein coding sequence, or have at least five codons replaced with less frequently used synonymous codons, or has an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence; and the recoded E protein has a reduced codon pair bias compared to its parent E protein coding sequence, or has at least five codons replaced with less frequent synonymous codons; or having an increased number of CpG or UpA dinucleotides compared to its parent E protein coding sequence, Modified dengue virus. [The present invention 1002] 1001. The modified dengue virus of the present invention, wherein expression of said prM protein or E protein or both is reduced compared to its parent dengue virus. [The present invention 1003] 1001. The modified dengue virus of the present invention, wherein said recoded prM protein has reduced codon pair bias compared to its parent prM protein coding sequence. [The present invention 1004] 1001. The modified dengue virus of the present invention, wherein said recoded prM protein has at least five codons replaced with less frequently used synonymous codons. [The present invention 1005] 1001. The modified dengue virus of the present invention, wherein said recoded prM protein has an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence. [The present invention 1006] 1001. The modified dengue virus of the present invention, wherein said recoded E protein has reduced codon pair bias compared to its parent E protein coding sequence. [The present invention 1007] 1001. The modified dengue virus of the present invention, wherein said recoded E protein has at least five codons replaced with less frequently used synonymous codons. [The present invention 1008] 1001. The modified dengue virus of the present invention, wherein said recoded E protein has an increased number of CpG or UpA dinucleotides compared to its parent E protein coding sequence. [The present invention 1009] 1001. The modified dengue virus of the present invention, wherein each of said recoded prM or E protein coding sequences has a codon pair bias of less than -0.05. [The present invention 1010] 1001. The modified dengue virus of the present invention, wherein the codon pair bias of each of the recoded prM or E protein coding sequences is reduced by at least 0.05. [The present invention 1011] The modified dengue virus of the present invention 1001, selected from types 1, 2, 3, 4 or a combination thereof. [The present invention 1012] 1001. A modified dengue virus of the present invention, which is a modified tetravalent dengue virus. [The present invention 1013] A modified dengue virus according to any one of claims 1001 to 1012 of the present invention, A pharma- ceutically acceptable excipient or carrier; 23. A dengue vaccine composition for inducing a protective immune response in a subject, comprising: [The present invention 1014] 1. A method of eliciting an immune response in a subject, comprising: A step of administering to the subject an effective dose of a composition comprising any one of the modified dengue viruses of the present inventions 1001 to 1012. A method comprising: [The present invention 1015] The method of claim 1014, wherein said immune response is a protective immune response and a prophylactically or therapeutically effective dose of the vaccine composition of the invention is administered. [The present invention 1016] The method of claim 1014, further comprising administering to said subject at least one adjuvant. [The present invention 1017] The method of claim 1014, wherein said immune response is cross-protective against a heterologous dengue virus. [The present invention 1018] 1. A method for inducing an immune response in a subject in need thereof, comprising the steps of: Prime dose of (i) an attenuated dengue virus produced by a method other than codon pair deoptimization or codon deoptimization or the increase in CpG or UpA dinucleotides; or (ii) a modified dengue virus comprising a recoded prM protein, a recoded envelope (E) protein, or both, the recoded prM protein has a reduced codon pair bias compared to its parent prM protein coding sequence, or has at least five codons replaced with less frequently used synonymous codons; or has an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence; and the recoded E protein has a reduced codon pair bias compared to its parent E protein coding sequence, or has at least five codons replaced with less frequent synonymous codons, or having an increased number of CpG or UpA dinucleotides compared to its parent E protein coding sequence, The modified dengue virus administering one or more boost doses, (i) an attenuated dengue virus produced by a method other than codon pair deoptimization or codon deoptimization or the increase in CpG or UpA dinucleotides; or (ii) The modified dengue virus to said subject in need thereof. Including, The method, wherein at least said prime dose or said one or more boost doses is said modified dengue virus. [The present invention 1019] The method of claim 1018, wherein a first of said one or more boost doses is administered about 2 weeks after said prime dose. [The present invention 1020] The method of claim 1018, wherein expression of said prM protein or E protein or both is reduced compared to its parent dengue virus. [The present invention 1021] The method of any one of claims 10 to 18, wherein said recoded prM protein has a reduced codon pair bias compared to its parent prM protein coding sequence. [The present invention 1022] The method of any one of claims 10 to 19, wherein said recoded prM protein has at least five codons replaced with less frequently used synonymous codons. [The present invention 1023] The method of claim 1018, wherein said recoded prM protein has an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence. [The present invention 1024] The method of any one of claims 10 to 18, wherein said recoded E protein has reduced codon pair bias compared to its parent E protein coding sequence. [The present invention 1025] The method of any one of claims 10 to 19, wherein said recoded E protein has at least five codons replaced with less frequently used synonymous codons. [The present invention 1026] The method of any one of claims 10 to 18, wherein said recoded E protein has an increased number of CpG or UpA dinucleotides compared to its parent E protein coding sequence. [The present invention 1027] The method of any one of claims 10 to 18, wherein each of said recoded prM or E protein coding sequences has a codon pair bias of less than -0.05. [The present invention 1028] The method of any one of claims 10 to 15, wherein the codon pair bias of each of the recoded prM or E protein coding sequences is reduced by at least 0.05. [The present invention 1029] The method of claim 1018, wherein said modified dengue virus is selected from type 1, type 2, type 3, type 4, or a combination thereof. [The present invention 1030] The method of any one of claims 1 to 8, wherein said modified dengue virus is a modified tetravalent dengue virus. [The present invention 1031] 1. A method for making a modified dengue virus genome, comprising: obtaining a nucleotide sequence encoding an envelope protein of the dengue virus; recoding said envelope-encoding nucleotide sequence to reduce protein expression; and substituting a nucleic acid having said recoded envelope-encoding nucleotide sequence into a parent dengue virus genome to create a modified dengue virus genome. wherein expression of the recoded envelope-encoding nucleotide sequence is reduced compared to the parent virus. [Brief description of the drawings]

[0029] [Figure 1A]Figure 1 shows the rapid construction of SAVE-deoptimized live attenuated dengue vaccine candidates using growth in Vero cells under animal component-free conditions. Codon pair bias of dengue prM / E genes and their SAVE-deoptimized counterparts with respect to the human ORFeome. Codon pair bias (CPB) is expressed as the average codon pair score of a given gene open reading frame (ORF). Positive and negative CPB values ​​represent the predominance of statistically over- or under-represented codon pairs, respectively, in the ORF. Red circles indicate the CPBs of each of the 14,795 human ORFs, which represent the majority of known annotated human genes (ORFeome) at the time of our analysis. The CPB of the wild-type E gene falls within the normal range for genes in human host cells. After SAVE-mediated codon pair "deoptimization," the resulting deoptimized prM / E gene segments are predominantly encoded by underrepresented human codon pairs, as evidenced by their extremely negative CPBs, and are dramatically different from any human genes. B. cDNA genomes of wild-type and synthetically "deoptimized" chimeric dengue vaccine variants. SAVE-deoptimized synthetic E was de novo synthesized and individually subcloned into the WT DENV genome using overlapping PCR to obtain eight independent cDNA genomes, each containing a synthetically "deoptimized" fragment. We constructed infectious cDNA genomes of wt DENV1-4 and then recovered fully infectious replicative viruses for deoptimized DENV vaccine candidates via RNA transfection into Vero (all DENV WT, EW / MIN, and E-MIN viruses) or BHK cells. [Figure 1B] See legend to Figure 1A. [Diagram 2]A diagram of the subcloning strategy to reduce attenuation or increase immunogenicity by reducing the length of the deoptimized sequence in the E coding region is shown. The second generation dengue vaccine candidate exploits the flexibility of the SAVE platform to reduce the deoptimized region from full length (E-min) to approximately half the length (WE-Min) while keeping the amino acid sequence 100% identical. [Diagram 3] Growth of heterologous backbone dengue vaccine candidates in Vero cells under animal component-free conditions. DENV1-4 E-Min was used to infect Vero cells under animal component-free conditions at an MOI of 0.01 and supernatants were titrated daily in a multi-step growth curve over the course of 10 days post-infection. DENV2, DENV3, and DENV4 candidates reached titers of 1-2x105 FFU / ml, while the titer of DENV1 E-Min was reduced by approximately 1 log10 compared to the others. [Figure 4] Figures 4A-4C show multi-step growth curves performed in Vero cells for the synthetic DENV1 WT virus in the full-length DENV1 backbone (Figure 4A) and the live attenuated vaccine candidates DENV1 EW / MIN (Figure 4B) and E-MIN (Figure 4C) derived from DENV1 WT. The synthetic DENV1 WT grows well in Vero cells at 33°C and 37°C. Typical for DENV, a transient reduction in virus yield was observed at 39°C, but titers recovered to 37°C levels by 7 dpi. DENV1 EW / MIN, however, reached usable titers about 10-fold lower than DENV1 WT at both 33°C and 37°C. DENV1 E-MIN was highly attenuated in Vero cells, only reaching detectable titers at 33°C from days 10-14 post-infection. [Diagram 5]Multi-step growth curves performed in Vero cells for the synthetic DENV2 WT virus (in the full-length DENV2 backbone) and the live attenuated vaccine candidates DENV2 EW / MIN and E-MIN derived from DENV2 WT are shown. Both DENV2 EW / MIN and E-MIN were attenuated in vitro, with a 1-2 log10 FFU / ml reduction for EW / MIN and, more significantly, a 2-3 log10 FFU / ml reduction for DENV2 E-MIN (Figure 3). Although growth kinetics were delayed, with regular medium changes every 1-2 days, virus titers reached >106 FFU / ml for DENV2 EW / MIN. Importantly, attenuation correlated with the degree of deoptimization in the E region. Temperature sensitivity (difference in titers at 37°C and 39°C) was similar for DENV2-WT and DENV2 EW / MIN through 4 days post-infection, but there were significant differences on days 5, 6, and 7. No temperature sensitivity was observed for DENV2 E-MIN, as there was no replication at any temperature through 6 DPI, however, from days 7 to 14, DENV2 E-MIN began to produce detectable virus (but only at 33°C and 37°C). [Figure 6] Multi-step growth curves performed in Vero cells for the synthetic DENV3 WT virus (in the full-length DENV3 backbone) and the live attenuated vaccine candidates DENV3 EW / MIN and E-MIN derived from DENV3 WT are shown. DENV3 EW / MIN was clearly more temperature sensitive than WT at 39°C, with significantly greater differences observable at days 3, 4, 5, and 7 post-infection. Since DENV3 E-MIN had minimal levels of replication in Vero cells at 39°C, the difference in virus yield from 37°C to 39°C was as large as 107 FFU / ml. However, when grown at permissive temperatures, DENV3 E-MIN reached high titers promising for cGMP manufacturing. [Figure 7]Multi-step growth curves performed in Vero cells are shown for the synthetic DENV4 WT virus (in the full-length DENV4 backbone) and the live attenuated vaccine candidates DENV4 EW / MIN and E-MIN derived from DENV4 WT. Both DENV4 WT and EW / MIN grew to high titers in Vero cells, with peak DENV4 EW / MIN titers being approximately 10-fold lower but still high (approximately 107 FFU / ml) at 33°C and 37°C. Both viruses were partially restricted at 39°C, but not to the extent seen with DENV1-3. A small but significant (approximately 5-fold) change in titer was observed from 37°C to 39°C compared to WT at day 5. DENV4 E-MIN was attenuated in Vero cells, with peak titers 100-fold lower than WT. Replication of DENV4 E-MIN at 33°C and 37°C was similar, but DENV4 E-MIN was not viable at 39°C and no virus was detectable from days 1 to 14 postinfection. [Figure 8A] Figures 8A-8B show the evaluation of attenuation, immunogenicity, and efficacy of DENV2 vaccine candidates in AG129 mice. DENV-2 E-min, based on the DENV2 16681 backbone, was tested in an immunogenicity / dose escalation study in AG129 mice lacking interferon alpha or gamma receptors. Animals were immunized with DENV-2 E-min or DENV-2 16681 at two different concentrations. The resulting neutralizing antibody titers were determined and the protective efficacy conferred against a mouse-adapted lethal strain of DENV2, D2S10, was evaluated. [Figure 8B] See legend to Figure 8A. [Figure 9]Figure 1 shows the constructed D2-E-min and D2-1 / 2-E-min with E sequences from DENV-2 / NI / BID-V533 / 2005 in a heterologous DENV2 16681 backbone to improve the clinical relevance of our DENV2 candidates. We tested D2-1 / 2-E-min with an E region consisting of half the wt DENV-2 / NI / BID-V533 / 2005 sequence and half the CPD to improve the immunogenicity of our viruses. AG129 and BALB / c mice (n=5) were vaccinated with 106 or 104 FFU of D2-E-min, D2-1 / 2-E-min, or D2-WTE (DENV2 16681 backbone with WT DENV-2 / NI / BID-V533 / 2005 E region). No mortality or morbidity was observed with DENV2 D2-E-min, and the D2-1 / 2-E-min group showed at least 100-fold attenuation. The immunogenicity of our candidate was improved by reducing the degree of CPD, and the PRNT50 values ​​of serum from D2-1 / 2-E-min were higher compared to D2-E-Min vaccinated AG129 mice. Additionally, high neutralizing antibody titers were maintained with D2-1 / 2-E-min vaccination, and no significant difference was observed between the 106 (GM=3447) and 104 (GM=2867) FFU vaccinated groups. In immune-competent BALB / c mice, both D2-E-min and D2-1 / 2-E-min produced levels of neutralizing antibodies comparable to wild type. [Figure 10A]10A-10B show AG129 mice used to test the immunogenicity and protective efficacy of DENV-3 vaccine candidate DENV D2 / D3-E-min, which contains a DENV-2 strain 16681 backbone with a codon-deoptimized prME cassette from DENV-3 / VE / BID-V2268 / 2008. This study was designed to evaluate the immunogenicity and efficacy of this candidate vaccine against challenge with pathogenic DENV3 CO360 / 94. The first immunization was well tolerated by all animals, with no significant associated weight loss and no animals developed clinical signs. Following lethal challenge with 1.2×107 PFU DENV-3 CO360 / 94, animals in the medium control group developed a rapidly progressive infection that was fatal in 8 / 9 animals, with a mean day of death (MDD) of 4.0±0.8 days for animals that died. Immunization with the higher inoculum of DENV D2 / D3-E-min provided significant protection, with no mortality or morbidity (as measured by >10% weight loss) in animals in the group. [Figure 10B] See legend to Figure 10A. [Figure 11A]Figures 11A-11B show AG129 mice used to test the immunogenicity and protective efficacy of DENV-4 vaccine candidates DENV D2 / D4-E-min and D2 / D4-1 / 2-E-min, which contain a DENV-2 strain 16681 backbone with a codon-deoptimized prME cassette from DENV4. AG 129 mice were vaccinated with 106 D2 / D4-E-min (N=12), 106 D2 / D4-1 / 2-E-min (N=12), 106 D2 / D4-WTE, or media as a control (N=9). This study was designed to evaluate the immunogenicity and efficacy of this candidate vaccine against challenge with virulent DENV4 703 / 4. The first immunization with all strains was well tolerated by animals in all groups with no sustained weight loss over the 5-day observation period, except for one mouse in the D2 / D4-WTE group that had to be euthanized before the second immunization and an additional animal death after the second immunization. No animals in any of the other vaccine groups experienced progressive weight loss. Serum was collected on day 35 to test for neutralizing antibodies before challenge and from all surviving animals on day 30 after challenge to test for final titers by FRNT50. Using a 96-well plate method with Vero cells, each serum sample was tested in duplicate using serum dilutions from 1:64 to 1:32784 incubated with 50 FFU of DENV4 WT virus. Each vaccine candidate was immunogenic at 35 DPI, with vaccination with D2 / D4-E-min (GMT 332.9) and D2 / D4-1 / 2-E-min (GMT 738.4) lower than vaccination with D2 / D4-WTE (GMT 1640). At 2 days post-challenge (DPC), viremia was detected in 4 / 12 mice vaccinated with D2 / D4-WTE. In all mice immunized with the other two viruses, viremia levels were below the detection limit of the assay (500 genome equivalents / ml). DENV-4 703 / 4 challenge produced a rapidly progressive infection that was uniformly lethal in the medium control group.Additionally, three animals immunized with D2 / D4-E-min experienced fatal infections, while the other animals in this group remained healthy for the duration of the study. Importantly, there was no mortality or evidence of morbidity as determined by weight loss in any of the D2 / D4-1 / 2-E-min immunized animals following DENV-4 703 / 4 challenge. [Figure 11B] See legend to Figure 11A. [Figure 12] We present vaccine candidates of DENV2-4 in addition to DENV1-4 WT that were tested for immunogenicity in IFNα receptor knockout mice. All WT viruses were highly immunogenic in these mice, with neutralizing antibody titers >1024. DENV2-EW / MIN was equally immunogenic to DENV2 WT virus at both doses of 106 and 104 FFU. The immunogenicity of DENV3 virus was attenuated with decreasing doses and increasing deoptimization, but DENV3 EW / MIN was still highly immunogenic at a dose of 104 FFU (FRNT50 of approximately 1024). We noted a significant improvement in the immunogenicity of DENV4 EW / MIN and WT compared to the heterologous DENV4 WT in the DENV2 16681 backbone, which was poorly immunogenic in mice. [Figure 13]Immunogenicity and efficacy of quadrivalent vaccination in AG129 mice against lethal challenge with DENV3 CO360 / 94 are shown. AG129 mice were vaccinated on days 0 and 21 with 106 IFU of monovalent D2 / D3-E-min vaccine (N=16; 10 males and 6 females), vaccinated with quadrivalent vaccines containing 106 IFU of each of the four monovalent DENV E-min viruses (N=16; 10 males and 6 females), or mock vaccinated with media (N=13; 8 males and 5 females). Serum was collected prior to challenge for measurement of neutralizing antibodies by FRNT50. All of the mice immunized with the monovalent vaccine developed detectable neutralizing antibody titers (range 20-160). In mice immunized with the quadrivalent vaccine formulation, titers were generally lower (range <20-80). On day 35, all remaining mice were challenged with 107 IFU of DENV3 CO360 / 94 delivered by the intraperitoneal route. As expected, animals in the medium control group developed a rapidly progressive infection that was fatal in 8 / 9 (89%) animals, with a mean day of death (MDD) of 4.1 ± 0.4 days in animals that died. Immunization with the monovalent DENV D2 / D3-E-min or quadrivalent vaccines provided significant protection, with no mortality or morbidity (as measured by >10% weight loss) in animals in either group. [Figure 14]Attenuation of the homologous backbone virus - vaccine strain vs. wt focus size - was demonstrated, with the vaccine virus spreading smaller in vitro compared to the wt. Individual focus areas (mm2) were calculated for Vero cells infected with DENV1-4 WT, EW / MIN, or E-MIN and incubated for 3 days at temperatures of 33°C, 37°C, or 39°C. We completed the analysis of focus-forming unit (FFU) size for each virus using ImageJ software (NIH, v1.52a). Each image of the FFA plate was converted to a binary 8-bit image using ImageJ (an example is presented in Figure 2), and the scale for measuring the FFU area was set to the diameter of a well in a 24-well plate (15.5 mm). The area of ​​each FFU was calculated by ImageJ using the "analyze particles" function, and the results were compared by Sidak's multiple comparison test (GraphPad Prism 8.1.2). [Figure 15] In vivo immunogenicity data for the tetravalent homologous backbone vaccines are shown. The tetravalent and monovalent homologous backbone dengue vaccine candidates were immunogenic following vaccination with 10 or 10 FFU delivered by subcutaneous route on days 0 and 21 in IFNα receptor knockout mice. Neutralizing antibodies were tested using a focus reduction neutralization 50% test on days 0, 21, and 35 post-vaccination against each strain of DENV1-4 wt. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description All references referred to in this specification are incorporated by reference in their entirety as if fully set forth. Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. Indeed, the present invention is not limited to the methods and materials described in any way. For the purposes of the present invention, the following terms are defined below.

[0031] As used herein, the term "about", when used in conjunction with a referenced numerical representation, means the referenced numerical representation plus or minus up to 5% of the referenced numerical representation, unless otherwise specifically provided herein. For example, the expression "about 50%" covers a range of 45% to 55%. In various embodiments, the term "about", when used in conjunction with a referenced numerical representation, and when specifically provided in the claims, can mean the referenced numerical representation plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the referenced numerical representation.

[0032] Codon pair bias (CPB) is expressed as the average codon pair score for a given gene open reading frame (ORF).

[0033] "Subject" refers to any animal or artificially modified animal. Animals include, but are not limited to, humans, non-human primates, cows, horses, sheep, pigs, dogs, cats, rabbits, ferrets, rodents, such as mice, rats and guinea pigs, and birds. Artificially modified animals include, but are not limited to, SCID mice with human immune systems. In a preferred embodiment, the subject is a human. Bird embodiments are domesticated poultry species, including, but are not limited to, chickens, turkeys, ducks, and geese.

[0034] "Virus host" refers to any animal or artificially modified animal, or insect that the virus can infect. Animals include, but are not limited to, humans, non-human primates, cattle, horses, sheep, pigs, dogs, cats, rabbits, ferrets, rodents, such as mice, rats and guinea pigs, and birds. Artificially modified animals include, but are not limited to, SCID mice with a human immune system. In a specific embodiment, the virus host is a human. Bird embodiments are domesticated poultry species, including, but are not limited to, chickens, turkeys, ducks, and geese. Insects include, but are not limited to, mosquitoes.

[0035] "Prophylactically effective dose" is any amount of vaccine that, when administered to a subject susceptible to viral infection or virus-related disorder, induces an immune response in the subject that protects the subject from being infected with the virus or suffering from the disorder.To "protect" a subject means to reduce the possibility of the subject being infected with the virus or to reduce the possibility of the subject starting the disorder by at least 2 times, preferably at least 10 times, 25 times, 50 times, or 100 times.For example, if a subject has a 1% chance of being infected with a virus, then a 2-fold reduction in the possibility of the subject being infected with the virus results in the subject having a 0.5% chance of being infected with the virus.

[0036] As used herein, "therapeutically effective dose" is any amount of vaccine that, when administered to a subject suffering from a disease for which the vaccine is effective, induces an immune response in the subject, causing the subject to experience a reduction, remission or regression of the disease and / or its symptoms.In a preferred embodiment, the recurrence of the disease and / or its symptoms is prevented.In another preferred embodiment, the subject is cured of the disease and / or its symptoms.

[0037] The present invention relates to the creation of attenuated dengue viruses and attenuated dengue viruses that can be used to protect against viral infection and disease. A fundamental premise in vaccination is the sufficient delivery of protective antigens to the vaccine recipient, assuming that very high doses ("peptides or virus-like particles") or doses corresponding to live viral infections of these traditionally predominant antigenic polypeptides ("ChimeriVax") are sufficient for full vaccine efficacy alone. Apart from those expectations, the present invention benefits from the opposite approach. The present invention provides attenuated dengue viruses with reduced expression of viral proteins and excellent growth characteristics useful for vaccine production, but with exceptional safety profiles and enhanced protective features. The attenuated viruses grow almost as well as wild-type viruses and exhibit LD 50 As evidenced by the values, these antibodies have a highly attenuated phenotype, are remarkably effective in providing protective immunity against challenge with dengue virus of the same strain, and also provide protective immunity against challenge with dengue virus of other strains.

[0038] In certain embodiments of the present invention, the attenuated dengue virus of the present invention comprises a recoded pre-membrane (prM) / envelope (E) coding region. In embodiments where the C, NS1, NS2, NS3, NS4, or NS5 protein coding regions are not recoded, this does not exclude mutations and other variations in their sequences, but only means that any mutations or variations made in their sequences have little or no effect on attenuation. Little or no effect on attenuation includes one or both of the following: (1) the mutations or variations in the C, NS1, NS2, NS3, NS4, or NS5 coding regions do not reduce viral replication or viral infectivity by more than 20% when the variant C, NS1, NS2, NS3, NS4, or NS5 coding region is the only variant in the tested dengue virus; (2) the mutations or variations in any of the C, NS1, NS2, NS3, NS4, or NS5 coding regions are less than 10% of the nucleotides in the coding sequence. As specifically provided in the claims, little or no effect on attenuation includes one or both of the following: (1) mutations or variations in the C, NS1, NS2, NS3, NS4, or NS5 coding regions do not reduce viral replication or viral infectivity by more than 10% when the variant C, NS1, NS2, NS3, NS4, or NS5 coding region is the only variant in the test dengue virus; (2) mutations or variations in any of the C, NS1, NS2, NS3, NS4, or NS5 coding regions account for fewer than 5% of the nucleotides in that coding sequence.

[0039] In various embodiments, the virus of the present invention is attenuated.In an embodiment of the present invention, compared to wild type, the virus is at least 10 times attenuated in AG129 mouse model, at least 50 times attenuated, or at least 100 times attenuated, or at least 200 times attenuated, or at least 500 times attenuated, or at least 1000 times attenuated, or at least 2000 times attenuated, compared to wild type virus having the same amino acid sequence but protein encoded by different nucleotide sequence.

[0040] The attenuated virus is also highly protective against wild-type virus of the same strain. In an embodiment of the invention, the protective dose (PD 50 ) as measured, for example, in a mouse model as exemplified herein.

[0041] The attenuated viruses of the invention also provide a large margin of safety (i.e., LD 50 and P.D. 50 (difference between 50 / PD 50 In certain embodiments of the invention, the safety factor is at least 10 2 , or at least 10 3 , or at least 10 4 , or at least 10 5 , or at least 2 × 10 5 , or at least 3 × 10 5 , or at least 4 × 10 5 Or at least 5×10 5 , or at least 10 6 , or at least 2 × 10 6 , or at least 3 × 10 6 , or at least 4 × 10 6 , or at least 5 × 10 6 In certain embodiments, the safety factor is 10 2 ~10 3 , or 103 ~10 4 , or 10 4 ~10 5 , or 10 5 ~10 6 It is.

[0042] The attenuated viruses of the invention are also highly protective against heterologous strains of dengue virus within the same serotype. In certain embodiments of the invention, the protective dose (PD 50 ) is less than 1000 PFU, or less than 750 PFU, or less than 500 PFU, or less than 200 PFU, or less than 100 PFU, or less than 50 PFU, as measured by a mouse model, for example as exemplified herein.

[0043] Recoding of the E protein coding sequence of the attenuated virus of the present invention has been or can be done by those skilled in the art in light of the disclosure discussed herein. According to the present invention, nucleotide substitutions are engineered at multiple positions in the E coding sequence, introducing multiple synonymous codons into the genome through the substitutions. In certain embodiments, the synonymous codon substitutions change the codon bias, codon pair bias, density of rare codons or rare codon pairs, RNA secondary structure, CG and / or TA (or UA) dinucleotide content, C+G content, translation frameshift sites, translation pause sites, presence or absence of microRNA recognition sequences, or any combination thereof, in the genome. Codon substitutions may be engineered at multiple positions distributed throughout the E coding sequence or at multiple positions constrained to a portion of the E coding sequence. Due to the large number of defects (i.e., nucleotide substitutions) involved, the present invention provides a means to produce stably attenuated viruses and live vaccines.

[0044] As discussed further below, in some embodiments, the viral coding sequence is recoded by replacing one or more codons with synonymous codons that are used less frequently in dengue hosts (e.g., mammals, humans, mosquitoes). In some embodiments, the viral coding sequence is recoded by replacing one or more codons with synonymous codons that are used less frequently in dengue viruses. In certain embodiments, the number of codons replaced with synonymous codons is at least 5. In some embodiments, at least 10, or at least 20 codons are replaced with synonymous codons. In some embodiments, the number of codons replaced with synonymous codons is at least 30, or at least 40, or at least 50, or at least 75, or at least 100, or at least 125, or at least 150, or at least 175.

[0045] In some embodiments, the viral codon pairs are recoded to reduce (i.e., lower) the codon pair bias. In certain embodiments, the codon pair bias is reduced by identifying codon pairs in the E coding sequence that have a codon pair score that can be reduced, and reducing the codon pair bias by replacing the codon pair with a codon pair with a lower codon pair score. In some embodiments, this replacement of codon pairs takes the form of rearranging the existing codons of the sequence. In some such embodiments, a subset of codon pairs is replaced by rearranging a subset of synonymous codons. In other embodiments, the codon pairs are replaced by maximizing the number of rearranged synonymous codons. It is noted that the rearrangement of codons leads to a reduced (more negative) codon pair bias for the viral coding sequence overall, and that while the rearrangement results in a reduced CPS at many positions, there may be a concomitant increase in CPS at other positions, but on average, the codon pair score of the modified sequence, and therefore the CPB, is reduced. In some embodiments, recoding a codon or codon pair may take into consideration altering the G+C content of the E coding sequence. In some embodiments, recoding a codon or codon pair may take into consideration altering the frequency of CG and / or TA dinucleotides in the E coding sequence.

[0046] In certain embodiments, the recoded E protein coding sequence has a codon pair bias of less than -0.05, or less than -0.06, or less than -0.07, or less than -0.08, or less than -0.09, or less than -0.1, or less than -0.11, or less than -0.12, or less than -0.13, or less than -0.14, or less than -0.15, or less than -0.16, or less than -0.17, or less than -0.18, or less than -0.19, or less than -0.2, or less than -0.25, or less than -0.3, or less than -0.35, or less than -0.4, or less than -0.45, or less than -0.5. In certain embodiments, the codon pair bias of the recoded E protein coding sequence is reduced by at least 0.05, or at least 0.06, or at least 0.07, or at least 0.08, or at least 0.09, or at least 0.1, or at least 0.11, or at least 0.12, or at least 0.13, or at least 0.14, or at least 0.15, or at least 0.16, or at least 0.17, or at least 0.18, or at least 0.19, or at least 0.2, or at least 0.25, or at least 0.3, or at least 0.35, or at least 0.4, or at least 0.45, or at least 0.5, compared to the E protein coding sequence for which the calculation is being made, e.g., the E protein coding sequence of a wild-type virus.

[0047] In certain embodiments, the rearrangement of synonymous codons in the E protein coding sequence provides a reduction in codon pair bias of at least 0.05, or at least 0.06, or at least 0.07, or at least 0.08, or at least 0.09, or at least 0.1, or at least 0.11, or at least 0.12, or at least 0.13, or at least 0.14, or at least 0.15, or at least 0.16, or at least 0.17, or at least 0.18, or at least 0.19, or at least 0.2, or at least 0.25, or at least 0.3, or at least 0.35, or at least 0.4, or at least 0.45, or at least 0.5, compared to the E protein coding sequence for which the calculation is to be made, e.g., the E protein coding sequence of a wild-type virus.

[0048] Usually, these substitutions and changes are made without changing the amino acid sequence of the encoded protein, and reduce the expression of the encoded viral protein. In certain embodiments, the present invention also includes changes in the E coding sequence that result in non-synonymous codon substitutions and amino acid substitutions in the encoded protein, which may or may not be conservative. In various embodiments, there are up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotide substitutions.

[0049] Most amino acids are coded by more than one codon. See the genetic code in Table 1. For example, alanine is coded by GCU, GCC, GCA, and GCG. Three amino acids (Leu, Ser, and Arg) are coded by six different codons, with only Trp and Met having unique codons. "Synonymous" codons are those that code for the same amino acid. Thus, for example, CUU, CUC, CUA, CUG, UUA, and UUG are synonymous codons that code for Leu. Synonymous codons are not used with equal frequency. In general, the most frequently used codons in a particular organism are those for which cognate tRNAs are abundant, and the use of these codons enhances the rate and / or accuracy of protein translation. Conversely, tRNAs for rarely used codons are found at relatively low levels, and the use of rare codons is believed to reduce the rate and / or accuracy of translation.

[0050] (Table 1) Genetic code TIFF2025041836000002.tif79128 a The first nucleotide in each codon that encodes a particular amino acid is shown in the left-most column, the second nucleotide is shown in the line above, and the third nucleotide is shown in the right-most column.

[0051] Codon Bias As used herein, a "rare" codon is one of at least two synonymous codons that code for a particular amino acid that occurs in mRNA at a significantly lower frequency than the most frequently used codon for that amino acid. Thus, a rare codon may occur at about two times lower frequency than the most frequently used codon for that amino acid. Preferably, a rare codon occurs at least three times, more preferably at least five times, less frequently than the most frequently used codon for that amino acid. Conversely, a "high frequency" codon is one of at least two synonymous codons that code for a particular amino acid that occurs in mRNA at a significantly higher frequency than the least frequently used codon for that amino acid. A high frequency codon may occur at about two times, preferably at least three times, more preferably at least five times, more frequently than the least frequently used codon for that amino acid. For example, human genes use the leucine codon CTG 40% of the time, but the synonymous CTA only 7% of the time (see Table 2). Thus, CTG is a high frequency codon, while CTA is a rare codon. Roughly in line with these usage frequencies, there are six copies in the genome for the gene for the tRNA that recognizes CTG, while there are only two copies for the gene for the tRNA that recognizes CTA. Similarly, human genes use the frequent codons TCT and TCC for serine 18% and 22% of the time, respectively, but the rare codon TCG only 5% of the time. TCT and TCC are read via wobble by the same tRNA that has its gene in 10 copies in the genome, while TCG is read by a tRNA that has only four copies. It is well known that highly actively translated mRNAs are strongly biased to use only the most frequent codons. This includes genes for ribosomal proteins and glycolytic enzymes. On the other hand, mRNAs for relatively less abundant proteins may use rare codons.

[0052] (Table 2) Codon usage in Homo sapiens (Source: www.kazusa.or.jp / codon / ) TIFF2025041836000003.tif84128TIFF2025041836000004.tif21987

[0053] The tendency of highly expressed genes to use frequent codons is called "codon bias". Genes for ribosomal proteins may use only the 20-25 most frequent of the 61 codons and have a high codon bias (a codon bias close to 1). Poorly expressed genes may use all 61 codons and have little or no codon bias (a codon bias close to 0). Frequently used codons are those for which a greater amount of cognate tRNAs are expressed, and the use of these codons is thought to allow translation to proceed more quickly, or more accurately, or both. For example, the PV capsid protein is very actively translated and has a high codon bias.

[0054] Codon Pair Bias Additionally, a given organism has a preference for the nearest codon neighbor of a given codon A, which is referred to as a bias in codon pair usage. Altering the codon pair bias without changing the existing codon can affect the rate of protein synthesis and the production of the protein.

[0055] Codon pair bias can be explained by considering the amino acid pair Ala-Glu, which can be coded for by eight different codon pairs. If no factors other than the frequency of each individual codon (as shown in Table 2) contribute to the frequency of a codon pair, the predicted frequency of each of the eight encodings can be calculated by multiplying the frequencies of the two related codons. For example, this calculation predicts that the codon pair GCA-GAA occurs with a frequency of 0.097 (0.23 x 0.42; based on the frequencies in Table 2) among all Ala-Glu coding pairs. To relate the predicted (hypothetical) frequency of each codon pair to the frequency actually observed in the human genome, the Consensus CDS of Consistently Annotated Human Coding Regions (CCDS) database was used, which contains a total of 14,795 human genes. This set of genes is the most comprehensive representation of human coding sequences. Using this set of genes, the frequency of codon usage was recalculated by dividing the number of occurrences of the codon by the number of all synonymous codons that code for the same amino acid. As expected, the frequencies correlated closely with those reported previously, e.g., those given in Table 2. The minor frequency variations may be due to oversampling effects in the data provided by the codon usage database at Kazusa DNA Research Institute (www.kazusa.or.jp / codon / codon.html), in which 84949 human coding sequences (much more than the actual number of human genes) were included in the calculation. The codon frequencies so calculated were then used to calculate the predicted codon pair frequencies by first multiplying the frequencies of the two related codons together (see predicted frequencies in Table 3), and then multiplying this result by the observed frequency (in the entire CCDS dataset) at which the amino acid pair encoded by the codon pair in question occurs. In the example of the codon pair GCA-GAA, this second calculation gives a predicted frequency of 0.098 (compared to 0.097 in the first calculation using the Kazusa dataset).Finally, the actual codon pair frequencies as observed in the set of 14,795 human genes were determined by counting the total number of occurrences of each codon pair in the set and dividing it by the number of all synonymous coding pairs in the set that code for the same amino acid pair (Table 3; observed frequencies). Based on the set of 14,795 human genes, 3721 (61. 2 The frequencies and observed / expected values ​​for the complete set of 100 codon pairs are provided herein as Table 3.

[0056] Table 3 Codon pair scores exemplified by the amino pair Ala-Glu TIFF2025041836000005.tif57128

[0057] A codon pair is said to be over-represented if the ratio of observed / expected frequency of the codon pair is greater than 1. If the ratio is less than 1, it is said to be under-represented. In the example, the codon pair GCA-GAA is over-represented by 1.65-fold, and the coding pair GCC-GAA is under-represented by more than 5-fold.

[0058] Many other codon pairs show very strong bias; some pairs are under-represented and others are over-represented. For example, the codon pair GCCGAA (Ala Glu) and GATCTG (Asp Leu) is under-represented by 3-6 times (the preferred pairs are GCAGAG and GACCTG, respectively), and the codon pair GCCAAG (Ala Lys) and AATGAA (Asn Glu) is over-represented by about 2 times. It is noteworthy that the codon pair bias is not related to the frequency of the amino acid pair, nor to the frequency of the individual codons. For example, the under-represented pair GATCTG (Asp Leu) uses the most frequent Leu codon (CTG).

[0059] As discussed more fully below, codon pair bias takes into account the score of each codon pair in a coding sequence, averaged over the entire length of the coding sequence. According to the present invention, codon pair bias is Determined by TIFF2025041836000006.tif17128.

[0060] Thus, a similar codon pair bias for a coding sequence can be obtained, for example, by a minimized codon pair score for a subsequence, or a moderately reduced codon pair score for the entire length of the coding sequence.

[0061] Calculating codon pair bias Every individual codon pair (e.g., GTT-GCT) of the 3721 possible non-"stop" containing codon pairs has an assigned "codon pair score," or "CPS," that is unique to a given "training set" of genes. The CPS of a given codon pair is defined as the log ratio of the observed number of occurrences to the expected number in this set of genes (in this example, the human genome). Determining the actual number of occurrences of a particular codon pair (or in other words the likelihood that a particular amino acid pair is coded by a particular codon pair) is simply a matter of counting the actual number of occurrences of the codon pair in a particular set of coding sequences. However, determining the expected number requires additional calculations. The expected number is calculated to be independent of both amino acid frequency and codon bias, similar to Gutman and Hatfield. That is, the expected frequency is calculated based on the relative proportion of times that an amino acid is coded by a particular codon. A positive CPS value indicates that a given codon pair is statistically over-represented, whereas a negative CPS indicates that the pair is statistically under-represented in the human genome.

[0062] To perform these calculations in the human context, we used the most recent Consensus CDS (CCDS) database of consistently annotated human coding regions, containing a total of 14,795 genes. This data set provides the frequencies of codons and codon pairs, and therefore amino acids and amino acid pairs, on a genome-scale.

[0063] The approach of Gutman and Hatfield (1989) was further enhanced using the paradigm of Federov et al. (2002), which allows the calculation of the expected frequency of a given codon pair independent of the codon frequency and non-random associativity of the adjacent codons that code for a particular amino acid pair. The detailed formulas used to calculate CPB are disclosed in WO 2008 / 121992 and WO 2011 / 044561, which are incorporated by reference. TIFF2025041836000007.tif11128

[0064] In the calculation, P ij is N in the synonym group. O (P ij ) is a codon pair that occurs with a frequency of C i and C j are the frequencies F(C i ) and F(C j ), P ij More specifically, F(C i ) is the corresponding amino acid X i Codon C is found throughout the entire coding region. i is the frequency coded by F(C i )=N O (C j ) / N O (X i ), where N O (C i ) and N O (X i ) are codon C i and amino acid X i is the observed number of occurrences of F(Cj ) is calculated accordingly. Furthermore, N O (X ij ) is the amino acid pair X throughout the entire coding region. ij is the number of occurrences of P ij The codon pair bias score S(P ij ) to N e (P ij ) versus the expected number of occurrences of N o (P ij ) was calculated as the log odds ratio.

[0065] Using the above formula, the corresponding genomic N calculated by using the entire human CCDS dataset e (P ij We then determined whether individual codon pairs in individual coding sequences were over- or under-represented when compared to the S(P) values. This calculation yielded a positive S(P) for over-represented codon pairs in human coding regions. ij ) score value, and negative values ​​for underrepresented codon pairs.

[0066] The following formula: The “combined” codon pair bias of each coding sequence was calculated by averaging all codon pair scores according to TIFF2025041836000008.tif13128.

[0067] The codon pair bias of an entire coding region is therefore calculated by adding all of the individual codon pair scores that make up the region and dividing this sum by the length of the coding sequence.

[0068] Calculating codon pair bias, running algorithms to change the codon pair bias An algorithm was developed to quantify codon pair bias. Every possible individual codon pair was given a "codon pair score," or "CPS," defined as the natural logarithm of the ratio of the observed to expected number of occurrences of each codon pair across all human coding regions, humans being the host species for the vaccine virus to be recoded. TIFF2025041836000009.tif18128

[0069] While the calculation of the observed occurrence of a particular codon pair is straightforward (the actual number in a gene set), the expected number of occurrences of a codon pair requires additional calculation.Similar to Gutman and Hatfield, we calculate this expected number to be independent of both amino acid frequency and codon bias.That is, the expected frequency is calculated based on the relative proportion of times that an amino acid is coded by a particular codon.A positive CPS value represents that a given codon pair is statistically over-represented, while a negative CPS indicates that the pair is statistically under-represented in the human genome.

