Dengue Serotype 2 Attenuated Strain

Abstract

The invention relates to live attenuated VDV2 (VERO-Derived Vaccine Dengue serotype 2) strains which have been derived from the wild-type dengue-2 strain 16681 by passaging on PDK and Vero cells and nucleic acids thereof. The invention further relates to a vaccine composition which comprises a VDV2 strain.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. application Ser. No. 12/633,459, filed Dec. 8, 2009, which is a divisional of U.S. application Ser. No. 11/453,344, filed Jun. 15, 2006, which claims the benefit of U.S. provisional application 60/691,274, filed on Jun. 17, 2005, all of said references incorporated herein by reference.

[0002] The invention relates to new live attenuated VDV2 (VERO-Derived Dengue serotype 2 virus) strains which are derived from the wild-type dengue-2 strain 16681 by passaging on PDK and Vero cells sanitization. The invention further relates to a vaccine composition which comprises such VDV2 strain.

[0003] Dengue diseases are caused by four closely related, but antigenically distinct, virus serologic types (Gubler, 1988; Kautner et al., 1997; Rigau-Perez et al., 1998; Vaughn et al., 1997), of the genus Flavivirus (Gubler, 1988). Infection with a dengue virus serotype can produce a spectrum of clinical illnesses ranging from a non-specific viral syndrome to severe, fatal haemorrhagic disease. The incubation period of dengue fever (DF) after the mosquito bite averages 4 days (range 3-14 days). DF is characterised by biphasic fever, headache, pain in various parts of the body, prostration, rash, lymphadenopathy and leukopenia (Kautner et al., 1997; Rigau-Perez et al., 1998). The viremic period is the same as of febrile illness (Vaughn et al., 1997). Recovery from DF is usually complete in 7 to 10 days but prolonged asthenia is common. Leukocytes and platelets counts decreases are frequent.

[0004] Dengue haemorrhagic fever (DHF) is a severe febrile disease characterised by abnormalities of homeostasis and increased vascular permeability that can lead to hypovolemia and hypotension (dengue shock syndrome, DSS) often complicated by severe internal bleeding. The case fatality rate of DHF can be as high as 10% without therapy, but below 1% in most centres with therapeutic experience (WHO Technical Guide, 1986).

[0005] Routine laboratory diagnosis of dengue infections are based on virus isolation and/or the detection of dengue virus-specific antibodies.

[0006] Dengue disease is the second most important tropical infectious disease after malaria, with over half of the world's population (2.5 billion) living in areas at risk for epidemic transmission. An estimated 50 to 100 million cases of dengue, 500,000 hospitalised DHF patients and 25,000 deaths occur each year. Dengue is endemic in Asia, the Pacific, Africa, Latin America, and the Caribbean. More than 100 tropical countries have endemic dengue virus infections, and DHF have been documented in more than 60 of these (Gubler, 2002; Monath, 1994). A number of well described factors appear to be involved in dengue infections: population growth, unplanned and uncontrolled urbanisation particularly in association with poverty, increased air travel, lack of effective mosquito control, and the deterioration of sanitary and public health infrastructure (Gubler, 2002). The awareness of dengue in travelers and expatriates is increasing (Shirtcliffe et al., 1998). Dengue has proven to be a major cause of febrile illness among US troops during deployments in dengue-endemic tropical areas (DeFraites et al., 1994).

[0007] The viruses are maintained in a cycle that involves humans and Aedes aegypti, a domestic, day-biting mosquito that prefers to feed on humans. Human infection is initiated by the injection of virus during blood feeding by an infected Aedes aegypti mosquito. Salivary virus is deposited mainly in the extravascular tissues. The primary cell subset infected after inoculation is dendritic cells, which subsequently migrate to draining lymph nodes (Wu et al., 2000). After initial replication in the skin and draining lymph nodes, virus appears in the blood during the acute febrile phase, generally for 3 to 5 days.