[0070] Using these calculated CPS, any coding region can then be assessed as using either over- or under-represented codon pairs by taking the average of the codon pair scores, thus giving the codon pair bias (CPB) for the entire gene. TIFF2025041836000010.tif13128

[0071] The CPB was calculated for all annotated human genes using the formula shown and plotted (Figure 1). Each point in the graph corresponds to the CPB of a single human gene. The peak of the distribution has a positive codon pair bias of 0.07, which is the average score for all annotated human genes. There are also very few genes with negative codon pair bias. We then accounted for this bias using the formula established to define and calculate CPB.

[0072] An algorithm for reducing codon pair bias and attenuation Recoding of protein coding sequences can be performed with or without computer assistance, for example, using gradient descent, or simulated annealing, or other minimization routines. An example of a procedure for rearranging codons present in a starting sequence is represented by the following steps: (1) Obtain the wild-type viral genome sequence. (2) Select a protein coding sequence to target for attenuation engineering. (3) Locking down DNA segments that have known or suspected non-coding functions. (4) Select the desired codon distribution for the remaining amino acids in the redesigned protein. (5) Randomly shuffle the positions of at least two synonymous unlocked codons and calculate the codon pair score. (6) Optionally, a simulated annealing procedure is used to further reduce (or increase) the codon pair score. (7) Inspect the resulting designs for excessive secondary structure and unwanted restriction sites: If so, go to step (5) or correct the design by replacing the problematic region with a wild type sequence and go to step (8). (8) Synthesize the DNA sequence corresponding to the virus design. (9) Generate viral constructs and evaluate viral phenotypes: If too attenuated, prepare subclone constructs and go to 9; If attenuation is insufficient, go to 2.

[0073] Viral attenuation by reducing codon pair bias is disclosed in WO 2008 / 121992 and WO 2011 / 044561, which are incorporated by reference.

[0074] Methods for obtaining full-length flavivirus or dengue genomic sequences or codon pair deoptimized sequences embedded in wild-type flavivirus or dengue genomic sequences can include constructing infectious cDNA clones using, for example, an overlap extension PCR strategy or a long PCR-based fusion strategy.

[0075] Modified Flavivirus Various embodiments of the present invention provide modified Flavivirus viruses in which expression of viral proteins is reduced compared to the parent virus. The reduced expression is the result of recoding the prM or envelope (E) region or both. In some embodiments, the parent virus is a wild-type Flavivirus, and thus the comparison is made to the wild-type virus or sequences in the wild-type virus.

[0076] In various embodiments, the E protein coding sequence is recoded by reducing the codon pair bias or codon usage bias of the protein coding sequence.In various embodiments, reducing the codon pair bias comprises identifying the codon pair in the parent protein coding sequence that has a codon pair score that can be reduced, and reducing the codon pair bias by replacing the codon pair with a codon pair with a lower codon pair score.In other embodiments, reducing the codon pair bias comprises rearranging the codons of the parent protein coding sequence.

[0077] In various embodiments, each of the recoded prM / E protein coding sequences has a codon pair bias of less than -0.05, less than -0.1, or less than -0.2, or less than -0.3, or less than -0.4.

[0078] In certain embodiments, the recoded prM protein coding sequence has a codon pair bias of less than -0.05, or less than -0.06, or less than -0.07, or less than -0.08, or less than -0.09, or less than -0.1, or less than -0.11, or less than -0.12, or less than -0.13, or less than -0.14, or less than -0.15, or less than -0.16, or less than -0.17, or less than -0.18, or less than -0.19, or less than -0.2, or less than -0.25, or less than -0.3, or less than -0.35, or less than -0.4, or less than -0.45, or less than -0.5.

[0079] In certain embodiments, the recoded E protein coding sequence has a codon pair bias of less than -0.05, or less than -0.06, or less than -0.07, or less than -0.08, or less than -0.09, or less than -0.1, or less than -0.11, or less than -0.12, or less than -0.13, or less than -0.14, or less than -0.15, or less than -0.16, or less than -0.17, or less than -0.18, or less than -0.19, or less than -0.2, or less than -0.25, or less than -0.3, or less than -0.35, or less than -0.4, or less than -0.45, or less than -0.5.

[0080] In certain embodiments, the codon pair bias of the recoded prM protein coding sequence is reduced by at least 0.05, or at least 0.06, or at least 0.07, or at least 0.08, or at least 0.09, or at least 0.1, or at least 0.11, or at least 0.12, or at least 0.13, or at least 0.14, or at least 0.15, or at least 0.16, or at least 0.17, or at least 0.18, or at least 0.19, or at least 0.2, or at least 0.25, or at least 0.3, or at least 0.35, or at least 0.4, or at least 0.45, or at least 0.5, compared to the parent prM protein coding sequence from which it is derived.

[0081] In certain embodiments, the codon pair bias of the recoded E protein coding sequence is reduced by at least 0.05, or at least 0.06, or at least 0.07, or at least 0.08, or at least 0.09, or at least 0.1, or at least 0.11, or at least 0.12, or at least 0.13, or at least 0.14, or at least 0.15, or at least 0.16, or at least 0.17, or at least 0.18, or at least 0.19, or at least 0.2, or at least 0.25, or at least 0.3, or at least 0.35, or at least 0.4, or at least 0.45, or at least 0.5, compared to the E protein coding sequence for which the calculation is being made, e.g., the E protein coding sequence of a wild-type virus.

[0082] In various embodiments, the E protein coding sequence is recoded by increasing the number of CpG or UpA dinucleotides compared to its parent virus, hi various embodiments, the E protein coding sequence is recoded by altering the G+C content compared to its parent virus.

[0083] In various embodiments, the E protein coding sequence is recoded by replacing one or more codons with synonymous codons that are less frequent in the viral host, eg, humans.

[0084] In various embodiments, the E protein coding sequence is recoded by replacing one or more codons with synonymous codons that are less frequent in the virus itself.

[0085] In some embodiments, the number of codons replaced with synonymous codons in the prM protein coding sequence is at least 5, or at least 10, or at least 30, or at least 30, or at least 40, or at least 50, or at least 75, or at least 100, or at least 150.

[0086] In some embodiments, the number of codons replaced with synonymous codons in the E protein coding sequence is at least 5, or at least 10, or at least 30, or at least 30, or at least 40, or at least 50, or at least 75, or at least 100, or at least 125, or at least 150, or at least 175.

[0087] In various embodiments, the parent virus is a flavivirus selected from the group consisting of Dengue virus, West Nile virus, Yellow fever virus, Japanese encephalitis virus, Spondweni virus, Zika virus, St. Louis encephalitis virus, and Powassan virus. In various embodiments, the parent virus is a natural isolate. In various embodiments, the parent virus is a mutant of a natural isolate.

[0088] Modified dengue virus Various aspects of the present invention provide modified dengue viruses as modified flaviviruses.

[0089] In various embodiments, modified dengue viruses are provided, in which the expression of viral proteins is reduced compared to the parent virus, and the reduced expression is the result of recoding prM or envelope (E) region, or both.In some embodiments, the parent dengue virus is a wild type dengue virus.Therefore, comparison can be made to wild type dengue virus.

[0090] In various embodiments, one or both of the E protein coding sequences are recoded by reducing the codon pair bias or codon usage bias of the protein coding sequence.In various embodiments, reducing the codon pair bias comprises identifying the codon pair in the parent protein coding sequence that has a codon pair score that can be reduced, and reducing the codon pair bias by replacing the codon pair with a codon pair with a lower codon pair score.In various embodiments, reducing the codon pair bias comprises rearranging the codons of the parent protein coding sequence.

[0091] Various embodiments of the present invention provide modified dengue viruses comprising a recoded prM protein, a recoded envelope (E) protein, or both, wherein the recoded prM protein has reduced codon pair bias compared to its parent prM protein coding sequence, or has at least five codons replaced with less frequently used synonymous codons, or has an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence, and the recoded E protein has reduced codon pair bias compared to its parent E protein coding sequence, or has at least five codons replaced with less frequently used synonymous codons, or has an increased number of CpG or UpA dinucleotides compared to its parent E protein coding sequence. In various embodiments, the "parent protein coding sequence" is a "wild-type dengue protein coding sequence", e.g., a "wild-type dengue E protein coding sequence", a "wild-type dengue prM protein coding sequence".

[0092] In various embodiments, expression of the prM protein or the E protein, or both, is reduced compared to its parent dengue virus.

[0093] In various embodiments, the recoded prM protein has a reduced codon pair bias compared to its parent prM protein coding sequence. In various embodiments, the codon pair bias of the recoded prM coding sequence is reduced by at least 0.05. In certain embodiments, the codon pair bias of the recoded prM protein coding sequence of a dengue virus is reduced by at least 0.06, or at least 0.07, or at least 0.08, or at least 0.09, or at least 0.1, or at least 0.11, or at least 0.12, or at least 0.13, or at least 0.14, or at least 0.15, or at least 0.16, or at least 0.17, or at least 0.18, or at least 0.19, or at least 0.2, or at least 0.25, or at least 0.3, or at least 0.35, or at least 0.4, or at least 0.45, or at least 0.5 compared to the prM protein coding sequence of the parent dengue virus from which it is derived. In certain embodiments, it is relative to the prM protein coding sequence against which the calculation is to be made, for example, the prM protein coding sequence of a wild-type dengue virus.

[0094] In various embodiments, the recoded prM protein has at least 5 codons replaced with less frequently used synonymous codons.In various embodiments, the recoded prM protein can have at least 10, 20, 25, 30, 35, 40, 45, 50 or 55 codons replaced with less frequently used synonymous codons.In some embodiments, the replacement with less frequently used synonymous codons is less frequently used in the virus host, e.g., human, mosquito.In some embodiments, the replacement with less frequently used synonymous codons is less frequently used in the virus itself.

[0095] In various embodiments, the recoded prM protein has an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence. In various embodiments, the recoded prM protein has an increase of 15 to 55 CpG or UpA dinucleotides compared to its parent prM protein coding sequence. In various embodiments, the recoded prM protein has an increase of about 15, 20, 25, 30, 35, 40, 45 or 55 CpG or UpA dinucleotides compared to its parent prM protein coding sequence.

[0096] In various embodiments, the recoded E protein has a reduced codon pair bias compared to its parent E protein coding sequence. In various embodiments, the codon pair bias of the E protein coding sequence is reduced by at least 0.05 compared to its parent E protein coding sequence. In certain embodiments, the codon pair bias of the recoded E protein coding sequence of the dengue virus is reduced by at least 0.06, or at least 0.07, or at least 0.08, or at least 0.09, or at least 0.1, or at least 0.11, or at least 0.12, or at least 0.13, or at least 0.14, or at least 0.15, or at least 0.16, or at least 0.17, or at least 0.18, or at least 0.19, or at least 0.2, or at least 0.25, or at least 0.3, or at least 0.35, or at least 0.4, or at least 0.45, or at least 0.5 compared to the E protein coding sequence of the parent dengue virus from which it is derived. In certain embodiments, it is relative to the E protein coding sequence for which the calculation is to be made, for example, the E protein coding sequence of a wild-type dengue virus.

[0097] In various embodiments, recoded E protein has at least 5 codons replaced with less frequently used synonymous codons.In various embodiments, recoded E protein can have at least 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150 or 175 codons replaced with less frequently used synonymous codons.In some embodiments, the replacement with less frequently used synonymous codons is less frequently used in virus host, e.g., human, mosquito.In some embodiments, the replacement with less frequently used synonymous codons is less frequently used in virus itself.

[0098] In various embodiments, the recoded E protein has an increased number of CpG or UpA dinucleotides compared to its parent E protein coding sequence. In various embodiments, the recoded prM protein has an increase of 5 to 12 CpG or UpA dinucleotides compared to its parent E protein coding sequence. In various embodiments, the recoded prM protein has an increase of about 5, 6, 7, 8, 9, 10, 11 or 12 CpG or UpA dinucleotides compared to its parent E protein coding sequence.

[0099] In various embodiments, each of the recoded prM or E protein coding sequences has a codon pair bias of less than -0.05. In certain embodiments, the recoded prM protein coding sequence has a codon pair bias of less than -0.05, or less than -0.06, or less than -0.07, or less than -0.08, or less than -0.09, or less than -0.1, or less than -0.11, or less than -0.12, or less than -0.13, or less than -0.14, or less than -0.15, or less than -0.16, or less than -0.17, or less than -0.18, or less than -0.19, or less than -0.2, or less than -0.25, or less than -0.3, or less than -0.35, or less than -0.4, or less than -0.45, or less than -0.5. In certain embodiments, the recoded E protein coding sequence has a codon pair bias of less than -0.05, or less than -0.06, or less than -0.07, or less than -0.08, or less than -0.09, or less than -0.1, or less than -0.11, or less than -0.12, or less than -0.13, or less than -0.14, or less than -0.15, or less than -0.16, or less than -0.17, or less than -0.18, or less than -0.19, or less than -0.2, or less than -0.25, or less than -0.3, or less than -0.35, or less than -0.4, or less than -0.45, or less than -0.5.

[0100] In various embodiments, the modified dengue virus is selected from type 1, type 2, type 3, type 4, or combinations thereof. For example, types 1 and 2, types 1 and 3, types 1 and 4, types 2 and 3, types 2 and 4, or types 3 and 4. Additional examples are types 1, 2 and 3; types 1, 3 and 4; or types 2, 3 and 4. In various embodiments, the modified dengue virus is a modified tetravalent dengue virus (i.e., types 1, 2, 3 and 4).

[0101] method Various aspects provide a method of inducing an immune response in a subject, comprising administering to the subject an effective dose of a composition comprising a modified dengue virus of the invention as described herein above.

[0102] In various embodiments, the immune response is a protective immune response and 3 ~10 7 In various embodiments, the immune response is a protective immune response and is administered in a prophylactically or therapeutically effective dose of 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 A prophylactically or therapeutically effective dose of the claimed vaccine composition is administered. In various embodiments, the method further comprises administering to the subject at least one adjuvant. In various embodiments, the immune response is cross-protective against heterologous dengue viruses.

[0103] Various embodiments include a method of eliciting an immune response in a subject in need thereof, comprising administering to a subject in need thereof a prime dose of (i) an attenuated dengue virus produced by a method other than codon pair deoptimization or codon deoptimization, or an increase in CpG or UpA dinucleotides, or (ii) a modified dengue virus comprising a recoded prM protein, a recoded envelope (E) protein, or both, wherein the recoded prM protein has a reduced codon pair bias compared to its parent prM protein coding sequence, or has at least five codons replaced with less frequently used synonymous codons, or has an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence. and administering one or more boost doses of (i) an attenuated dengue virus made by a method other than codon pair deoptimization or codon deoptimization, or an increase in CpG or UpA dinucleotides, or (ii) a modified dengue virus, wherein at least the prime dose or one or more boost doses are modified dengue virus.

[0104] In various embodiments, the first of the one or more boost doses is administered about two weeks after the prime dose.

[0105] In various embodiments, expression of the prM protein or the E protein or both is reduced compared to its parent dengue virus, hi various embodiments, the parent dengue virus is a wild-type dengue virus.

[0106] In various embodiments, the recoded prM protein has a reduced codon pair bias compared to its parent prM protein coding sequence. In various embodiments, the codon pair bias of the recoded prM coding sequence is reduced by at least 0.05. In certain embodiments, the codon pair bias of the recoded prM protein coding sequence of a dengue virus is reduced by at least 0.06, or at least 0.07, or at least 0.08, or at least 0.09, or at least 0.1, or at least 0.11, or at least 0.12, or at least 0.13, or at least 0.14, or at least 0.15, or at least 0.16, or at least 0.17, or at least 0.18, or at least 0.19, or at least 0.2, or at least 0.25, or at least 0.3, or at least 0.35, or at least 0.4, or at least 0.45, or at least 0.5 compared to the prM protein coding sequence of the parent dengue virus from which it is derived. In certain embodiments, it is relative to the prM protein coding sequence against which the calculation is to be made, for example, the prM protein coding sequence of a wild-type dengue virus.

[0107] In various embodiments, the recoded prM protein has at least 5 codons replaced with less frequently used synonymous codons.In various embodiments, the recoded prM protein can have at least 10, 20, 25, 30, 35, 40, 45, 50 or 55 codons replaced with less frequently used synonymous codons.In some embodiments, the replacement with less frequently used synonymous codons is less frequently used in the virus host, e.g., human, mosquito.In some embodiments, the replacement with less frequently used synonymous codons is less frequently used in the virus itself.

[0108] In various embodiments, the recoded prM protein has an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence. In various embodiments, the recoded prM protein has an increase of 15 to 55 CpG or UpA dinucleotides compared to its parent prM protein coding sequence. In various embodiments, the recoded prM protein has an increase of about 15, 20, 25, 30, 35, 40, 45 or 55 CpG or UpA dinucleotides compared to its parent prM protein coding sequence. In certain embodiments, it is compared to the prM protein coding sequence for which the calculation is being made, e.g., the prM protein coding sequence of a wild-type dengue virus.

[0109] In various embodiments, the recoded E protein has a reduced codon pair bias compared to its parent E protein coding sequence. In various embodiments, the codon pair bias of the E protein coding sequence is reduced by at least 0.05 compared to its parent E protein coding sequence. In certain embodiments, the codon pair bias of the recoded E protein coding sequence of the dengue virus is reduced by at least 0.06, or at least 0.07, or at least 0.08, or at least 0.09, or at least 0.1, or at least 0.11, or at least 0.12, or at least 0.13, or at least 0.14, or at least 0.15, or at least 0.16, or at least 0.17, or at least 0.18, or at least 0.19, or at least 0.2, or at least 0.25, or at least 0.3, or at least 0.35, or at least 0.4, or at least 0.45, or at least 0.5 compared to the E protein coding sequence of the parent dengue virus from which it is derived. In certain embodiments, it is relative to the E protein coding sequence for which the calculation is to be made, for example, the E protein coding sequence of a wild-type dengue virus.

[0110] In various embodiments, recoded E protein has at least 5 codons replaced with less frequently used synonymous codons.In various embodiments, recoded E protein can have at least 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150 or 175 codons replaced with less frequently used synonymous codons.In some embodiments, the replacement with less frequently used synonymous codons is less frequently used in virus host, e.g., human, mosquito.In some embodiments, the replacement with less frequently used synonymous codons is less frequently used in virus itself.

[0111] In various embodiments, the recoded E protein has an increased number of CpG or UpA dinucleotides compared to its parent E protein coding sequence. In various embodiments, the recoded prM protein has an increase of 5-12 CpG or UpA dinucleotides compared to its parent E protein coding sequence. In various embodiments, the recoded prM protein has an increase of about 5, 6, 7, 8, 9, 10, 11 or 12 CpG or UpA dinucleotides compared to its parent E protein coding sequence. In certain embodiments, it is compared to the E protein coding sequence for which the calculation is being made, e.g., the E protein coding sequence of a wild-type dengue virus.

[0112] In various embodiments, each of the recoded prM or E protein coding sequences has a codon pair bias of less than -0.05. In certain embodiments, the recoded prM protein coding sequence has a codon pair bias of less than -0.05, or less than -0.06, or less than -0.07, or less than -0.08, or less than -0.09, or less than -0.1, or less than -0.11, or less than -0.12, or less than -0.13, or less than -0.14, or less than -0.15, or less than -0.16, or less than -0.17, or less than -0.18, or less than -0.19, or less than -0.2, or less than -0.25, or less than -0.3, or less than -0.35, or less than -0.4, or less than -0.45, or less than -0.5. In certain embodiments, the recoded E protein coding sequence has a codon pair bias of less than -0.05, or less than -0.06, or less than -0.07, or less than -0.08, or less than -0.09, or less than -0.1, or less than -0.11, or less than -0.12, or less than -0.13, or less than -0.14, or less than -0.15, or less than -0.16, or less than -0.17, or less than -0.18, or less than -0.19, or less than -0.2, or less than -0.25, or less than -0.3, or less than -0.35, or less than -0.4, or less than -0.45, or less than -0.5.

[0113] In various embodiments, the modified dengue virus is selected from type 1, type 2, type 3, type 4, or combinations thereof. For example, types 1 and 2, types 1 and 3, types 1 and 4, types 2 and 3, types 2 and 4, or types 3 and 4. Additional examples are types 1, 2 and 3; types 1, 3 and 4; or types 2, 3 and 4. In various embodiments, the modified dengue virus is a modified tetravalent dengue virus (i.e., types 1, 2, 3 and 4).

[0114] Methods for producing modified flavivirus genomes Various aspects of the present invention provide a method for making a modified Flavivirus virus genome, comprising obtaining a nucleotide sequence encoding a Flavivirus virus envelope protein and a nucleotide sequence encoding a Flavivirus virus nonstructural 3 protein; recoding the envelope-coding nucleotide sequence to reduce protein expression, and recoding the nonstructural protein 3 coding nucleotide sequence to reduce protein expression, and substituting a nucleic acid having the recoded envelope-coding nucleotide sequence and a nucleic acid having the recoded nonstructural protein 3 coding nucleotide sequence into a parent Flavivirus virus genome to make a modified Flavivirus virus genome, wherein expression of the recoded envelope-coding nucleotide sequence and expression of the recoded nonstructural protein 3 coding nucleotide sequence are reduced compared to the parent virus.

[0115] Various embodiments of the present invention provide methods for making a modified dengue virus genome, comprising obtaining a nucleotide sequence encoding a dengue virus envelope protein and a nucleotide sequence encoding a dengue virus nonstructural 3 protein; recoding the envelope-coding nucleotide sequence to reduce protein expression, and recoding the nonstructural protein 3 coding nucleotide sequence to reduce protein expression, and substituting nucleic acids having the recoded envelope-coding nucleotide sequence and the recoded nonstructural protein 3 coding nucleotide sequence into a parent dengue virus genome to make a modified dengue virus genome, wherein expression of the recoded envelope-coding nucleotide sequence and expression of the recoded nonstructural protein 3 coding nucleotide sequence are reduced compared to the parent virus.

[0116] Various aspects of the present invention provide a method of making a modified dengue virus genome, comprising the steps of obtaining a nucleotide sequence encoding an envelope protein of a dengue virus; recoding the envelope-coding nucleotide sequence to reduce protein expression, and substituting a nucleic acid having the recoded envelope-coding nucleotide sequence into a parent dengue virus genome to make a modified dengue virus genome, wherein expression of the recoded envelope-coding nucleotide sequence is reduced compared to the parent virus.

[0117] According to various aspects of the present invention, virus attenuation is achieved by reducing the expression of viral proteins through codon pair deoptimization of E coding sequence. One way to reduce the expression of coding sequence is to reduce codon pair bias, but other methods can also be used alone or in combination. Although codon bias can be changed, it is particularly advantageous to adjust codon pair bias. For example, attenuating a virus through codon bias generally requires the elimination of common codons, thus reducing the complexity of nucleotide sequence. In contrast, reducing or minimizing codon pair bias can be achieved while maintaining much greater sequence diversity, and consequently greater control over nucleic acid secondary structure, annealing temperature, and other physical and biochemical properties.

[0118] Codon pair bias of protein coding sequences (i.e., open reading frames) is calculated as described above and in Coleman et al., 2008.

[0119] Viral attenuation and induction or protective immune responses can be confirmed in ways well known to those of skill in the art, including but not limited to the methods and assays disclosed herein, non-limiting examples of which include plaque assays, growth measurements, reduced mortality in test animals, and protection from subsequent infection with wild-type virus.

[0120] In various embodiments, the present invention provides viruses that are highly attenuated and induce immunity against multiple dengue types and / or subtypes, including viruses of serogroups 1, 2, 3, and 4. Examples of attenuated dengue protein coding sequences are provided below.

[0121] Table 4. Reduced expression of dengue virus genes TIFF2025041836000011.tif67128

[0122] composition Various aspects provide flavivirus compositions for inducing an immune response in a subject, comprising a modified flavivirus of the invention as described herein.

[0123] Various aspects provide flavivirus vaccine compositions for inducing a protective immune response in a subject, comprising a modified flavivirus of the invention as described herein.

[0124] Various aspects provide a modified dengue virus composition for inducing an immune response in a subject comprising a modified dengue virus of the invention as described herein above and a pharma- ceutically acceptable excipient or carrier.

[0125] Various aspects provide a dengue vaccine composition for inducing a protective immune response in a subject comprising a modified dengue virus of the invention as described herein above and a pharma- ceutically acceptable excipient or carrier.

[0126] In various embodiments, the modified dengue virus is selected from type 1, type 2, type 3, type 4, or a combination thereof.

[0127] In various embodiments, the modified dengue viruses are type 1 and type 2 modified dengue viruses; type 1 and type 3 modified dengue viruses; type 1 and type 4 modified dengue viruses; type 2 and type 3 modified dengue viruses; type 2 and type 4 modified dengue viruses; type 3 and type 4 modified dengue viruses.

[0128] In various embodiments, the modified dengue virus is a type 1, type 2 and type 3 modified dengue virus; a type 1, type 2 and type 4 modified dengue virus; a type 1, type 3 and type 4 modified dengue virus; or a type 2, type 3 and type 4 modified dengue virus.

[0129] In various embodiments, the modified dengue virus is a modified tetravalent dengue virus; (types 1, 2, 3 and 4).

[0130] Non-limiting examples of wild-type and modified dengue viruses are described herein.

[0131] TIFF2025041836000012.tif84165TIFF2025041836000013.tif244165TIFF2025041836000014.tif244165TIFF2025041836000015.tif244165TIFF2025041836000016.tif244165TIFF2025041836000017.tif244165TIFF2025041836000018.tif244165TIFF2025041836000019.tif244165TIFF2025041836000020.tif244165TIFF2025041836000021.tif238165TIFF2025041836000022.tif244165TIFF2025041836000023.tif244165TIFF2025041836000024.tif244165TIFF2025041836000025.tif244165TIFF2025041836000026.tif244165TIFF2025041836000027.tif244165TIFF2025041836000028.tif244165TIFF2025041836000029.tif244165TIFF2025041836000030.tif244165TIFF2025041836000031.tif244165TIFF2025041836000032.tif244165TIFF2025041836000033.tif244165TIFF2025041836000034.tif244165TIFF2025041836000035.tif244165TIFF2025041836000036.tif238165TIFF2025041836000037.tif244165TIFF2025041836000038.tif244165TIFF2025041836000039.tif244165TIFF2025041836000040.tif244165TIFF2025041836000041.tif244165TIFF2025041836000042.tif244165TIFF2025041836000043.tif244165TIFF2025041836000044.tif244165TIFF2025041836000045.tif244165TIFF2025041836000046.tif244165TIFF2025041836000047.t if244165TIFF2025041836000048.tif244165TIFF2025041836000049.tif238165TIFF2025041836000050.ti f244165TIFF2025041836000051.tif244165TIFF2025041836000052.tif244165TIFF2025041836000053.tif 244165TIFF2025041836000054.tif244165TIFF2025041836000055.tif244165TIFF2025041836000056.tif24 4165TIFF2025041836000057.tif244165TIFF2025041836000058.tif244165TIFF2025041836000059.tif244 165TIFF2025041836000060.tif244165TIFF2025041836000061.tif244165TIFF2025041836000062.tif2441 65TIFF2025041836000063.tif244165TIFF2025041836000064.tif238165TIFF2025041836000065.tif24416 5TIFF2025041836000066.tif244165TIFF2025041836000067.tif244165TIFF2025041836000068.tif157165.

[0132] The present invention provides a composition for inducing an immune response in a subject comprising any of the attenuated viruses described herein and a pharma- ceutically acceptable carrier, in various embodiments, the composition is a vaccine composition and the immune response is a protective immune response.

[0133] It should be understood that when the attenuated viruses of the present invention are used to induce an immune response (or a protective immune response) in a subject or to prevent or reduce the likelihood of a subject suffering from a virus-associated disease, they are administered to the subject in the form of a composition additionally comprising a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, one or more of 0.01-0.1M and preferably 0.05M phosphate buffer, phosphate buffered saline (PBS), or 0.9% saline. Such carriers also include aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, saline, and buffered media. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose. Solid compositions may include non-toxic solid carriers, such as glucose, sucrose, mannitol, sorbitol, lactose, starch, magnesium stearate, cellulose or cellulose derivatives, sodium carbonate and magnesium carbonate. For administration in aerosol, such as for pulmonary and / or intranasal delivery, the agent or composition is preferably formulated with a non-toxic surfactant, such as an ester or partial ester of C6-C22 fatty acid or natural glyceride, and a propellant. Additional carriers, such as lecithin, may be included to facilitate intranasal delivery. Pharmaceutically acceptable carriers may further include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, and other additives, such as antimicrobial agents, antioxidants, and chelating agents, which enhance the shelf life and / or effectiveness of the active ingredient. The compositions can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to a subject, as is well known in the art.

[0134] In various embodiments of the compositions or vaccine compositions, the attenuated virus (i) does not substantially alter viral protein synthesis and processing in infected cells; (ii) produces similar amounts of virions per infected cell as the wt virus; and / or (iii) exhibits a substantially lower virion-specific infectivity than the wt virus. In further embodiments, the attenuated virus induces an immune response in a host animal substantially similar to the corresponding wt virus.

[0135] The present invention also provides modified host cell lines that are specifically isolated or engineered to be permissive for attenuated viruses that are not viable in wild-type host cells. In embodiments where the attenuated virus cannot grow in normal (wild-type) host cells, it is absolutely dependent on a specific helper cell line for growth. This provides a very high level of safety for the production of viruses for vaccine production. Various embodiments of the modified cell lines allow the growth of attenuated viruses, and the genome of the cell line has been modified to increase the number of genes encoding rare tRNAs.

[0136] Methods for Inducing an Immune Response Various embodiments provide methods of inducing an immune response in a subject, comprising administering to the subject an effective dose of a composition comprising a modified flavivirus of the invention. Certain embodiments provide methods of inducing a protective immune response in a subject, comprising administering to the subject a prophylactically or therapeutically effective dose of a vaccine composition comprising a modified flavivirus of the invention. In various embodiments, the immune response is cross-protective against heterologous Flaviviruses.

[0137] In various embodiments, the method further comprises administering to the subject at least one adjuvant.Non-limiting examples of adjuvants are discussed herein.Certain embodiments provide a method of inducing an immune response in a subject, comprising administering to the subject a prophylactically or therapeutically effective dose of a vaccine composition comprising the modified dengue virus of the present invention.

[0138] Various embodiments provide a method of eliciting an immune response in a subject, comprising administering to the subject an effective dose of a composition comprising a modified dengue virus of the invention.

[0139] Various embodiments provide a method of eliciting a protective immune response in a subject, comprising administering to the subject a prophylactically or therapeutically effective dose of a vaccine composition comprising a modified dengue virus of the invention.

[0140] In various embodiments, the method further comprises administering to the subject at least one adjuvant, non-limiting examples of which are described herein.

[0141] In various embodiments, the immune response is cross-protective against a heterologous dengue virus.

[0142] Various aspects of the present invention provide a method of inducing an immune response in a subject in need thereof, comprising administering to the subject in need thereof a prime dose of an attenuated flavivirus produced by methods other than codon pair deoptimization or codon deoptimization or the increase in CpG or UpA dinucleotides, or a modified flavivirus, in which the expression of viral proteins is reduced compared to the parent virus, and the reduced expression is the result of recoding the prM or envelope (E) region; and administering one or more boost doses of an attenuated flavivirus produced by methods other than codon pair deoptimization or codon deoptimization or the increase in CpG or UpA dinucleotides, or a modified flavivirus, wherein at least the prime dose or one or more boost doses are modified viruses. In various aspects, the first dose of the one or more boost doses is administered about 2 weeks after the prime dose.

[0143] In various embodiments, the flavivirus is a dengue virus.

[0144] In various embodiments, one or both of the E protein coding sequences are recoded by reducing the codon pair bias or codon usage bias of the protein coding sequence. In various embodiments, reducing the codon pair bias comprises identifying a codon pair in the parent protein coding sequence that has a codon pair score that can be reduced, and reducing the codon pair bias by replacing the codon pair with a codon pair that has a lower codon pair score.

[0145] In various embodiments, reducing codon pair bias comprises rearranging the codons of the parental protein coding sequence. In various embodiments, the E protein coding sequence is recoded by increasing the number of CpG or UpA dinucleotides compared to the parental virus. In various embodiments, each of the recoded prM / E protein coding sequences has a codon pair bias of less than -0.05, or less than -0.06, or less than -0.07, or less than -0.08, or less than -0.09, or less than -0.1, or less than -0.11, or less than -0.12, or less than -0.13, or less than -0.14, or less than -0.15, or less than -0.16, or less than -0.17, or less than -0.18, or less than -0.19, or less than -0.2, or less than -0.25, or less than -0.3, or less than -0.35, or less than -0.4, or less than -0.45, or less than -0.5. In various embodiments, one or both of the E protein coding sequences are recoded by replacing one or more codons with synonymous codons that are less frequent in the viral host (e.g., humans). In some embodiments, the number of codons replaced with synonymous codons is at least 5, or at least 10, or at least 30, or at least 30, or at least 40, or at least 50, or at least 75, or at least 100, or at least 200, or at least 300, or at least 400, or at least 500.

[0146] Additionally, the present invention provides a method of inducing a protective immune response in a subject, comprising administering to the subject a prophylactically or therapeutically effective dose of any of the vaccine compositions described herein. The present invention also provides a method of preventing a subject from contracting a virus-associated disease, comprising administering to the subject a prophylactically effective dose of any of the vaccine compositions. In an embodiment of the above method, the subject is exposed to a pathogenic virus. "Exposed" to a pathogenic virus means contact with the virus such that infection can result.

[0147] The invention further provides a method for delaying the onset or slowing the rate of progression of a virus-associated disease in a subject infected with a virus, comprising administering to the subject a therapeutically effective dose of any of the present vaccine compositions.

[0148] As used herein, "administering" means delivering using any of a variety of methods and delivery systems known to those skilled in the art.Administering can be performed, for example, intranasally, intraperitoneally, intracerebrally, intravenously, orally, transmucosally, subcutaneously, transdermally, intradermally, intramuscularly, topically, parenterally, via implant, intrathecally, intralymphatically, intralesional, pericardially, or epidurally.The agent or composition can also be administered in an aerosol, such as for pulmonary and / or intranasal delivery.Administering can be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0149] Inducing a protective immune response in a subject can be achieved, for example, by administering a first dose of vaccine to the subject, followed by one or more subsequent administrations of the vaccine after a suitable time period.The suitable time period between administrations of the vaccine can be easily determined by those skilled in the art, and is usually on the order of several weeks to several months.However, the present invention is not limited to any particular method, route or frequency of administration.

[0150] In various aspects, the present invention provides a method of inducing an immune response in a subject in need thereof, comprising administering a prime dose of an attenuated flavivirus produced by a method other than codon pair deoptimization or codon deoptimization, or an increase in CpG or UpA dinucleotides, or a modified flavivirus in which expression of viral proteins is reduced compared to the parent virus, the reduced expression being the result of recoding the prM, or the envelope (E) region, or both; and administering one or more boost doses of an attenuated flavivirus produced by a method other than codon pair deoptimization or codon deoptimization, or an increase in CpG or UpA dinucleotides, or a modified flavivirus to a subject in need thereof, wherein at least the prime dose or one or more boost doses are modified viruses.

[0151] In various embodiments, one or more boost doses are administered about 2 weeks after the prime dose. In various embodiments, 2, 3, 4, or 5 boost doses are administered. In various embodiments, the interval between boost doses can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In additional embodiments, the interval between boost doses can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. As a non-limiting example, a prime dose can be administered, followed by a first boost dose about 2 weeks later, a second boost dose about 1 month after the first boost dose, and a third boost dose about 6 months after the second boost dose. As another non-limiting example, a prime dose may be administered, followed about two weeks later by a first boost dose, about six months after the first boost dose, and about twelve months after the second boost dose by a third boost dose. In further embodiments, additional boost doses may be administered periodically, e.g., every five years, every ten years, etc.

[0152] In various embodiments, the flavivirus is a dengue virus. In various embodiments, the flavivirus is selected from the group consisting of dengue virus, Zika virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, Spondweni virus, St. Louis encephalitis virus, and Powassan virus.

[0153] In various embodiments, one or both of the E protein coding sequences are recoded by reducing the codon pair bias or codon usage bias of the protein coding sequence, hi various embodiments, the E protein coding sequence is recoded by increasing the number of CpG or UpA dinucleotides compared to the parent virus.

[0154] In various embodiments, reducing codon pair bias involves identifying codon pairs in a parent protein coding sequence that have a codon pair score that can be reduced, and reducing the codon pair bias by replacing the codon pair with a codon pair having a lower codon pair score.

[0155] In various embodiments, reducing codon pair bias comprises rearranging the codons of the parent protein coding sequence.

[0156] In various embodiments, it comprises an increase in CpG dinucleotides in the modified virus.

[0157] In various embodiments, it comprises an increase in the UpA dinucleotide in the modified virus.

[0158] In various embodiments, the recoded prM / E protein coding sequence has a codon pair bias of less than -0.05, or less than -0.06, or less than -0.07, or less than -0.08, or less than -0.09, or less than -0.1, or less than -0.11, or less than -0.12, or less than -0.13, or less than -0.14, or less than -0.15, or less than -0.16, or less than -0.17, or less than -0.18, or less than -0.19, or less than -0.2, or less than -0.25, or less than -0.3, or less than -0.35, or less than -0.4, or less than -0.45, or less than -0.5, respectively.

[0159] In certain embodiments, the codon pair bias of the recoded prM protein coding sequence, the E protein coding sequence, or both, is reduced by at least 0.05, or at least 0.06, or at least 0.07, or at least 0.08, or at least 0.09, or at least 0.1, or at least 0.11, or at least 0.12, or at least 0.13, or at least 0.14, or at least 0.15, or at least 0.16, or at least 0.17, or at least 0.18, or at least 0.19, or at least 0.2, or at least 0.25, or at least 0.3, or at least 0.35, or at least 0.4, or at least 0.45, or at least 0.5, compared to the parent prM / E protein coding sequence from which it is derived.

[0160] In various embodiments, the prM protein coding sequence, the E protein coding sequence, or both, are recoded by replacing one or more codons with synonymous codons that are less frequent in the viral host. In some embodiments, the number of codons replaced with synonymous codons is at least 5, or at least 10, or at least 30, or at least 30, or at least 40, or at least 50, or at least 75, or at least 100, or at least 200, or at least 300, or at least 400, or at least 500.

[0161] In various embodiments, the prime dose is administered subcutaneously, intramuscularly, intradermally, or intranasally.

[0162] In various embodiments, one or more boost doses are administered intratumorally, intravenously, or intrathecally.

[0163] The timing between the prime dose and the boost dose can vary depending on, for example, the stage of infection or disease (e.g., non-infected, infected, days after infection) and the health condition of the patient.In various embodiments, one or more boost doses are administered about 2 weeks after the prime dose.That is, the prime dose is administered, and the boost dose is administered about 2 weeks later.

[0164] In various embodiments, dosages may vary between the prime and boost doses. As a non-limiting example, the prime dose may contain fewer copies of virus compared to the boost dose.

[0165] In other embodiments, the type of attenuated virus or modified virus of the present invention made by methods other than codon pair deoptimization or codon deoptimization, or the increase in CpG or UpA dinucleotides, may be varied between the prime and boost doses. In one non-limiting example, a modified virus of the present invention may be used in the prime dose, and an attenuated virus (made by methods other than codon pair deoptimization or codon deoptimization, or the increase in CpG or UpA dinucleotides) of the same or different family, genus, species, group, or order may be used in the boost dose.

[0166] In other embodiments, the route of administration may vary between the prime and boost doses. In a non-limiting example, the prime dose may be administered subcutaneously and the boost dose may be administered via injection into the tumor, and for accessible or difficult to access tumors, the boost dose may be administered intravenously.

[0167] Certain embodiments of any of the present immunization and treatment methods further include administering at least one adjuvant to the subject. "Adjuvant" refers to any agent suitable for enhancing the immunogenicity of an antigen and enhancing the immune response in a subject. Numerous adjuvants, including particulate adjuvants, suitable for use with both protein-based and nucleic acid-based vaccines, as well as methods of combining adjuvants with antigens, are well known to those skilled in the art. Suitable adjuvants for nucleic acid-based vaccines include, but are not limited to, Quil A, imiquimod, resiquimod, and interleukin-12 delivered in the form of purified protein or nucleic acid. Suitable adjuvants for use with protein immunization include, but are not limited to, alum, Freund's incomplete adjuvant (FIA), saponin, Quil A, and QS-21.

[0168] The present invention also provides a kit for immunizing a subject with the attenuated virus of the present invention. The kit includes an attenuated virus, a pharma- ceutically acceptable carrier, an applicator, and instructions for its use. In further embodiments, the attenuated virus may be one or more dengue viruses, one or more Japanese encephalitis viruses, one or more West Nile viruses, one or more yellow fever viruses, one or more Zika viruses, etc. More than one virus may be preferred when it is desired to immunize a host against multiple different isolates of a particular virus. The present invention includes other aspects of the kit known to those skilled in the art. Instructions for use may provide any information directing the administration of the attenuated virus.

[0169] Throughout this application, various publications, references, textbooks, technical manuals, patents, and patent applications have been referenced. The entire teachings and disclosures of these publications, patents, patent applications, and other documents are incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. However, the citation of a reference herein should not be construed as an admission that such reference is prior art to the present invention.