[0008] Monocytes and macrophages are with dendritic cells among the primary target of dengue virus. Protection against homotypic reinfection is complete and probably lifelong, but cross-protection between dengue types lasts less than 12 weeks (Sabin, 1952). Consequently a subject can experience a second infection with a different serotype. A second dengue infection is a theoretical risk factor of developing severe dengue disease. However, DHF is multifactorial including: the strain of the virus involved, as well as the age, immune status, and genetic predisposition of the patient. Two factors play a major role in the occurrence of DHF: a rapid viral replication with high viremia (the severity of the disease being related to the level of viremia (Vaughn et al., 2000) and an important inflammatory response with release of high levels of inflammatory mediators (Rothman and Ennis, 1999).

[0009] There is no specific treatment against Dengue diseases. The management of DF is supportive with bed rest, control of fever and pain with antipyretics and analgesics, and adequate fluid intake. The treatment of DHF needs correction of fluid loss, replacement of coagulation factors, and infusion of heparin.

[0010] Preventive measures presently rely on vector control and personal protection measures, which are difficult to enforce and expensive. No vaccine against dengue is currently registered. Since the 4 serotypes of dengue are circulating worldwide and since they are reported to be involved in cases of DHF, vaccination should ideally confer protection against all 4 dengue virus serotypes.

[0011] Live attenuated vaccines (LAVs), which reproduce natural immunity, have been used for the development of vaccines against many diseases, including some viruses belonging to the same genus as dengue (examples of commercially available flavivirus live-attenuated vaccines include yellow fever and Japanese encephalitis vaccines). The advantages of live-attenuated virus vaccines are their capacity of replication and induction of both humoral and cellular immune responses. In addition, the immune response induced by a whole virion vaccine against the different components of the virus (structural and non-structural proteins) reproduced those induced by natural infection.

[0012] A dengue vaccine project was initiated in Thailand at the Centre for Vaccine Development, Institute of Sciences and Technology for Development Mahidol University. Candidate live-attenuated vaccines were successfully developed, at a laboratory scale, for dengue serotype 1 (strain 16007, passage 13), serotype 2 (strain 16681, passage 53=LAV2), and serotype 4 (strain 1036, passage 48) viruses in Primary Dog Kidney (PDK) Cells, and for serotype 3 (strain 16562) in Primary Green Monkey Kidney (PGMK) cells (passage 30) and Fetal Rhesus Lung (FRhL) cells (passage 3). These vaccines have been tested as monovalent (single serotype), bivalent (two serotypes), trivalent (three serotypes), and tetravalent (all four serotypes) vaccines in Thai volunteers. Those vaccines were found to be safe and immunogenic in children and in adults (Gubler, 1997). These LAV 1-4 strains have been described in EP 1159968 in the name of the Mahidol University and were deposited before the CNCM (CNCM 1-2480; CNCM 1-2481; CNCM 1-2482 and CNCM 1-2483 respectively).

[0013] The Den-2 strain 16681 was recovered from serum of a DHF (Dengue Hemorrhagic Fever) patient in Bangkok in 1964 (Halstead et al., 1970). The original viremic serum had been passaged 4 times on BSC-1 cells (African Green Monkey kidney cells) and 5 times on continuous LLC-MK.sub.2 cells (Rhesus Monkey kidney cells). In 1977, the virus was passaged once in vivo, in susceptible monkeys (Macaca Mulatta), and then again in LLC-MK.sub.2 cells. Two additional passages in mosquitoes (Toxorhynchites amboinensis) were conducted in 1980. Virus attenuation was performed by passages at 32.degree. C. on PDK cells (Primary Dog Kidney cells). Attenuation of the strain was checked according to several in vitro and in vivo markers. Passage 50 fulfilled all these attenuation criteria and was chosen as master seed for vaccine production (1982), at passage 53. DEN-2 PDK53 vaccine candidate was evaluated in humans and found to be strongly immunogenic with no untoward clinical signs and symptoms (Bhamarapravati et al., 1989).

[0014] The complete sequence of the Dengue 2 Live-Attenuated Virus strain (LAV2) was established by R. Kinney et al. (CDC, Fort Collins) in 2001. Sequence differences between parent DEN-2 strain 16681 (SEQ ID No.3) and LAV2 (SEQ ID No.38) strain are described in Table 1. Thus, genetic comparison of the wild-type virus strain 16681 and LAV2 strain showed a set of 9 point mutations which could be linked to LAV2 attenuation.