[0170] It should be understood and expected that variations in the principles of the invention disclosed herein can be made by those skilled in the art, and such modifications are intended to be included within the scope of the present invention. The following examples further illustrate the present invention, but should not be construed as limiting the scope of the present invention in any way. Detailed descriptions of conventional methods used in, for example, constructing recombinant plasmids, transfecting viral constructs into host cells, polymerase chain reaction (PCR), and immunological techniques can be obtained from numerous publications, including Sambrook et al. (1989) and Coligan et al. (1994). All references mentioned herein are incorporated by reference in their entirety into this application. The contents of WO 2008 / 121992 and WO 2011 / 044561 are incorporated by reference. EXAMPLES

[0171] Example 1 SAVE - Rapid design and construction of a deoptimized live-attenuated dengue vaccine candidate To select the target sequence, we started with all prM-E sequences of all DENV virus isolates from the last 10 years and performed amino acid consensus sequence analysis available in the NCBI Virus Variation Database. Since the consensus sequence is usually never present as a real virus, it is unwise to use it as the basis for our design. We prefer natural virus evolution to teach us what works and what does not. Therefore, the consensus sequence for each serotype was next searched against the entire Genbank using the tblastn algorithm to identify the real genome of the replicating virus that is most identical to the consensus sequence, and we then used this sequence as our target sequence. Additionally, the target sequence must not be rare in the database (a possible sequencing artifact), and if it is rare, we select the sequence with the next highest identity to the consensus represented by two or more independent virus isolates. This search algorithm to determine the most related and replicative human isolates results in sequences that are (1) authentic human isolates and (2) as homologous to the rest of the DENV sequence space as a single sequence can be. Using this approach, we have identified the following viruses to select target prM-E antigenic sequences: DENV-1 / VN / BID-V1774 / 2007, DENV-2 / NI / BID-V533 / 2005, DENV-3 / VE / BID-V2268 / 2008, DENV-4 / US / BID-V2448 / 1999. We have used the E-Min of DENV-2 / NI / BID-V533 / 2005 to initiate a DENV vaccine program and have generated live attenuated strains containing the other three E genes selected through this process (Figure 1). The CPB of the wild-type prM / E genes falls within the normal range of genes in human host cells.After SAVE-mediated codon pair "deoptimization," the resulting deoptimized prM / E gene segments are predominantly encoded by underrepresented human codon pairs, as evidenced by their extremely negative CPBs, and are dramatically different from any human genes. The SAVE-deoptimized synthetic prM / Es from FIG. 1 were de novo synthesized and individually subcloned into the WT DENV genome using overlapping PCR to obtain six independent cDNA genomes, each containing a synthetically "deoptimized" fragment. We constructed infectious cDNA genomes of wt DENV1-4 and then recovered fully infectious replicating viruses for deoptimized DENV vaccine candidates via RNA transfection into Vero or BHK cells.

[0172] Example 2 Leveraging the flexibility of the SAVE platform to generate second-generation dengue virus vaccine candidates To fine-tune attenuation and immunogenicity, a second-generation dengue virus vaccine candidate was constructed using the SAVE platform (Figure 2). The second-generation dengue vaccine candidate leverages the flexibility of the SAVE platform to reduce the deoptimized region from 2014 bp (E-min) to 997 bp (WE-Min) or even to 664 bp (WWE-Min) while keeping the amino acid sequence 100% identical in the homologous backbone.

[0173] Example 3 Growth of a heterologous backbone dengue vaccine candidate in Vero cells under animal component-free conditions To optimize manufacturing conditions for future cGMP manufacturing and in vitro attenuation testing, DENV1-4 E-Min was used to infect Vero cells under animal component-free conditions at an MOI of 0.01 and supernatants were titrated daily in a multi-step growth curve over the course of 10 days post-infection. DENV2, DENV3, and DENV4 candidates were titrated at 1-2 × 10 5 Although the titer reached 100 FFU / ml, the titer of DENV1 E-Min was approximately 1 log lower than the others.10 was reduced (Figure 3).

[0174] Example 4 Growth of a homologous skeletal dengue virus type 1 vaccine candidate in Vero cells and testing temperature sensitivity as a mechanism of attenuation Growth kinetics performed at low (33°C), intermediate (37°C), and high (39°C) temperatures were used to optimize manufacturing conditions for the deoptimized candidates and tested for in vitro attenuation, examining CPD variants for increased temperature sensitivity as a means of explaining the mechanism of attenuation. Multi-step growth curves were performed in Vero cells for the synthetic DENV1 WT virus (in the full-length DENV1 backbone) and the DENV1 WT-derived live attenuated vaccine candidates DENV1 EW / MIN and E-MIN. Synthetic DENV1 WT grows well in Vero cells at 33°C and 37°C. Typical for DENV, a transient reduction in virus yield was observed at 39°C, but titers recovered to 37°C levels by 7 dpi. DENV1 EW / MIN, however, reached usable titers about 10-fold lower than DENV1 WT at both 33°C and 37°C. DENV1 E-MIN was highly attenuated in Vero cells and only reached detectable titers at 33°C from days 10–14 postinfection.

[0175] Example 5 Growth of a homologous skeletal dengue virus type 2 vaccine candidate in Vero cells and testing temperature sensitivity as a mechanism of attenuation Growth kinetics performed at low (33°C), intermediate (37°C), and high (39°C) temperatures were used to optimize manufacturing conditions for the deoptimized candidates and tested for in vitro attenuation, examining CPD variants for increased temperature sensitivity as a means of delineating the mechanism of attenuation. Multi-step growth curves were performed in Vero cells for the synthetic DENV2 WT virus (in the full-length homologous DENV2 backbone) and the attenuated live vaccine candidates DENV2 EW / MIN and E-MIN derived from DENV2 WT. Both DENV2 EW / MIN and E-MIN were attenuated in vitro, with a 1-2 log increase for EW / MIN. 10 Reduction in FFU / ml, and more significantly, 2-3 log for DENV2 E-MIN 10 The growth kinetics was slowed, but the virus titer was >10 for DENV2 EW / MIN by regular medium changes every 1-2 days. 6 FFU / ml. Importantly, attenuation correlated with the degree of deoptimization in the E region. Temperature sensitivity (difference in titers at 37°C and 39°C) was similar for DENV2-WT and DENV2 EW / MIN through 4 days post-infection, but there were significant differences on days 5, 6, and 7. No temperature sensitivity was observed for DENV2 E-MIN, as there was no replication at any temperature through 6 DPI. However, from days 7 to 14, DENV2 E-MIN began to produce detectable virus (but only at 33°C and 37°C).

[0176] Example 6 Growth of a homologous skeletal dengue virus type 3 vaccine candidate in Vero cells and testing temperature sensitivity as a mechanism of attenuation Growth kinetics performed at low (33°C), intermediate (37°C), and high (39°C) temperatures were used to optimize manufacturing conditions for the deoptimized candidates and tested for in vitro attenuation, examining CPD variants for increased temperature sensitivity as a means of explaining the mechanism of attenuation. Multi-step growth curves were performed in Vero cells for the synthetic DENV3 WT virus (in the full-length DENV3 backbone) and the DENV3 WT-derived live-attenuated vaccine candidates DENV3 EW / MIN and E-MIN. It was apparent that DENV3 EW / MIN was more temperature sensitive than WT at 39°C, with significantly greater differences observable at days 3, 4, 5, and 7 post-infection. DENV3 E-MIN had minimal levels of replication in Vero cells at 39°C, so the difference in virus yield from 37°C to 39°C was 10. 7 However, when grown at permissive temperatures, DENV3 E-MIN reached high titers promising for cGMP manufacturing.

[0177] Example 7 Growth of a homologous skeletal dengue virus type 4 vaccine candidate in Vero cells and testing temperature sensitivity as a mechanism of attenuation Growth kinetics performed at low (33°C), intermediate (37°C), and high (39°C) temperatures were used to optimize manufacturing conditions for the deoptimized candidates and tested for in vitro attenuation, examining CPD variants for increased temperature sensitivity as a means of delineating the mechanism of attenuation. Multi-step growth curves were performed in Vero cells for the synthetic DENV4 WT virus (in the full-length DENV4 backbone) and the live-attenuated vaccine candidates DENV4 EW / MIN and E-MIN derived from DENV4 WT. Both DENV4 WT and EW / MIN grew to high titers in Vero cells, with peak DENV4 EW / MIN titers being approximately 10-fold lower at 33°C and 37°C but still high (approximately 10 7Both viruses were partially restricted at 39°C, but not to the extent seen with DENV1-3. A small but significant (approximately 5-fold) change in titer was observed from 37°C to 39°C compared to WT at day 5. DENV4 E-MIN was attenuated in Vero cells, with peak titers 100-fold lower than WT. Replication of DENV4 E-MIN at 33°C and 37°C was similar, but DENV4 E-MIN was not viable at 39°C and no virus was detectable from days 1 to 14 postinfection.

[0178] Example 8 Evaluation of attenuation, immunogenicity, and efficacy of DENV2 vaccine candidates in AG129 mice As mentioned above, the AG129 adult mouse model has been accepted for the study of flavivirus vaccines and therapeutics. We used AG129 mice to: (1) 6 (1) each synthetically derived wild-type virus DENV (positive control) at a dose of 10 4 and 10 6Vaccine candidates were tested at 10 ... Viral RNA was detected in 55% (5 / 9) of animals immunized with the higher inoculum of DENV-2 16681 and in 11% (1 / 9) of animals given the lower inoculum, but not in any of the mice immunized with DENV-2 E-min. The RT-PCR primers used do not target the viral E gene, which is codon-deoptimized in this virus, and they successfully amplified the DENV-2 E-min viral RNA included in the assay. As a result, the failure to detect viremia in this study is unlikely to be the result of suboptimal RT-PCR amplification, indicating that the vaccine virus produced very low levels of viremia in vivo. Once the immunization regimen was completed, serum was collected from all mice prior to viral challenge. All immunized animals had detectable DENV-2 neutralizing antibody titers prior to viral challenge with D2S10.Scatter plots of the data for each vaccine group demonstrate that there was an increase in neutralizing antibody titers with increasing vaccine dose for both DENV-2 E-min and DENV-2 16681, and that the titers seen in E-min immunized mice were lower than those in mice immunized with the corresponding dose of DENV-2 16681. Animals in the medium control group developed a rapidly progressive infection and all animals died. Mice immunized with the low dose of DENV-2 E-min vaccine were not protected, experiencing high mortality (67%) and mean days to death (MDD), which was not significantly different from that seen in the medium control. In contrast, animals immunized with the high dose of DENV-2 E-min vaccine and both doses of DENV-2 16681 experienced significant protection from DENV-2 D2S10 challenge. There were no fatalities or morbidity as indicated by loss of more than 10% of body weight in any of these groups during the 30-day observation period. As expected, high levels of viral RNA were detected in the serum of all medium control animals on both sampled days. Viral RNA was also detected in all of the animals immunized with the lower dose of E-min on both days. The levels in these animals were comparable to those in the controls on day 2, but significantly lower on day 3. For animals immunized with the high dose of Emin, viral RNA was detected in the serum of 2 / 4 animals on day 2, and RNA levels in viremic animals were significantly lower than controls. On day 3, viral RNA was not detected in the serum of any of the high dose E-min animals. Overall, immunization with the high dose of Emin significantly reduced the number of animals with detected viremia (2 / 8 vs. 8 / 8; p<0.01). For mice immunized with DENV-2 16681, viral RNA was not detected in the serum from any of the animals at either immunization dose on any of the two sampled days.

[0179] Example 9 Titration of codon pair deoptimization leads to improved DENV2 vaccine candidates in AG129 mice To improve the clinical relevance of our DENV2 candidates, we next constructed D2-E-min and D2-1 / 2-E-min with E sequences derived from DENV-2 / NI / BID-V533 / 2005 in the heterologous DENV2 16681 backbone. Since previous studies have shown that the degree of attenuation can be titrated in DENV2 by reducing the CPD sequence, we tested D2-1 / 2-E-min with an E region consisting of half the wt DENV-2 / NI / BID-V533 / 2005 sequence and half the CPD to improve the immunogenicity of our virus. 10 6 or 10 4 Vaccination with FFU of D2-E-min, D2-1 / 2-E-min, or D2-WTE (DENV2 16681 backbone carrying the WT DENV-2 / NI / BID-V533 / 2005 E region). Interestingly, introduction of the experimental DENV-2 / NI / BID-V533 / 2005 E region resulted in increased virulence, with a 10 6 or 10 4 All AG129 mice infected with FFU required euthanasia. No mortality or morbidity was observed in infected BALB / c mice or in any of the CPD DENV2 groups.

[0180] Reduction of the CPD sequence in the E region was associated with a significant, approximately 4-fold increase (p=-.0002, Student's t-test) in the PRNT50 values ​​of sera from D2-1 / 2-E-min compared to D2-E-Min vaccinated AG129 mice. Additionally, high neutralizing antibody titers were maintained with D2-1 / 2-E-min vaccination and were increased by 10 6 (GM=3447) and 10 4 (GM=2867) No significant differences were observed between the FFU vaccinated groups. In immune-competent BALB / c mice, both D2-E-min and D2-1 / 2-E-min produced levels of neutralizing antibodies comparable to wild type.

[0181] Example 10 Evaluation of immunogenicity and protective efficacy of DENV3 vaccine candidates in AG129 mice AG129 mice were used to test the immunogenicity and protective efficacy of DENV-3 vaccine candidate DENV D2 / D3-E-min, which contains the DENV-2 strain 16681 backbone with a codon-deoptimized prME cassette from DENV-3 / VE / BID-V2268 / 2008. This study was designed to evaluate the immunogenicity and efficacy of this candidate vaccine against challenge with virulent DENV-3 CO360 / 94. The first immunization was well tolerated by all animals, with no significant associated weight loss and no animals developing clinical signs. Similar results were seen after the second immunization, but unexpectedly, Group 3 (10 6 One animal with FFU DENV D2 / D3-WT developed rapid progressive weight loss and became moribund necessitating euthanasia on day 2 after immunization. A second animal from the same group then unexpectedly died 2 weeks after immunization and prior to challenge with virulent DENV-3. 7 After lethal challenge with PFU DENV-3 CO360 / 94, animals in the medium control group developed a rapidly progressive infection that was fatal in 8 / 9 animals, with a mean day of death (MDD) of 4.0 ± 0.8 days for animals that died. Immunization with the higher inoculum of DENV D2 / D3-E-min provided significant protection, with no mortality or morbidity (as measured by >10% weight loss) in animals in the group. Immunization with the lower inoculum of DENV D2 / D3-E-min was also protective, with only a single animal developing a fatal infection. Animals given DENV D2 / D3-WT virus prior to DENV-3 challenge were also protected, with no mortality in either group, although two animals in the higher inoculum group lost >10% of their original body weight.

[0182] Example 11 Evaluation of immunogenicity and protective efficacy of DENV4 vaccine candidates in AG129 mice AG129 mice (n=12) were used to administer 10 mg of 10 ...6 The immunogenicity and protective efficacy of heterologous D2 / D4-E-min and D2 / D4-1 / 2-E-Min viruses were tested at doses of FFU compared to D2 / D4-WTE (n=12) and medium-immunized mock control mice (n=9). All mice were immunized on days 0 and 21 of the study, and sera were collected on day 2 to determine viremia and on day 35 for titration of neutralizing antibodies. On day 35, 10 7 Mice were challenged by intraperitoneal injection of PFU DENV4 703 / 4. Challenged mice were weighed and monitored for morbidity to assess the impact of viral load. Additionally, five animals from each group were anesthetized and blood was collected by retro-orbital bleeding on day 2 post-challenge, and the remaining animals had viremia levels determined by qRT-PCR on day 3. The study was terminated 30 days post-challenge, and serum was collected from all surviving animals for determination of post-challenge PRNT50 values. The first immunization with all strains was well tolerated by animals in all groups, with no sustained weight loss over the 5-day observation period, except for one mouse in the D2 / D4-WTE group that had to be euthanized prior to the second immunization, and there was an additional animal death after the second immunization. No animals in any of the other vaccine groups experienced progressive weight loss. At 2 days post-challenge (DPC), viremia was detected in 4 / 12 mice inoculated with D2 / D4-WTE. In all mice immunized with the other two viruses, viremia levels were below the detection limit of the assay (500 genome equivalents / ml). DENV-4 703 / 4 challenge produced a rapidly progressive infection that was uniformly lethal in the medium control group. Additionally, three animals immunized with D2 / D4-E-min experienced fatal infections, while the other animals in this group remained healthy for the duration of the study. Importantly, there was no lethality or evidence of morbidity as determined by weight loss in any of the D2 / D4-1 / 2-E-min immunized animals following DENV-4 703 / 4 challenge.

[0183] Example 12 Immunogenicity of homologous DENV1-4 vaccine candidates in A129 mice We tested DENV1-4 WT as well as DENV2-4 vaccine candidates for immunogenicity in IFNα receptor knockout mice. All WT viruses were highly immunogenic in these mice, with neutralizing antibody titers >10. DENV2-EW / MIN was 10 6 and 10 4 DENV3 EW / MIN was equally immunogenic to DENV2 WT virus at both doses of FFU. The immunogenicity of DENV3 virus diminished with decreasing dose and increasing deoptimization, whereas DENV3 EW / MIN was attenuated at 10 4 The DENV4 EW / MIN and WT were still highly immunogenic at doses of 10 FFU (FRNT50 of approximately 1024). We noted a significant improvement in immunogenicity of DENV4 EW / MIN and WT compared to the heterologous DENV4 WT in the DENV2 16681 backbone, which was poorly immunogenic in A129 mice.

[0184] Example 13 Quadrivalent vaccination in AG129 mice and efficacy against DENV3 challenge AG129 mice, which are homozygous for a double knockout of the IFNα / β and IFNγ receptors, are used to test flavivirus vaccine candidates due to their increased susceptibility to infection. We used AG129 mice to inoculate them with 10% IFNα / β and IFNγ at days 0 and 21. 6 IFU monovalent D2 / D3-E-min vaccine (N=16; 10 males and 6 females), 10 6We tested our tetravalent formulation of DENV vaccine by vaccinating mice with the tetravalent vaccine (N=16; 10 males and 6 females) containing IFU of each of the four monovalent DENV E-min viruses, or with medium (N=13; 8 males and 5 females). Both immunizations were well tolerated by all animals, and none of the animals developed clinical signs or significant weight loss (≥10%). Serum was collected prior to challenge for measurement of neutralizing antibodies by FRNT50. All of the mice immunized with the monovalent vaccine developed detectable neutralizing antibody titers (range 20-160). In mice immunized with the tetravalent vaccine formulation, titers were generally lower (range <20-80). On day 35, all remaining mice were vaccinated with 10 mAb delivered by intraperitoneal route. 7 Immunization with monovalent DENV D2 / D3-E-min or quadrivalent vaccines provided significant protection, with no mortality or morbidity (as measured by >10% weight loss) in animals in either group. Viral RNA was detected in 8 / 9 control animals, with titers comparable on days 2 and 3 post-challenge.

[0185] Example 14 In vivo LD50 data for the tetravalent and each strain of the homologous vaccine demonstrates neuroattenuation. Week-old mice were injected with 10 μl inoculum containing synthetic wt DENV1-4 virus as indicated doses. Each mouse was weighed daily for up to 2 weeks, and death was determined by humane early endpoints (lack of weight gain, paresis, hind limb paralysis) whenever possible. TIFF2025041836000069.tif52128

[0186] Various aspects of the present invention are described above in the detailed description. Although these descriptions directly describe the above aspects, it is understood that those skilled in the art can imagine modifications and / or variations to the specific aspects shown and described herein. Any such modifications or variations that fall within the scope of this specification are also intended to be included herein. Unless otherwise stated, it is the intention of the inventors that the words and phrases in the specification and claims are given the ordinary and customary meaning to those skilled in the applicable technical field.

[0187] The above description of various aspects of the invention known to the applicant at the time of filing this application is presented and is intended for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, as many modifications and variations are possible in light of the above teachings. The described aspects serve to explain the principles of the invention and its practical application, and to enable those skilled in the art to utilize the invention in various aspects and with various modifications as appropriate for the particular use envisioned. Therefore, it is not intended that the invention be limited to the specific aspects disclosed for carrying out the invention.

[0188] While particular embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that, based on the teachings herein, changes and modifications may be made without departing from the present invention and its broader aspects, and therefore, the appended claims encompass within their scope all such changes and modifications as are within the true spirit and scope of the present invention. In general, it will be understood by those skilled in the art that the terms used in this specification are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including but not limited to," etc.).

[0189] As used herein, the term "comprising" or "comprises" is used in reference to compositions, methods, and their respective components that are useful for an embodiment, but is open to the inclusion of unspecified components, whether useful or not. In general, it will be understood by those skilled in the art that the terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including but not limited to," etc.). Although the open-ended term "comprising" as a synonym for terms such as including, containing, or having is used herein to describe and claim the present invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as "consisting of" or "consisting essentially of."

[0190] Sequence information SEQUENCE LISTING <110> CODAGENIX INC. <120> ATTENUATED DENGUE VIRUSES <150> US 62 / 866,477 <151> 2019-06-25 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 10735 <212> DNA <213> Dengue virus <400> 1 agttgttagt ctacgtggac cgacaagaac agtttcgaat cggaagcttg cttaacgtag 60 ttctaacagt ttttattag agagcagatc tctgatgaac aaccaacgga aaaagacggc 120 tcgaccgtct ttcaatatgc tgaaacgcgc gagaaaccgc gtgtcaactg tttcacagtt 180 ggcgaagaga ttctcaaaag gattgctctc aggccaagga cccatgaaat tggtgatggc 240 tttcatagca ttcctaagat ttctagccat acccccaaca gcaggaattt tggctagatg 300 gggttcattc aagaagagtg gagcgatcaa agtgctacgg ggtttcaaga aagaaatctc 360 aaacatgttg aataatga atagaaggaa aagatctgtg accatgctcc ttatgctgat 420 gcctacagcc ttggcgttcc atttgactac acgaggggga gagccgcaca tgatagtcag 480 caagcaggaa agaggaaagt cactcttgtt taagacctca gcaggtgtca acatgtgcac 540 ccttatagcg atggatttgg gagagttatg tgaggacaca atgacttaca aatgccctcg 600 aatcactgaa actgaaccag atgacgttga ttgttggtgt aatgccacag acacatgggt 660 gacctatgga acatgttccc aaactggcga gcaccgacga gacaaacgtt ccgtcgcact 720 ggccccacac gtgggacttg gtttggaaac aagaaccgaa acgtggatgt cctctgaagg 780 cgcttggaaa cagatacaa gagtggagac ttggccctg agacacccag gattcacggt 840 gatagccctt ttcttagcac atgccatagg aacatccatc acccagaaag ggattatttt 900 cattttgtta atgctggtaa caccatccat ggccatgcga tgcgtggga taggcagcag 960 ggactcgtg gaaggactgt caggagcaac ttgggtagat gtggtactgg aacatggaag 1020 ttgcgtcacc accatggcaa agacaacc aacattggac attgactct tgagacgga 1080 agtcacaaac cctgccgtcc tgcgcaact gtgcattgaa gctaaaatat caacaccac 1140 caccgactca agatgtccaa caaggaga agccacacta gtggagaac aagacgcgaa 1200 ctttgtgtgt cgacgaacgt tgtggacag aggctggggc aatggctgtg gcctcttcgg 1260 aaaaggaagc ctaataacgt gtgcaagtt caagtgtgtg aaaactgg aaggaagat 1320 agttcaatt gagaacttga atattcagt atagtcacc gttcacaccg gagaccagca 1380 tcaagtggga atgaaagca cagaacatgg cakaactgca actatacac ctcaagctcc 1440 tacgacggaa atacagctga ccgactacgg agctcttaca tggattgtt cacctagaac 1500 aggactagac tttaatgaaa tggtgttgtt gatagaaa gaaaatcat ggctagtcca 1560 caaacaatgg ttttagacc taccactgcc ttggactcg ggagctca catcacaaga 1620 gacttggaac agacagatt tgctggtgac atttagaca gctcatgcaa agaagcagga 1680 agtagtcgta ctaggatcac aagaaggagc aatgcacact gcgttgaccg gagcgacaga 1740 aatccaaacg tctgaacga tgcaattt tgcaacc tgaatgca tgcaagat 1800 ggacaaactg actctaaag ggatgtcata tgtgatgtgc acagctcat tcaagctaga 1860 gaagaagtg gctgagaccc agcatggaac cgttctagtg cagattaat acgaggaac 1920 agatgcacca tgcagatcc ctttttcgac ccagatgaa aaggagtaa cccagaatgg 1980 gagattgata acagctacc ccatagttac tgacaagaa aaaccagtca acatgaggc 2040 agaaccgccc ttggtgaga gttacatcgt ataggagca ggtgaaaag ctttgaaact 2100 aagctggttc aagaaggaa gcagcatagg gaaatgttt gaggcaactg ccagaggagc 2160 acgaaggatg gccatactgg gagacaccgc atgggacttt ggttctatag gaggagtgtt 2220 cacgtctgtt ggaaaattag tacaccagat ttttggact gcatatggag tttgttcag 2280 cggtgtttcc tggaccatga aaataggaat aggttctg ctgacatggc tgggattaa 2340 ctcaggagc acgtcccttt cgatgacgtg cattgcagtt ggcctagtaa cactatacct 2400 aggagtcatg gttcaggcgg attcaggatg tgtattaat tggaaggta gagaactca 2460 gtgtggaagt ggcattttg tcaccaatga agttcacact tggacagc atacaatt 2520 tcaagctgac tcccctaaga gactcagc agccatcggg aaggcatggg aggaggttgt 2580 gtgtggaatt cgatcagcaa ctcgtctcga gaacatcatg tggagcaaa tatcaatga 2640 actgaatcac atcttacttg aaatgatat gaattcaca gtggttgtag gagatgttgc 2700 tgggatttg gctcaggaa agaaatgat taggccacaa cccatggaat acaatactc 2760 gtggaaaagc tggggaagg ctaaaatcat aggggcagat gtacagaaca ccaccttcat 2820 catcgatggc ccaacaccc cagaatgccc tgatgaccaa agagcatgga acatttggga 2880 agttgaggac tattggattg gattttcac gandaacata tggctgaat tgcgtgattc 2940 ctacacccaa gtgtgtgacc accggctaat gtcagctgcc atcaggaca gcaggcagt 3000 tcacgctgac atggggtact ggatagaaag tgaaagac gagacctgga agctggcaag 3060 agcctcattc atagaagtta aaacatgtat ctggccaaaa tcccactc tatggagcaa 3120 tggagttctg gaagtgaaa tgataattcc aagatctat ggaggaccaa tatctcagca 3180 caactacaga ccaggatatt ttacacaagc agcaggggccg tggcacctag gcaagttgga 3240 actggatttt gatttgtgtg agggtaccac agttgttgtg gatgaacatt gtggaaatcg 3300 aggaccatct cttaggacca cacagtcac aggaagata attcatgaat ggtgttgcag 3360 atcttgcacg ctaccaccct tacgtttcag aggagagat gggtgctggt acggtatgga 3420 aatcagacca gtcaggaaa aggaaaaa tctagtcaa tcaatggtct ctgcaggggtc 3480 3540 gatgagatcc agatggagta gaagaatgct gatgactgga acactggctg tgttctctcct 3600 tctcataatg ggacaattga catggaacga tctgatcaga ttgcatca tggttggagc 3660 caacgcttcc gacaggatgg ggatgggaac gacgtaccta gccctgatgg ccacttttaa 3720 aatgagaccg atgttcgctg tagggctatt atttcgcaga ctaacatcca gagaagttct 3780 tcttctaaca attggattga gtctagtggc atctgtggag ttaccaaatt ccttggagga 3840 gctgggggat ggacttgcaa tgggcattat gattttaaaa ttattgactg actttcaatc 3900 acatcagctg tgggctacct tgctgtcctt gacatttatc aaaacaacgt tttccttgca 3960 ctacgcatgg aagacaatgg ctatggtact gtcaattgta tctctcttcc ctttatgcct 4020 gtccacgacc tcccaaaaaa caacatggct tccggtgcta ttgggatccc ttggatgcaa 4080 accactaacc atgtttctta tagcagaaaa caaaatctgg ggaaggagaa gttggcccct 4140 caatgaagga atcatggctg ttggaatagt cagcatccta ctaagttcac tcctcaaaaa 4200 tgatgtaccg ctagctgggc cactaatagc tggaggcatg ctaatagcat gttatgttat 4260 atctggaagc tcagccgacc tatcattaga gaaagcggct gaggtctcct gggaagaaga 4320 agcagaacac tctggtgcct cacacaacat attagtggaa gtccaagatg atggaaccat 4380 gagataag gatgagaga gagatgacac gctaccatt ctccttaag caacctgtt 4440 agcagttca ggggtgtacc cattatcaat accagcgacc ctttcgtgt ggtacttttg 4500 gcagaaaaag aaacagagat ctggagtgtt atgggacaca cccagccctc cagaagtgga 4560 aagagcagtt cttgatgatg gtatctatag aattatgcag agaggactgt tgggcaggtc 4620 ccaagtagggg gtaggagttt tccaagaaaa cgtgttccac acatgtggc atgtcaccag 4680 gggagctgta ctcatgtatc aagggaag attggaaccg agctgggcca gtgtcaaaaa 4740 agacctgatc tcatatggag gaggttggag gcttcagga tcctggaca caggagaga 4800 agtgcaggtg attgctgttg aaccagggaa aaccccaaa atgtacaa cagcgccggg 4860 caccttaag acccctgaag gtgaagttgg agccattgcc ctagacttta aacctggcac 4920 atctggatct cccatcgtga acagagaagg aaaatagta ggtctttatg gaatggagt 4980 agtgacaca agtggaacct acgtcagtgc catagctca gccaaagcat cacaagaagg 5040 gcccctacca gagattgaag acgaggtgtt taggaaag aacttaaca taatggacct 5100 acatccagga tcgggggaaaa caagaagata tcttccagcc atagtccgtg aggccataaa 5160 aaggaagctg cgcacactaa tcctggctcc cacaagggtt gtcgcttccg aaatggcaga 5220 ggcgctcaag ggaatgccaa taaggtacca aacaacagca gtgaagagtg aacatacagg 5280 aaaagagata gttgacctca tgtgtcacgc cactttcacc atgcgcctcc tgtctcccgt 5340 aagagttccc aattacaaca tgatcatcat ggatgaagca catttcaccg atccatccag 5400 tatagcggcc agagggtaca tctcaacccg agtgggcatg ggtgaagcag ctgcaatctt 5460 catgacagcc actcccccag gatcagtgga ggcctttcca cagagcaacg cagttatcca 5520 agatgaggaa agagacattc ctgagagatc atggaactca ggctatgagt ggatcactga 5580 cttcccaggt aaaacagttt ggtttgttcc aagcattaaa tcaggaaatg acattgccaa 5640 ctgcttaaga aagaatggga aacgggtgat tcaattgagc aggaaaacct ttgatacaga 5700 gtaccaaaaa acaaaaaaca acgactggga ctacgtcgtc acaacagaca tctccgaaat 5760 gggagcaaat ttccgagccg acagggtgat agacccaaga cggtgtctga aaccggtaat 5820 actaaaagat gtccagagc gtgtcatttt agcaggacca atgccagtga ctgtggccag 5880 tgccgctcag aggaggaa gattggaag gaaccacaat aaggaggtg atcagtacat 5940 ctacatggga cagcctttaa acacgatga agatcacgct cactggacag aagcaaaat 6000 gctccttgat ataataaca caccagaagg gatttacccca gccctctttg agccagagag 6060 agaaaagagt gcagcaatag acgggaata cagactgcgg ggtgaagcaa ggaaaacgtt 6120 tgtggagctc atgagagag gagatctacc tgtctggcta tcctacaag tgcctcaga 6180 aggcttccag tactctgaca gagatggtg ctttgacggg gaaggaca accaggtgtt 6240 ggaggagac atggacgtgg agatctggac aaaagaagg gaagaaga aactacgacc 6300 ccgctggctg gatgccagaa catactcaga cccactagcc ctgcgcgagt ttaaagagtt 6360 tgcagcagggg agaagaagcg tctcaggtga tttaatatta gaaataggga agcttccaca 6420 acacttgacg caaagggccc agaatgccct ggacaacctg gttatgttgc acaactccga 6480 aaaggaggc agagcctata gatgcaat ggaagaactg ccagacacca tagaacgtt 6540 gatgctccta gctttgatag ctgtgttaac tggtggagtg acactgttct tcctatcagg 6600 aaggggccta gggaaaacat ctatcggcct actctgcgta atggcttcaa gcgtactgct 6660 atggatggcc agtgtggagc cccattggat agcggcctcc atcatactgg agttcttcct 6720 gatggtgctg cttattccag agccagacag acaacgcact ccgcaggaca accagctggc 6780 atatgtggtg ataggtttgt tattcatgat actgacagta gcagccaatg agatgggact 6840 gctggaaacc acaaagaaag acttagggat tggccatgtg gctgttgaaa atcaccacca 6900 tgccgcaatg ctggacgtag acttacatcc agcttcagcc tggaccctct atgcagtggc 6960 cacaacaatt atcactccca tgatgaggca cacaatcgaa aacacaacgg caaacatttc 7020 cctgacagcc attgcaaacc aggcagctat attgatggga cttgacaaag gatggccaat 7080 atcgaagatg gacataggag ttccacttct cgccttgggg tgctattccc aggtgaatcc 7140 actgacgctg acagcggcgg tattgatgct agtggctcat tacgccataa ttggacctgg 7200 actgcaagca aaagctacta gagaagctca aaaaaggaca gcggccggaa taatgaaaaa 7260 tccaaccgtt gatggaattg ttgcaataga tttggaccct gtggtttatg atgcaaaatt 7320 tgaaaaaaaa ctaggccaaa taatgttgtt gatactgc acatcacaga tcctcttgat 7380 gcggactaca tgggccttgt gcgagtccat cacactggcc actggacctc tgaccacgct 7440 ttgggaggga tctccaggaa aattttggaa caccacgata gcggttttcca tggcaacat 7500 ttcagagga agttatctag caggagcagg tctggccttc tcattaatga aatctctagg 7560 aggaggtagg agaggcacgg gagcccagg ggaacactg ggagagaat ggaaaagaca 7620 gctgaaccaa ctgagcaagt cagaatttaa cacctataaa aggagtggga ttggaagt 7680 ggacagatcc gaagccaag agggattga aagaggaaaaccaaac atgcagtgtc 7740 gagaggaacc gctaactga ggtggttcgt gaggaggaac cttgtgaac cagaagggaa 7800 agtcatagac ctcggttgtg gaagaggtgg ctggtcatac tattgcgctg ggctgaagaa 7860 agtcacagaa gtgaaggggt atacaaagg aggacctgga catgaggaac caatcccaat 7920 ggcgacctat ggatggaacc tagtaagct gcactctggg aaagacgtat ttttattacc 7980 acctgagaaa tgtgacacccc ttttgtgtga tattggtgag tcctctccaa acccaactat 8040 agaggaagga agaacgctac gcgtcctaaa gatggtggaa ccatggctca gaggaaacca 8100 atttgcata aaaattctga atccctacat gccaagtgtg gtggaaactc tggagcaaat 8160 gcaaagaaaa catggaggaa tgctagtgcg aaatccactt tcaagaaatt ctactcatga 8220 aatgtattgg gtttcatgtg gaacaggaaa cattgtgtca gcagtaaaca tgacatctag 8280 aatgttgcta aatcgattca caatggctca caggaaacca acatatgaaa gagacgtgga 8340 cctaggcgcc ggaacaagac atgtggcagt ggaaccagag gtagccaacc tagatatcat 8400 tggccagagg atagagaaca taaaacatga acataagtca acatggcatt atgatgagga 8460 caatccatat aaaacatggg cctatcatgg atcatatgag gtcaagccat caggatcagc 8520 ctcatccatg gtcaatggcg tggtgaaact gctcaccaaa ccatgggatg tcatccctat 8580 ggtcacacaa atagccatga ctgacactac accctttgga caacagaggg tgtttaaaga 8640 gaaagttgac acacgcacac caaaagcaaa acggggcaca gcacaaatca tggaggtgac 8700 agccaagtgg ttatggggtt ttctttctag aaacaagaaa ccaagaattt gcacaagaga 8820. ggagttcaca agaaaagtta ggtcaaacgc agccattgga gcagtgttcg ttgatgaaaa tcaatggaac tcagcaaaag aagcagtgga agatgagcgg ttctgggacc ttgtgcatag aggagggag cttcacaaac agggaaaatg tgctacgtgt gtttacaaca tgatgggga gagagagaa aagctaggag agttcggaa ggcaaaagga agtcgtgcaa tatggtacat gtggttggga gcacgctttc taggttcga agctcttggt ttcatgaacg aagatcactg gttcagcaga gagaattcac tcagcggagt ggaggaga ggactccaca aacttggata 9180. the father of the gacatatcaa agattccagg gggaaacatg the father of the acacagccgg atgggataca aggataacag aggatgacct tcagaatgag gccagaatta ctgacatcat ggaacccga catgccctcc tggctaagtc aatcttcaag ttaacctacc aaaataaggt ggtaagggta cagagaccag caaaaaatgg aaccgtgatg gatgtcatat ccagacgtga ccagagga agtggtcagg tcggaactta tggcttaaac actttcacca acatggaagc ccagctgata agacaaatgg agtctgaggg aatcttttca cccagcgaat tagagacccc aaatttagcc gagagagttc tcgactggct ggaaaaatat ggcgtcgaaa ggctgaaaag aatggcaatc agcggagatg actgcgtagt gaaaccaatt gatgataggt ttgcaacagc cttgacagct ctgaatgata tgggaaaagt aagaaaagat ataccacaat gggaaccttc aaaaggatgg aatgattggc aacaagtgcc tttttgttca caccacttcc accagctgat tatgaaggat gggagggaaa tagtggtgcc atgccgcaac caagatgaac ttgtgggtag ggctagagta tcacaaggtg ctggatggag cctgagaga actgcatgcc tagcaagtc atatgcacaa atgtggcagc tgatgtactt ccacaggaga gacctgagac tagccgctaa tgctatctgt tcagccgttc cagttgattg gatcccaacc agccgtacca cctggtcgat 9960. ccatgcccat caccaatgga tgacaacaga agacatgctg tcagtgtgga atagggtttg gatagagga aacccatgga tggaggaca aacccacata tccagttggg gagatgttcc attack aaaagggaag accaatggtg tggatccctg attackctaa cagcaagggc cacctgggcc accacatac aagtggccat aaaccaagtg agagacta ttgggaatga 10200 catcatcta gattacatga catcatgaa gagatcaag aacgagagtg atcccgaagg 10260 ggcactctgg tgagtcaaca catttacaa aaaggaaa ataagaatc aaaaaggca 10320 agaagtcagg ccggattaag ccatagtacg gtaagagcta tgctgcctgt gagccccgtc 10380 taaggacgta aaatgaagtc aggccgaaag ccacggcttg agcaaccgt gctgcctgta 10440 gctccatcgt ggggatgtaa aaacctggga ggctgcacc catggaagct gtacgcatgg 10500 ggtagcagac tagtggttag aggagacccc tccgaaaca taacgcagca gcggggccca 10560 acaccagggg aagctgtacc ctggtgtaa ggactagagg ttagaggaga cccccgcat 10620 aacaataaac agcatattga cgctgggaga gaccagagat cctgctgtct ctacagcatc 10680 attccaggca cagaacgcca gaaaatggaa tggtgctgtt gatcacag gttct 10735 <210> 2 <211> 10735 <212> DNA <213> Dengue Virus <400> 2 agttgttagt ctacgtggac cgacagaac agtttcgaat cggaagcttg cttaacgtag 60 ttctacagt tttttag agagcagatc tctgatgaac aaccaacgga aaagacggc 120 tcgaccgtct ttcaatgc tgaacgcgc gagaaccgc gtgtcaactg ttcacagtt 180 ggcgaagaga ttctcaaag gattgctctc aggccaagga cccatgaat tggtgatggc 240 ttcatagca ttcctaagat tttagccat acccccaaca gcaggaattt tggctagatg 300 gggttcattc aagagagtg gagcgatcaa agtgctacgg ggtttcaaga aagaaatctc 360 aaacatgttg atataatga atagaggaa aagatctgtg accatgctcc ttatgctgat 420 gcctacagcc ttggcgttcc atttgactac acgaggggga gagccgcaca tgagtcag 480 caagcaggaa agaggaaagt cactcttgtt tagacctca gcaggtgtca acatgtgcac 540 ccttatagcg atggattgg gagagttatg tgaggacaca atgacttaca atgccctcg 600 aatcactgaa actgaaccag atgacgttga tgttggtgt aatgccacag accatgggt 660 gacctatgga acatgttccc aaactggcga gcaccgacga gandaacgtt ccgtcgcact 720 ggccccacac gtgggacttg gtttggaac aagaaccgaa acgtggatgt cctctgaagg 780 cgcttggaaa cagatacaa gagtggagac ttggccctg agacacccag gattcacggt 840 gatagccctt ttcttagcac atgccatagg aacatccatc acccagaaag ggattatttt 900 cattttgtta atgctggtaa caccatccat ggccatgcga tgcgtagg taggtcacg 960 cgatttcgtc gaaggactat ccggagcgac atgggtcgac gtcgtactcg aacacggatc 1020 atgcgttacg actatgcta agacaacc tacactagac atagagttac tgaaaaccga 1080 agttacgaat cccgccgtac tgcgaaaatt gtgtacgaa gcgaaaatta gcaatacgac 1140 tacagactct aggtgtccta cacaggcga agcgacattg gtcgagac aggacgcta 1200 cttcgtatgt agacgaacat tcgtcgatag ggggtgggga aacggatgcg gattgttcgg 1260 taaaggatca ctgattacat gcgcaagtt aaatgcgtt acgaactgg aaggtaaat 1320 agtgcaatac gaaaacctaa agtatagcgt atcgttaca gtgcataccg gagaccaaca 1380 tcaggtcgga aacgaatcaa ccgaacacgg aaaaccgca acattacac cacaggcacc 1440 tacaaccgaa attcaactaa ccgattacgg agctctttaca ctagattgct cacctagaac 1500 cggattggac tttaacgaaa tggtactgtt gactatgaag gagaaatcat ggttagtgca 1560 1620 aacatggaat agacaggacc tattggtgac atttaagacc gcacacgcta agaagcagga 1680 agtcgtagtg ttagggtcac aagagggagc aatgcatacc gcactaaccg gagcaaccga 1740 1800 ggaaactg acacttaaag gaatgtcata cgttatgtgt acgggatcct ttaagctcga 1860 aaaagaggtt gccgaaacgc aacacggaac agtgctagtg caaattaaat atgagggaac 1920 cgacgcacca tgtaagatac cgtttagtac cgaagacgaa aagggcgtta cgcaaaacgg 1980 aagactgatt accgctaacc ctatcgttac agaaagag aaacccgtta acatagaggc 2040 cgaaccacct tttggcgaat catatatagt gataggcgca ggcgaaaaag cactaaagct 2100 gtcatggttc aaaaaaggat ctagcatagg gaagatgttc gaagcaaccg ctaggggagc 2160 tagacgaatg gcaatactgg gagataccgc atgggacttc ggatcgatag ggggagtgtt 2220 tactagcgta ggtaagttgg tgcatcagat attcggaacc gcatacggag tgttgtttag 2280 cggagtgtca tggactga agataggt aggagtgcta tgacatggc tcggactgaa 2340 ttcgagatcg actagcctat ctatgacatg catagccgtc ggactggtta cactgtatct 2400 aggcgtaatg gtgcaagcag attcaggatg tgtattaat tggaaggta gagaactca 2460 gtgtggaagt ggcattttg tcaccaatga agttcacact tggacagc atacaatt 2520 tcaagctgac tcccctaaga gactcagc agccatcggg aaggcatggg aggaggttgt 2580 gtgtggaatt cgatcagcaa ctcgtctcga gaacatcatg tggagcaaa tatcaatga 2640 actgaatcac atcttacttg aaatgatat gaattcaca gtggttgtag gagatgttgc 2700 tgggatttg gctcaggaa agaaatgat taggccacaa cccatggaat acaatactc 2760 gtggaaaagc tggggaagg ctaaaatcat aggggcagat gtacagaaca ccaccttcat 2820 catcgatggc ccaacaccc cagaatgccc tgatgaccaa agagcatgga acatttggga 2880 agttgaggac tattggattg gattttcac gandaacata tggctgaat tgcgtgattc 2940 ctacacccaa gtgtgtgacc accggctaat gtcagctgcc atcaggaca gcaggcagt 3000 tcacgctgac atggggtact ggatagaaag tgaaagac gagacctgga agctggcaag 3060 agcctcattc atagaagtta aaacatgtat ctggccaaaa tcccactc tatggagcaa 3120 tggagttctg gaagtgaaa tgataattcc aagatctat ggaggaccaa tatctcagca 3180 caactacaga ccaggatatt ttacacaagc agcaggggccg tggcacctag gcaagttgga 3240 actggatttt gatttgtgtg agggtaccac agttgttgtg gatgaacatt gtggaaatcg 3300 aggaccatct cttaggacca cacagtcac aggaagata attcatgaat ggtgttgcag 3360 atcttgcacg ctaccaccct tacgtttcag aggagagat gggtgctggt acggtatgga 3420 aatcagacca gtcaggaaa aggaaaaa tctagtcaa tcaatggtct ctgcaggggtc 3480 3540 gatgagatcc agatggagta gaagaatgct gatgactgga acactggctg tgttctctcct 3600 tctcataatg ggacaattga catggaacga tctgatcaga ttatgcatca tggttggagc 3660 caacgcttcc gacaggatgg ggatgggaac gacgtaccta gccctgatgg ccacttttaa 3720 aatgagaccg atgttcgctg tagggctatt atttcgcaga ctaacatcca gagaagttct 3780 tcttctaaca attggattga gtctagtggc atctgtggag ttaccaaatt ccttggagga 3840 gctgggggat ggacttgcaa tgggcattat gattttaaaa ttattgactg actttcaatc 3900 acatcagctg tgggctacct tgctgtcctt gacatttatc aaaacaacgt tttccttgca 3960 ctacgcatgg aagacaatgg ctatggtact gtcaattgta tctctcttcc ctttatgcct 4020 gtccacgacc tcccaaaaaa caacatggct tccggtgcta ttgggatccc ttggatgcaa 4080 accactaacc atgtttctta tagcagaaaa caaaatctgg ggaaggagaa gttggcccct 4140 caatgaagga atcatggctg ttggaatagt cagcatccta ctaagttcac tcctcaaaaa 4200 tgatgtaccg ctagctgggc cactaatagc tggaggcatg ctaatagcat gttatgttat 4260 atctggaagc tcagccgacc tatcattaga gaaagcggct gaggtctcct gggaagaaga 4320 agcagaacac tctggtgcct cacacaacat attagtggaa gtccaagatg atggaaccat 4380 gaagataaag gatgaagaga gagatgacac gctaaccatt ctccttaaag caactctgtt 4440 agcagtttca ggggtgtacc cattatcaat accagcgacc cttttcgtgt ggtacttttg 4500 gcagaaaaag aaacagagat ctggagtgtt atgggacaca cccagccctc cagaagtgga 4560 aagagcagtt cttgatgatg gtatctatag aattatgcag agaggactgt tgggcaggtc 4620 ccaagtaggg gtaggagttt tccaagaaaa cgtgttccac acaatgtggc atgtcaccag 4680 gggagctgta ctcatgtatc aagggaagag attggaaccg agctgggcca gtgtcaaaaa 4740 agacctgatc tcatatggag gaggttggag gcttcaagga tcctggaaca caggagaaga 4800 agtgcaggtg attgctgttg aaccagggaa aaaccccaaa aatgtacaaa cagcgccggg 4860 cacctttaag acccctgaag gtgaagttgg agccattgcc ctagacttta aacctggcac 4920 atctggatct cccatcgtga acagagaagg aaaaatagta ggtctttatg gaaatggagt 4980 agtgacaaca agtggaacct acgtcagtgc catagctcaa gccaaagcat cacaagaagg 5040 gcccctacca gagattgaag acgaggtgtt taggaaaaga aacttaacaa taatggacct 5100 acatccagga tcgggggaaaa aaagagata tcttccagcc atagtccgtg aggccataaa 5160 aaagctg cgcacactaa tcctggctcc cacaagggtt gtcgcttccc aaatggcaga 5220 ggcgctcaag ggaatgccaa taagtcacca aacaacagca gtgaagagtg aacatacagg 5280 aaaagagata gttgacctca tgtgtcacgc cactttcacc atgcgcctcc tgtctcccgt 5340 aagagttccc aattacaaca tgatcatcat ggatgaagca catttcaccg atccatccag 5400 tatagcggcc agagggtaca tctcaacccg agtgggcatg ggtgaagcag ctgcaatctt 5460 catgacagcc actcccccag gatcagtgga ggcctttcca cagagcaacg cagttatcca 5520 agatgagaa agagacattc ctgagagatc atggaactca ggctatgagt ggatcactga 5580 cttcccaggt aaaacagttt ggtttgttcc aagcattaaa tcaggaaatg acattgccaa 5640 ctgcttaaga aagaatggga aacgggtgat tcaattgagc aggaaaacct ttgatacaga 5700 gtaccaaaaa acaaaaaaca acgactggga ctacgtcgtc acaacagaca tctccgaaat 5760 gggagcaaat ttccgagccg acagggtgat agacccaga cggtgtctga aaccggtaat 5820 actaaaagat gtccagagc gtgtcatttt agcaggacca atgccagtga ctgtggccag 5880 tgccgctcag aggaggaa gattggaag gaaccacaat aaggaggtg atcagtacat 5940 ctacatggga cagcctttaa acacgatga agatcacgct cactggacag aagcaaaat 6000 gctccttgat ataataaca caccagaagg gatttacccca gccctctttg agccagagag 6060 agaaaagagt gcagcaatag acgggaata cagactgcgg ggtgaagcaa ggaaaacgtt 6120 tgtggagctc atgagagag gagatctacc tgtctggcta tcctacaag tgcctcaga 6180 aggcttccag tactctgaca gagatggtg ctttgacggg gaaggaca accaggtgtt 6240 ggaggagac atggacgtgg agatctggac aaaagaagg gaagaaga aactacgacc 6300 ccgctggctg gatgccagaa catactcaga cccactagcc ctgcgcgagt ttaaagagtt 6360 tgcagcagggg agaagaagcg tctcaggtga tttaatatta gaaataggga agcttccaca 6420 acacttgacg caaagggccc agaatgccct ggacaacctg gttatgttgc acaactccga 6480 acaaggaggc agagcctata gacatgcaat ggaagaactg ccagacacca tagaaacgtt 6540 gatgctccta gctttgatag ctgtgttaac tggtggagtg acactgttct tcctatcagg 6600 aaggggccta gggaaaacat ctatcggcct actctgcgta atggcttcaa gcgtactgct 6660 atggatggcc agtgtggagc cccattggat agcggcctcc atcatactgg agttcttcct 6720 gatggtgctg cttattccag agccagacag acaacgcact ccgcaggaca accagctggc 6780 atatgtggtg ataggtttgt tattcatgat actgacagta gcagccaatg agatgggact 6840 gctggaaacc acaaagaaag acttagggat tggccatgtg gctgttgaaa atcaccacca 6900 tgccgcaatg ctggacgtag acttacatcc agcttcagcc tggaccctct atgcagtggc 6960 cacaacaatt atcactccca tgatgaggca cacaatcgaa aacacaacgg caaacatttc 7020 cctgacagcc attgcaaacc aggcagctat attgatggga cttgacaaag gatggccaat 7080 atcgaagatg gacataggag ttccacttct cgccttgggg tgctattccc aggtgaatcc 7140 actgacgctg acagcggcgg tattgatgct agtggctcat tacgccataa ttggacctgg 7200 actgcaagca aaagctacta gagaagctca aaaaaggaca gcggccggaa taatgaaaaa 7260 tccaaccgtt gatggaattg ttgcaataga tttggaccct gtggtttatg atgcaaaatt 7320 tgaaaaaaaa ctaggccaaa taatgttgtt gatactgc acatcacaga tcctcttgat 7380 gcggactaca tgggccttgt gcgagtccat cacactggcc actggacctc tgaccacgct 7440 ttgggaggga tctccaggaa aattttggaa caccacgata gcggttttcca tggcaacat 7500 ttcagagga agttatctag caggagcagg tctggccttc tcattaatga aatctctagg 7560 aggaggtagg agaggcacgg gagcccagg ggaacactg ggagagaat ggaaaagaca 7620 gctgaaccaa ctgagcaagt cagaatttaa cacctataaa aggagtggga ttggaagt 7680 ggacagatcc gaagccaag agggattga aagaggaaaaccaaac atgcagtgtc 7740 gagaggaacc gctaactga ggtggttcgt gaggaggaac cttgtgaac cagaagggaa 7800 agtcatagac ctcggttgtg gaagaggtgg ctggtcatac tattgcgctg ggctgaagaa 7860 agtcacagaa gtgaaggggt atacaaagg aggacctgga catgaggaac caatcccaat 7920 ggcgacctat ggatggaacc tagtaaagct gcactctggg aaagacgtat tttttatacc 7980 acctgagaaa tgtgacacccc ttttgtgtga tattggtgag tcctctccaa acccaactat 8040 agaggaagga agaacgctac gcgtcctaaa gatggtggaa ccatggctca gaggaaacca 8100 atttgcata aaaattctga atccctacat gccaagtgtg gtggaaactc tggagcaaat 8160 gcaaagaaaa catggaggaa tgctagtgcg aaatccactt tcaagaaatt ctactcatga 8220 aatgtattgg gtttcatgtg gaacaggaaa cattgtgtca gcagtaaaca tgacatctag 8280 aatgttgcta aatcgattca caatggctca caggaaacca acatatgaaa gagacgtgga 8340 cctaggcgcc ggaacaagac atgtggcagt ggaaccagag gtagccaacc tagatatcat 8400 tggccagagg atagagaaca taaaacatga acataagtca acatggcatt atgatgagga 8460 caatccatat aaaacatggg cctatcatgg atcatatgag gtcaagccat caggatcagc 8520 ctcatccatg gtcaatggcg tggtgaaact gctcaccaaa ccatgggatg tcatccctat 8580 ggtcacacaa atagccatga ctgacactac accctttgga caacagaggg tgtttaaaga 8640 gaaagttgac acacgcacac caaaagcaaa acggggcaca gcacaaatca tggaggtgac agccaagtgg ttatggggtt ttctttctag aaacaagaaa ccaagaattt gcacaagaga 8820. ggagttcaca agaaaagtta ggtcaaacgc agccattgga gcagtgttcg ttgatgaaaa tcaatggaac tcagcaaaag aagcagtgga agatgagcgg ttctgggacc ttgtgcatag aggagggag cttcacaaac agggaaaatg tgctacgtgt gtttacaaca tgatgggga gagagagaa aagctaggag agttcggaa ggcaaaagga agtcgtgcaa tatggtacat gtggttggga gcacgctttc taggttcga agctcttggt ttcatgaacg aagatcactg gttcagcaga gagaattcac tcagcggagt ggaggaga ggactccaca aacttggata 9180. the father of the gacatatcaa agattccagg gggaaacatg the father of the acacagccgg atgggataca aggataacag aggatgacct tcagaatgag gccagaatta ctgacatcat ggaacccga catgccctcc tggctaagtc aatcttcaag ttaacctacc aaaataaggt ggtaagggta cagagaccag caaaaaatgg aaccgtgatg gatgtcatat ccagacgtga ccagagga agtggtcagg tcggaactta tggcttaac actttcacca acatggaagc ccagctgata agacaaatgg agtctgaggg aatcttttca cccagcgaat tagagacccc aaatttagcc gagagagttc tcgactggct ggaaaaatat ggcgtcgaaa ggctgaaaag aatggcaatc agcggagatg actgcgtagt gaaaccaatt gatgataggt ttgcaacagc cttgacagct ctgaatgata tgggaaaagt aagaaaagat ataccacaat gggaaccttc aaaaggatgg aatgattggc aacaagtgcc tttttgttca caccacttcc accagctgat tatgaaggat gggagggaaa tagtggtgcc atgccgcaac caagatgaac ttgtgggtag ggctagagta tcacaaggtg ctggatggag cctgagaga actgcatgcc tagcaagtc atatgcacaa atgtggcagc tgatgtactt ccacaggaga gacctgagac tagccgctaa tgctatctgt tcagccgttc cagttgattg gatcccaacc agccgtacca cctggtcgat 9960. ccatgcccat caccaatgga tgacaacaga agacatgctg tcagtgtgga atagggtttg gatagagga aacccatgga tggaggaca aacccacata tccagttggg gagatgttcc atattagga aaagggaag accaatggtg tggatccctg ataggcttta cagcaagggc 10140 cacctgggcc accacatac aagtggccat aaaccaagtg agagacta ttgggaatga 10200 catcatcta gattacatga catcatgaa gagatcaag aacgagagtg atcccgaagg 10260 ggcactctgg tgagtcaaca catttacaa aaaggaaa ataagaatc aaaaaggca 10320 agaagtcagg ccggattaag ccatagtacg gtaagagcta tgctgcctgt gagccccgtc 10380 taaggacgta aaatgaagtc aggccgaaag ccacggcttg agcaaccgt gctgcctgta 10440 gctccatcgt ggggatgtaa aaacctggga ggctgcacc catggaagct gtacgcatgg 10500 ggtagcagac tagtggttag aggagacccc tccgaaaca taacgcagca gcggggccca 10560 acaccagggg aagctgtacc ctggtgtaa ggactagagg ttagaggaga cccccgcat 10620 aacaataaac agcatattga cgctgggaga gaccagagat cctgctgtct ctacagcatc 10680 attccaggca cagaacgcca gaaaatggaa tggtgctgtt gatcacag gttct 10735 <210> 3 <211> 10725 <212> DNA <213> Dengue Virus <400> 3 agttgttagt ctacgtggac cgacaagac agattctttg agggagctaa gctcaacgta 60 gttctacag ttttaatt agagagcaga tctctgatga ataccaacg aaaaaaggcg 120 agaagtacgc ctttcaat gctgaaacgc gagagaaacc gcgtgtcaac tgtgcaacag 180 ctgacaaga gattctcact tggaatgctg caaggacgcg gaccattaaa actgttcatg 240 gcccttgtgg cgttccttcg tttcctaaca atcccacca cagcagggat actaaaaga 300 tggggaacga tcaaaaatc aaagctatc aatgttttga gaggttcag gaagagatt 360 ggaaggatgc tgacatctt gaacaagaga cgcaggacag caggcgtgat tgttatgttg 420 attccaacag cgatggcgtt ccattaacc acacgcaatg gagaaccaca catgatcgtt 480 ggtaggcagg agaaagggaa aagtcttctg ttcaaacag aggatggtgt taacatgtgt 540 actctcatgg ccatagacct tggtgaattg tgtgaagata caatcacgta caagtgtcct 600 ctcctcagac aaaatgacc agagacata gattgttggt gcacttac gtccacatgg 660 gtaacttatg ggacatgtac caccacagga gaacacagaa gagaaaaag atcagtggcg 720 ctcgttccac atgtaggtat gggactggag acacgaactg aaacatggat gtcatcagaa 780 ggggcctgga aacatgttca gagaattgaa acctggatct tgagacatcc aggttttacc 840 ataatggcag caatcctggc atacaccata ggaacgacac atttccaag ggccttgatt 900 ttcattttac tgacagctgt cgctccttca atgacaatgc gctgcatagg atatcaaat 960 agagacttcg aggaggtt ttcaggagga agctggttg acatagtctt agaacatgga 1020 agttgtgtga cgacgatggc aaaaaaaaaaacattgg attttgaact gataaaaaaca 1080 gaagccaaac aacctgccac tctaggaag tactgtatag aagcaagct gaccacaca 1140 acacagaat cgcgttgccc aacacaaggg gaacccagtc taatgagga gcaggacaaa 1200 aggttcatct gcaacactc catggtagac agaggatggg gaatggatg tggatttt 1260 ggaaagggag gcattgtgac ctgtgctatg tttacatgca aaagaacat ggaaggaaaa 1320 gtcgtgcagc cagaaaattt ggaatacacc atcgtgataa cacctcactc aggaggagg 1380 cacgctgtag gtaatgacac aggaaagcat ggcaaggaaa tcaaaaataac accacagagc 1440 tccatcacag aagcagaact gacaggctat ggcactgtca cgatggagtg ctctccgaga 1500 acgggcctcg acttcaatga gatggtactg ctgcagatgg aagaacaaagc ttggctggtg 1560 cacaggcaat ggttcctaga cctgccgtta ccatggctac ccggagcgga cacaagga 1620 tcaaattgga tacagaaaga gacattggtc actttcaaaa atccccacgc gaaaacag 1680 gatgtcgttg tcttagggtc tcaagaaggg gccatgcaca cggcactcac aggggccaca 1740 gaaatccaga tgtcatcagg aaacttactg ttcacaggac atctcaagtg caggctgaga 1800 atggacaaac tacagctcaa aggaatgtca tactctatgt gtacaggaaa gtttaaaaatt 1860 gtgaaggaaa tagcagaaac acaacatgga acaatagtta tcagagtaca atatgaaggg 1920 gacggttctc catgcaagat cccttttgag ataacagatt tggaaaaaag acacgtctta 1980 ggtcgcttga ttacagttaa cccaatcgta acagaaaaag atagcccagt caacatagaa 2040 gcagaacctc cattcggaga cagctacatc attataggag tagagccggg aaattgaaa 2100 ctcaatttggt ttaagaaggg aagttccatc ggccaatgt ttgagacac atgagaga 2160 gcaagagaa tggccattt aggtgacaca gctgggact ttggatccct gggaggagtg 2220 tttacatcta taggaagggc tctccaccaa gtttcggag caatctatgg ggctgctttt 2280 agtggggtct catggactat gaaaatccctc ataggagtca tcatcacatg gatgaatg 2340 aattcacgta gcacctcact gtctgtgtcg ctagtattgg tgggcgttgt jagactgtac 2400 ctgggagcta tggtgcaagc tgagtggt tgcgttgtga gctggaaaaa taaagaactg 2460 aaatgtggca gcgggatctt catcacagat aacgtacaca catggacaga acatatatag 2520 ttccaaccag aatccccttc aaaactagct tcagctatcc aaaaagctca tgagaggggc 2580 atttgtggaa tccgctcagt aacagattg gagaatctga tgtggaaaaacacca 2640 gattgaatc atattctatc agaaatgag gtaagttga ccattatgac aggacatt 2700 agaggaatca tgcaggcagg aaacgatcc tgcggcccc agcccactga gctgaagtac 2760 tcatggaaaa catggggaaa ggcgaaaatg ctctccacag agtctcacaa tcagaccttt 2820 cttattgatg gccctgaac agcagaatgc cccacacaa acagagccctg gaactcgctg 2880 gaagttgaag actatggttt tggagttttc accaccaata tattggctgaa attgagagaa 2940 aaacaggatg tattttgtga ctcaaactc atgtcagcgg ccattaaga cacagagcc 3000 gttcatgctg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatggag 3060 aaagcctcct tcattgaagt taaagctgc cactggccaa agtcacac ccttggagc 3120 aatggagtat tagaagtga gatgataatc ccaaaaaattt ttgccgggcc agtgtcacaa 3180 cacaactaca gaccaggcta ccatacacaa acagcaggac cttggcatct aggtaagctt 3240 gagatgact tgatctctg cgaggact acagtggtgg tgactgagga ctgtggaat 3300 agaggaccct ctttaagac gaccactgcc tctggaaac tcatacaga atggtgctgc 3360 cgatcctgca cactaccacc tctaagatac agaggtgagg atggatgctg gtacgggatg 3420 gaatcagac cattgaagaga gaagagag aatttggtca actccttggt cacagccgga 3480 catgggcaga ttgacaactt ttcactagga gtcttgggaa tggcactgtt cctggaagaa 3540 atgctcagga cccgaatagg aacgaaacat gcaatactgc tagttgcagt atcttttgtg 3600 acattgatta ctgggaacat gtcttttaga gacctgggaa gagtgatggt tatggtgggc 3660 gctaccatga cggatgacat aggtatggga gtgacttatc ttgccctact agcagctttt 3720 aaggttagac caacttttgc agctggacta ctcttaagaa aactgacctc caaggaattg 3780 3840 gaactgactg atgcattagc cctgggcatg atggtcctca aaatagtgag aaatatggaa 3900 aaataccaat tggcagtgac tatcatggct atttcatgtg tcccaaatgc agtgatactg 3960 caaaacgcat ggaaggtgag ttgcacaata ttggcagcgg tgtccgtttc accactgctc 4020 ttaacatcct your car agcggattgg ataccactgg cattgacgat aaaaggtctc 4080 aatccaacag ccattttttt aacaactctc tcgaggacca gcaagaaaag gagctggccg 4140 ctaaatgaag ctatcatggc agttgggatg gtgagcattt tagccagttc tctcctaaag 4200 aatgatattc ctatgacagg tccattagtg gctggaggac tcctcaccgt atgttacgtg 4260 ctcactggac gatcggccga tttggaactg gagagagctg ccgatgtaaa atgggaagat caggcagaaa tatcaggaag cagcccaatc ctgtcaata caatatcaga agatggcagc atgtcgataa aaaatgaaga ggaagaacaa acactgacca tactcatcag aacgggattg ttggtgatct caggagtctt tccagtatcg ataccaatta cggcagcagc atggtacctg 4560. tgggagtaa agaaacaacg ggctggagta ctgtgggacg tcccttcacc cccaccagtg gaaaaagccg aactggagga tggagcctac agaatcaagc aaagagggat ccttggatat tctcagattg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcacgtcaca cgtggtgctg ttctgatgca taggaggag aggattgac catcatgggc agatgtcaag aaagacctaa tatcatatgg aggaggctgg aagctagaag gagaatgga ggaaggagag gagtccag tcctggcatt gagtgga aaaaatcca gagccgtcca aacgaaacct ggaatattca aaaccaacac cggaaccata ggcgccgtat ctctggactt ttcccctgga acgtcaggat ctccaatcgt cgacagaaaa ggaaaagttg tgggtcttta cggtaatggt gttgtcacaa ggagtggagc atatgtaagt gctatagccc agaccgaaaa aagcattgaa 5040 gacaatccag agatcgaaga tgacatttc cgaaagaaaa gattgaccat catggacctc 5100 catccaggag caggaaagac aaaaagatac cttccagcca tagttagaga agccataaaa 5160 cgtggcttga gaacattgat cctggctccc actagagtag tggcagctga aatggaggaa 5220 gctcttagag gacttccaat aagataccaa actacagcca tcaaaaccga gcataccggg 5280 cgggagatcg tggacctaat gtgtcatgcc acatttacta tgaggctgtt atcaccagtc 5340 agagtgccaa attacaacct gatcatcatg gacgaagccc acttcacaga cccagcaagt 5400 atagcagcta gaggatacat ttcaactcga gtagagatgg gtgaagcagc cgggattttc 5460 atgacagcca ctcctccggg aagtagac ccatttcctc agagcaatgc accaattatg 5520 gatgaggaaa gagaaatccc tgagcgttca tggaattcag gacacgaatg ggtcacggat 5580 tttaagggga agactgtttg gtttgttcca agtataaaag caggaaatga tatagcagct 5640 tgtcttagga aaaatggaaa gaaagtgata caactcagta ggaagacttt tgactctgag 5700 tatgttaaga ctagagccaa tgattgggac tttgtggtca caactgacat ttcagaaatg 5760 ggtgccaact tcaaggctga gagggttata gaccccagac gttgcatgaa accagttata 5820 ctaacagatg gcgaggagcg ggtgatcttg gctggaccta tgccagtgac ccactctagt 5880 gcagcgcaaa gaagggag aataggaaga aatccaaaaa atgaaaatga ccagtacata 5940 6000 ctcctagata acatcaacac acctgaagga atcattccta gtatgttcga accagagcgt 6060 gaaaaagtgg atgccattga tggtgaatac cgtttgagag gagaagcaag gaaaaccttt 6120 gtggacctaa tgagaagagg ggacttacca gtctggttgg cctacaaagt ggcagctgaa 6180 ggcatcaact acgcagacag aaagtggtgt tttgatggaa ttaagaacaa ccaaatactg 6240 gaaaaata tggaaggtgga aatctggaca aaagaagggg aaaggaaaaa attaaaaccc 6300 agatggttgg atgctaggat ctattctgac ccactagcac taaaagaatt caaggaattt 6360 gcagctggaa gaaaatcttt gaccctgaac ctaatcacag aaatgggtag gcttccaact 6420 ttcatgactc agaaagcaag aaacgcactg gacaacctgg ctgtgctgca tacggctgag 6480 gcaggtggaa gggcgtacaa tcatgctctc agtgaactgc cggagaccct ggagacactg 6540 ctcctactga cacttctggc aacagtcaca ggaggaatct tcttattctt aatgagcgga 6600 aaaggtatag ggaagatgac cctgggaatg tgttgcataa tcacggctag tatcctccta 6660 tggtatgcac agatacaacc acactggata gcagcttcaa taatactgga gttttttctc 6720 atagttttgc tcattccaga accagaaaaa cagagaacac cccaagacaa ccaattgacc 6780 tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840 ctggaaaaaa ccaagaaaga cctcggattg ggaagcatta caacccagga atctgagagc 6900 aatatcctgg acatagatct acgccctgca tcagcatgga cgctgtatgc cgtagctaca 6960 acatttgtca caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtctcccta 7020 acagccattg ctaaccaagc tacagtgcta atgggtcttg ggaaaggatg gccattgtca 7080 aagatggaca ttggagttcc cctccttgcc attggatgct attcacaagt caaccctata 7140 actctcacag cagctctcct tttattgta gcacattatg ccattatagg gccaggactt 7200 caaghaaag aaccaaaaaaaaaaaaaaaaaccaaaaaaaaacca 7260 acagtcgatg gataacagt gattgaccta gaaccatac cctatgatcc aaaatttgaa 7320 aagcagttag vakaagtaat gctcctaatc ctctgcgtga ctcaagtatt atgatgagg 7380 actacatggg ctttatgtga ggctctacc ctagcgaccg ggcccactc cacactgtgg 7440 gaaggaaatc cagggaggtt ttggacact accattgcag tgtcaatggc tacacacttt 7500 agggggagct acttggccgg agctggactt ctctttcca tcatgaaaaa cacaacaac 7560 acagagag gaactggcaa cataggagag acacttggag aaaatggaa aagtcgatta 7620 aacgcactgg ggaaaagtga atttcaaatc tacaagaaa gtggaatcca ggaagtggat 7680 agaaccctag caaagaagg tatcaaaga ggagaaacgg accaccatgc tgtgtcgcga 7740 gttcagcaa aactgagatg gttcgtcgag agaatatgg tcacaccgga agggaggtg 7800 gtggatctcg gttgcggcag agggggctgg tcatactatt gcgggggact aaagaatgta 7860 agagaagtca aaggcctaac aaaaggagga ccaggacatg aagaacccat ccccatgtca 7920 acatagggt ggaatctagt gcgtctgcaa agtggagttg acgttttctt caccccgcca 7980 gaaaagtgtg atacattgtt gtgtgacata ggggagtcgt caccaaatcc cacgatagaa 8040 gcaggacgaa cactcagagt cctcactta gtggaaatt ggttgacaa taacaccca 8100 ttttgcataa aggtcctcaa cccatatatg ccttcagtca tagaaaaat ggaagcatta 8160 haaggaaat atggaggagc cttagtgagg aatccactct cacgaactc cacgcatgaa 8220 atgtactggg tatctaatgc caccgggaac atagtgtcat cagtgaacat gatttcagg 8280 atgttgatta acagattcac aatgaacac aagaaagcca cttacgagcc agatgttgac 8340 ctaggaagtg gaacccgcaa cattggaatt gaagtgaga taccaatct agacatata 8400 ggaagagaa tagagaaat aaaaaaaaacag catgaaacat catggcacta tgatxagac 8460 cacccataca aaacgtgggc ttaccatggc agctatgaaaaaaaaac tggatcagca 8520 tcatctatgg tgacggagt ggtcagatg ctgacaaac cttgggacgt cgtccctatg 8580 gtgacacaga tggcaatgac agacacgact ccttttggac aacagcgcgt ttcaagag 8640 aaagtagaca cgagaaccca agaaccgaag gaggcaca agaactgat gaaaattacg 8700 gcagagtggc tttggaaga actaggaaag aaaagacac ctaggatgtg taccagagaa 8760 gattcacaa gaaagtgag aagcaatgca gccttgggggg ccatattcac tgatgagaac 8820 aaatggaaat cggcacgtga ggctgttga gatggtaggt tctgggagct ggttgacagg 8880 gaaagaaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940 agagagaga aactagggga gttcggcaag gcaaaggta gcagagccat atggtacatg 9000 tggcttggag cacgctctt agagtttga gccctaggat tcctgaatga agatcactgg 9060 ttctccagag ggaactccct gagtggagtg gaaggagaag ggctgcacag gctaggctac 9120 attttagag acgtgggcaa gaaggaaggg ggggcaatgt acgccgatga tacagcagga 9180 tgggacacaa gatcacacact agaagactta aaaatgaag aaatggtaac gaaccacatg 9240 aaaggagaac acaagaact agccgaggcc atattcaaac taacgtacca aaaacaggtg 9300 gtgcgtgtgc aaagaccaac accaagaggt acagtaatgg atatcatatc gagaagagac 9360 caaagaggca gtgggcaagt cggcacctat ggccttaata ccttcaccaa tatggaagcc 9420 caattaatta gacagatgga gggagaagga atcttcaaaa gcattcagca cctgacagcc 9480 acagaagaaa tcgctgtaca gaactggcta gcaagagtgg ggcgtgaaag gctatcaaga 9540 atggcaatca gtggagatga ttgtgttgta aaacctatag atgacagatt tgcaagtgct 9600 ttaacagctc taaatgacat gggaaaagtt agaaaagata tacaacaatg ggaaccttca 9660 agaggatgga acgattggac acaagtgcct ttctgttcac accactttca tgagttagtc 9720 atgaaagatg gtcgcgtgct cgtagtccca tgcagaaacc aagatgaact gattggtaga 9780 gcccgaattt cccagggagc tgggtggtct ttgaaagaga cggcctgttt gggaaagtct 9840 tacgcccaaa tgtggactct gatgtacttc cacagacgtg acctcagact ggcggcaaat 9900 gccatctgct cggcagtccc gtcacattgg gttccaacaa gtcgaacaac ctggtccata 9960 cacgctaagc atgaatggat gacgacggaa gacatgctgg cagtctggaa cagggtgtgg 10020 atccaagaaa acccgtggat ggaagataaa actccagtgg aatcatggga agaagtccca 10080 tacttgggaa aaagagaaga ccaatggtgc ggctcattga ttgggctaac aagcagggct 10140 acctgggcaa agaacatcca aacagcaata aatcaagtca gatcccttat aggcaatgag 10200 gaatacacag actacatgcc atccatgaag agatttagaa gggaagagga agaggcaggt 10260 gtcctgtggt agaaggcaaa actaacatga aacaaggcta aaagtcaggt cggattaagc 10320 catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380 ggccatcaca aaatgccaca gcttgagtaa actgtgcagc ctgtagctcc acctgaggag 10440 gtgtaaaaaa cctgggaggc cacaaaccat ggaagctgta cgcatggcgt agtggactag 10500 cggttagagg agacccctcc cttacaaatc gcagcaaaca acgggggccc aaggtgagat 10560 gaagctgtaa tctcactgga aggactagag gttagaggag acccccccaa aacaaaaaac 10620 agcatattga cgctgggaaa gaccagagat cctgctgtct cctcagcatc attccaggca 10680 cagaacgcca gaaaatggaa