TABLE-US-00001 TABLE 1 DEN-2 16681 and DEN-2 16681/PDK53 (LAV2) Sequence Differences coordinates LAV2 16681 Gene-aa position Nt Aa nt aa Non coding Nt-57 T -- C -- PrM-29 Nt-524 T Val A Asp E-373 Nt-2055 T Phe C Phe NS1-53 Nt-2579 A Asp G Gly NS2A-181 Nt-4018 T Phe C Leu NS3-250 Nt-5270 A/T Val/Glu A Glu NS3-342 Nt-5547 C Arg T Arg NS4A-75 Nt-6599 C Ala G Gly NS5-334 Nt-8571 T Val C Val

Nucleotide changes modifying the corresponding codon are indicated in bold.

[0015] The LAV2 strain which was initially established in 1983 was further rapidly identified as potential vaccine candidate (Bhamarapravati and Yoksan, 1997).

[0016] However, at that time, transmission to humans of Spongiform Encephalitis through mammalian cultures was not perceived as a risk and the virus was routinely maintained in Primary Dog Kidney cells (PDK). Furthermore, this LAV2 strain corresponds to a heterogeneous population. This heterogeneity represents an additional risk due to a potential in vitro or in vivo selection of one of the strain present in the composition.

[0017] In view of these increasing concerns, the Applicant decided to set up a sanitization process in order to get rid of any such risks. By transfecting Vero cells with the purified genomic RNA of LAV2, followed by three cycles of amplification in Vero cells, and two successive steps of virus plaque purification the Applicant produced a new Vero-Derived serotype 2 virus (VDV2).

[0018] This new VDV2 strain which has been thus derived by transfer to VERO cells and biological cloning differs from the LAV2 strain by sequence, an homogenous plaque size and temperature sensitivity but importantly has conserved some phenotypic and genotypic features of the LAV2 such as e.g. attenuation spots, small plaque phenotype, growth restriction at high temperature and has conserved the immunogenic features of the LAV2 strains. These features make this new strain a valuable vaccine candidate for prophylactic immunization in humans.

DEFINITIONS

[0019] "Dengue viruses" are positive-sense, single-stranded RNA viruses belonging to the Flavivirus genus of the flaviridae family. In the case of dengue serotype 2 (DEN-2) strain 16681, the entire sequence is 10723 nucleotides long (SEQ ID No.3). The RNA genome contains a type I cap at the 5'-end but lacks a 3'-end poly (A)-tail. The gene organization is 5'-noncoding region (NCR), structural protein (capsid (C), premembrane/membrane (prM/M), envelope (E)) and non structural protein (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) and 3' NCR. The viral RNA genome is associated with the C proteins to form nucleocapsid (icosahedral symmetry). As with other flaviviruses, the DEN viral genome encodes the uninterrupted open reading frame (ORF) which is translated to a single polyprotein.

[0020] Serial passaging of a virulent (disease-causing) strain of dengue-2 results in the isolation of modified virus which are "live attenuated", i.e., infectious, yet not capable of causing disease. These modified viruses are usually tested in monkeys to evaluate their attenuation. However, Humans are the only primates that exhibit signs of clinical disease. The viruses that cause mild (i.e. acceptable in terms of regulatory purposes as presenting a positive benefit/risk ratio) to low or no secondary effects (i.e. systemic events and/or biological abnormalities and/or local reactions) in the majority of the tested humans but still infect and induce an immune response are called "live attenuated".

[0021] The term "LAV" denotes live attenuated Dengue viral strains. In the context of the invention "LAVs" are live attenuated strains initially derived from the Dengue serotype 2 (DEN-2) strain 16681 by passages in Primary Dog Kidney (PDK) Cells. For instance "LAV2/PDK53" is the attenuated strain established after 53 passages of strain 16681 in PDK cells (DEN-2 16681/PDK53). "LAV2/PDK50" is the attenuated strain established after 50 passages of strain 16681 in PDK cells (DEN-2 16681/PDK50). LAV2/PDK53 nucleotide sequence is shown in SEQ ID No.38.