tggtgctgtt gaatcaacag gttct 10725 <210> 4 <211> 10725 <212> DNA <213> Dengue Virus <400> 4 agttgttagt ctacgtggac cgacaagac agattctttg agggagctaa gctcaacgta 60 gttctacag ttttaatt agagagcaga tctctgatga ataccaacg aaaaaaggcg 120 agaagtacgc ctttcaat gctgaaacgc gagagaaacc gcgtgtcaac tgtgcaacag 180 ctgacaaga gattctcact tggaatgctg caaggacgcg gaccattaaa actgttcatg 240 gcccttgtgg cgttccttcg tttcctaaca atcccacca cagcagggat actaaaaga 300 tggggaacga tcaaaaatc aaagctatc aatgttttga gaggttcag gaagagatt 360 ggaaggatgc tgacatctt gaacaagaga cgcaggacag caggcgtgat tgttatgttg 420 attccaacag cgatggcgtt ccattaacc acacgcaatg gagaaccaca catgatcgtt 480 ggtaggcagg agaaagggaa aagtcttctg ttcaaacag aggatggtgt taacatgtgt 540 actctcatgg ccatagacct tggtgaattg tgtgaagata caatcacgta caagtgtcct 600 ctcctcagac aaaatgaacc agaagacata gattgttggt gcaactctac gtccacatgg 660 gtaacttatg ggacatgtac cacacagga gaacagaa gagaaaaag atcagtggcg 720 ctcgttccac atgtaggtat gggactggag acacgaactg aaacatggat gtcatcagaa 780 ggggcctgga aacatgttca gagaattgaa acctggatct tgagacatcc aggttttacc 840 ataatggcag caatcctggc atacaccata ggaacgacac atttccaaag ggccttgatt 900 ttcattttac tgacagctgt cgctccttca atgacaatga gatgcatagg gatatcgaat 960 cgcgatttcg tcgaaggcgt atccggaggg tcatgggtcg acatcgtact cgaacacgga 1020 tcatgcgtta cgactatggc tagagaataag cctacactag acttcgaact gattaagaca 1080 gaggctaagc aaccggcaac attgcgtaag tactgtatcg aagcgaaact gactaacact 1140 acaaccgaat ctagatgccc tacacagggc gaacctagtc tgaacgaaga cgaagacaaa 1200 aggtttat gcaacactc tatggtcgat cgcggatggg gaacggatg cggattgttc 1260 ggtaaggggg gaatcgttac atgcgctatg tttacatgta aaaaaaaatat ggagggaaag 1320 gtcgtgcaac cagagaatct cgaataca atcgtaatca caccgcatag cggagaggaa 1380 cacgcagtcg gaaacgaac cggaaaacac ggaaagaga ttaagattac accacagagc 1440 tccataccg aagccgaact ggaggggtac ggaacagtga caatggaatg ctcacctaga 1500 acaggcctag actttaacga aatggtgtta ctgcaatgg agacaaagc atggttagtg 1560 cataggcaat ggttttaga cctaccacta ccatggttgc ccggagccga tacacaggga 1620 tcgaactgga tacagaaaga vakactcgtt acgtttaaaa acccacacgc taaaaaacag 1680 gacgtagtcg tactcggatc acaggaaggc gcaatgcata ccgcattgac aggcgctaca 1740 gagatacaga tgtctagcgg aaatctgttg tttacagggc atctgaatg tagactgaga 1800 atggacaac tgcaattgaa gggaatgtca tactctgt gtacgggtaa gtttaagata 1860 gtcaaagaga tagccgaaac acacacgga acaatcgtaa ttaggtgca atacgaaggc 1920 gacgggtcac catgtagat accattcgaa attacagacc tcgaaaaag acacgtactc 1980 ggaagactga taacagtgaa tccgatcgtt acggaaaag actcacccgt taatatcgaa 2040 gccgaaccac cattcggaga ctcatacata atatcggag tcgaacccgg acaattgaaa 2100 ctgaattggt ttaaaaaagg gtcatcaatc ggacaatgt tcgaacaac tatgagaggc 2160 gctaagcgta tggctatact cggagacaca gcatgggact tcggatcctt aggggagtg 2220 tttacgtcaa tcggtaggc actacaccag gtattcggag cgatatacgg agccgcattt 2280 agcggagtgt cgtggacaat gagatactg atcggagtga taatcacatg gatcggaatg 2340 atagtaggt ctacaagtct atccgttagc ttagtgttag tcggagtcgt tacactgtat 2400 ctaggcgcta tggtgcaagc cgatagtggt tgcgttgtga gctggaaaaa taaagaactg 2460 aaatgtggca gcgggatctt catcacagat aacgtacaca catggacaga acatatatag 2520 ttccaaccag aatccccttc aaaactagct tcagctatcc aaaaagctca tgagaggggc 2580 atttgtggaa tccgctcagt aacagattg gagaatctga tgtggaaaaacacca 2640 gattgaatc atattctatc agaaatgag gtaagttga ccattatgac aggacatt 2700 agaggaatca tgcaggcagg aaacgatcc tgcggcccc agcccactga gctgaagtac 2760 tcatggaaaa catggggaaa ggcgaaatg ctctccacag agtctcacaa tcagaccttt 2820 cttattgatg gccctgaac agcagaatgc cccacacaa acagagccctg gaactcgctg 2880 gaagttgaag actatggttt tggagttttc accaccaata tattggctgaa attgagagaa 2940 aaacaggatg tattttgtga ctcaaactc atgtcagcgg ccattaaga cacagagcc 3000 gttcatgctg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatggag 3060 aaagcctcct tcattgaagt taaagctgc cactggccaa agtcacac ccttggagc 3120 aatggagtat tagaagtga gatgataatc ccaaaaaattt ttgccgggcc agtgtcacaa 3180 cacaactaca gaccaggcta ccatacacaa acagcaggac cttggcatct aggtaagctt 3240 gagatgact tgatctctg cgaggact acagtggtgg tgactgagga ctgtggaat 3300 agaggaccct ctttaagac gaccactgcc tctggaaac tcatacaga atggtgctgc 3360 cgatcctgca cactaccacc tctaagatac agaggtgagg atggatgctg gtacgggatg 3420 gaatcagac cattgaagaga gaagagag aatttggtca actccttggt cacagccgga 3480 3540 atgctcagga cccgaatagg aacgaaacat gcaatactgc tagttgcagt atcttttgtg 3600 acattgatta ctgggaacat gtcttttaga gacctgggaa gagtgatggt tatggtgggc 3660 gctaccatga cggatgacat aggtatggga gtgacttatc ttgccctact agcagctttt 3720 aaggttagac caacttttgc agctggacta ctcttaagaa aactgacctc caaggaattg 3780 3840 gaactgactg atgcattagc cctgggcatg atggtcctca aaatagtgag aaatatggaa 3900 aaataccaat tggcagtgac tatcatggct atttcatgtg tcccaaatgc agtgatactg 3960 caaaacgcat ggaaggtgag ttgcacaata ttggcagcgg tgtccgtttc accactgctc 4020 ttaacatcct your car agcggattgg ataccactgg cattgacgat aaaaggtctc 4080 aatccaacag ccattttttt aacaactctc tcgaggacca gcaagaaaag gagctggccg 4140 ctaaatgaag ctatcatggc agttgggatg gtgagcattt tagccagttc tctcctaaag 4200 aatgatattc ctatgacagg tccattagtg gctggaggac tcctcaccgt atgttacgtg 4260 ctcactggac gatcggccga tttggaactg gagagagctg ccgatgtaaa atgggaagat caggcagaaa tatcaggaag cagcccaatc ctgtcaata caatatcaga agatggcagc atgtcgataa aaaatgaaga ggaagaacaa acactgacca tactcatcag aacgggattg ttggtgatct caggagtctt tccagtatcg ataccaatta cggcagcagc atggtacctg 4560. tgggagtaa agaaacaacg ggctggagta ctgtgggacg tcccttcacc cccaccagtg gaaaaagccg aactggagga tggagcctac agaatcaagc aaagagggat ccttggatat tctcagattg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcacgtcaca cgtggtgctg ttctgatgca taggaggag aggattgac catcatgggc agatgtcaag aaagacctaa tatcatatgg aggaggctgg aagctagaag gagaatgga ggaaggagag gagtccag tcctggcatt gagtgga aaaaatcca gagccgtcca aacgaaacct ggaatattca aaaccaacac cggaaccata ggcgccgtat ctctggactt ttcccctgga acgtcaggat ctccaatcgt cgacagaaaa ggaaaagttg tgggtcttta cggtaatggt 4980 gttgtcacaa ggagtggagc atatgtaagt gctatagccc agaccgaaaa aagcattgaa 5040 gacaatccag agatcgaaga tgacatttc cgaaagaaaa gattgaccat catggacctc 5100 catccaggag caggaaagac aaaaagatac cttccagcca tagttagaga agccataaaa 5160 cgtggcttga gaacattgat cctggctccc actagagtag tggcagctga aatggaggaa 5220 gctcttagag gacttccaat aagataccaa actacagcca tcaaaaccga gcataccggg 5280 cgggagatcg tggacctaat gtgtcatgcc acatttacta tgaggctgtt atcaccagtc 5340 agagtgccaa attacaacct gatcatcatg gacgaagccc acttcacaga cccagcaagt 5400 atagcagcta gaggatacat ttcaactcga gtagagatgg gtgaagcagc cgggattttc 5460 atgacagcca ctcctccggg aagtagac ccatttcctc agagcaatgc accaattatg 5520 gatgaggaaa gagaaatccc tgagcgttca tggaattcag gacacgaatg ggtcacggat 5580 tttaagggga agactgtttg gtttgttcca agtataaaag caggaaatga tatagcagct 5640 5700 tatgttaaga ctagagccaa tgattgggac tttgtggtca caactgacat ttcagaaatg 5760 ggtgccaact tcaaggctga gagggttata gaccccagac gttgcatgaa accagttata 5820 ctaacagatg gcgaggagcg ggtgatcttg gctggaccta tgccagtgac ccactctagt 5880 gcagcgcaaa gaagggag aataggaaga aatccaaaaa atgaaaatga ccagtacata 5940 6000 ctcctagata acatcaacac acctgaagga atcattccta gtatgttcga accagagcgt 6060 gaaaaagtgg atgccattga tggtgaatac cgtttgagag gagaagcaag gaaaaccttt 6120 gtggacctaa tgagaagagg ggacttacca gtctggttgg cctacaaagt ggcagctgaa 6180 ggcatcaact acgcagacag aaagtggtgt tttgatggaa ttaagaacaa ccaaatactg 6240 gaaaaata tggaaggtgga aatctggaca aaagaagggg aaaggaaaaa attaaaaccc 6300 agatggttgg atgctaggat ctattctgac ccactagcac taaaagaatt caaggaattt 6360 gcagctggaa gaaaatcttt gaccctgaac ctaatcacag aaatgggtag gcttccaact 6420 ttcatgactc agaaagcaag aaacgcactg gacaacctgg ctgtgctgca tacggctgag 6480 gcaggtggaa gggcgtacaa tcatgctctc agtgaactgc cggagaccct ggagacactg 6540 ctcctactga cacttctggc aacagtcaca ggaggaatct tcttattctt aatgagcgga 6600 aaaggtatag ggaagatgac cctgggaatg tgttgcataa tcacggctag tatcctccta 6660 tggtatgcac agatacaacc acactggata gcagcttcaa taatactgga gttttttctc 6720 atagttttgc tcattccaga accagaaaaa cagagaacac cccaagacaa ccaattgacc 6780 tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840 ctggaaaaaa ccaagaaaga cctcggattg ggaagcatta caacccagga atctgagagc 6900 aatatcctgg acatagatct acgccctgca tcagcatgga cgctgtatgc cgtagctaca 6960 acatttgtca caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtctcccta 7020 acagccattg ctaaccaagc tacagtgcta atgggtcttg ggaaaggatg gccattgtca 7080 aagatggaca ttggattcc cctccttgcc attggatgct attcacaagt caaccctata 7140 actctcacag cagctctcct tttattgta gcacattatg ccattatagg gccaggactt 7200 caaghaaag aaccaaaaaaaaaaaaaaaaaccaaaaaaaaacca 7260 acagtcgatg gataacagt gattgaccta gaaccatac cctatgatcc aaaatttgaa 7320 aagcagttag vakaagtaat gctcctaatc ctctgcgtga ctcaagtatt atgatgagg 7380 actacatggg ctttatgtga ggctctacc ctagcgaccg ggcccactc cacactgtgg 7440 gaaggaaatc cagggaggtt ttggacact accattgcag tgtcaatggc tacacacttt 7500 agggggagct acttggccgg agctggactt ctctttcca tcatgaaaaa cacaacaac 7560 acagagag gaactggcaa cataggagag acacttggag aaaatggaa aagtcgatta 7620 aacgcactgg ggaaaagtga atttcaaatc tacaagaaa gtggaatcca ggaagtggat 7680 agaaccctag caaagaagg tatcaaaga ggagaaacgg accaccatgc tgtgtcgcga 7740 gttcagcaa aactgagatg gttcgtcgag agaatatgg tcacaccgga agggaggtg 7800 gtggatctcg gttgcggcag agggggctgg tcatactatt gcgggggact aaagaatgta 7860 agagaagtca aaggcctaac aaaaggagga ccaggacatg aagaacccat ccccatgtca 7920 acatagggt ggaatctagt gcgtctgcaa agtggagttg acgttttctt caccccgcca 7980 gaaaagtgtg atacattgtt gtgtgacata ggggagtcgt caccaaatcc cacgatagaa 8040 gcaggacgaa cactcagagt cctcactta gtggaaatt ggttgacaa taacaccca 8100 ttttgcataa aggtcctcaa cccatatatg ccttcagtca tagaaaaat ggaagcatta 8160 haaggaaat atggaggagc cttagtgagg aatccactct cacgaactc cacgcatgaa 8220 atgtactggg tatctaatgc caccgggaac atagtgtcat cagtgaacat gatttcagg 8280 atgttgatta acagattcac aatgaacac aagaaagcca cttacgagcc agatgttgac 8340 ctaggaagtg gaacccgcaa cattggaatt gaagtgaga taccaatct agacatata 8400 ggaagagaa tagagaaat aaaaaaaaacag catgaaacat catggcacta tgatxagac 8460 cacccataca aaacgtgggc ttaccatggc agctatgaaaaaaaaac tggatcagca 8520 tcatctatgg tgacggagt ggtcagattg ctgacaaac cttgggacgt cgtccctatg 8580 gtgacacaga tggcaatgac agacacgact ccttttggac aacagcgcgt ttcaagag 8640 aaagtagaca cgagaaccca agaaccgaag gaggcaca agaactgat gaaaattacg 8700 gcagagtggc tttggaaga actaggaaag aaaagacac ctaggatgtg taccagagaa 8760 gattcacaa gaaagtgag aagcaatgca gccttgggggg ccatattcac tgatgagaac 8820 aaatggaaat cggcacgtga ggctgttga gatggtaggt tctgggagct ggttgacagg 8880 gaaagaaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940 agagagaga aactagggga gttcggcaag gcaaaggta gcagagccat atggtacatg 9000 tggcttggag cacgctctt agagtttga gccctaggat tcctgaatga agatcactgg 9060 ttctccagag ggaactccct gagtggagtg gaaggagaag ggctgcacag gctaggctac 9120 attttagag acgtgggcaa gaaggaaggg ggggcaatgt acgccgatga tacagcagga 9180 tgggacacaa gatcacacact agaagactta aaaatgaag aaatggtaac gaaccacatg 9240 aaaggagaac acaagaaact agccgaggcc atattcaaac taacgtacca aaacaaggtg 9300 gtgcgtgtgc aaagaccaac accaagaggt acagtaatgg atatcatatc gagaagagac 9360 caaagaggca gtgggcaagt cggcacctat ggccttaata ccttcaccaa tatggaagcc 9420 caattaatta gacagatgga gggagaagga atcttcaaaa gcattcagca cctgacagcc 9480 acagaagaaa tcgctgtaca gaactggcta gcaagagtgg ggcgtgaaag gctatcaaga 9540 atggcaatca gtggagatga ttgtgttgta aaacctatag atgacagatt tgcaagtgct 9600 ttaacagctc taaatgacat gggaaaagtt agaaaagata tacaacaatg ggaaccttca 9660 agaggatgga acgattggac acaagtgcct ttctgttcac accactttca tgagttagtc 9720 atgaaagatg gtcgcgtgct cgtagtccca tgcagaaacc aagatgaact gattggtaga 9780 gcccgaattt cccagggagc tgggtggtct ttgaaagaga cggcctgttt gggaaagtct 9840 tacgcccaaa tgtggactct gatgtacttc cacagacgtg acctcagact ggcggcaaat 9900 gccatctgct cggcagtccc gtcacattgg gttccaacaa gtcgaacaac ctggtccata 9960 cacgctaagc atgaatggat gacgacggaa gacatgctgg cagtctggaa cagggtgtgg 10020 atccaagaaa acccgtggat ggaagataaa actccagtgg aatcatggga agaagtccca 10080 tacttgggaa aaagagaaga ccaatggtgc ggctcattga ttgggctaac aagcagggct 10140 acctgggcaa agaacatcca aacagcaata aatcaagtca gatcccttat aggcaatgag 10200 gaatacacag actacatgcc atccatgaag agatttagaa gggaagagga agaggcaggt 10260 gtcctgtggt agaaggcaaa actaacatga aacaaggcta aaagtcaggt cggattaagc 10320 catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380 ggccatcaca aaatgccaca gcttgagtaa actgtgcagc ctgtagctcc acctgaggag 10440 gtgtaaaaaa cctgggaggc cacaaaccat ggaagctgta cgcatggcgt agtggactag 10500 cggttagagg agacccctcc cttacaaatc gcagcaaaca acgggggccc aaggtgagat 10560 gaagctgtaa tctcactgga aggactagag gttagaggag acccccccaa aacaaaaaac 10620 agcatattga cgctgggaaa gaccagagat cctgctgtct cctcagcatc attccaggca 10680 cagaacgcca gaaatggaa tggtgctgtt gatcacag gttct 10725 <210> 5 <211> 10708 <212> DNA <213> Dengue Virus <400> 5 agttgttagt ctacgtggac cgacagaac agtttcgact cggaagcttg cttaacgtag 60 tgctaacagt ttttag agagcagatc tctgatgaac aaccaacgga agaagacggg 120 aaaaccgtct atcaatgc tgaacgcgt gagaaaccgt gtgtcaactg gatcacagtt 180 ggcgaagaga ttctcaaag gactgctgaa cggccaggga ccaatgaat tggttatggc 240 gttcatagct ttcctcagat ttcatagct tccaccaca gcaggagtct tggtagatg 300 gggaaccttc agaagtcgg gggccattaa ggtcctgaaa ggctttaaga aggagatctc 360 aaacatgctg agcataatca aaacggaa aagacatcg ctctgtctca tgatgatatt 420 gccagcagca cttgctttcc acttgactc acgagatgga gagccgcgca tgattgtggg 480 gagaatgaa agaggaaat ccctacttt taagacagcc tctggaatta acatgtgcac 540 actcatagcc atggacttgg gagagatgtg tgatgacacg gtcacttaca aatgccccca 600 tattaccgaa gtggaacctg aagacattga ctgctggtgc aacctcacat caacatgggt 660 gacttatgga acgtgcaatc aagccggaga gcatagacgc gataagagat cagtggcgtt 720 agctccccat gtcggcatgg gactggatac acgcacccaa acttggatgt cggctgaagg 780 agcttggagg caagtcgaga aggtagagac atgggccctt aggcacccag ggttcaccat 840 actagcccta tttcttgccc attacatagg cacttccttg acccagaagg tggttatttt 900 tatactgcta atgctggtca ccccatccat gacaatgaga tgtgtgggag taggaaacag 960 agattttgtg gaaggactat caggagctac gtgggttgac gtggtgctcg agcacggggg 1020 gtgtgtgacc accatggcta agaacaagcc cacgttggat atagagcttc agaagaccga 1080 ggccacccaa ctggcgaccc taaggaagct atgcattgag gggaaaatca ccaacataac 1140 aactgactca agatgtccta cccaagggga agcggttttg cctgaggagc aggaccaaaa 1200 ctacgtgtgt aagcatacat acgtagacag aggctggggg aacggatgtg gttttgtttgg 1260 taagggaagc ttggtaacat gtgcgaatt tcaatgcctg gaaccaatag agggaaagt 1320 ggtgcaat gagaacctca atacaccgt tatcatcaca gtgcacacag gagaccaca 1380 ccaggtggga atgaaacgc agggagtcac ggctgagata acacctcagg catcaccac 1440 tgagccatc tgcctgaat atggacct tgtcaccac tgctcaccac tgcaggtt 1500 ggacttcaat gaatgatct tgctacaat gagacaaa gcatggatgg tacatagaca 1560 atggttttt gacctaccctc taccatggac atcaggagct acacggaaa caccaacctg 1620 gacaggaag gagctcttg tgacatta aaacgcacat gcgagaaac aagaagtagt 1680 tgttcttggg tcgcaagagg gagcaatgca taccgcattg acaggagcca cagaaatcca 1740 aaactcagga gcacaagca ttttgcggg gcacttaaaa tgtagactta agatggacaa 1800 attagaactc aagggatga gctatgcaat gtgcacgaat accttgtgt tgagaaaga 1860 agtctcagaa acgcagcatg ggacaatact tatcaggtc gagtacaag gggagatgt 1920 accttgcaag attccttct ccacagagga tggacaaggg aaagctcaca atggcagact 1980 gattacagcc aacccagtgg tgactaagaa ggaggagcct gtcaatattg aggctgaacc 2040 tccttttggg gaagcaata tagtattgg aattggac aacgccttga aaatcactg 2100 gtacaagaag ggaagctcta ttgggaagat gttcgaggcc actctagag gtgcaaggcg 2160 catggccatc ttgggagaca cagcttggga ctttggatca gtggtggtg ttctgaactc 2220 attaggcaa atggtgcacc aaatattcgg aagtgcttac acagccctat tcagtggagt 2280 ctcttgggta atgaaaatttg gataggagt tctcttgact tggataggt tgaattcaa 2340 aaacacatcc atgtcatttt catgcattgc gatgaatc atcacactct atctggggc 2400 tgtggtgcaa gctgacatgg ggtgtgttat aactggaaa ggcaagaac tcaagtgtgg 2460 aagtggaatc ttcgtcacca acgaggtcca tacctggaca gagcaataca aattccaagc 2520 agactcccca aaagattgg cgacagccat tgcaggcgct tgggagaatg gagtgtgcgg 2580 aattaggtca aaccagaa tggaatct cctgtggaag aattagcca atgaactgaa 2640 ctacatattg tgggaaaaca atcaatt aacggttgtt gtgggcgata taattggggt 2700 cttagagcaa gggaaaagaa cactacacc acaaccatg gagctaaaat actcatggaa 2760 aacgtgggga aaggcaaaa tagtgacagc tgaacaca aattctctt tcatataga 2820 tggaccaac acacggagt gtccaagtgc ctcaagagca tggaatgtgt gggaggtgga 2880 agattacgggg ttcggagtct tcacaccaa catatggctg aaactccgag aggtgtatac 2940 ccaactatgt gaccataggt taatgtcggc agccgtcaag gatgaaaggg ccgtacatgc 3000 cgacatgggc tattggatag aaagtcaaaa gatggaagt tggagctag aaaagcatc 3060 cctcatagag gtgaaaacct gcacatggcc aaatcacat acccttgga gtaatggtgt 3120 gttagagagt gatagatca ttccaaaag tctagctggt cctatctcgc aacacaacta 3180 caggcccgggg taccaccc agacggcggg accttggcat ttaggaaat tagagctgga 3240 cttcaactat tgtgaggaa cacagttgt catcacagaa aactgtggga caagaggccc 3300 atcattgaga acaacag tgtcagggaa gttaatacac gatggtgct gccgctcgtg 3360 cacacttcct cccctgcgat acatgggaga agacggttgc tggtatggca tggatcag 3420 acccatcagt gagaaagaag aaaacatggt aaagtcttta gtctcagcgg gaagtggaga 3480 ggtggacaac ttcacaatgg gtgtcttgtg tttggcaatc ctctttgaag aggtgatgag 3540 aggaaaattt gggaagaaac acatgattgc gggggttttc ttcacgtttg tactccttct 3600 ctcagggcaa ataacatgga gagatatggc gcacacacta ataatgattg ggtccaatgc 3660 atctgacagg atgggaatgg gcgtcaccta cctagcttta attgcaacat ttaaaatcca 3720 gccatttttg gctttgggat ttttcctaag aaaactgaca tccagagaaa atttattgtt 3780 aggagttggg ctggctatgg caacaacgtt acaactgcca gaggacattg aacaaatggc 3840 aaatggaatc gctctggggc tcatggctct caaactgata acacaatttg aaacatacca 3900 actatggaca gcattaatct ccttaacgtg ttcaaataca atttttacgt tgactgttgc 3960 ctggagaaca gccaccctga ttttggctgg agtttcactt ttaccagtgt gccagtcttc 4020 gagtatgagg aaaacagact ggcttccaat gacagtggca gctatgggag ttccacctct 4080 accacttttt atttttagct taaaagacac actcaaaagg agaagctggc cactgaatga 4140 agggtgatg gctgttgggc tgtgagcat tctggccagt tctctcctta gaatgatgt 4200 acccatggct ggaccattag tggccgggg cttgctgata gcgtgctacg tcataactgg 4260 cacgtcagca gacccaccg tagaaaagc agcagatgta acatgggagg aagaggctga 4320 gcaaacagga gtgtcccaca acttaatgat cacagttgat gatgatgaa caatgagaat 4380 aaagatgat gagactgaga atacttac agtgctttg aaaacagcat tactatagt 4440 gtcaggaatc tttccatact ccataccccgc aacattgttg gtctggcata cttggcaaaa 4500 gcaaacccaa agatccggcg ttctatggga tgtacccagc cctccagaga cacagaaagc 4560 agaactggaa gaaggggtct ataggatca acaghagga atttggga aaacccaagt 4620 aggggttgga gtacagaaag aaggttctt tcacacatg tggcacgtta caaggggc 4680 agtgttgaca tataatggga aagactgga accaattgg gctagcgtga aaaagatct 4740 gatttcatac ggaggatt ggagattgag cgcacaatgg caaaggggg aggaggtgca 4800 ggttattgcc gtagagcctg ggagaaccc aaagaacttt caaccatgc caggcacttt 4860 tcagactaca acagggaaa taggagcaat tgcactggat ttcaagcccg gaacttcagg 4920 atctcctatc ataacagag agggaaggt agtgggactg tatggcaatg gagtggttac 4980 aaagaatggt gggctacgtca gcggaatagc gcaacgaat gcagaaccag atggaccgac 5040 accagaattg gagagaga tgttcaaaa gcgaaatcta accataatgg atcttcatcc 5100 tgggtcagga agacacgga ataccttcc agctattgtt aggaggca tcaagagacg 5160 tttgagaact ctaattctgg caccaacaag ggtggttgca gctgagatgg aagaagcatt 5220 gaaagggctc cchaataggt accaacaac agcaaaaa tctgaacaca caggagaga 5280 gattgttgat ctgatgtgcc acgcaacgtt cacaatgcgt ctgctgtcac cagttaggt 5340 tccaaactat aacttgataa taatggatga agcccatttc acagacccag ccagtatagc 5400 tgctagagggg tacatatcaa ctcgtgttgg aatgggaa gcagccgcaa tattcatgac 5460 agcaacgccc cctggaacag ctgatgcctt tcccagagc aacgctccaa ttcagatga 5520 agaaagggac ataccagaac gctcatggaa ttcaggcaat gatggataa ccgacttcgc 5580 tgggaaaacg gtgtggtttg tcccagcat taaagccgga atgacatag caattgcct 5640 gcggaaaaac gggaaaaagg tcattcact tagtaggag acttttgaca cagaatca 5700 gaaaaccaaa ctgaatgat gggactttgt ggtgacgact gatttcag aaatgggggc 5760 caatttcaa gcagatagag tgatcgaccc aagagatgt ctcaaccag tgatcctgac 5820 agatggacca gagcgggtga tcctggctgg accaatgcca gtcaccgcag cgagtgctgc 5880 gcaaggaga gggagagttg gcaggaaccc aaaaaaa atgaccagt acatattcac 5940 gggccagcct ctcacaatg atgaagacca tgctcactgg acagaagcaa aaatgctgct 6000 ggacaacatt aacacaccag aagggatcat accagctctc tttgagccag aaagggagaa 6060 gtcagccgcc atagacggtg agtatcgcct gaaggtgag tccaggaaga ctttcgtgga 6120 actcatgagg aggggtgacc ttccagtctg gttagcccat aaagtagcat cagaggt 6180 haaataca gatagaaat ggtgctttga tggacaacgt aaatcaa ttttagaga 6240 gacaatgt gtggaaatct gacaaagga aggaaaag aaaaaattga gacctaggtg 6300 gcttgatgct cgcacctatt cagatccctt agcactcaag gaattcaagg actttgcggc 6360 tggcaggaag tcaatagccc ttgatcttgt gacagaaata ggaagagtgc cttcacacct 6420 agctcataga acgagaaacg ctctggacaa tctggtgatg ctgcatacgt cagaacatgg 6480 cggtaaggcc tacaggcatg cggtggagga actaccagag acaatggaaa cactcctact 6540 cttgggactc atgatcttgt tgacaggtgg agcaatgctt ttcttaatat caggtaaagg 6600 gattggaaag acttcaatag gactcatttg tgtaattgct tccagcggca tgttgtggat 6660 ggccgaaatc ccactccaat ggatcgcgtc ggccatagtc ctggagtttt ttatgatggt 6720 gttgcttata ccagaaccag agaagcagag aaccccccaa gacaaccaac tcgcatatgt 6780 cgtgataggc atacttacac tggcagcaat aatagcagcc aatgaaatgg gattgttgga 6840 aactacaaag agagatttag gaatgtctaa ggagccaggt gttgtttctc caaccagcta 6900 tttagatgtg gacttgcacc cagcatcagc ctggacattg tacgctgtgg ccactacagt 6960 aataacacca atgttaagac ataccataga gaattccaca gcaaatgtgt ccttggcagc 7020 tatagccaac caggcagtgg tcctgatggg tttggacaaa ggatggccaa tatcaaaaaat 7080 ggacttagga gtacccctgc tggcattggg ttgctattca caagtgaacc cactgactct 7140 aacagcggca gtactcttgc tgatcacaca ttatgccatt ataggtccag gattgcaggc 7200 aaaagccacc cgtgaagctc agaaaaggac agctgctgga ataatgaaga atccaacagt 7260 ggatgggata atgacaatag acctagatcc tgtaatatat gattcaaaat ttgaaaagca 7320 actgggacag gttatgctcc tggttttgtg tgcagttcaa cttttgttaa tgagaacatc 7380 atgggccttg tgtgaagctt taaccctagc tacaggacca ataacaacac tctgggaagg 7440 atcacctgg aagttttgga acaccacgat agctgtttcc atggcgaaca tttttagagg 7500 gagctattta gcaggagctg ggcttgcttt ctctattatg aaatcagttg gaagagggaa 7560 aaggagaaca ggctcacagg gtgaaacttt gggagaaaaa tggaaaaaga agtaaatca 7620 7680 agaagccaaa gaagggggtga aagaggga aataacacat catgccgtgt ccagaggtag 7740 tgcaaaactt caatggtttg tggagagaaa catggtcatt cccgaaggaa gagttataga 7800 cttgggctgt ggaagaggag gctggtcata ttactgtgca ggactgaaaa aagtcacaga 7860 agtgcgagga tacacaaaag gcggtccagg acacgaagaa ccagtaccta tgtccacata 7920 tggatggaac atagttaagt taatgagtgg aaaggatgtg tttatcttc cacctgaaaa 7980 gtgtgacacc ctgttgtgcg acattggaga atcttcacca agcccaacag tggagaagaaag 8040 cagaactata agagttttga agatggttga accatggcta aagaacaacc aattttgtat 8100 taaagtattg aacccttaca tgccaactgt gattgagcac ctagaaagac tacaaaggaa 8160 acatggagga atgcttgtga gaaatccact ttcacgaaac tccacgcacg aaatgtactg 8220 gatatccaat ggcacaggta acattgtcgc ttcagtcaac atggtatcta gactgctact 8280 gaacaggttc acgatgacac acagaagacc caccattgag aaagatgtgg atttaggagc 8340 aggaactcga catgttaatg cggaaccaga aacacccaac atggatgtca ttgggggaaag 8400 aataaaaagg atcaaggagg agcataattc aacatggcac tacgatgacg aaaaccccta 8460 caaaacgtgg gcttaccatg gatcttatga agtcaaagcc acaggctcag cctcctccat 8520 gataaatgga gtcgtgaaac tcctcactaa accatgggat gtggtgccca tggtgacaca 8580 gatggcaatg acagatacaa ctccattgg ccagcagaga gtctttaaag agaagtgga 8640 caccaggaca cccaggtcca tgccaggaac aagaagggtt atgggatca cagcggagtg 8700 gctctggaga accctgggaa ggataaaaa acccaggtta tgcacaaggg aagagtttac 8760 aaaaaaggtc agaactaacg cagccatggg agctgttttc acagaggaga accaatggga 8820 cagcgcgaaa gctgctgttg aggatgagga ttttggaaa cttgtggaca gagaacgtga 8880 actccacaaa ctgggcaagt gtggaagctg tgtttacaac atgatgggta aggagagaa 8940 gaaacttgga gagtttggca aagcaaagg cagtagagct atatggtaca tgtggttggg 9000 agccaggtac cttgagttcg aagccttgg attctttaat gagaccact ggttctcgcg 9060 tgagaactct tacagtggag tggaggaga aggactgcac aagctaggct attattag 9120 ggacattcc aagatacccg gaggagctat gtatgctgat gatacagctg gttgggacac 9180 aagaataca gaagagatgacc tgcacaatga ggaaagagatc acacagcaa tggaccctga 9240 accacaggcag tagcgaacg ctatatta gctcacatac caaaaaag tggtcaaagt 9300 tcaacgaccg actccacag gcacggtaat ggatatcata tctaggaag accaaagagg 9360 cagtggacag gtaggaactt atggtctgaa tacattcacc aacatggaag cccagttaat 9420 cagacaatg gaaggagaag gtgtgctgtc aaaggcagac ctcgagaacc ctcatctgcc 9480 aggagaaa attacacaat ggttggaaac aaggagtg gagaggtta aaagaatggc 9540 cataagtggg gatgactgcg tggtgaaacc aatcgatgac aggttcgcta atgccctgct 9600 cgctctgaac agamatgggaa aggttcggaa agacatacct caatggcagc catcaaggg 9660 atggcatgat tggcacagg ttccttctg ctcccaccac ttcatgaat tgatcatgaa 9720 agatgaga aagttggtgg ttccctgcag accccaggac gaactaatag gaagggcaag 9780 aatctctcaa ggagcgggat ggagccttag agaaccgca tgtctgggga aagcctacgc 9840 tcaaatgtgg agtctcatgt atttcacag agagaccctc agactagcat ccaacgccat 9900 atgttcagca gtaccagtcc actgggtccc acgacatggt ctattcacgc 9960 tcaccatcag tggatgacca cagaagacat gcttactgtc tggagaagag tgtggatcga 10020 ggacaatcca tggatggaag acaaaactcc agtcacaacc tgggaaaatg ttccatatct 10080 agggaagaga gaagaccaat ggtgcggatc acttattggt ctcacttcca gagcaacctg 10140 ggcccagaac atacccacag caattcaaca ggttagaagc ctttataggca atgaaggtt 10200 tctggactac atgccttcaa tgaagagatt taggaaggag gagagtcgg agggagccat 10260 ttggtaaaac gtaggaagtg aaaaagaggt taactgtcag gcatattaa gccacagtac 10320 ggaagaagct gtgctgcctg tgagccccgt ccaaggacgt taaaagaaga agtcaggccc 10380 caaagccacg gtttgagcaa accgtgctgc ctgtagctcc gtcgtgggga cgtaaaacct 10440 gggaggctgc aaactgtgga agctctacgc acggtgtagc agactagcgg ttaggagaga 10500 cccctcccat gacacaacgc agcaggcgggg cccgagcact gaggaagct gtacctcctt 10560 gcaaaggact agaggttaga ggagaccccc cgcaataaa aacagcatat tgacgctggg 10620 agagaccaga gatcctgctg tctcctcagc atcattccag gcacagaacg ccagaaaatg 10680 gatggtgct gttgaatcaa caggttct 10708 <210> 6 <211> 10708 <212> DNA <213> Dengue Virus <400> 6 agttgttagt ctacgtggac cgacagaac agtttcgact cggaagcttg cttaacgtag 60 tgctaacagt ttttag agagcagatc tctgatgaac aaccaacgga agaagacggg 120 aaaaccgtct atcaatgc tgaacgcgt gagaaaccgt gtgtcaactg gatcacagtt 180 ggcgaagaga ttctcaaag gactgctgaa cggccaggga ccaatgaat tggttatggc 240 gttcatagct ttcctcagat ttcatagct tccaccaca gcaggagtct tggtagatg 300 gggaaccttc agaagtcgg gggccattaa ggtcctgaaa ggctttaaga aggagatctc 360 aaacatgctg agcataatca aaacggaa aagacatcg ctctgtctca tgatgatatt 420 gccagcagca cttgctttcc acttgactc acgagatgga gagccgcgca tgattgtggg 480 gagaatgaa agaggaaat ccctacttt taagacagcc tctggaatta acatgtgcac 540 actcatagcc atggacttgg gagagatgtg tgatgacacg gtcacttaca aatgccccca 600 tattaccgaa gtggaacctg aagacattga ctgctggtgc aacctcacat caacatgggt 660 gacttatgga acgtgcaatc aagccggaga gcatagacgc gataagagat cagtggcgtt 720 agctccccat gtcggcatgg gactggatac acgcacccaa acttggatgt cggctgaagg 780 agcttggagg caagtcgaga aggtagagac atgggccctt aggcacccag ggttcaccat 840 actagcccta tttcttgccc attacatagg cacttccttg acccagaagg tggttatttt 900 tatactgcta atgctggtca ccccatccat gacaatgaga tgcgtgggcg tagggaatag 960 agactttgta gagggattga gcggagcgac atgggtcgac gtcgtactcg aacacggggg 1020 gtgcgtgacg actatgcta agaataagcc tacactcgat atcgaactgc aaaaaccga 1080 agcgacacaa ttggcgacat tgcgcaaact atgtatcgaa ggtaagatta cgaacattac 1140 aaccgatagt agatgcccta cacaaggcga agccgttcta cctgaggagc aagaccaaaa 1200 ctacgtatgt aagcatacat acgttgatag ggggtggggg aacggatgcg gattgttcgg 1260 taaggggtca ttggtcacat gcgctaagtt ccaatgctta gagcctatcg agggtaaggt 1320 ggtacaatac gaaaacctta agtataccgt gataattaca gttcataccg gagaccaaca 1380 ccaagtggga aacgagacac agggagtgac agccgaaatt acgcctcagg ctagtacaac 1440 cgaagcgata ctacccgaat acggaacatt ggggttggag tgttcaccta gaaccggatt 1500 ggactttaac gaaatgatac tgttgactat gaaaaacaag gcatggatgg tgcataggca 1560 atggtttttt gacctaccat tgccttggac tagcggagcg acaaccgaaa cacctacatg 1620 gaatagaaaa gagctcctag tgacatttaa aaacgctcac gctaagaagc aggaagtggt 1680 cgttttaggg tcacaggagg gagctatgca taccgcacta accggagcga ctgagataca 1740 gaatagcgga gggacatcaa tcttcgccgg acaccttaag tgtagattga aaatggacaa 1800 acttgagctt aaggggatgt catacgctat gtgtacgaac acattcgtgc tgaaaaaaga 1860 ggtaagcgaa acgcaacacg gaacgatact gattaaggtt gagtataagg gcgaagacgt 1920 cccatgtaag ataccttttt cgacagagga cggacagggt aaggcccata acggaagact 1980 gattaccgct aacccagtgg tgaccaaaaa agaggaacca gtcaatcg aagccgaacc 2040 tccattcgga gagtcaaca tagtgatagg gataggcgat aacgcactta agattactg 2100 gtacaaaaaa gggtcatcaa tcggtaagat gtttgaggca accgctaggg gggctagacg 2160 gatggccata ctcggagaca ccgcatggga cttcggatcc gtgggggggg tacttactc 2220 actcggtaag atggtgcacc aaatttcgg atccgcatat accgctat ttagcggagt 2280 gtcatgggtg atgaaatcg gatcggagt tctattgaca tggatcggat tgaactctaa 2340 gatacatct atgtcatttt catgtatcgc aatcggaatt atctcggagc 2400 cgtagtgcaa gccgacatgg ggtgtgttat aactggaaa gccaagaac tcaagtgtgg 2460 aagtggaatc ttcgtcacca acgaggtcca tacctggaca gagcaataca aattccaagc 2520 agactcccca aaagattgg cgacagccat tgcaggcgct tgggagaatg gagtgtgcgg 2580 aattaggtca aaccagaa tggaatct cctgtggaag aattagcca atgaactgaa 2640 ctacatattg tgggaaaaca atcaatt aacggttgtt gtgggcgata taattggggt 2700 cttagagcaa gggaaaagaa cactacacc acaaccatg gagctaaaat actcatggaa 2760 aacgtgggga aaggcaaaa tagtgacagc tgaacaca aattctctt tcatataga 2820 tggaccaac acacggagt gtccaagtgc ctcaagagca tggaatgtgt gggaggtgga 2880 agattacgggg ttcggagtct tcacaccaa catatggctg aaactccgag aggtgtatac 2940 ccaactatgt gaccataggt taatgtcggc agccgtcaag gatgaaaggg ccgtacatgc 3000 cgacatgggc tattggatag aaagtcaaaa gatggaagt tggagctag aaaagcatc 3060 cctcatagag gtgaaaacct gcacatggcc aaatcacat acccttgga gtaatggtgt 3120 gttagagagt gatagatca ttccaaaag tctagctggt cctatctcgc aacacaacta 3180 caggcccgggg taccaccc agacggcggg accttggcat ttaggaaat tagagctgga 3240 cttcaactat tgtgaggaa cacagttgt catcacagaa aactgtggga caagaggccc 3300 atcattgaga acaacag tgtcagggaa gttaatacac gatggtgct gccgctcgtg 3360 cacacttcct cccctgcgat acatgggaga agacggttgc tggtatggca tggatcag 3420 acccatcagt gagaaagaag aaaacatggt aaagtcttta gtctcagcgg gaagtggaga 3480 ggtggacaac ttcacaatgg gtgtcttgtg tttggcaatc ctctttgaag aggtgatgag 3540 aggaaaattt gggaagaaac acatgattgc gggggttttc ttcacgtttg tactccttct 3600 ctcagggcaa ataacatgga gagatatggc gcacacacta ataatgattg ggtccaatgc 3660 atctgacagg atgggaatgg gcgtcaccta cctagcttta attgcaacat ttaaaatcca 3720 gccatttttg gctttgggat ttttcctaag aaaactgaca tccagagaaa atttattgtt 3780 aggagttggg ctggctatgg caacaacgtt acaactgcca gaggacattg aacaaatggc 3840 aaatggaatc gctctggggc tcatggctct caaactgata acacaatttg aaacatacca 3900 actatggaca gcattaatct ccttaacgtg ttcaaataca atttttacgt tgactgttgc 3960 ctggagaaca gccaccctga ttttggctgg agtttcactt ttaccagtgt gccagtcttc 4020 gagtatgagg aaaacagact ggcttccaat gacagtggca gctatgggag ttccacctct 4080 accacttttt atttttagct taaaagacac actcaaaagg agaagctggc cactgaatga 4140 agggtgatg gctgttgggc tgtgagcat tctggccagt tctctcctta gaatgatgt 4200 acccatggct ggaccattag tggccgggg cttgctgata gcgtgctacg tcataactgg 4260 cacgtcagca gacccaccg tagaaaagc agcagatgta acatgggagg aagaggctga 4320 gcaaacagga gtgtcccaca acttaatgat cacagttgat gatgatgaa caatgagaat 4380 aaagatgat gagactgaga attackacttac agtgctttg aaaacagcat tactatagt 4440 gtcaggaatc tttccatact ccataccccgc aacattgttg gtctggcata cttggcaaaa 4500 gcaaacccaa agatccggcg ttctatggga tgtacccagc cctccagaga cacagaaagc 4560 agaactggaa gaaggggtct ataggatca acaghagga atttggga aaacccaagt 4620 aggggttgga gtacagaaag aaggttctt tcacacatg tggcacgtta caaggggc 4680 agtgttgaca tataatggga aagactgga accaattgg gctagcgtga aaaagatct 4740 gatttcatac ggaggatt ggagattgag cgcacaatgg caaaggggg aggaggtgca 4800 ggttattgcc gtagagcctg ggagaaccc aaagaacttt caaccatgc caggcacttt 4860 tcagactaca acagggaaa taggagcaat tgcactggat ttcaagcccg gaacttcagg 4920 atctcctatc ataacagag agggaaggt agtgggactg tatggcaatg gagtggttac 4980 aaagaatggt gggctacgtca gcggaatagc gcaacgaat gcagaaccag atggaccgac 5040 accagaattg gagagaga tgttcaaaa gcgaaatcta accataatgg atcttcatcc 5100 tgggtcagga agacacgga ataccttcc agctattgtt aggaggca tcaagagacg 5160 tttgagaact ctaattctgg caccaacaag ggtggttgca gctgagatgg aagaagcatt 5220 gaaagggctc cchaataggt accaacaac agcaaaaa tctgaacaca caggagaga 5280 gattgttgat ctgatgtgcc acgcaacgtt cacaatgcgt ctgctgtcac cagttaggt 5340 tccaaactat aacttgataa taatggatga agcccatttc acagacccag ccagtatagc 5400 tgctagagggg tacatatcaa ctcgtgttgg aatgggaa gcagccgcaa tattcatgac 5460 agcaacgccc cctggaacag ctgatgcctt tcccagagc aacgctccaa ttcagatga 5520 agaaagggac ataccagaac gctcatggaa ttcaggcaat gatggataa ccgacttcgc 5580 tgggaaaacg gtgtggtttg tcccagcat taaagccgga atgacatag caattgcct 5640 gcggaaaaac gggaaaaagg tcattcact tagtaggag acttttgaca cagaatca 5700 gaaaaccaaa ctgaatgat gggactttgt ggtgacgact gatttcag aaatgggggc 5760 caatttcaa gcagatagag tgatcgaccc aagagatgt ctcaaccag tgatcctgac 5820 agatggacca gagcgggtga tcctggctgg accaatgcca gtcaccgcag cgagtgctgc 5880 gcaaggaga gggagagttg gcaggaaccc aaaaaaa atgaccagt acatattcac 5940 gggccagcct ctcacaatg atgaagacca tgctcactgg acagaagcaa aaatgctgct 6000 ggacaacatt aacacaccag aagggatcat accagctctc tttgagccag aaagggagaa 6060 gtcagccgcc atagacggtg agtatcgcct gaaggtgag tccaggaaga ctttcgtgga 6120 actcatgagg aggggtgacc ttccagtctg gttagcccat aaagtagcat cagaggt 6180 haaataca gatagaaat ggtgctttga tggacaacgt aaatcaa ttttagaga 6240 gacaatgt gtggaaatct gacaaagga aggaaaag aaaaaattga gacctaggtg 6300 gcttgatgct cgcacctatt cagatccctt agcactcaag gaattcaagg actttgcggc 6360 tggcaggaag tcaatagccc ttgatcttgt gacagaaata ggaagagtgc cttcacacct 6420 agctcataga acgagaaacg ctctggacaa tctggtgatg ctgcatacgt cagaacatgg 6480 cggtaaggcc tacaggcatg cggtggagga actaccagag acaatggaaa cactcctact 6540 cttgggactc atgatcttgt tgacaggtgg agcaatgctt ttcttaatat caggtaaagg 6600 gattggaaag acttcaatag gactcatttg tgtaattgct tccagcggca tgttgtggat 6660 ggccgaaatc ccactccaat ggatcgcgtc ggccatagtc ctggagtttt ttatgatggt 6720 gttgcttata ccagaaccag agaagcagag aaccccccaa gacaaccaac tcgcatatgt 6780 cgtgataggc atacttacac tggcagcaat aatagcagcc aatgaaatgg gattgttgga 6840 aactacaaag agagatttag gaatgtctaa ggagccaggt gttgtttctc caaccagcta 6900 tttagatgtg gacttgcacc cagcatcagc ctggacattg tacgctgtgg ccactacagt 6960 aataacacca atgttaagac ataccataga gaattccaca gcaaatgtgt ccttggcagc 7020 tatagccaac caggcagtgg tcctgatggg tttggacaaa ggatggccaa tatcaaaaaat 7080 ggacttagga gtacccctgc tggcattggg ttgctattca caagtgaacc cactgactct 7140 aacagcggca gtactcttgc tgatcacaca ttatgccatt ataggtccag gattgcaggc 7200 aaaagccacc cgtgaagctc agaaaaggac agctgctgga ataatgaaga atccaacagt 7260 ggatgggata atgacaatag acctagatcc tgtaatatat gattcaaaat ttgaaaagca 7320 actgggacag gttatgctcc tggttttgtg tgcagttcaa cttttgttaa tgagaacatc 7380 atgggccttg tgtgaagctt taaccctagc tacaggacca ataacaacac tctgggaagg 7440 atcacctgg aagttttgga acaccacgat agctgtttcc atggcgaaca tttttagagg 7500 gagctattta gcaggagctg ggcttgcttt ctctattatg aaatcagttg gaagagggaa 7560 aaggagaaca ggctcacagg gtgaaacttt gggagaaaaa tggaaaaaga agtaaatca 7620 7680 agaagccaaa gaagggggtga aagaggga aataacacat catgccgtgt ccagaggtag 7740 tgcaaaactt caatggtttg tggagagaaa catggtcatt cccgaaggaa gagttataga 7800 cttgggctgt ggaagaggag gctggtcata ttactgtgca ggactgaaaa aagtcacaga 7860 agtgcgagga tacacaaaag gcggtccagg acacgaagaa ccagtaccta tgtccacata 7920 tggatggaac atagttaagt taatgagtgg aaaggatgtg tttatcttc cacctgaaaa 7980 gtgtgacacc ctgttgtgcg acattggaga atcttcacca agcccaacag tggagaagaaag 8040 cagaactata agagttttga agatggttga accatggcta aagaacaacc aattttgtat 8100 taaagtattg aacccttaca tgccaactgt gattgagcac ctagaaagac tacaaaggaa 8160 acatggagga atgcttgtga gaaatccact ttcacgaaac tccacgcacg aaatgtactg 8220 gatatccaat ggcacaggta acattgtcgc ttcagtcaac atggtatcta gactgctact 8280 gaacaggttc acgatgacac acagaagacc caccattgag aaagatgtgg atttaggagc 8340 aggaactcga catgttaatg cggaaccaga aacacccaac atggatgtca ttgggggaaag 8400 aataaaaagg atcaaggagg agcataattc aacatggcac tacgatgacg aaaaccccta 8460 caaaacgtgg gcttaccatg gatcttatga agtcaaagcc acaggctcag cctcctccat 8520 gataaatgga gtcgtgaaac tcctcactaa accatgggat gtggtgccca tggtgacaca 8580 gatggcaatg acagatacaa ctccattgg ccagcagaga gtctttaaag agaagtgga 8640 caccaggaca cccaggtcca tgccaggaac aagaagggtt atgggatca cagcggagtg 8700 gctctggaga accctgggaa ggataaaaa acccaggtta tgcacaaggg aagagtttac 8760 aaaaaaggtc agaactaacg cagccatggg agctgttttc acagaggaga accaatggga 8820 cagcgcgaaa gctgctgttg aggatgagga ttttggaaa cttgtggaca gagaacgtga 8880 actccacaaa ctgggcaagt gtggaagctg tgtttacaac atgatgggta aggagagaa 8940 gaaacttgga gagtttggca aagcaaagg cagtagagct atatggtaca tgtggttggg 9000 agccaggtac cttgagttcg aagccttgg attctttaat gagaccact ggttctcgcg 9060 tgagaactct tacagtggag tggaggaga aggactgcac aagctaggct attattag 9120 ggacattcc aagatacccg gaggagctat gtatgctgat gatacagctg gttgggacac 9180 aagaataca gaagagatgacc tgcacaatga ggaaagagatc acacagcaa tggaccctga 9240 accacaggcag tagcgaacg ctatatta gctcacatac caaaaaag tggtcaaagt 9300 tcaacgaccg actccacag gcacggtaat ggatatcata tctaggaag accaaagagg 9360 cagtggacag gtaggaactt atggtctgaa tacattcacc aacatggaag cccagttaat 9420 cagacaatg gaaggagaag gtgtgctgtc aaaggcagac ctcgagaacc ctcatctgcc 9480 aggagaaa attacacaat ggttggaaac aaggagtg gagaggtta aaagaatggc 9540 cataagtggg gatgactgcg tggtgaaacc aatcgatgac aggttcgcta atgccctgct 9600 cgctctgaac agamatgggaa aggttcggaa agacatacct caatggcagc catcaaggg 9660 atggcatgat tggcacagg ttccttctg ctcccaccac ttcatgaat tgatcatgaa 9720 agatgaga aagttggtgg ttccctgcag accccaggac gaactaatag gaagggcaag 9780 aatctctcaa ggagcgggat ggagccttag agaaccgca tgtctgggga aagcctacgc 9840 tcaaatgtgg agtctcatgt atttcacag agagaccctc agactagcat ccaacgccat 9900 atgttcagca gtaccagtcc actgggtccc acgacatggt ctattcacgc 9960 tcaccatcag tggatgacca cagaagacat gcttactgtc tggagaagag tgtggatcga 10020 ggacaatcca tggatggaag acaaaactcc agtcacaacc tgggaaaatg ttccatatct 10080 agggaagaga gaagaccaat ggtgcggatc acttattggt ctcacttcca gagcaacctg 10140 ggcccagaac atacccacag caattcaaca ggttagaagc ctttataggca atgaaggtt 10200 tctggactac atgccttcaa tgaagagatt taggaaggag gagagtcgg agggagccat 10260 ttggtaaaac gtaggaagtg aaaaagaggt taactgtcag gcatattaa gccacagtac 10320 ggaagaagct gtgctgcctg tgagccccgt ccaaggacgt taaaagaaga agtcaggccc 10380 caaagccacg gtttgagcaa accgtgctgc ctgtagctcc gtcgtgggga cgtaaaacct 10440 gggaggctgc aaactgtgga agctctacgc acggtgtagc agactagcgg ttaggagaga 10500 cccctcccat gacacaacgc agcaggcgggg cccgagcact gaggaagct gtacctcctt 10560 gcaaaggact agaggttaga ggagaccccc cgcaataaa aacagcatat tgacgctggg 10620 agagaccaga gatcctgctg tctcctcagc atcattccag gcacagaacg ccagaaaatg 10680 gaatggtgct gttgaatcaa caggttct 10708 <210> 7 <211> 10649 <212> DNA <213> Dengue virus <400> 7 agttgttagt ctgtgtggac cgacaaggac agttccaaat cggaagcttg cttaacacag 60 ttctaacagt ttgtttaaat agagagcaga tctctggaaa aatgaaccaa cgaaaaaagg 120 tggttagacc acctttcaat atgctgaaac gcgagagaaa ccgcgtatca acccctcaag 180 ggttggtgaa gagattctca accggacttt tttctgggaa aggaccctta cggatggtgc 240 tagcattcat cacgtttttg cgagtccttt ccatcccacc aacagcaggg attctgaaga 300 gatggggaca gttgaagaaa aataaggcca tcaagatact gattggattc aggaaggaga 360 taggccgcat gctgaacatc ttgaacggga gaaaaaggtc aacgataaca ttgttgtgct 420 tgattcccac cgtaatggcg tttcacttgt caacaagaga tggcgaaccc ctcatgatag 480 tggcaaaaca tgaaaggggg agacctctct tgtttaagac aacagagggg atcaacaaat 540 gcactctcat tgccatggac ttgggtgaaa tgtgtgagga cactgtcacg tataaatgcc 600 ccctactggt caataccgaa cctgaagaca ttgattgctg gtgcaacctc acgtctacct 660 gggtcatgta tgggacatgc acccagagcg gagaacggag acgagagaag cgctcagtag 720 ctttgacacc acattcagga atgggattgg aaacaagagc tgagacatgg atgtcatcgg 780 aaggggcttg gaagcatgct cagagagtag agagctggat actcagaaac ccaggatttg 840 cgctcttggc aggatttatg gcttatatga ttgggcaaac aggaatccag cgaactgtct 900 tctttgtcct aatgatgctg gtcgccccat cctacggaat gcggtgcgta ggagtaggaa 960 acagagactt tgtggaagga gtctcaggtg gagcatgggt cgacctggtg ctagaacatg 1020 gaggatgcgt cacaaccatg gcccagggaa aaccaacctt ggattttgaa ctgactaaga 1080 caacagctaa ggaagtggct ctgttaagaa cctattgcat tgaagcctca atatcaaaca 1140 taactacggc aacaagatgt ccaacgcaag gagagcctta tctgaaagag gaacaggacc 1200 aacagtacat ctgccggaga gatgtggtag acagaggatg gggcaatggc tgtggcttgt 1260 ttggaaaagg aggagttgtg acatgtgcga agttttcatg ttcggggaag atacaggca 1320 atctggtcca aattgagaac cttgaataca cagtggttgt aacagtccac atggagaca 1380 cccatgcagt aggaatgac acatccaatc atggagttac agccacgata actcccaggt 1440 caccatcggt agaagtcaaa ttgccggact atggagact aacactcgat tgtgaaccca 1500 ggtctggaat tgactttaat gagatgatcc taatgaaat gaaaaagaa acatggctcg 1560 tgcataagca atggttttg gatctgccctc ttccatggac agcaggagca gandacatcag 1620 aggttcactg gattacaa gagagaatgg tgacgttcaa ggttcctcat gccagagac 1680 aggatgtgac agtgctggga tctcaggaag gagccatgca ttctgccctc gctggagcca 1740 cagaagtgga ctccggtgat ggaatcaca tgtttgcagg acatctcaag tgcaagtcc 1800 gtatggagaa attgagaatc aagggaatgt catacacgat gtgttcagga aagttttca 1860 ttgacaaga gatggcagaa accagcatg ggacacagt ggtgaaagtc aagtgaag 1920 gtgctggagc tccgtgtaaa gtccccatag agataagaga tgtaacaag gaaaaagtgg 1980 ttgggcgcgt tatctcatcc acccctttgg ctgagaatac caacagtgta accaacatag 2040 aattagaacc cccctttggg gacagctaca ttgtgatagg tgttggaaac agcgcattaa 2100 cactccattg gttcaggaaa gggagttcca ttggcaagat gtttgagtcc acatacagag 2160 gtgcaaaacg aatggccatt ctaggtgaaa cagcttggga ttttggttcc gttggtggac 2220 tgttcacatc attgggaaag gctgtacacc aggtttttgg aagcgtgtat acaaccatgt 2280 ttggaggagt ctcatggatg attagaatcc taattgggtt cttagtgttg tggattggca 2340 cgaattcaag gaacacttca atggccatga cgtgcatagc tgttggagga attactctgt 2400 ttttgggctt cacagttcaa gcggacatgg gttgtgtggt gtcatggagt gggagagaat 2460 tgaagtgtgg aagcggaatt tttgtggttg acaacgtgca cacttggaca gaacagtaca 2520 aattccaacc agagtcccca gcgagactag cgtctgcaat attaaatgcc cacaaagatg 2580 gggtctgtgg aattagatca accacgaggc tggaaaatgt tatgtggaag caaataacca 2640 atgagctaaa ctatgttctc tgggaaggag gacatgatct cactgtagtg gctggggatg 2700 tgaaaggggt gttgaccaag gggcagagag cactcacacc cccagcgagt gatctgaaat 2760 attcatggaa gatagggggg aaagcaaaaa tctcacccc tgagcaag aacaccacat 2820 ttttaataga cggaccagac acctctgaat gccccaatga acgaagggca tggaatttctt 2880 ttgaggtgga agactatgga ttgaggtgt tcacgaccaa catatggatg aaattccgag 2940 aaggaagttc agaagtgtgt gaccaggt taatgtcagc tgcaattaaa gaccagaaag 3000 ctgtgcatgc tgacatgggt tattggatag agagctcaaa aaaccagacc tggcagatg 3060 agagagcatc tcttattgaa gtgaaaacat gtctgtggcc caacccat acactgtgga 3120 gcaatggagt gctggaaagc cagatgctta ttccaaatc atatgcaggc cctttttcac 3180 agcacaatta ccgccaggggc tatgctacgc aaaccgtggg tccatggcac ttaggcaac 3240 tagagataga ctttggagaa tgccccggaa cacagtcac attcaggag attgtgacc 3300 atagaggccc atctttgagg accaccactg catctggaaa actagtcacg caatggtgtt 3360 gccgctcctg cacgatgccc cccttaaggt tcttaggaga agatgggtgc tggtaggga 3420 tggagattag gcccttgagt gaaaaagaag agaacatggt caaatcacag gtgacggccg 3480 gacagggcac atcggaaact ttttcaatgg gtctgttgtg cctgaccttg tttgtggaag 3540 aatgcttgag gagaagagtc accaggaaac acatgatatt agctgtggta atcactcttt 3600 gtgctatcat cctggggggc ctcacatgga tggacttgct acgagccctc atcatgttgg 3660 gggacactat gtctggtaga ataggaggac agacccacct agccatcatg gcagtgttca 3720 agatgtcacc gggatacgtg ctgggtgtgt ttttaaggaa actcacttca agagagacag 3780 cactaatggt aataggaatg gccatgacaa caacactttc aattccacat gacctcatgg 3840 aactcattga tggaatatca ctaggactaa ttttgctaaa aatagtaaca cagtttgaca 3900 acacccaagt gggaacctta gctctttcct tgactttcat aagatcaaca atgtcattgg 3960 tcatggcttg gaggaccatt atggctgtgt tgtttgtggt cacactcatt cctttgtgca 4020 ggacaagctg tcttcaaaaa cagtctcatt gggtagaaat aacagcactc atcctaggag 4080 cccaagctct gccagtgtac ctaatgactc ttatgaaagg agcctcaaga agatcttggc 4140 ctcttaacga aggcataatg gctgtgggtt tggttagtct cttaggaagc gctcttttaa 4200 agaatgatgt ccctttagct ggcccaatgg tggcaggagg cttacttctg gcggcttacg 4260 taatgagtgg cagctcagca gatctgtcac tagagaaggc cgctaatgtg cagtgggatg 4320 aaatggcaga cataacaggc tcaagtccaa tcatagaagt gaagcaagat gaggatggct 4380 ctttctccat acgggacgtc gaggaaacca atatgataac ccttttggtg aaactggcac 4440 tgataacggt gtcaggtctc taccccttgg caattccaat cacaatgacc ttatggtaca 4500 tgtggcaagt gaaaacacaa agatcaggag ccctgtggga cgtcccttca cccgccgcca 4560 ctcaaaaagc cgcactgtct gaaggagtgt acaggatcat gcaaagaggg ttattcggga 4620 aaactcaggt tggagtaggg atacacatgg aaggtgtatt tcacacaatg tggcatgtta 4680 caagaggatc ggtgatctgc cacgagactg ggagattgga gccatcttgg gctgatgtca 4740 ggaatgacat gatatcatac ggtgggggat ggaggcttgg agataaatgg gacaaagaag 4800 aagacgttca ggtcctcgct atagaaccag ggaaaaatcc caaacatgtc caaacgaaac 4860 ctggcctttt caacccta actggagaaa ttggagcagt aacattagat ttcaacccg 4920 gaacgtctgg ttctcccatt atcacagga aaggaaagt catcggactc tatggaatg 4980 gagtggtcac aaatcaggt gattacgtca gtgccataac acagccgaa agaattggag 5040 agccagatta tgaagtggat gaggacatttt ttcgagaa agactact atatggact 5100 tacaccccgg agccggaaag aaaaagaa ttctccatc atagtgaga gaagccttaa 5160 aaaggaggct gcgaactttg attttggctc ccacgagagt ggtggcggcc gagatggaag 5220 aggcctacg tggactgcca atccgttacc aaaccccagc tgtaaatca gaacacacag 5280 gaagagagat tgtagaccctc atgtgccatg caaccttcac aacaagactt ttgtcatca 5340 ccagagttcc aaactacaac cttatagtaa tggatgaagc acatttcacc gatccttcca 5400 gtgtcgcggc tagaggatac atttcgacca gggtggaaat gggagaggca gcagccatct 5460 tcatgaccgc aacccctccc ggagcgacag atccctttcc ccagagcac agcccaatag 5520 aagacatcga gagagagatt cggaaggt catggacac agggttcgac tggatacag 5580 actaccaagg gaaaactgtg tggtttgttc ccagcataaa agctggaaat gacattgcaa 5640 attgtttgag aaagtcggga aagaaagtta tccagttgag taggaaaacc tttgatacag 5700 aatatccaaa aacgaaactc acggactggg actttgtggt cactacagac atatctgaaa 5760 tggggctaa ctttagagct gggagagtga tagaccctag aagatgcctc aagccagtta 5820 tcctaacaga tgggccagag agagtcatct tagcaggtcc tattccagtg actccagcaa 5880 gcgctgctca gagaagagg cgaataggaa ggaacccagc acaagaagac gaccaatacg 5940 ttttctccgg agacccacta aaaaatgatg aagaccatgc ccactggaca gaagcaaaga 6000 tgctgcttga caatatctac accccagaag ggatcattcc aacattgttt ggtccggaaa 6060 gggaaaaaac ccaagctatt gatggagagt ttcgcctcag agggaacaa aggaagactt 6120 ttgtggaatt aatgaggaga ggagaccttc cggtgtggct gagttataag gtagcttctg 6180 ctggcatttc ttacaaagat cgggaatggt gctttactgg ggaaagaaat aaccaaattt 6240 tagagaaaa catggaggtt gaaatttgga ctagagagg agaaaagaaa aaactgaggc 6300 caaaatggtt agatgcacgt gtatacgctg accccacatggc tttgaaggat ttcaaggagt 6360 ttgccagtgg aaagaagagt ataactctcg acatcctaac agagatcgcc agtttgccaa 6420 cttacctttc ctctagggcc aagctcgccc ttgacaacat agttatgctc cacaacag 6480 aaaaggagg gagggcctat caacatgccc tgaacgaact tccggagtca ctggaaacac 6540 tcatgcttgt agccttacta ggagctatga cagcaggtat cttcctgttt ttcatgcaag 6600 ggaaggaat agggaaattg tcaatgggtt tgataaccat tgcggtggct agtggcttgc 6660 tctgggtagc agaaattcaa ccccagtgga tagcggcctc aatcatactg gagttttttc 6720 tcatggtact gttgatacca gaaccagaaa aacaaaggac cccacaagac aatcaattga 6780 tctacgtcat attgaccatt ctcaccatta ttggtctaat agcagccaac gagatggggc 6840 tgatagaaaa aacaaaaacg gattttgggt tttaccaggt aaaaacagaa accaccatcc 6900 tcgatgtgga cctgagacca gcttcagcat ggacgctcta tgcggtagcc accacaattc 6960 tgactcccat gctgagacac accatagaaa atacgtcggc caacctatct ttagcagcca 7020 tcgccaacca ggcagccgtc ctaatggggc ttggaaaagg atggccactc cacagaatgg 7080 acctcggtgt gccgctgtta gcaatgggat gctattctca agtgaaccca acaaccttga 7140 cagcatcctt agtcatgctt ttagtccatt atgcaataat aggcccagga ttgcaggcaa 7200 aagccacaag agaggcccag aaaaggacag ctgctggaat catgaaaaat cccacagtgg 7260 acgggataac agtgatagat ctagaaccaa tatcctatga cccaaaattt gaaaagcaat 7320 tagggcaggt catgttactc gtcttgtgtg ctggacaact actcttgatg agaacaacat 7380 gggctttctg tgaagttttg actttggcca caggaccaat cttgaccttg tgggagggca 7440 acccgggaag gttttggaac acgaccatag ccgtatctac cgccaacatt ttcaggggaa 7500 gttatttggc aggagctgga ctggcttttt cactcataaa gaatgcacaa acccccagga 7560 ggggaactgg gaccacagga gagacactgg gagagaagtg gaagagacag ctaaactcat 7620 tagacaggaa agagtttgaa gagtataaaa gaagtggaat actagaagtg gacaggactg 7680 aagccaagtc cgccctgaaa gatgggtcta aaatcaagca tgcagtatct agagggtcca 7740 gtaagatcag atggattgtt gagagaggga tggtaaagcc aaaggggaa gttgtagatc ttggctgtgg gagaggaga tggtcttatt acatggcgac actcaagaac gtgactgaag tgaaagggta tacaaaagga ggtccaggac atgaagaacc gattcccatg gctacttatg gctggaattt ggtcaaactc cattcagggg ttgacgtgtt ctacaaaccc acagagcaag tggacaccct gctctgtgat attggggagt catcttctaa tggacacaata 8040 attggggagt 8100. catggctctc ttcaaaacct catggctgca tcaaagtcct caacccctac atgccaacag tcatagagga gctggagaaa ctgcagagaa aacacggtgg gaaccttgtc agatgcccgc tgtccagga ctccacccat gagatgtatt gggtgtcagg agcgtcggga aatattgtga gctctgtgaa cacaacatca aagatgttgt tgaacaggtt cacaacaagg cataggaaac ccacttatga gaggacgta gatcttgggg 8400. 8400. 8400. 8400. 8400. 8400. 8400. 8400. 8400 ggcttcagcg attgcaagag gagcacaaag aaacatggca ttatgatcag gaaaacccat acagaacctg ggcgtatcat ggaagctatg aagctccttc zgaggctct gcatcctcca 8520 tggtgaacgg gtggtgaaa ctgctaacaa aaccctggga tgtgattcca atggtgactc 8580 agttagccat gabataca accccttttg ggcacaaag agtgttcaa gagaaggtgg 8640 ataccagaac gccgcaacca aaaccaggca cacgaatggt tatgaccacg acagccaatt 8700 ggctatgggc cctccttgga aagagaaaa atcccagact gtgtacagg gagagttca 8760 tctcaaagt tagatcaac gcagccatag gcgcagctt tcaggagaa caggatgga 8820 catcagccag tgaagctgtg aatgacagcc ggttttggga actggttgac aaagaaaggg 8880 cccttcacca ggaagggaaa tgtgaatcgt gtgtctataa catgatggga aaacgtgaga 8940 aaagttagg agagtttggc agagccaagg gaagccgagc aatctggtac atgtggctgg 9000 gagcgcggtt tctggaattt gaagccctgg gttttgaa tgagatcac tggttttggca 9060 gagaaaatttc atggagtgga gtggaagggg aaggtctgca cagattggga tacatcctgg 9120 aggagataga caagaaggat ggagacctaa tgtagctga tgacacagca ggttgggaca 9180 caagaatcac tgaggatgac cttcaatg aggactgat cacggacag atggctcccc 9240 accacagat cctagccaaa gccattttca aactaaccta tcaaaacaa gtggtgaaag 9300 tcctcagacc cacaccgaga ggagcggtga tggatatcat atccaggaa gaccaaagg 9360 gtagtggaca agttggaaca tatggtttga acacattcac siacatggaa gttcaactca 9420 tccgccaaat ggaagctgaa ggagtcatca cacagatga catgcagaac ccaaagggt 9480 tgaaagaag agttgagaaa tggctgagag agtgtggtgt cgacaggtta aagaggatgg 9540 caatcagtgg agacgattgc gtggtgaagc ccctagatga gaggttttggc acttccctcc 9600 tcttcttgaa cgacatggga aaggtgagga aagacattcc gcagtgggaa ccatctagg 9660 gatggaaaaa ctggcaagag gttccttttt gctcccatca ctttcacaag atctttatga 9720 aggatggccg ctcactagtt gttccatgta gaaaccagga tgaactgata gggagagcca 9780 gatctcgca gggggctgga tggagcttaa gagaaacagc ctgcctgggc aaagcttacg 9840 cccagatgtg gtcgcttatg tactccaca gaagggatct gcgtttagcc tccatggcca 9900 tatgctcagc agttccaacg gatggtttc caacaagcag aacaatgg tcaatccatg 9960 ctcatcacca atggatgacc actgagaca tgctcaagt gtggaacaga gtgtggatag 10020 aagacaaccc caataccttacc cagaagactc cagtccattc gtgggagat ataccttacc 10080 taggaaaag agaggattg tggtgtgat ccctgattgg actttctct agagccacct 10140 gggcgaagaa cattcacacg gccatactc aggtcaggaa cttgatcgga aaagaggaat 10200 acgtggatta catgccagta atgaaagat acagtgctcc ttcagagt gaaggagttc 10260 tgtaattacc aacaacaac accaaaggct attgaagtca ggccacttgt gccacggttt 10320 gagcaaaccg tgctgcctgt agctccgcca ataatgggag gcgtaataat ccctagggag 10380 gccatgcgcc acggaagctg tacgcgtggc atattggact agcggttaga ggagacccct 10440 cccatcactg acaaaacgca gcaaagggg gcccgaagcc aggaggagc tgtactccctg 10500 gtggaaggac tagaggttag aggacccc cccaacacaa aaacagcata ttgacgctgg 10560 gaagaccag agatcctgct gtctctacaa catcaatcca ggcacagagc gccgcagat 10620 ggattggtgt tgttgatcca acaggttct 10649 <210> 8 <211> 10649 <212> DNA <213> Dengue virus <400> 8 agttgttagt ctgtgtggac cgacaaggac agttccaaat cggaagcttg cttaacacag 60 ttctaacagt ttgtttaaat agagagcaga tctctggaaa aatgaaccaa cgaaaaaagg 120 tggttagacc acctttcaat atgctgaaac gcgagagaaa ccgcgtatca acccctcaag 180 ggttggtgaa gagattctca accggacttt tttctgggaa aggaccctta cggatggtgc 240 tagcattcat cacgtttttg cgagtccttt ccatcccacc aacagcaggg attctgaaga 300 gatggggaca gttgaagaaa aataaggcca tcaagatact gattggattc aggaaggaga 360 taggccgcat gctgaacatc ttgaacggga gaaaaaggtc aacgataaca ttgttgtgct 420 tgattcccac cgtaatggcg tttcacttgt caacaagaga tggcgaaccc ctcatgatag 480 tggcaaaaca tgaaaggggg agacctctct tgtttaagac aacagagggg atcaacaaat 540 gcactctcat tgccatggac ttgggtgaaa tgtgtgagga cactgtcacg tataaatgcc 600 ccctactggt caataccgaa cctgaagaca ttgattgctg gtgcaacctc acgtctacct 660 gggtcatgta tgggacatgc acccagagcg gagaacggag acgagagaag cgctcagtag 720 ctttgacacc acattcagga atgggattgg aaacaagagc tgagacatgg atgtcatcgg 780 aaggggcttg gaagcatgct cagagagtag agagctggat actcagaaac ccaggatttg 840 cgctcttggc aggatttatg gcttatatga ttgggcaaac aggaatccag cgaactgtct 900 tctttgtcct aatgatgctg gtcgccccat cctacggaat gagatgcgta ggcgtaggta 960 atcgcgattt cgttgaggga gtgtcaggcg gagcatgggt cgatctggta ctcgaacacg 1020 gaggatgcgt tacgactatg gcacagggaa aaccgacatt ggactttgag ttgactaaga 1080 caaccgctaa agaggtcgca ctattgcgaa catactgtat cgaagcgtca atttcgaata 1140 ttacaaccgc tactaggtgt cctacacagg gcgaaccata ccttaaagag gaacaggacc 1200 aacagtatat ttgtagaaga gacgtagtcg atagggggtg gggaaacgga tgcggattgt 1260 tcggtaaggg gggagtcgtt acatgcgcta agttctcatg ttccggtaag attaccggta 1320 acttagtgca aattgagaat ctcgaatata ccgtagtcgt tacagtgcat aacggagaca 1380 cacacgcagt cggaaacgat acatctaacc atggcgtaac cgctacaatt acacctaggt 1440 caccatccgt tgaggttaag ttgccccgatt acggcgaact gacactcgat tgcgaaccta 1500 gatccggaat agactttaac gaaatgatac tgatgaaaat gaaaaaaaag acatggttag 1560 tgcataagca atggttctc gacttacccc taccttggac cgccggagcc gatactagcg 1620 aagtgcactg gaattacaaa gagagaatgg ttacatttaa agtgccacac gctaagagac 1680 aggacgttac agtgttaggg tcacaggagg gagctatgca tagcgcacta gccggagcaa 1740 ccgaagtcga tagcggagac ggaaatcata tgttcgccgg acatctgaaa tgcaaagtga 1800 gaatggagaa attgcggatt aagggaatgt catacactat gtgttccggt aagttttcga 1860 ttgacaaaga gatggccgaa acgcaacacg gaacaacagt cgttaaggtt aagtacgaag 1920 gagccggagc tccgtgtaaa gtgccaatcg aaattagaga cgttaacaaa gagaaagtgg 1980 tcggaagagt gatatctagt acacccctag ccgaaaatac gaattccgtt acgaatatcg 2040 aactcgaacc cccctttgga gactcataca tagtgatagg agtgggtaat tccgcactca 2100 cgttgcactg gttcagaaag ggatcatcaa tcgggaagat gttcgaatca acatataggg 2160 gagcgaaaag aatggctata ttgggcgaaa ccgcatggga cttcggatca gtcggaggac 2220 tgtttacgag tctgggtaag gccgtacatc aggtattcgg atcagtgtat acaacaatgt 2280 ttggcggagt gtcatggatg atacggatac tgatagggtt tctagtgttg tggatcggaa 2340 cgaactcacg taatacgtct atggctatga catgtattgc cgtagggggg attacactgt 2400 ttctgggatt tacagtgcaa gcagacatgg gttgtgtggt gtcatggagt gggagagaat 2460 tgaagtgtgg aagcggaatt tttgtggttg acaacgtgca cacttggaca gaacagtaca 2520 aattccaacc agagtcccca gcgagactag cgtctgcaat attaaatgcc cacaaagatg 2580 gggtctgtgg aattagatca accacgaggc tggaaaatgt tatgtggaag caaataacca 2640 atgagctaaa ctatgttctc tgggaaggag gacatgatct cactgtagtg gctggggatg 2700 tgaaaggggt gttgaccaag ggcaagagag cactcacacc cccagcgagt gatctgaaat 2760 attcatggaa gatagggggg aaagcaaaaa tctcacccc tgagcaag aacaccacat 2820 ttttaataga cggaccagac acctctgaat gccccaatga acgaagggca tggaatttctt 2880 ttgaggtgga agactatgga ttgaggtgt tcacgaccaa catatggatg aaattccgag 2940 aaggaagttc agaagtgtgt gaccaggt taatgtcagc tgcaattaaa gaccagaaag 3000 ctgtgcatgc tgacatgggt tattggatag agagctcaaa aaaccagacc tggcagatg 3060 agagagcatc tcttattgaa gtgaaaacat gtctgtggcc caacccat acactgtgga 3120 gcaatggagt gctggaaagc cagatgctta ttccaaatc atatgcaggc cctttttcac 3180 agcacaatta ccgccaggggc tatgctacgc aaaccgtggg tccatggcac ttaggcaac 3240 tagagataga ctttggagaa tgccccggaa cacagtcac attcaggag attgtgacc 3300 atagaggccc atctttgagg accaccactg catctggaaa actagtcacg caatggtgtt 3360 gccgctcctg cacgatgccc cccttaaggt tcttaggaga agatgggtgc tggtaggga 3420 tggattag gcccttgagt gaaaagaag agaacatggt caatcacag gtgacggccg 3480 gacagggcac atcggaaact ttttcaatgg gtctgttgtg cctgaccttg tttgtggaag 3540 aatgcttgag gagaagagtc accaggaaac acatgatatt agctgtggta atcactcttt 3600 gtgctatcat cctggggggc ctcacatgga tggacttgct acgagccctc atcatgttgg 3660 gggacactat gtctggtaga ataggaggac agacccacct agccatcatg gcagtgttca 3720 agatgtcacc gggatacgtg ctgggtgtgt ttttaaggaa actcacttca agagagacag 3780 cactaatggt aataggaatg gccatgacaa caacactttc aattccacat gacctcatgg 3840 aactcattga tggaatatca ctaggactaa ttttgctaaa aatagtaaca cagtttgaca 3900 acacccaagt gggaacctta gctctttcct tgactttcat aagatcaaca atgtcattgg 3960 tcatggcttg gaggaccatt atggctgtgt tgtttgtggt cacactcatt cctttgtgca 4020 ggacaagctg tcttcaaaaa cagtctcatt gggtagaaat aacagcactc atcctaggag 4080 cccaagctct gccagtgtac ctaatgactc ttatgaaagg agcctcaaga agatcttggc 4140 ctcttaacga aggcataatg gctgtgggtt tggttagtct cttaggaagc gctcttttaa 4200 agaatgatgt ccctttagct gcccaatgg tggcaggagg cttactctg gcggcttacg 4260 taatgagtgg cagctcagca gatctgtcac taggagaaggc cgctaatgtg cagtgggatg 4320 aaatggcaga cataacaggc tcaagtccaa tcatagaagt gagcagat gaggatggct 4380 ctttctccat acgggacgtc gaggaaacca atatgataac ccttttggtg aactggcac 4440 tgataacggt gtcaggtctc taccccttgg cattccaat cacaatgacc ttatgtaca 4500 tgtggcaagt gaaaacacaa agatcaggag ccctgtggga cgtcccttca cccgccgcca 4560 ctcaaaaagc cgcactgtct gaggagtgt acaggatcat gcaagagggg ttattcggga 4620 aaactcaggt tggagtaggg atacacatgg aaggtgtatt tcacacaatg tggcatgtta 4680 cagaggatc ggtgatctgc cacgagactg ggagattgga gccatcttgg gctgatgtca 4740 ggaatgacat gatatcatac ggtgggggat ggaggctttgg agataatgg vakaagaag 4800 aagacgttca ggtcctcgct atagaaccag ggaaaaatcc siacatgtc caacgaaac 4860 ctggcctttt caacccta actggagaaa ttggagcagt aacattagat ttcaacccg 4920 gaacgtctgg ttctcccatt atcacagga aaggaaagt catcggactc tatggaatg 4980 gagtggtcac aaatcaggt gattacgtca gtgccataac acagccgaa agaattggag 5040 agccagatta tgaagtggat gaggacatttt ttcgagaa agactact atatggact 5100 tacaccccgg agccggaaag aaaaagaa ttctccatc atagtgaga gaagccttaa 5160 aaaggaggct gcgaactttg attttggctc ccacgagagt ggtggcggcc gagatggaag 5220 aggcctacg tggactgcca atccgttacc aaaccccagc tgtaaatca gaacacacag 5280 gaagagagat tgtagaccctc atgtgccatg caaccttcac aacaagactt ttgtcatca 5340 ccagagttcc aaactacaac cttatagtaa tggatgaagc acatttcacc gatccttcca 5400 gtgtcgcggc tagaggatac atttcgacca gggtggaaat gggagaggca gcagccatct 5460 tcatgaccgc aacccctccc ggagcgacag atccctttcc ccagagcac agcccaatag 5520 aagacatcga gagagagatt cggaaggt catggacac agggttcgac tggatacag 5580 actaccaagg gaaaactgtg tggtttgttc ccaccataa agctggaat gatatgcaa 5640 attgtttgag aaagtcggga aagaaagtta tccagttgag taggaaaacc tttgatacag 5700 aatatccaaa aacgaaactc acggactggg actttgtggt cactacagac atatctgaaa 5760 tggggctaa ctttagagct gggagagtga tagaccctag aagatgcctc aagccagtta 5820 tcctaacaga tgggccagag agagtcatct tagcaggtcc tattccagtg actccagcaa 5880 gcgctgctca gagaagagg cgaataggaa ggaacccagc acaagaagac gaccaatacg 5940 ttttctccgg agacccacta aaaaatgatg aagaccatgc ccactggaca gaagcaaaga 6000 tgctgcttga caatatctac accccagaag ggatcattcc aacattgttt ggtccggaaa 6060 gggaaaaaac ccaagctatt gatggagagt ttcgcctcag agggaacaa aggaagactt 6120 ttgtggaatt aatgaggaga ggagaccttc cggtgtggct gagttataag gtagcttctg 6180 ctggcatttc ttacaaagat cgggaatggt gctttactgg ggaaagaaat aaccaaattt 6240 tagagaaaa catggaggtt gaaatttgga ctagagagg agaaaagaaa aaactgaggc 6300 caaaatggtt agatgcacgt gtatacgctg accccatggc tttgaaggat ttcaaggagt 6360 ttgccagtgg aaagaagagt ataactctcg acatcctaac agagatcgcc agtttgccaa 6420 cttacctttc ctctagggcc aagctcgccc ttgacaacat agttatgctc cacaacag 6480 aaaaggagg gagggcctat caacatgccc tgaacgaact tccggagtca ctggaaacac 6540 tcatgcttgt agccttacta ggagctatga cagcaggtat cttcctgttt ttcatgcaag 6600 ggaaggaat agggaaattg tcaatgggtt tgataaccat tgcggtggct agtggcttgc 6660 tctgggtagc agaaattcaa ccccagtgga tagcggcctc aatcatactg gagttttttc 6720 tcatggtact gttgatacca gaaccagaaa aacaaaggac cccacaagac aatcaattga 6780 tctacgtcat attgaccatt ctcaccatta ttggtctaat agcagccaac gagatggggc 6840 tgatagaaaa aacaaaaacg gattttgggt tttaccaggt aaaaacagaa accaccatcc 6900 tcgatgtgga cctgagacca gcttcagcat ggacgctcta tgcggtagcc accacaattc 6960 tgactcccat gctgagacac accatagaaa atacgtcggc caacctatct ttagcagcca 7020 tcgccaacca ggcagccgtc ctaatggggc ttggaaaagg atggccactc cacagaatgg 7080 acctcggtgt gccgctgtta gcaatgggat gctattctca agtgaaccca acaaccttga 7140 cagcatcctt agtcatgctt ttagtccatt atgcaataat aggcccagga ttgcaggcaa 7200 aagccacaag agaggcccag aaaaggacag ctgctggaat catgaaaaat cccacagtgg 7260 acgggataac agtgatagat ctagaaccaa tatcctatga cccaaaattt gaaaagcaat 7320 tagggcaggt catgttactc gtcttgtgtg ctggacaact actcttgatg agaacaacat 7380 gggctttctg tgaagttttg actttggcca caggaccaat cttgaccttg tgggagggca 7440 acccgggaag gttttggaac acgaccatag ccgtatctac cgccaacatt ttcaggggaa 7500 gttatttggc aggagctgga ctggcttttt cactcataaa gaatgcacaa acccccagga 7560 ggggaactgg gaccacagga gagacactgg gagagaagtg gaagagacag ctaaactcat 7620 tagacaggaa agagtttgaa gagtataaaa gaagtggaat actagaagtg gacaggactg 7680 aagccaagtc cgccctgaaa gatgggtcta aaatcaagca tgcagtatct agagggtcca 7740 gtaagatcag atggattgtt gagagaggga tggtaaagcc aaaggggaaa gttgtagatc 7800 ttggctgtgg gagaggagga tggtcttatt acatggcgac actcaagaac gtgactgaag 7860 tgaaagggta tacaaaagga ggtccaggac atgaagaacc gattcccatg gctacttatg 7920 gctggaattt ggtcaaactc cattcagggg ttgacgtgtt ctacaaaccc acagagcaag 7980 tggacaccct gctctgtgat attggggagt catcttctaa tccaacaata gaggaaggaa 8040 gaacattaag agttttgaag atggtggagc catggctctc ttcaaaacct gaattctgca 8100 tcaaagtcct caacccctac atgccaacag tcatagagga gctggagaaa ctgcagagaa 8160 aacacggtgg gaaccttgtc agatgcccgc tgtccaggaa ctccacccat gagatgtatt 8220 gggtgtcagg agcgtcggga aatattgtga gctctgtgaa cacaacatca aagatgttgt 8280 tgaacaggtt cacaacaagg cataggaaac ccacttatga gaaggacgta gatcttgggg 8340 caggaacgag aagtgtctct actgaaacag aaaaaccaga catgacaatc attgggagaa 8400 ggcttcagcg attgcaagag gagcacaaag aaacatggca ttatgatcag gaaaacccat 8460 acagaacctg ggcgtatcat ggaagctatg aagctccttc gacaggctct gcatcctcca 8520 tggtgaacgg gtggtgaaa ctgctaacaa aaccctggga tgtgattcca atggtgactc 8580 agttagccat gabataca accccttttg ggcacaaag agtgttcaa gagaaggtgg 8640 ataccagaac gccgcaacca aaaccaggca cacgaatggt tatgaccacg acagccaatt 8700 ggctatgggc cctccttgga aagagaaaa atcccagact gtgtacagg gagagttca 8760 tctcaaagt tagatcaac gcagccatag gcgcagctt tcaggagaa caggatgga 8820 catcagccag tgaagctgtg aatgacagcc ggttttggga actggttgac aaagaaaggg 8880 cccttcacca ggaagggaaa tgtgaatcgt gtgtctataa catgatggga aaacgtgaga 8940 aaagttagg agagtttggc agagccaagg gaagccgagc aatctggtac atgtggctgg 9000 gagcgcggtt tctggaattt gaagccctgg gttttgaa tgagatcac tggttttggca 9060 gagaaaatttc atggagtgga gtggaagggg aaggtctgca cagattggga tacatcctgg 9120 aggagataga caagaaggat ggagacctaa tgtagctga tgacacagca ggttgggaca 9180 caagaatcac tgaggatgac cttcaatg aggactgat cacggacag atggctcccc 9240 accacagat cctagccaaa gccattttca aactaaccta tcaaaacaa gtggtgaaag 9300 tcctcagacc cacaccgaga ggagcggtga tggatatcat atccaggaa gaccaaagg 9360 gtagtggaca agttggaaca tatggtttga acacattcac siacatggaa gttcaactca 9420 tccgccaaat ggaagctgaa ggagtcatca cacagatga catgcagaac ccaaagggt 9480 tgaaagaag agttgagaaa tggctgagag agtgtggtgt cgacaggtta aagaggatgg 9540 caatcagtgg agacgattgc gtggtgaagc ccctagatga gaggttttggc acttccctcc 9600 tcttcttgaa cgacatggga aaggtgagga aagacattcc gcagtgggaa ccatctagg 9660 gatggaaaaa ctggcaagag gttccttttt gctcccatca ctttcacaag atctttatga 9720 aggatggccg ctcactagtt gttccatgta gaaaccagga tgaactgata gggagagcca 9780 gatctcgca gggggctgga tggagcttaa gagaaacagc ctgcctgggc aaagcttacg 9840 cccagatgtg gtcgcttatg tactccaca gaagggatct gcgtttagcc tccatggcca 9900 tatgctcagc agttccaacg gatggtttc caacaagcag aacaatgg tcaatccatg 9960 ctcatcacca atggatgacc actgagaca tgctcaagt gtggaacaga gtgtggatag 10020 aagacaaccc caataccttacc cagaagactc cagtccattc gtgggagat ataccttacc 10080 taggaaaag agaggattg tggtgtgat ccctgattgg actttctct agagccacct 10140 gggcgaagaa cattcacacg gccatactc aggtcaggaa cttgatcgga aaagaggaat 10200 acgtggatta catgccagta atgaaagat acagtgctcc ttcagagt gaaggagttc 10260 tgtaattacc aacaacaac accaaaggct attgaagtca ggccacttgt gccacggttt 10320 gagcaaaccg tgctgcctgt agctccgcca ataatgggag gcgtaataat ccctagggag 10380 gccatgcgcc acggaagctg tacgcgtggc atattggact agcggttaga ggagacccct 10440 cccatcactg acaaaacgca gcaaagggg gcccgaagcc aggaggagc tgtactccctg 10500 gtggaaggac tagaggttag aggacccc cccaacacaa aaacagcata ttgacgctgg 10560 gaagaccag agatcctgct gtctctacaa catcaatcca ggcacagagc gccgcagat 10620 ggattggtgt tgttgatcca acaggttct 10649 <210> 9 <211> 10735 <212> DNA <213> Dengue Virus <400> 9 agttgttagt ctacgtggac cgacagaac agtttcgaat cggaagcttg cttaacgtag 60 ttctacagt tttttag agagcagatc tctgatgaac aaccaacgga aaagacggc 120 tcgaccgtct ttcaatgc tgaacgcgc gagaaccgc gtgtcaactg ttcacagtt 180 ggcgaagaga ttctcaaag gattgctctc aggccaagga cccatgaat tggtgatggc 240 ttcatagca ttcctaagat tttagccat acccccaaca gcaggaattt tggctagatg 300 gggttcattc aagagagtg gagcgatcaa agtgctacgg ggtttcaaga aagaaatctc 360 aaacatgttg atataatga atagaggaa aagatctgtg accatgctcc ttatgctgat 420 gcctacagcc ttggcgttcc atttgactac acgaggggga gagccgcaca tgagtcag 480 caagcaggaa agaggaaagt cactcttgtt tagacctca gcaggtgtca acatgtgcac 540 ccttatagcg atggattgg gagagttatg tgaggacaca atgacttaca atgccctcg 600 aatcactgaa actgaaccag atgacgttga tgttggtgt aatgccacag accatgggt 660 gacctatgga acatgttccc aaactggcga gcaccgacga gandaacgtt ccgtcgcact 720 ggccccacac gtgggacttg gtttggaac aagaaccgaa acgtggatgt cctctgaagg 780 cgcttggaaa cagatacaa gagtggagac ttggccctg agacacccag gattcacggt 840 gatagccctt ttcttagcac atgccatagg aacatccatc acccagaaag ggattatttt 900 cattttgtta atgctggtaa caccatccat ggccatgcga tgcgtggga taggcagcag 960 ggactcgtg gaaggactgt caggagcaac ttgggtagat gtggtactgg aacatggaag 1020 ttgcgtcacc accatggcaa agacaacc aacattggac attgactct tgagacgga 1080 agtcacaaac cctgccgtcc tgcgcaact gtgcattgaa gctaaaatat caacaccac 1140 caccgactca agatgtccaa caaggaga agccacacta gtggagaac aagacgcgaa 1200 ctttgtgtgt cgacgaacgt tgtggacag aggctggggc aatggctgtg gcctcttcgg 1260 aaaaggaagc ctaataacgt gtgcaagtt caagtgtgtg aaaactgg aaggaagat 1320 agttcaatt gagaacttga atattcagt atagtcacc gttcacaccg gagaccagca 1380 tcaagtggga atgaaagca cagaacatgg cakaactgca actatacac ctcaagctcc 1440 tacgacggaa atacagctga ccgactacgg agctcttaca tggattgtt cacctagaac 1500 aggactagac tttaatgaaa tggtgttgtt gatagaaa gaaaatcat ggctagtcca 1560 caaacaatgg ttttagacc taccactgcc ttggactcg ggagctca catcacaaga 1620 gacttggaac agacagatt tgctggtgac atttagaca gctcatgcaa agaagcagga 1680 agtagtagtg ttaggtcac agagggagc aatgcatacc gcactaccg gagcaaccga 1740 atacagact agcggaacta caacgattt tgccggtcac ctgaatgta gactgagat 1800 ggacaaactg acacttaag gatgtcata cgttatgtgt acgggatcct ttaagctcga 1860 aaagaggtt gccgaacgc aacacggaac agtgctagtg caatttaat atgaggac 1920 cgacgcacca tgtaagatac cgtttagtac gcaagacgaa aagggcgtta cgcaaaacgg 1980 aagactgatt accgctaacc ctacgttac agacaaag aaacccgtta acatagaggc 2040 cgaaccacct tttggcgaat catatatagt gataggcgca ggcgaaaag cactaaagct 2100 gtcatggttc aaaaaaggat ctagcatagg gagatgttc gaagcaccg ctaggggagc 2160 tagacgaatg gcaatactgg gagataccgc atgggacttc ggatcgatag ggggagtgtt 2220 tactagcgta ggtaagttgg tgcatcagat attcggaacc gcatacggag tgttgtttag 2280 cggagtgtca tggactga agataggt aggagtgcta tgacatggc tcggactgaa 2340 ttcgagatcg actagcctat ctatgacatg catagccgtc ggactggtta cactgtatct 2400 aggcgtaatg gtgcaagcag attcaggatg tgtattaat tggaaggta gagaactca 2460 gtgtggaagt ggcattttg tcaccaatga agttcacact tggacagc atacaatt 2520 tcaagctgac tcccctaaga gactcagc agccatcggg aaggcatggg aggaggttgt 2580 gtgtggaatt cgatcagcaa ctcgtctcga gaacatcatg tggagcaaa tatcaatga 2640 actgaatcac atcttacttg aaatgatat gaattcaca gtggttgtag gagatgttgc 2700 tgggatttg gctcaggaa agaaatgat taggccacaa cccatggaat acaatactc 2760 gtggaaaagc tggggaagg ctaaaatcat aggggcagat gtacagaaca ccaccttcat 2820 catcgatggc ccaacaccc cagaatgccc tgatgaccaa agagcatgga acatttggga 2880 agttgaggac tattggattg gattttcac gandaacata tggctgaat tgcgtgattc 2940 ctacacccaa gtgtgtgacc accggctaat gtcagctgcc atcaggaca gcaggcagt 3000 tcacgctgac atggggtact ggatagaaag tgaaagac gagacctgga agctggcaag 3060 agcctcattc atagaagtta aaacatgtat ctggccaaaa tcccactc tatggagcaa 3120 tggagttctg gaagtgaaa tgataattcc aagatctat ggaggaccaa tatctcagca 3180 caactacaga ccaggatatt ttacacaagc agcaggggccg tggcacctag gcaagttgga 3240 actggatttt gatttgtgtg agggtaccac agttgttgtg gatgaacatt gtggaaatcg 3300 aggaccatct cttaggacca cacagtcac aggaagata attcatgaat ggtgttgcag 3360 atcttgcacg ctaccaccct tacgtttcag aggagagat gggtgctggt acggtatgga 3420 aatcagacca gtcaggaaa aggaaaaa tctagtcaa tcaatggtct ctgcaggggtc 3480 3540 gatgagatcc agatggagta gaagaatgct gatgactgga acactggctg tgttcttcct 3600 tctcataatg ggacaattga catggaacga tctgatcaga ttatgcatca tggttggagc 3660 caacgcttcc gacaggatgg ggatgggaac gacgtaccta gccctgatgg ccacttttaa 3720 aatgagaccg atgttcgctg tagggctatt atttcgcaga ctaacatcca gagaagttct 3780 tcttctaaca attggattga gtctagtggc atctgtggag ttaccaaatt ccttggagga 3840 gctgggggat ggacttgcaa tgggcattat gattttaaaa ttattgactg actttcaatc 3900 acatcagctg tgggctacct tgctgtcctt gacatttatc aaaacaacgt tttccttgca 3960 ctacgcatgg aagacaatgg ctatggtact gtcaattgta tctctcttcc ctttatgcct 4020 gtccacgacc tcccaaaaaa caacatggct tccggtgcta ttgggatccc ttggatgcaa 4080 accactaacc atgtttctta tagcagaaaa caaaatctgg ggaaggagaa gttggcccct 4140 caatgaagga atcatggctg ttggaatagt cagcatccta ctaagttcac tcctcaaaaa 4200 tgatgtaccg ctagctgggc cactaatagc tggaggcatg ctaatagcat gttatgttat 4260 atctggaagc tcagccgacc tatcattaga gaaagcggct gaggtctcct gggaagaaga 4320 agcagaacac tctggtgcct cacacaacat attagtggaa gtccaagatg atggaaccat 4380 gaagataaag gatgaagaga gagatgacac gctaaccatt ctccttaaag caactctgtt 4440 agcagtttca ggggtgtacc cattatcaat accagcgacc cttttcgtgt ggtacttttg 4500 gcagaaaaag aaacagagat ctggagtgtt atgggacaca cccagccctc cagaagtgga 4560 aagagcagtt cttgatgatg gtatctatag aattatgcag agaggactgt tgggcaggtc 4620 ccaagtaggg gtaggagttt tccaagaaaa cgtgttccac acaatgtggc atgtcaccag 4680 gggagctgta ctcatgtatc aagggaagag attggaaccg agctgggcca gtgtcaaaaa 4740 agacctgatc tcatatggag gaggttggag gcttcaagga tcctggaaca caggagaaga 4800 agtgcaggtg attgctgttg aaccagggaa aaaccccaaa aatgtacaaa cagcgccggg 4860 cacctttaag acccctgaag gtgaagttgg agccattgcc ctagacttta aacctggcac 4920 atctggatct cccatcgtga acagagaagg aaaaatagta ggtctttatg gaaatggagt 4980 agtgacaaca agtggaacct acgtcagtgc catagctcaa gccaaagcat cacaagaagg 5040 gcccctacca gagattgaag acgaggtgtt taggaaaaga aacttaacaa taatggacct 5100 acatccagga tcgggggaaaa aaagagata tcttccagcc atagtccgtg aggccataaa 5160 aaagctg cgcacactaa tcctggctcc cacaagggtt gtcgcttccc aaatggcaga 5220 ggcgctcaag ggaatgccaa taagtcacca aacaacagca gtgaagagtg aacatacagg 5280 aaaagagata gttgacctca tgtgtcacgc cactttcacc atgcgcctcc tgtctcccgt 5340 aagagttccc aattacaaca tgatcatcat ggatgaagca catttcaccg atccatccag 5400 tatagcggcc agagggtaca tctcaacccg agtgggcatg ggtgaagcag ctgcaatctt 5460 catgacagcc actcccccag gatcagtgga ggcctttcca cagagcaacg cagttatcca 5520 agatgagaa agagacattc ctgagagatc atggaactca ggctatgagt ggatcactga 5580 cttcccaggt aaaacagttt ggtttgttcc aagcattaaa tcaggaaatg acattgccaa 5640 ctgcttaaga aagaatggga aacgggtgat tcaattgagc aggaaaacct ttgatacaga 5700 gtaccaaaaa aaaaaaaca acgactggga ctacgtcgtc acaacaca tctccgaat 5760 gggagcaaat ttccgagccg acagggtgat agacccaga cggtgtctga aaccggtaat 5820 actaaaagat gtccagagc gtgtcatttt agcaggacca atgccagtga ctgtggccag 5880 tgccgctcag aggaggaa gattggaag gaaccacaat aaggaggtg atcagtacat 5940 ctacatggga cagcctttta acacgatga agatcacgct cactggacag aagcaaaat 6000 gctccttgat ataataaca caccagaagg gatttacccca gccctctttg agccagagag 6060 agaaaagagt gcagcaatag acgggaata cagactgcgg ggtgaagcaa ggaaaacgtt 6120 tgtggagctc atgagaagg gagatctacc tgtctggcta tcctacaag tgcctcaga 6180 aggcttccag tactctgaca gagatggtg ctttgacggg gaaggaca accaggtgtt 6240 ggaggagac atggacgtgg agatctggac aaaagaagg gaagaaga aactacgacc 6300 ccgctggctg gatgccagaa catactcaga cccactagcc ctgcgcgagt ttaaagagtt 6360 tgcagcagggg agagaagcg tctcaggtga tttaatatta gaaataggga agcttccaca 6420 acacttgacg caaagggccc agaatgccct ggacaacctg gttatgttgc acaactccga 6480 acaaggaggc agagcctata gacatgcaat ggaagaactg ccagacacca tagaaacgtt 6540 gatgctccta gctttgatag ctgtgttaac tggtggagtg acactgttct tcctatcagg 6600 aaggggccta gggaaaacat ctatcggcct actctgcgta atggcttcaa gcgtactgct 6660 atggatggcc agtgtggagc cccattggat agcggcctcc atcatactgg agttcttcct 6720 gatggtgctg cttattccag agccagacag acaacgcact ccgcaggaca accagctggc 6780 atatgtggtg ataggtttgt tattcatgat actgacagta gcagccaatg agatgggact 6840 gctggaaacc acaaagaaag acttagggat tggccatgtg gctgttgaaa atcaccacca 6900 tgccgcaatg ctggacgtag acttacatcc agcttcagcc tggaccctct atgcagtggc 6960 cacaacaatt atcactccca tgatgaggca cacaatcgaa aacacaacgg caaacatttc 7020 cctgacagcc attgcaaacc aggcagctat attgatggga cttgacaaag gatggccaat 7080 atcgaagatg gacataggag ttccacttct cgccttgggg tgctattccc aggtgaatcc 7140 actgacgctg acagcggcgg tattgatgct agtggctcat tacgccata tggacctgg 7200 actgcaagca aaagctacta gagaagctca aaaaaggaca gcggccggaa taatgaaaaa 7260 tccaaccgtt gatggaattg ttgcaataga tttggaccct gtggtttatg atgcaaaatt 7320 tgaaaaaaaa ctaggccaaa taatgttgtt gatactgc acatcacaga tcctcttgat 7380 gcggactaca tgggctgt gcgagtccat cacactggcc actggacctc tgaccacgct 7440 ttgggaggga tctccaggaa aattttggaa caccacgata gcggttttcca tggcaacat 7500 ttcagagga agttatctag caggagcagg tctggccttc tcattaatga aatctctagg 7560 aggaggtagg agaggcacgg gagcccagg ggaacactg ggagagaat ggaaaagaca 7620 gctgaaccaa ctgagcaagt cagaatttaa cacctataaa aggagtggga ttggaagt 7680 ggacagatcc gaagccaag agggattga aagaggaaaaccaaac atgcagtgtc 7740 gagaggaacc gctaactga ggtggttcgt gaggaggaac cttgtgaac cagaagggaa 7800 agtcatagac ctcggttgtg gaagaggtgg ctggtcatac tattgcgctg ggctgaagaa 7860 agtcacagaa gtgaaggggt atacaaaagg aggacctgga catgaggaac caatcccaat 7920 ggcgacctat ggatggaacc tagtaaagct gcactctggg aaagacgtat tttttatacc 7980 acctgagaaa tgtgacacccc ttttgtgtga tattggtgag tcctctccaa acccaactat 8040 agaggaagga agaacgctac gcgtcctaaa gatggtggaa ccatggctca gaggaaacca 8100 atttgcata aaaattctga atccctacat gccaagtgtg gtggaaactc tggagcaaat 8160 gcaaagaaaa catggaggaa tgctagtgcg aaatccactt tcaagaaatt ctactcatga 8220 aatgtattgg gtttcatgtg gaacaggaaa cattgtgtca gcagtaaaca tgacatctag 8280 aatgttgcta aatcgattca caatggctca caggaaacca acatatgaaa gagacgtgga 8340 cctaggcgcc ggaacaagac atgtggcagt ggaaccagag gtagccaacc tagatatcat 8400 tggccagagg atagagaaca taaaacatga acataagtca acatggcatt atgatgagga 8460 caatccatat aaaacatggg cctatcatgg atcatatgag gtcaagccat caggatcagc 8520 ctcatccatg gtcaatggcg tggtgaaact gctcaccaaa ccatgggatg tcatccctat 8580 ggtcacacaa atagccatga ctgacactac accctttgga caacagaggg tgtttaaaga gaaagttgac acacgcacac caaaagcaaa acggggcaca gcacaaatca tggaggtgac agccaagtgg ttatggggtt ttctttctag aaacaagaaa ccaagaattt gcacaagaga 8820. ggagttcaca agaaaagtta ggtcaaacgc agccattgga gcagtgttcg ttgatgaaaa tcaatggaac tcagcaaaag aagcagtgga agatgagcgg ttctgggacc ttgtgcatag aggagggag cttcacaaac agggaaaatg tgctacgtgt gtttacaaca tgatgggga gagagagaa aagctaggag agttcggaa ggcaaaagga agtcgtgcaa tatggtacat gtggttggga gcacgctttc taggttcga agctcttggt ttcatgaacg aagatcactg gttcagcaga gagaattcac tcagcggagt ggaggaga ggactccaca aacttggata 9180. the father of the gacatatcaa agattccagg gggaaacatg the father of the acacagccgg atgggataca aggataacag aggatgacct tcagaatgag gccagaatta ctgacatcat ggaacccga catgccctcc tggctaagtc aatcttcaag ttaacctacc aaaataaggt ggtaagggta cagagaccag caaaaaatgg aaccgtgatg gatgtcatat ccagacgtga ccagagga agtggtcagg tcggaactta tggcttaaac actttcacca acatggaagc ccagctgata agacaaatgg agtctgaggg aatcttttca cccagcgaat tagagacccc aaatttagcc gagagagttc tcgactggct ggaaaaatat ggcgtcgaaa ggctgaaaag aatggcaatc agcggagatg actgcgtagt gaaaccaatt gatgataggt ttgcaacagc cttgacagct ctgaatgata tgggaaaagt aagaaaagat ataccacaat gggaaccttc aaaaggatgg aatgattggc aacaagtgcc tttttgttca caccacttcc accagctgat tatgaaggat gggagggaaa tagtggtgcc atgccgcaac caagatgaac ttgtgggtag ggctagagta tcacaaggtg ctggatggag cctgagaga actgcatgcc tagcaagtc atatgcacaa atgtggcagc tgatgtactt ccacaggaga gacctgagac tagccgctaa tgctatctgt tcagccgttc cagttgattg gatcccaacc agccgtacca cctggtcgat 9960. ccatgcccat caccaatgga tgacaacaga agacatgctg tcagtgtgga atagggtttg gatagaggaa aacccatgga tggaggacaa aaccacata tccagttggg gagatgttcc 10080 atattagga aaagggaag accaatggtg tggatccctg ataggcttta cagcaagggc 10140 cacctgggcc accacatac aagtggccat aaaccaagtg agagacta ttgggaatga 10200 catcatcta gattacatga catcatgaa gagatcaag aacgagagtg atcccgaagg 10260 ggcactctgg tgagtcaaca catttacaa aaaggaaa ataagaatc aaaaaggca 10320 agaagtcagg ccggattaag ccatagtacg gtaagagcta tgctgcctgt gagccccgtc 10380 taaggacgta aaatgaagtc aggccgaaag ccacggcttg agcaaccgt gctgcctgta 10440 gctccatcgt ggggatgtaa aaacctggga ggctgcacc catggaagct gtacgcatgg 10500 ggtagcagac tagtggttag aggagacccc tccgaaaca taacgcagca gcggggccca 10560 acaccagggg aagctgtacc ctggtgtaa ggactagagg ttagaggaga cccccgcat 10620 aacaataaac agcatattga cgctgggaga gaccagagat cctgctgtct ctacagcatc 10680 attccaggca cagaacgcca gaaaatggaa tggtgctgtt gatcacag gttct 10735 <210> 10 <211> 10724 <212> DNA <213> Dengue Virus <400> 10 agttgttagt ctacgtggac cgacaagac agattctttg agggagctaa gctcaacgta 60 gttctacag ttttaatt agagagcaga tctctgatga ataccaacg aaaaaaggcg 120 agaagtacgc ctttcaat gctgaaacgc gagagaaacc gcgtgtcaac tgtgcaacag 180 ctgacaaga gattctcact tggaatgctg caaggacgcg gaccattaaa actgttcatg 240 gcccttgtgg cgttccttcg tttcctaaca atcccacca cagcagggat actaaaaga 300 tggggaacga tcaaaaatc aaagctatc aatgttttga gaggttcag gaagagatt 360 ggaaggatgc tgacatctt gaacaagaga cgcaggacag caggcgtgat tgttatgttg 420 attccaacag cgatggcgtt ccattaacc acacgcaatg gagaaccaca catgatcgtt 480 ggtaggcagg agaaagggaa aagtcttctg ttcaaacag aggatggtgt taacatgtgt 540 actctcatgg ccatagacct tggtgaattg tgtgaagata caatcacgta caagtgtcct 600 ctcctcagac aaaatgacc agagacata gattgttggt gcacttac gtccacatgg 660 gtaacttatg ggacatgtac caccacagga gaacacagaa gagaaaaag atcagtggcg 720 ctcgttccac atgtaggtat gggactggag acacgaactg aaacatggat gtcatcagaa 780 ggggcctgga aacatgttca gagaattgaa acctggatct tgagacatcc aggttttacc 840 ataatggcag caatcctggc atacaccata ggaacgacac atttccaag ggccttgatt 900 ttcattttac tgacagctgt cgctccttca atgacaatgc gctgcatagg atatcaaat 960 agagactcg aggaggtt ttcaggagga agctggttg acatagtctt agaacatgga 1020 agttgtgtga cgacgatggc aaaaaaaaaaacattgg attttgaact gataaaaaaca 1080 gaagccaaac aacctgccac tctaggaag tactgtatag aagcaagct gaccacaca 1140 acacagaat cgcgttgccc aacacaaggg gaacccagtc taatgaggg caggaaaaa 1200 ggttcatctg siaacactcc atggtagaca gaggatgggg aaatggatgt ggattattg 1260 gaaagggagg cattgtgacc tgtgctatgt ttacatgca aaagaacatg gaaggaaag 1320 tcgtgcagcc agaaaatttg gaatacacca tcgtgataac acctcactca ggagaggagc 1380 acgctgtagg taatgacaca ggaaagcatg gcaaggaaat caaaataaca ccacagagct 1440 ccatcacaga agcagaactg acaggctatg gcactgtcac gatggagtgc tctccgagaa 1500 cgggcctcga cttcaatgag atggtactgc tgcagatgga agacaaagct tggctggtgc 1560 acaggcaatg gttcctagac ctgccgttac catggctacc cggagcggac acacaaggat 1620 caaattggat acagaaagag acattggtca ctttcaaaaa tccccacgcg aagaaacagg 1680 acgtagtcgt actcggatca caggaaggcg caatgcatac cgcattgaca ggcgctacag 1740 agatacagat gtctagcgga aatctgttgt ttacagggca tctgaaatgt agactgagaa 1800 tggacaaact gcaattgaag ggaatgtcat actctatgtg tacgggtaag tttaagatag 1860 tcaaagagat agccgaaaca caacacggaa caatcgtaat tagggtgcaa tacgaaggcg 1920 acgggtcacc atgtaagata ccattcgaaa ttacagacct cgaaaaaaga cacgtactcg 1980 gaagactgat aacagtgaat ccgatcgtta cggaaaaaga ctcacccgtt aatatcgaag 2040 ccgaaccacc attcggagac tcatacataa taatcggagt cgaacccgga cattgaac 2100 tgaattggtt taaaaaaggg tcatcaatcg gatagtt cgaacaact atgagaggcg 2160 ctaagcgtat ggctatactc ggagacacag catgggactt cggatcctta gggggagtgt 2220 ttacgtcaat cggtaggca ctacaccagg tattcggagc gatatacgga gccgcattta 2280 gcggagtgtc gtggacaatg agatactga tcggagtgat atcacatgg atcggaatga 2340 atagtaggtc tacaagtcta tccgttagct tagtgtt cggagtcgtt acactgtatc 2400 taggcgctat ggtgcaagcc gatagtggtt gcgttgtgag ctggaaaaat aaagaactga 2460 aatgtggcag cgggatctc atcacagata acgtacacac atggacagaa caataagt 2520 tccaccaga atccccttca aaactagctt cagctatcca aaagctcat gagaggggca 2580 tttgtggaat ccgctcagta acagattgg agaatctgat gtggaacaa atacaccag 2640 aattgaatca tattctca gaaatgagg taagttgac cattatgaca ggagacatta 2700 gaggaatcat gcaggcagga aaacgatcct tgcggcccca gcccactgag ctgaagtact 2760 catggaaaac atggggaaag gcgaaaatgc tctccacaga gtctcacaat cagacctttc 2820 ttattgatgg ccctgaaaca gcagaatgcc ccaacacaaa cagagcctgg aactcgctgg 2880 aagttgaaga ctatggtttt ggagttttca ccaccaatat atggctgaaa ttgagagaaa 2940 aacaggatgt atttgtgac tcaaaactca tgtcagcggc cattaaagac aacagagccg 3000 ttcatgctga tatgggttat tggatagaaa gtgcactcaa tgacacatgg aagatggaga 3060 aagcctcctt cattgaagtt aaaagctgcc actggccaaa gtcacacacc ctttggagca 3120 atggagtatt agaaagtgag atgataatcc caaaaaattt tgccgggcca gtgtcacaac 3180 acaactacag accaggctac catacacaaa cagcaggacc ttggcatcta ggtaagcttg 3240 agatggactt tgatctctgc gaaggaacta cagtggtggt gactgaggac tgtggaaata 3300 gaggaccctc tttaagaacg accactgcct ctggaaaact cataacagaa tggtgctgcc 3360 gatcctgcac actaccacct ctaagataca gaggtgagga tggatgctgg tacgggatgg 3420 aaatcagacc attgaaagag aaagaagaga atttggtcaa ctccttggtc acagccggac 3480 atgggcagat tgacaacttt tcactaggag tcttgggaat ggcactgttc ctggagaaa 3540 3600 cattgattac tgggaacatg tcttttagag acctgggaag agtgatggtt atggtgggcg 3660 ctaccatgac ggatgacata ggtatgggag tgacttatct tgccctacta gcagctttta 3720 aggttagacc aacttttgca gctggactac tcttaagaaa actgacctcc aaaggaattga 3780 tgatggccac cataggaatc gcactccttt cccaaagcac cataccagag accattcttg 3840 3900 aataccaatt ggcagtgact atcatggcta tttcatgtgt cccaaatgca gtgatactgc 3960 aaaacgcatg gaaggtgagt tgcacaatat tggcagcggt gtccgtttca ccactgctct 4020 4080 atccaacagc catttttta acaactctct cgaggaccag caagaaaagg agctggccgc 4140 taaatgaagc tatcatggca gttgggatgg tgagcatttt agccagttct ctcctaaaga 4200 atgatattcc tatgacaggt ccattagtgg ctggagact cctcaccgta tgttacgtgc 4260 tcactggacg atcggccgat ttggaactgg agagagctgc cgatgtaaaa tgggaagatc 4320 aggcagaaat atcaggaagc agcccaatcc tgtcaataac aatatcagaa gatggcagca 4380 tgtcgataaa aaatgaagag gaacaaa cactgaccat actcatcaga acgggattgt 4440 tggtgatctc aggagtcttt ccagtatcga taccaattac ggcagcagca tggtacctgt 4500 ggggaaa gaaaacgg gctggagagtac tgtgggacgt cccttcacccc ccaccagtgg 4560 aaaaagccga actggaggat ggagcctaca gaatcaagca aagagggatc cttggatatt 4620 ctcagattgg agccggagtt tacaaagaag gaacattcca tacaatgtgg cacgtcacac 4680 gtggtgctgt tctgatgcat agaggagaga ggattgaacc atcatgggca gatgtcaaga 4740 4800 aagtccaagt cctggcattg gaacctggaa aaaatccaag agccgtccaa acgaaacctg 4860 gaatattcaa aaccaacacc ggaaccatag gcgccgtatc tctggacttt tcccctggaa 4920 cgtcaggatc tccaatcgtc gacagaaaag gaaaagttgt gggtctttac ggtaatggtg ttgtcacaag gagtggagca tatgtaagtg ctatagccca gaccgaaaaa agcattgaag acaatccaga gatcgaagat gacattttcc gaaagaaaag attgaccatc atggacctcc atccaggagc aggaagaca aaaagatacc ttccagccat agttagaga gccataaaac gtggcttgag aacattgatc ctggctccca ctagagtagt ggcagctgaa atggaggaag ctcttagagg acttccaata agataccaaa ctacagccat caaaccgag cataccgggc gggagatcgt ggacctaatg tgtcatgcca catttactat gaggctgtta tcaccagtca gagtgccaaa ttacaacctg atcatcatgg acgaagccca cttcacagac ccagcaagta tagcagctag aggatacatt tcaactcgag tagggg tgaagcagcc gggattttca tgacagccac tcctccggga to take catttcctca gagcaatgca ccaattatgg atgaggaag agaaatccct gagcgttcat ggaattcagg acacgaatgg gtcacggatt ttaagggga gactgtttgg tttgttccaa gtataaaagc aggaaatgat atagcagctt 5700 atgttaagac tagagccaat gattgggact ttgtggtcac aactgacatt tcagaaatgg 5760 gtgccaactt caaggctgag agggttatag accccagacg ttgcatgaaa ccagttatac 5820 taacagatgg cgaggagcgg gtgatcttgg ctggacctat gccagtgacc cactctagtg 5880 cagcgcaaag aagagggaga ataggaaa atccaaaaaa tgaaaatgac cagtacatat 5940 acatggggga acctcttgaa aatgatgaag actgtgcaca ttggaaagaa gctaaaatgc 6000 tcctagataa catcaacaca cctgaaggaa tcattcctag tatgttcgaa ccagagcgtg 6060 aaaaagtgga tgccattgat ggtgaatacc gtttgagagg agaagcaagg aaaacctttg 6120 tggacctaat gagagaggg gacttaccag tctggttggc ctacaaagtg gcagctgaag 6180 gcatcaacta cgcagacaga aagtggtgtt ttgatggaat tagaacaac caaatactgg 6240 aagaaaatat ggaagtggaa atctggacaa aagaagggga aaagaaaa taaaaaccca 6300 gatggttgga tgctaggatc tattctgacc cactagcact aaaagaattc aaagaatttg 6360 cagctggaag aaaatctttg accctgaacc taatcacaga aatgggtagg cttccaactt 6420 tcatgactca gaagaagaa aacgcactgg acaacctggc tgtgctgcat acggctgagg 6480 caggtggaag ggcgtacaat catgctctca gtgaactgcc ggagaccctg gagacactgc 6540 tcctactgac acttctggca acagtcacag gaggaatctt cttattctta atgagcggaa 6600 aaggtatagg gaagatgacc ctgggaatgt gttgcataat cacggctagt atcctcctat 6660 ggtatgcaca gatacaacca cactggatag cagcttcaat aatactggag ttttttctca 6720 tagttttgct cattccagaa ccagaaaaac agagaacacc ccaagacaac caattgacct 6780 acgttgtcat agccatcctc acagtggtgg ccgcaaccat ggcaaacgag atgggtttcc 6840 tggaaaaaac caagaaagac ctcggattgg gaagcattac aacccaggaa tctgagagca 6900 atatcctgga catagatcta cgccctgcat cagcatggac gctgtatgcc gtagctacaa 6960 catttgtcac accaatgttg agacatagca ttgaaaattc ctcagtgaat gtctccctaa 7020 cagccattgc taaccaagct acagtgctaa tgggtcttgg gaaaggatgg ccattgtcaa 7080 agatggacat tggagttccc ctccttgcca ttggatgcta ttcacaagtc aaccctataa 7140 ctctcacagc agctctcctt ttatggtag cacattatgc cattataggg ccaggacttc 7200 aagcaaaagc aaccagagaa gcccagaaaa gagcagcagc aggcatcatg aaaaacccaa 7260 cagtcgatgg ataacagtg attgacctag aaccaatacc ctatgatcca aaatttgaaa 7320 agcagttagg acaagtaatg ctcctaatcc tctgcgtgac tcaagtatta atgatgagga 7380 ctacatgggc tttatgtgag gctctaaccc tagcgaccgg gcccatctcc acactgtggg 7440 aaggaaatcc agggaggttt tggaacacta ccattgcagt gtcaatggct aacatcttta 7500 gggggagcta cttggccgga gctggacttc tcttttccat catgaaaaac aacaaaca 7560 caagagaagg aactggcaac ataggagaga cacttggaga aaaatggaaa agtcgattaa 7620 7680 gaaccctagc aaaagaaggt atcaaaagag gagaacgga ccaccatgct gtgtcgcgag 7740 gttcagcaaa actgagatgg ttcgtcgaga gaatatggt caaccggaa gggaaggtgg 7800 tggatctcgg ttgcggcaga gggggctggt catactattg cggggacta aagaatgtaa 7860 gagaagtcaa aggcctaaca aaaggaggac caggacatga agaacccatc cccatgtcaa 7920 catatgggtg gaatctagtg cgtctgcaaa gtggagttga cgttttcttc accccgccag 7980 aaaagtgtga tacattgttg tgtgacatag gggagtcgtc accaaatccc acgatagaag 8040 caggaac actcagagtc ctcaacttag tggaaaattg gttgaacaat aacacccaat 8100 tttgcataaa ggtcctcaac ccatatatgc cttcagtcat agaaaaaatg gaagcattac 8160 aaaggaata tggaggagcc ttagtgagga atccactctc acgaaactcc acgcatgaaa 8220 tgtactgggt atctaatgcc accgggaaca tagtgtcatc agtgaacatg atttcaagga 8280 tgttgattaa cagattcaca atgaaacaca agaaagccac ttacgagcca gatgttgacc 8340 taggaagtgg aacccgcaac attggaattg aaagtgagat accaaatcta gacataatag 8400 gaagagaat agaaaaata aaacaagagc atgaaacatc atggcactat gatcaagacc 8460 acccatacaa aacgtgggct taccatggca gctatgaaac aaaacaaact ggatcagcat 8520 catctatggt gaacggagtg gtcagattgc tgacaaaacc ttgggacgtc gtccctatgg 8580 tgacacagat ggcaatgaca gacacgactc cttttggaca acagcgcgtt ttcaaagaga 8640 aagtagacac gagaacccaa gaaccgaagg aaggcacaaa gaaactgatg aaaattacgg 8700 cagagtggct ttggaaagaa ctaggaaaga aaaagacacc taggatgtgt accagagaag 8760 aattcacaag aaaagtgaga agcaatgcag ccttggggc catattcact gatgagacaa 8820 aatggaaatc ggcacgtgag gctgttgaag atggtaggtt ctgggagctg gttgacaggg 8880 aaagaaatct ccatcttgaa ggaaagtgtg aaacatgtgt gtacaacatg atgggaaaaa 8940 gagagagaa actaggggag ttcggcaagg caaaaggtag cagagccata tggtacatgt 9000 ggcttggagc acgcttctta gagtttgaag ccctaggatt cctgaatgaa gatcactggt 9060 tctccagagg gaactccctg agtggagtgg aaggagaagg gctgcacagg ctaggctaca 9120 ttttaagaga cgtgggcaag aaggaagggg gggcaatgta cgccgatgat acagcaggat 9180 gggacacaag aatcacacta gaacctaa aaaatgaaga aatggtaacg aaccacatga 9240 aaggaaca caagaaacta gccgaggcca tattcaaact aacgtaccaa aacaaggtgg 9300 tgcgtgtgca aagaccaaca ccaagaggta cattaatgga tatcatatcg agaagagacc 9360 aaagaggcag tgggcaagtc ggcacctatg gccttaatac cttcaccaat atggaagccc 9420 aattaattag acagatggag ggagaagagaa tcttcaaaag cattcagcac ctgacagcca 9480 cagaagaaat cgctgtacag aactggctag caagagtggg gcgtgaaagg ctatcaagaa 9540 tggcaatcag tggagatgat tgtgttgtaa aacctataga tgacagattt gcaagtgctt 9600 taacagctct aaatgacatg ggaaaagtta gaaagatat acaacaatgg gaaccttcaa 9660 gaggatggaa cgattggaca caagtgcctt tctgttcaca ccactttcat gagttagtca 9720 tgaaagatgg tcgcgtgctc gtagtcccat gcagaaacca agaatgaactg attggtagag 9780 cccgaatttc ccagggagct gggtggtctt tgaaagagac ggcctgtttg ggaaagtctt 9840 acgcccaaat gtggactctg atgtacttcc agagacgtga cctcagactg gcggcaaatg 9900 ccatctgctc ggcagtcccg tcacattggg ttccaacaag tcgaacaacc tggtccatac 9960 acgctaagca tgaatggatg acgacggaag acatgctggc agtctggaac agggtgtgga 10020 tccaagaaaa cccgtggatg gaagataaaa ctccagtgga atcatgggaa gaagtcccat 10080 acttgggaaa aagagaagac caatggtgcg gctcattgat tgggctaaca agcagggcta 10140 cctgggcaaa gaacatccaa acagcaataa atcaagtcag atcccttata ggcaatgagg 10200 aatacacaga ctacatgcca tccatgaaga gatttagaag ggaagaggaa gaggcaggtg 10260 tcctgtggta gaaggcaaaa ctaacatgaa acaaggctaa aagtcaggtc ggattaagcc 10320 atagtacgga aaaaactatg ctacctgtga gccccgtcca aggacgttaa aagaagtcag 10380 gccatcacaa aatgccacag cttgagtaaa ctgtgcagcc tgtagctcca cctgaggagg 10440 tgtaaaaaac ctgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500 ggttagagga gacccctccc ttacaaatcg cagcaaacaa cgggggccca aggtgagatg 10560 aagctgtaat ctcactggaa ggactagagg ttagaggaga cccccccaaa acaaaaaaca 10620 gcatattgac gctgggaaag accagagatc ctgctgtctc ctcagcatca ttccaggcac 10680 agaacgccag aaatggaat ggtgctgttg aatcacagg ttct 10724 <210> 11 <211> 10708 <212> DNA <213> Dengue Virus <400> 11 agttgttagt ctacgtggac cgacagaac agtttcgact cggaagcttg cttaacgtag 60 tgctaacagt ttttag agagcagatc tctgatgaac aaccaacgga agaagacggg 120 aaaaccgtct atcaatgc tgaacgcgt gagaaaccgt gtgtcaactg gatcacagtt 180 ggcgaagaga ttctcaaag gactgctgaa cggccaggga ccaatgaat tggttatggc 240 gttcatagct ttcctcagat ttcatagct tccaccaca gcaggagtct tggtagatg 300 gggaaccttc agaagtcgg gggccattaa ggtcctgaaa ggctttaaga aggagatctc 360 aaacatgctg agcataatca aaacggaa aagacatcg ctctgtctca tgatgatatt 420 gccagcagca cttgctttcc acttgactc acgagatgga gagccgcgca tgattgtggg 480 gagaatgaa agaggaaat ccctacttt taagacagcc tctggaatta acatgtgcac 540 actcatagcc atggacttgg gagagatgtg tgatgacacg gtcacttaca aatgccccca 600 tattaccgaa gtggaacctg aagacattga ctgctggtgc aacctcacat caacatgggt 660 gacttatgga acgtgcaatc aagccggaga gcatagacgc gataagagat cagtggcgtt 720 agctccccat gtcggcatgg gactggatac acgcacccaa acttggatgt cggctgaagg 780 agcttggagg caagtcgaga aggtagagac atgggccctt aggcacccag ggttcaccat 840 actagcccta tttcttgccc attacatagg cacttccttg acccagaagg tggttatttt 900 tatactgcta atgctggtca ccccatccat gacaatgaga tgtgtgggag taggaaacag 960 agattttgtg gaaggactat caggagctac gtgggttgac gtggtgctcg agcacggggg 1020 gtgtgtgacc accatggcta agaacaagcc cacgttggat atagagcttc agaagaccga 1080 ggccacccaa ctggcgaccc taaggaagct atgcattgag gggaaaatca ccaacataac 1140 aactgactca agatgtccta cccaagggga agcggttttg cctgaggagc aggaccaaaa 1200 ctacgtgtgt aagcatacat acgtagacag aggctggggg aacggatgtg gttttgtttgg 1260 taagggaagc ttggtaacat gtgcgaatt tcaatgcctg gaaccaatag agggaaagt 1320 ggtgcaat gagaacctca atacaccgt tatcatcaca gtgcacacag gagaccaca 1380 ccaggtggga atgaaacgc agggagtcac ggctgagata acacctcagg catcaccac 1440 tgagccatc tgcctgaat atggacct tgtcaccac tgctcaccac tgcaggtt 1500 ggacttcaat gaatgatct tgctacaat gagacaaa gcatggatgg tacatagaca 1560 atggttttt gacctaccctc taccatggac atcaggagct acacggaaa caccaacctg 1620 gacaggaag gagctcttg tgacatta aaacgcacat gcgagaaac aagaagtagt 1680 cgttttaggg tcaggagg gagctatgca taccgcacta accggagcga ctgagataca 1740 gatagcgga gggacatcaa tcttcgccgg acacttaag tgtagattga aaatggacaa 1800 acttgagctt aagggatgt catacgctat gtgtacgaac acatcgtgc tgaaaaaaga 1860 ggtaagcgaa acggcacacg gaacgatact gattaggtt gagtataagg gcgaagacgt 1920 cccatgtaag atacctttt cgacagagga cggacagggt aaggcccata acggagact 1980 gattaccgct aacccagtgg tgaccaaaaa agaggaacca gtcaatcg aagccgaacc 2040 tccattcgga gagtcaaca tagtgatagg gataggcgat aacgcactta agattactg 2100 gtacaaaaaa gggtcatcaa tcggtaagat gtttgaggca accgctaggg gggctagacg 2160 gatggccata ctcggagaca ccgcatggga cttcggatcc gtgggggggg tacttactc 2220 actcggtaag atggtgcacc aaatttcgg atccgcatat accgctat ttagcggagt 2280 gtcatgggtg atgaaatcg gatcggagt tctattgaca tggatcggat tgaactctaa 2340 gatacatct atgtcatttt catgtatcgc aatcggaatt atctcggagc 2400 cgtagtgcaa gccgacatgg ggtgtgttat aactggaaa gccaagaac tcaagtgtgg 2460 aagtggaatc ttcgtcacca acgaggtcca tacctggaca gagcaataca aattccaagc 2520 agactcccca aaagattgg cgacagccat tgcaggcgct tgggagaatg gagtgtgcgg 2580 aattaggtca aaccagaa tggaatct cctgtggaag aattagcca atgaactgaa 2640 ctacatattg tgggaaaaca atcaatt aacggttgtt gtgggcgata taattggggt 2700 cttagagcaa gggaaaagaa cactacacc acaaccatg gagctaaaat actcatggaa 2760 aacgtgggga aaggcaaaa tagtgacagc tgaacaca aattctctt tcatataga 2820 tggaccaac acacggagt gtccaagtgc ctcaagagca tggaatgtgt gggaggtgga 2880 agattacgggg ttcggagtct tcacaccaa catatggctg aaactccgag aggtgtatac 2940 ccaactatgt gaccataggt taatgtcggc agccgtcaag gatgaaaggg ccgtacatgc 3000 cgacatgggc tattggatag aaagtcaaaa gatggaagt tggagctag aaaagcatc 3060 cctcatagag gtgaaaacct gcacatggcc aaatcacat acccttgga gtaatggtgt 3120 gttagagagt gatagatca ttccaaaag tctagctggt cctatctcgc aacacaacta 3180 caggcccgggg taccaccc agacggcggg accttggcat ttaggaaat tagagctgga 3240 cttcaactat tgtgaggaa cacagttgt catcacagaa aactgtggga caagaggccc 3300 atcattgaga acaacag tgtcagggaa gttaatacac gatggtgct gccgctcgtg 3360 cacacttcct cccctgcgat acatgggaga agacggttgc tggtatggca tggatcag 3420 acccatcagt gagaaagaag aaaacatggt aaagtcttta gtctcagcgg gaagtggaga 3480 ggtggacaac ttcacaatgg gtgtcttgtg tttggcaatc ctctttgaag aggtgatgag 3540 aggaaaattt gggaagaaac acatgattgc gggggttttc ttcacgtttg tactccttct 3600 ctcagggcaa ataacatgga gagatatggc gcacacacta ataatgattg ggtccaatgc 3660 atctgacagg atgggaatgg gcgtcaccta cctagcttta attgcaacat ttaaaatcca 3720 gccatttttg gctttgggat ttttcctaag aaaactgaca tccagagaaa atttattgtt 3780 aggagttggg ctggctatgg caacaacgtt acaactgcca gaggacattg aacaaatggc 3840 aaatggaatc gctctggggc tcatggctct caaactgata acacaatttg aaacatacca 3900 actatggaca gcattaatct ccttaacgtg ttcaaataca atttttacgt tgactgttgc 3960 ctggagaaca gccaccctga ttttggctgg agtttcactt ttaccagtgt gccagtcttc 4020 gagtatgagg aaaacagact ggcttccaat gacagtggca gctatgggag ttccacctct 4080 accacttttt atttttagct taaaagacac actcaaaagg agaagctggc cactgaatga 4140 agggtgatg gctgttgggc tgtgagcat tctggccagt tctctcctta gaatgatgt 4200 acccatggct ggaccattag tggccgggg cttgctgata gcgtgctacg tcataactgg 4260 cacgtcagca gacccaccg tagaaaagc agcagatgta acatgggagg aagaggctga 4320 gcaaacagga gtgtcccaca acttaatgat cacagttgat gatgatgaa caatgagaat 4380 aaagatgat gagactgaga atacttac agtgctttg aaaacagcat tactatagt 4440 gtcaggaatc tttccatact ccataccccgc aacattgttg gtctggcata cttggcaaaa 4500 gcaaacccaa agatccggcg ttctatggga tgtacccagc cctccagaga cacagaaagc 4560 agaactggaa gaaggggtct ataggatca acaghagga atttggga aaacccaagt 4620 aggggttgga gtacagaaag aaggttctt tcacacatg tggcacgtta caaggggc 4680 agtgttgaca tataatggga aagactgga accaaattgg gctagcgtga aaaagatct 4740 gatttcatac ggaggatt ggagattgag cgcacaatgg caaaggggg aggaggtgca 4800 ggttattgcc gtagagcctg ggagaaccc aaagaacttt caaccatgc caggcacttt 4860 tcagactaca acagggaaa taggagcaat tgcactggat ttcaagcccg gaacttcagg 4920 atctcctatc ataacagag agggaaggt agtgggactg tatggcaatg gagtggttac 4980 aaagaatggt gggctacgtca gcggaatagc gcaacgaat gcagaaccag atggaccgac 5040 accagaattg gagagaga tgttcaaaa gcgaaatcta accataatgg atcttcatcc 5100 tgggtcagga agacacgga ataccttcc agctattgtt aggaggca tcaagagacg 5160 tttgagaact ctaattctgg caccaacaag ggtggttgca gctgagatgg aagaagcatt 5220 gaaagggctc cchaataggt accaacaac agcaaaaa tctgaacaca caggagaga 5280 gattgttgat ctgatgtgcc acgcaacgtt cacaatgcgt ctgctgtcac cagttaggt 5340 tccaaactat aacttgataa taatggatga agcccatttc acagacccag ccagtatagc 5400 tgctagagggg tacatatcaa ctcgtgttgg aatgggaa gcagccgcaa tattcatgac 5460 agcaacgccc cctggaacag ctgatgcctt tcccagagc aacgctccaa ttcagatga 5520 agaaagggac ataccagaac gctcatggaa ttcaggcaat gatggataa ccgacttcgc 5580 tgggaaaacg gtgtggtttg tcccagcat taaagccgga atgacatag caattgcct 5640 gcggaaaaac gggaaaaagg tcattcact tagtaggag acttttgaca cagaatca 5700 gaaaaccaaa ctgaatgat gggactttgt ggtgacgact gatttcag aaatgggggc 5760 caatttcaa gcagatagag tgatcgaccc aagagatgt ctcaaccag tgatcctgac 5820 agatggacca gagcgggtga tcctggctgg accaatgcca gtcaccgcag cgagtgctgc 5880 gcaaggaga gggagagttg gcaggaaccc aaaaagaa atgaccagt acatattcac 5940 gggccagcct ctcacaatg atgaagacca tgctcactgg acagaagcaa aaatgctgct 6000 ggacaacatt aacacaccag aagggatcat accagctctc tttgagccag aaagggagaa 6060 gtcagccgcc atagacggtg agtatcgcct gaaggtgag tccaggaaga ctttcgtgga 6120 actcatgagg aggggtgacc ttccagtctg gttagccat aaagtagcat cagaggt 6180 haaataca gatagaaat ggtgctttga tggacaacgt aaatcaa ttttagaga 6240 gacaatgt gtggaaatct gacaaagga aggaaaag aaaaaattga gacctaggtg 6300 gcttgatgct cgcacctatt cagatccctt agcactcaag gaattcaagg actttgcggc 6360 tggcaggaag tcaatagccc ttgatcttgt gacagaaata ggaagagtgc cttcacacct 6420 agctcataga acgagaaacg ctctggacaa tctggtgatg ctgcatacgt cagaacatgg 6480 cggtaaggcc tacaggcatg cggtggagga actaccagag acaatggaaa cactcctact 6540 cttgggactc atgatcttgt tgacaggtgg agcaatgctt ttcttaatat caggtaaagg 6600 gattggaaag acttcaatag gactcatttg tgtaattgct tccagcggca tgttgtggat 6660 ggccgaaatc ccactccaat ggatcgcgtc ggccatagtc ctggagtttt ttatgatggt 6720 gttgcttata ccagaaccag agaagcagag aaccccccaa gacaaccaac tcgcatatgt 6780 cgtgataggc atacttacac tggcagcaat aatagcagcc aatgaaatgg gattgttgga 6840 aactacaaag agagatttag gaatgtctaa ggagccaggt gttgtttctc caaccagcta 6900 tttagatgtg gacttgcacc cagcatcagc ctggacattg tacgctgtgg ccactacagt 6960 aataacacca atgttaagac ataccataga gaattccaca gcaaatgtgt ccttggcagc 7020 tatagccaac caggcagtgg tcctgatggg tttggacaaa ggatggccaa tatcaaaaaat 7080 ggacttagga gtacccctgc tggcattggg ttgctattca caagtgaacc cactgactct 7140 aacagcggca gtactcttgc tgatcacaca ttatgccatt ataggtccag gattgcaggc 7200 aaaagccacc cgtgaagctc agaaaaggac agctgctgga ataatgaaga atccaacagt 7260 ggatgggata atgacaatag acctagatcc tgtaatatat gattcaaaat ttgaaaagca 7320 actgggacag gttatgctcc tggttttgtg tgcagttcaa cttttgttaa tgagaacatc 7380 atgggccttg tgtgaagctt taaccctagc tacaggacca ataacaacac tctgggaagg 7440 atcacctgg aagttttgga acaccacgat agctgtttcc atggcgaaca tttttagagg 7500 gagctattta gcaggagctg ggcttgcttt ctctattatg aaatcagttg gaagagggaa 7560 aaggagaaca ggctcacagg gtgaaacttt gggagaaaaa tggaaaaaga agtaaatca 7620 7680 agaagccaaa gaagggggtga aagaggga aataacacat catgccgtgt ccagaggtag 7740 tgcaaaactt caatggtttg tggagagaaa catggtcatt cccgaaggaa gagttataga 7800 cttgggctgt ggaagaggag gctggtcata ttactgtgca ggactgaaaa aagtcacaga 7860 agtgcgagga tacacaaaag gcggtccagg acacgaagaa ccagtaccta tgtccacata 7920 tggatggaac atagttaagt taatgagtgg aaaggatgtg tttatcttc cacctgaaaa 7980 gtgtgacacc ctgttgtgcg acattggaga atcttcacca agcccaacag tggagaagaaag 8040 cagaactata agagttttga agatggttga accatggcta aagaacaacc aattttgtat 8100 taaagtattg aacccttaca tgccaactgt gattgagcac ctagaaagac tacaaaggaa 8160 acatggagga atgcttgtga gaaatccact ttcacgaaac tccacgcacg aaatgtactg 8220 gatatccaat ggcacaggta acattgtcgc ttcagtcaac atggtatcta gactgctact 8280 gaacaggttc acgatgacac acagaagacc caccattgag aaagatgtgg atttaggagc 8340 aggaactcga catgttaatg cggaaccaga aacacccaac atggatgtca ttgggggaaag 8400 aataaaaagg atcaaggagg agcataattc aacatggcac tacgatgacg aaaaccccta 8460 caaaacgtgg gcttaccatg gatcttatga agtcaaagcc acaggctcag cctcctccat 8520 gataaatgga gtcgtgaaac tcctcactaa accatgggat gtggtgccca tggtgacaca 8580 gatggcaatg acagatacaa ctccattgg ccagcagaga gtctttaaag agaagtgga 8640 caccaggaca cccaggtcca tgccaggaac aagaagggtt atgggatca cagcggagtg 8700 gctctggaga accctgggaa ggataaaaa acccaggtta tgcacaaggg aagagtttac 8760 aaaaaaggtc agaactaacg cagccatggg agctgttttc acagaggaga accaatggga 8820 cagcgcgaaa gctgctgttg aggatgagga ttttggaaa cttgtggaca gagaacgtga 8880 actccacaaa ctgggcaagt gtggaagctg tgtttacaac atgatgggta aggagagaa 8940 gaaacttgga gagtttggca aagcaaagg cagtagagct atatggtaca tgtggttggg 9000 agccaggtac cttgagttcg aagccttgg attctttaat gagaccact ggttctcgcg 9060 tgagaactct tacagtggag tggaggaga aggactgcac aagctaggct attattag 9120 ggacattcc aagatacccg gaggagctat gtatgctgat gatacagctg gttgggacac 9180 aagaataca gaagagatgacc tgcacaatga ggaaagagatc acacagcaa tggaccctga 9240 accacaggcag tagcgaacg ctatatta gctcacatac caaaaaag tggtcaaagt 9300 tcaacgaccg actccacag gcacggtaat ggatatcata tctaggaag accaaagagg 9360 cagtggacag gtaggaactt atggtctgaa tacattcacc aacatggaag cccagttaat 9420 cagacaatg gaaggagaag gtgtgctgtc aaaggcagac ctcgagaacc ctcatctgcc 9480 aggagaaa attacacaat ggttggaaac aaggagtg gagaggtta aaagaatggc 9540 cataagtggg gatgactgcg tggtgaaacc aatcgatgac aggttcgcta atgccctgct 9600 cgctctgaac agamatgggaa aggttcggaa agacatacct caatggcagc catcaaggg 9660 atggcatgat tggcacagg ttccttctg ctcccaccac ttcatgaat tgatcatgaa 9720 agatgaga aagttggtgg ttccctgcag accccaggac gaactaatag gaagggcaag 9780 aatctctcaa ggagcgggat ggagccttag agaaccgca tgtctgggga aagcctacgc 9840 tcaaatgtgg agtctcatgt atttcacag agagaccctc agactagcat ccaacgccat 9900 atgttcagca gtaccagtcc actgggtccc acgacatggt ctattcacgc 9960 tcaccatcag tggatgacca cagaagacat gcttactgtc tggagaagag tgtggatcga 10020 ggacaatcca tggatggaag acaaaactcc agtcacaacc tgggaaaatg ttccatatct 10080 agggaagaga gaagaccaat ggtgcggatc acttattggt ctcacttcca gagcaacctg 10140 ggcccagaac atacccacag caattcaaca ggttagaagc ctttataggca atgaaggtt 10200 tctggactac atgccttcaa tgaagagatt taggaaggag gagagtcgg agggagccat 10260 ttggtaaaac gtaggaagtg aaaaagaggt taactgtcag gcatattaa gccacagtac 10320 ggaagaagct gtgctgcctg tgagccccgt ccaaggacgt taaaagaaga agtcaggccc 10380 caaagccacg gtttgagcaa accgtgctgc ctgtagctcc gtcgtgggga cgtaaaacct 10440 gggaggctgc aaactgtgga agctctacgc acggtgtagc agactagcgg ttaggagaga 10500 cccctcccat gacacaacgc agcaggcgggg cccgagcact gaggaagct gtacctcctt 10560 gcaaaggact agaggttaga ggagaccccc cgcaataaa aacagcatat tgacgctggg 10620 agagaccaga gatcctgctg tctcctcagc atcattccag gcacagaacg ccagaaaatg 10680 gaatggtgct gttgaatcaa caggttct 10708 <210> 12 <211> 10649 <212> DNA <213> Dengue virus <400> 12 agttgttagt ctgtgtggac cgacaaggac agttccaaat cggaagcttg cttaacacag 60 ttctaacagt ttgtttaaat agagagcaga tctctggaaa aatgaaccaa cgaaaaaagg 120 tggttagacc acctttcaat atgctgaaac gcgagagaaa ccgcgtatca acccctcaag 180 ggttggtgaa gagattctca accggacttt tttctgggaa aggaccctta cggatggtgc 240 tagcattcat cacgtttttg cgagtccttt ccatcccacc aacagcaggg attctgaaga 300 gatggggaca gttgaagaaa aataaggcca tcaagatact gattggattc aggaaggaga 360 taggccgcat gctgaacatc ttgaacggga gaaaaaggtc aacgataaca ttgttgtgct 420 tgattcccac cgtaatggcg tttcacttgt caacaagaga tggcgaaccc ctcatgatag 480 tggcaaaaca tgaaaggggg agacctctct tgtttaagac aacagagggg atcaacaaat 540 gcactctcat tgccatggac ttgggtgaaa tgtgtgagga cactgtcacg tataaatgcc 600 ccctactggt caataccgaa cctgaagaca ttgattgctg gtgcaacctc acgtctacct 660 gggtcatgta tgggacatgc acccagagcg gagaacggag acgagagaag cgctcagtag 720 ctttgacacc acattcagga atgggattgg aaacaagagc tgagacatgg atgtcatcgg 780 aaggggcttg gaagcatgct cagagagtag agagctggat actcagaaac ccaggatttg 840 cgctcttggc aggatttatg gcttatatga ttgggcaaac aggaatccag cgaactgtct 900 tctttgtcct aatgatgctg gtcgccccat cctacggaat gcggtgcgta ggagtaggaa 960 acagagactt tgtggaagga gtctcaggtg gagcatgggt cgacctggtg ctagaacatg 1020 gaggatgcgt cacaaccatg gcccagggaa aaccaacctt ggattttgaa ctgactaaga 1080 caacagctaa ggaagtggct ctgttaagaa cctattgcat tgaagcctca atatcaaaca 1140 taactacggc aacaagatgt ccaacgcaag gagagcctta tctgaaagag gaacaggacc 1200 aacagtacat ctgccggaga gatgtggtag acagaggatg gggcaatggc tgtggcttgt 1260 ttggaaaagg aggagttgtg acatgtgcga agttttcatg ttcggggaag ataacaggca 1320 atctggtcca aattgagaac cttgaataca cagtggttgt aacagtccac aatggagaca 1380 cccatgcagt aggaaatgac acatccaatc atggagttac agccacgata actcccaggt 1440 caccatcggt agaagtcaaa ttgccggact atggagaact aacactcgat tgtgaaccca 1500 ggtctggaat tgactttaat gagatgatcc taatgaaaat gaaaaagaaa acatggctcg 1560 tgcataagca atggttttg gatctgcctc ttccatggac agcaggagca gacacatcag 1620 aggttcactg gaattacaaa gagagaatgg tgacgttcaa ggttcctcat gccaagagac 1680 aggacgttac agtgttaggg tcacaggagg gagctatgca tagcgcacta gccggagcaa 1740 ccgaagtcga tagcggagac ggaaatcata tgttcgccgg acatctgaaa tgcaaagtga 1800 gaatggagaa attgcggatt aagggaatgt catacactat gtgttccggt aagttttcga 1860 ttgacaaaga gatggccgaa acgcaacacg gaacaacagt cgttaaggtt aagtacgaag 1920 gagccggagc tccgtgtaaa gtgccaatcg aaattagaga cgttaacaaa gagaaagtgg 1980 tcggaagagt gatatctagt acacccctag ccgaaaatac gaattccgtt acgaatatcg 2040 aactcgaacc cccctttgga gactcataca tagtgatagg agtgggtaat tccgcactca 2100 cgttgcactg gttcagaaag ggatcatcaa tcgggaagat gttcgaatca acatataggg 2160 gagcgaaaag aatggctata ttgggcgaaa ccgcatggga cttcggatca gtcggaggac 2220 tgtttacgag tctgggtaag gccgtacatc aggtattcgg atcagtgtat acaacaatgt 2280 ttggcggagt gtcatggatg atacggatac tgatagggtt tctagtgttg tggatcggaa 2340 cgaactcacg taatacgtct atggctatga catgtattgc cgtagggggg attacactgt 2400 ttctgggatt tacagtgcaa gcagacatgg gttgtgtggt gtcatggagt gggagagaat 2460 tgaagtgtgg aagcggaatt tttgtggttg acaacgtgca cacttggaca gaacagtaca 2520 aattccaacc agagtcccca gcgagactag cgtctgcaat attaaatgcc cacaaagatg 2580 gggtctgtgg aattagatca accacgaggc tggaaaatgt tatgtggaag caaataacca 2640 atgagctaaa ctatgttctc tgggaaggag gacatgatct cactgtagtg gctggggatg 2700 tgaaaggggt gttgaccaag gggcagagag cactcacacc cccagcgagt gatctgaaat 2760 attcatggaa gatagggggg aaagcaaaaa tctcacccc tgagcaag aacaccacat 2820 ttttaataga cggaccagac acctctgaat gccccaatga acgaagggca tggaatttctt 2880 ttgaggtgga agactatgga ttgaggtgt tcacgaccaa catatggatg aaattccgag 2940 aaggaagttc agaagtgtgt gaccaggt taatgtcagc tgcaattaaa gaccagaaag 3000 ctgtgcatgc tgacatgggt tattggatag agagctcaaa aaaccagacc tggcagatg 3060 agagagcatc tcttattgaa gtgaaaacat gtctgtggcc caacccat acactgtgga 3120 gcaatggagt gctggaaagc cagatgctta ttccaaatc atatgcaggc cctttttcac 3180 agcacaatta ccgccaggggc tatgctacgc aaaccgtggg tccatggcac ttaggcaac 3240 tagagataga ctttggagaa tgccccggaa cacagtcac attcaggag attgtgacc 3300 atagaggccc atctttgagg accaccactg catctggaaa actagtcacg caatggtgtt 3360 gccgctcctg cacgatgccc cccttaaggt tcttaggaga agatgggtgc tggtaggga 3420 tggagattag gcccttgagt gaaaaagaag agaacatggt caaatcacag gtgacggccg 3480 gacagggcac atcggaaact ttttcaatgg gtctgttgtg cctgaccttg tttgtggaag 3540 aatgcttgag gagaagagtc accaggaaac acatgatatt agctgtggta atcactcttt 3600 gtgctatcat cctggggggc ctcacatgga tggacttgct acgagccctc atcatgttgg 3660 gggacactat gtctggtaga ataggaggac agacccacct agccatcatg gcagtgttca 3720 agatgtcacc gggatacgtg ctgggtgtgt ttttaaggaa actcacttca agagagacag 3780 cactaatggt aataggaatg gccatgacaa caacactttc aattccacat gacctcatgg 3840 aactcattga tggaatatca ctaggactaa ttttgctaaa aatagtaaca cagtttgaca 3900 acacccaagt gggaacctta gctctttcct tgactttcat aagatcaaca atgtcattgg 3960 tcatggcttg gaggaccatt atggctgtgt tgtttgtggt cacactcatt cctttgtgca 4020 ggacaagctg tcttcaaaaa cagtctcatt gggtagaaat aacagcactc atcctaggag 4080 cccaagctct gccagtgtac ctaatgactc ttatgaaagg agcctcaaga agatcttggc 4140 ctcttaacga aggcataatg gctgtgggtt tggttagtct cttaggaagc gctcttttaa 4200 agaatgatgt ccctttagct ggcccaatgg tggcaggagg cttacttctg gcggcttacg 4260 taatgagtgg cagctcagca gatctgtcac tagagaaggc cgctaatgtg cagtgggatg 4320 aaatggcaga cataacaggc tcaagtccaa tcatagaagt gaagcaagat gaggatggct 4380 ctttctccat acgggacgtc gaggaaacca atatgataac ccttttggtg aaactggcac 4440 tgataacggt gtcaggtctc taccccttgg caattccaat cacaatgacc ttatggtaca 4500 tgtggcaagt gaaaacacaa agatcaggag ccctgtggga cgtcccttca cccgccgcca 4560 ctcaaaaagc cgcactgtct gaaggagtgt acaggatcat gcaaagaggg ttattcggga 4620 aaactcaggt tggagtaggg atacacatgg aaggtgtatt tcacacaatg tggcatgtta 4680 caagaggatc ggtgatctgc cacgagactg ggagattgga gccatcttgg gctgatgtca 4740 ggaatgacat gatatcatac ggtgggggat ggaggcttgg agataaatgg gacaaagaag 4800 aagacgttca ggtcctcgct atagaaccag ggaaaaatcc caaacatgtc caaacgaaac 4860 ctggcctttt caacccta actggagaaa ttggagcagt aacattagat ttcaacccg 4920 gaacgtctgg ttctcccatt atcacagga aaggaaagt catcggactc tatggaatg 4980 gagtggtcac aaatcaggt gattacgtca gtgccataac acagccgaa agaattggag 5040 agccagatta tgaagtggat gaggacatttt ttcgagaa agactact atatggact 5100 tacaccccgg agccggaaag aaaaagaa ttctccatc atagtgaga gaagccttaa 5160 aaaggaggct gcgaactttg attttggctc ccacgagagt ggtggcggcc gagatggaag 5220 aggcctacg tggactgcca atccgttacc aaaccccagc tgtaaatca gaacacacag 5280 gaagagagat tgtagaccctc atgtgccatg caaccttcac aacaagactt ttgtcatca 5340 ccagagttcc aaactacaac cttatagtaa tggatgaagc acatttcacc gatccttcca 5400 gtgtcgcggc tagaggatac atttcgacca gggtggaaat gggagaggca gcagccatct 5460 tcatgaccgc aacccctccc ggagcgacag atccctttcc ccagagcac agcccaatag 5520 aagacatcga gagagagatt cggaaggt catggacac agggttcgac tggatacag 5580 actaccaagg gaaaactgtg tggtttgttc ccagcataaa agctggaaat gacattgcaa 5640 attgtttgag aaagtcggga aagaaagtta tccagttgag taggaaaacc tttgatacag 5700 aatatccaaa aacgaaactc acggactggg actttgtggt cactacagac atatctgaaa 5760 tggggctaa ctttagagct gggagagtga tagaccctag aagatgcctc aagccagtta 5820 tcctaacaga tgggccagag agagtcatct tagcaggtcc tattccagtg actccagcaa 5880 gcgctgctca gagaagagg cgaataggaa ggaacccagc acaagaagac gaccaatacg 5940 ttttctccgg agacccacta aaaaatgatg aagaccatgc ccactggaca gaagcaaaga 6000 tgctgcttga caatatctac accccagaag ggatcattcc aacattgttt ggtccggaaa 6060 gggaaaaaac ccaagctatt gatggagagt ttcgcctcag agggaacaa aggaagactt 6120 ttgtggaatt aatgaggaga ggagaccttc cggtgtggct gagttataag gtagcttctg 6180 ctggcatttc ttacaaagat cgggaatggt gctttactgg ggaaagaaat aaccaaattt 6240 tagagaaaa catggaggtt gaaatttgga ctagagagg agaaaagaaa aaactgaggc 6300 caaaatggtt agatgcacgt gtatacgctg accccacatggc tttgaaggat ttcaaggagt 6360 ttgccagtgg aaagaagagt ataactctcg acatcctaac agagatcgcc agtttgccaa 6420 cttacctttc ctctagggcc aagctcgccc ttgacaacat agttatgctc cacaacag 6480 aaaaggagg gagggcctat caacatgccc tgaacgaact tccggagtca ctggaaacac 6540 tcatgcttgt agccttacta ggagctatga cagcaggtat cttcctgttt ttcatgcaag 6600 ggaaggaat agggaaattg tcaatgggtt tgataaccat tgcggtggct agtggcttgc 6660 tctgggtagc agaaattcaa ccccagtgga tagcggcctc aatcatactg gagttttttc 6720 tcatggtact gttgatacca gaaccagaaa aacaaaggac cccacaagac aatcaattga 6780 tctacgtcat attgaccatt ctcaccatta ttggtctaat agcagccaac gagatggggc 6840 tgatagaaaa aacaaaaacg gattttgggt tttaccaggt aaaaacagaa accaccatcc 6900 tcgatgtgga cctgagacca gcttcagcat ggacgctcta tgcggtagcc accacaattc 6960 tgactcccat gctgagacac accatagaaa atacgtcggc caacctatct ttagcagcca 7020 tcgccaacca ggcagccgtc ctaatggggc ttggaaaagg atggccactc cacagaatgg 7080 acctcggtgt gccgctgtta gcaatgggat gctattctca agtgaaccca acaaccttga 7140 cagcatcctt agtcatgctt ttagtccatt atgcaataat aggcccagga ttgcaggcaa 7200 aagccacaag agaggcccag aaaaggacag ctgctggaat catgaaaaat cccacagtgg 7260 acgggataac agtgatagat ctagaaccaa tatcctatga cccaaaattt gaaaagcaat 7320 tagggcaggt catgttactc gtcttgtgtg ctggacaact actcttgatg agaacaacat 7380 gggctttctg tgaagttttg actttggcca caggaccaat cttgaccttg tgggagggca 7440 acccgggaag gttttggaac acgaccatag ccgtatctac cgccaacatt ttcaggggaa 7500 gttatttggc aggagctgga ctggcttttt cactcataaa gaatgcacaa acccccagga 7560 ggggaactgg gaccacagga gagacactgg gagagaagtg gaagagacag ctaaactcat 7620 tagacaggaa agagtttgaa gagtataaaa gaagtggaat actagaagtg gacaggactg 7680 aagccaagtc cgccctgaaa gatgggtcta aaatcaagca tgcagtatct agagggtcca 7740 gtaagatcag atggattgtt gagagaggga tggtaaagcc aaaggggaa gttgtagatc ttggctgtgg gagaggaga tggtcttatt acatggcgac actcaagaac gtgactgaag tgaaagggta tacaaaagga ggtccaggac atgaagaacc gattcccatg gctacttatg gctggaattt ggtcaaactc cattcagggg ttgacgtgtt ctacaaaccc acagagcaag tggacaccct gctctgtgat attggggagt catcttctaa tggacacaata 8040 attggggagt 8100. catggctctc ttcaaaacct catggctgca tcaaagtcct caacccctac atgccaacag tcatagagga gctggagaaa ctgcagagaa aacacggtgg gaaccttgtc agatgcccgc tgtccagga ctccacccat gagatgtatt gggtgtcagg agcgtcggga aatattgtga gctctgtgaa cacaacatca aagatgttgt tgaacaggtt cacaacaagg cataggaaac ccacttatga gaggacgta gatcttgggg 8400. 8400. 8400. 8400. 8400. 8400. 8400. 8400. 8400 ggcttcagcg attgcaagag gagcacaaag aaacatggca ttatgatcag gaaaacccat acagaacctg ggcgtatcat ggaagctatg aagctccttc zgaggctct gcatcctcca 8520 tggtgaacgg gtggtgaaa ctgctaacaa aaccctggga tgtgattcca atggtgactc 8580 agttagccat gabataca accccttttg ggcacaaag agtgttcaa gagaaggtgg 8640 ataccagaac gccgcaacca aaaccaggca cacgaatggt tatgaccacg acagccaatt 8700 ggctatgggc cctccttgga aagagaaaa atcccagact gtgtacagg gagagttca 8760 tctcaaagt tagatcaac gcagccatag gcgcagctt tcaggagaa caggatgga 8820 catcagccag tgaagctgtg aatgacagcc ggttttggga actggttgac aaagaaaggg 8880 cccttcacca ggaagggaaa tgtgaatcgt gtgtctataa catgatggga aaacgtgaga 8940 aaagttagg agagtttggc agagccaagg gaagccgagc aatctggtac atgtggctgg 9000 gagcgcggtt tctggaattt gaagccctgg gttttgaa tgagatcac tggttttggca 9060 gagaaaatttc atggagtgga gtggaagggg aaggtctgca cagattggga tacatcctgg 9120 aggagataga caagaaggat ggagacctaa tgtagctga tgacacagca ggttgggaca 9180 caagaatcac tgaggatgac cttcaatg aggactgat cacggacag atggctcccc 9240 accacagat cctagccaaa gccattttca aactaaccta tcaaaacaa gtggtgaaag 9300 tcctcagacc cacaccgaga ggagcggtga tggatatcat atccaggaa gaccaaagg 9360 gtagtggaca agttggaaca tatggtttga acacattcac siacatggaa gttcaactca 9420 tccgccaaat ggaagctgaa ggagtcatca cacagatga catgcagaac ccaaagggt 9480 tgaaagaag agttgagaaa tggctgagag agtgtggtgt cgacaggtta aagaggatgg 9540 caatcagtgg agacgattgc gtggtgaagc ccctagatga gaggttttggc acttccctcc 9600 tcttcttgaa cgacatggga aaggtgagga aagacattcc gcagtgggaa ccatctagg 9660 gatggaaaaa ctggcaagag gttccttttt gctcccatca ctttcacaag atctttatga 9720 aggatggccg ctcactagtt gttccatgta gaaaccagga tgaactgata gggagagcca 9780 gatctcgca gggggctgga tggagcttaa gagaaacagc ctgcctgggc aaagcttacg 9840 cccagatgtg gtcgcttatg tactccaca gaagggatct gcgtttagcc tccatggcca 9900 tatgctcagc agttccaacg gatggtttc caacaagcag aacaatgg tcaatccatg 9960 ctcatcacca atggatgacc actgagaca tgctcaagt gtggaacaga gtgtggatag 10020 aagacaaccc caataccttacc cagaagactc cagtccattc gtgggagat ataccttacc 10080 taggaaaag agaggattg tggtgtgat ccctgattgg actttctct agagccacct 10140 gggcgaagaa cattcacacg gccatactc aggtcaggaa cttgatcgga aaagaggaat 10200 acgtggatta catgccagta atgaaagat acagtgctcc ttcagagt gaaggagttc 10260 tgtaattacc aacaacaac accaaaggct attgaagtca ggccacttgt gccacggttt 10320 gagcaaaccg tgctgcctgt agctccgcca ataatgggag gcgtaataat ccctagggag 10380 gccatgcgcc acggaagctg tacgcgtggc atattggact agcggttaga ggagacccct 10440 cccatcactg acaaaacgca gcaaagggg gcccgaagcc aggaggagc tgtactccctg 10500 gtggaaggac tagaggttag aggacccc cccaacacaa aaacagcata ttgacgctgg 10560 gaagaccag agatcctgct gtctctacaa catcaatcca ggcacagagc gccgcagat 10620 ggattggtgt tgttgatcca acaggttct 10649

Claims

1. A modified dengue virus comprising: a recoded prM protein, a recoded envelope (E) protein, or both; the recoded prM protein has a reduced codon pair bias compared to its parent prM protein coding sequence, or have at least five codons replaced with less frequently used synonymous codons, or has an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence; and the recoded E protein has a reduced codon pair bias compared to its parent E protein coding sequence, or has at least five codons replaced with less frequent synonymous codons; or having an increased number of CpG or UpA dinucleotides compared to its parent E protein coding sequence, Modified dengue virus.

2. 2. The modified dengue virus of claim 1, wherein expression of the prM protein or the E protein or both is reduced compared to its parent dengue virus.

3. The modified dengue virus of claim 1, wherein the recoded prM protein has reduced codon pair bias compared to its parent prM protein coding sequence.

4. 2. The modified dengue virus of claim 1, wherein the recoded prM protein has at least five codons replaced with less frequently used synonymous codons.

5. 2. The modified dengue virus of claim 1, wherein the recoded prM protein has an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence.

6. 2. The modified dengue virus of claim 1, wherein the recoded E protein has reduced codon pair bias compared to its parent E protein coding sequence.

7. 2. The modified dengue virus of claim 1, wherein the recoded E protein has at least five codons replaced with less frequently used synonymous codons.

8. The modified dengue virus of claim 1, wherein the recoded E protein has an increased number of CpG or UpA dinucleotides compared to its parent E protein coding sequence.

9. The modified dengue virus of claim 1, wherein each of the recoded prM or E protein coding sequences has a codon pair bias of less than -0.

05.

10. 2. The modified dengue virus of claim 1, wherein the codon pair bias of each of the recoded prM or E protein coding sequences is reduced by at least 0.

05.

11. 2. The modified dengue virus of claim 1, selected from type 1, type 2, type 3, type 4 or a combination thereof.

12. 2. The modified dengue virus of claim 1, which is a modified tetravalent dengue virus.

13. A modified dengue virus according to any one of claims 1 to 12, A pharma- ceutically acceptable excipient or carrier; 23. A dengue vaccine composition for inducing a protective immune response in a subject, comprising:

14. 1. A method of eliciting an immune response in a subject, comprising: Administering to said subject an effective dose of a composition comprising the modified dengue virus of any one of claims 1 to 12. A method comprising:

15. 15. The method of claim 14, wherein the immune response is a protective immune response and a prophylactically or therapeutically effective dose of the claimed vaccine composition is administered.

16. 15. The method of claim 14, further comprising administering to the subject at least one adjuvant.

17. The method of claim 14, wherein the immune response is cross-protective against a heterologous dengue virus.

18. 1. A method for inducing an immune response in a subject in need thereof, comprising the steps of: Prime dose of (i) an attenuated dengue virus produced by a method other than codon pair deoptimization or codon deoptimization or an increase in CpG or UpA dinucleotides; or (ii) a modified dengue virus comprising a recoded prM protein, a recoded envelope (E) protein, or both, the recoded prM protein has a reduced codon pair bias compared to its parent prM protein coding sequence, or has at least five codons replaced with less frequently used synonymous codons; or has an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence; and the recoded E protein has a reduced codon pair bias compared to its parent E protein coding sequence, or has at least five codons replaced with less frequent synonymous codons, or having an increased number of CpG or UpA dinucleotides compared to its parent E protein coding sequence, The modified dengue virus and one or more boost doses, (i) an attenuated dengue virus produced by a method other than codon pair deoptimization or codon deoptimization or an increase in CpG or UpA dinucleotides; or (ii) The modified dengue virus to said subject in need thereof. Including, The method, wherein at least said prime dose or said one or more boost doses is said modified dengue virus.

19. 20. The method of claim 18, wherein a first of said one or more boost doses is administered about 2 weeks after said prime dose.

20. 20. The method of claim 18, wherein expression of the prM protein or the E protein, or both, is reduced compared to its parent dengue virus.

21. 20. The method of claim 18, wherein the recoded prM protein has reduced codon pair bias compared to its parent prM protein coding sequence.

22. 20. The method of claim 18, wherein the recoded prM protein has at least five codons replaced with less frequently used synonymous codons.

23. 20. The method of claim 18, wherein the recoded prM protein has an increased number of CpG or UpA dinucleotides compared to its parent prM protein coding sequence.

24. The method of claim 18, wherein the recoded E protein has reduced codon pair bias compared to its parent E protein coding sequence.

25. 20. The method of claim 18, wherein the recoded E protein has at least five codons replaced with less frequently used synonymous codons.

26. 20. The method of claim 18, wherein the recoded E protein has an increased number of CpG or UpA dinucleotides compared to its parent E protein coding sequence.

27. 20. The method of claim 18, wherein each of the recoded prM or E protein coding sequences has a codon pair bias of less than -0.

05.

28. 20. The method of claim 18, wherein the codon pair bias of each of the recoded prM or E protein coding sequences is reduced by at least 0.

05.

29. 19. The method of claim 18, wherein the modified dengue virus is selected from type 1, type 2, type 3, type 4, or a combination thereof.

30. 20. The method of claim 18, wherein the modified dengue virus is a modified tetravalent dengue virus.

31. 1. A method for making a modified dengue virus genome, comprising: obtaining a nucleotide sequence encoding an envelope protein of the dengue virus; recoding said envelope-encoding nucleotide sequence to reduce protein expression; and substituting a nucleic acid having said recoded envelope-encoding nucleotide sequence into a parent dengue virus genome to create a modified dengue virus genome. wherein expression of the recoded envelope-encoding nucleotide sequence is reduced compared to the parent virus.

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