[0022] "VDV2" is meant a LAV obtainable by the sanitization process disclosed in the present application. A VDV2 is thus a biological clone (homogeneous) VERO-adapted Dengue serotype 2 virus capable of inducing a specific humoral immune response including neutralizing antibodies in primate especially in humans. The VDV2 strains of the invention can be easily reconstructed starting directly from the here disclosed VDV2 sequences. The induction of a specific humoral immune response can be easily determined by an ELISA assay. The presence of neutralising antibody in the serum of a vaccinee is evaluated by the plaque reduction neutralization test as described in section 4.1.1.2.2. A serum is considered to be positive for the presence of neutralizing antibodies when the neutralizing antibody titer thus determined is at least superior or equal to 1:10.

[0023] The terms "mutation" means any detectable change in genetic material, e.g. DNA, RNA, cDNA, or any process, mechanism, or result of such a change. Mutations include substitution of one or more nucleotides. In the context of the instant application, mutations identified in dengue-2 virus genomic sequence or polyprotein are designated pursuant to the nomenclature of Dunnen and Antonarakis (2000). As defined by Dunnen and Antonarakis at the nucleic acid level, substitutions are designated by ">", e.g. "31A>G" denotes that at nucleotide 31 of the reference sequence a A is changed to a G.

[0024] Variations at the protein level describe the consequence of the mutation and are reported as follows. Stop codons are designated by X (e.g. R97X denotes a change of Arg96 to a termination codon). Amino acid substitutions a designated for instant by "59G", which means that Ser in position 9 is replaced by Gly.

VERO-Derived Dengue Serotype 2 Viruses (VD V2)

[0025] The composition of the previously developed dengue-2 vaccine candidate LAV2 was improved by a sanitization process.

[0026] The VERO-Derived Vaccine Dengue serotype 2 (VDV2) disclosed herein uses the DEN-2 16681 virus attenuated by serial passages on PDK cells. VDV2 contains the genomic sequence of the whole live-attenuated DEN-2 virus, and bears the same attenuation spots which have been linked to attenuation as the original LAV2 strain that was tested in humans.

[0027] Sanitization of the LAV2 vaccine was performed by removing proteins and introducing only purified viral genomic material into Vero cells. More specifically, sanitization of the strain was performed by purifying and transfecting viral RNA into Vero cells. The process comprises the following steps:

[0028] a) extracting and purifying viral RNA from plaque-purified LVA2 strain, e.g. DEN-2 16681/PDK50 viruses;

[0029] b) advantageously associating of the purified RNA with cationic lipids;

[0030] c) transfecting Vero cell, in particular Vero cell LS10;

[0031] d) recovering of the neo-synthesized virus; and

[0032] e) purifying a VDV strain by plaque purification and optionally amplifying it in host cells, especially Vero cells.

[0033] The Vero cell technology is a well-known technology which has been used for different commercial products (injectable and oral polio vaccines, rabies vaccine). In the present invention qualified Vero cells were advantageously used to guarantee the absence of any risks potentially linked to the presence of adventitious agents. By "qualified VERO cells" is meant cells or cell lines for which culture conditions are known and is such that the said cells are free from any adventitious agents. These include e.g. the VERO cell LS10 of Sanofi Pasteur.

[0034] The thus isolated VDV strains are classically stored either in the form of a freezed composition or in the form of a lyophilised product. For that purpose, the VDV can be mixed with a diluent classically a buffered aqueous solution comprising cryoprotective compounds such a sugar alcohol and stabilizer. The pH before freezing or lyophilisation is advantageously settled in the range of 6 to 9, e.g. around 7 such as a pH of 7.5.+-.0.2 as determined by a pH meter at RT. Before use, the lyophilised product is mixed with a pharmaceutically diluent or excipient such as a sterile NaCl 4% solution to reconstitute a liquid immunogenic composition or vaccine.

[0035] The Glu variant of LAV2 vaccine strain, at position NS3-250, was selected during transfection and cloning, and positions 5'NC-57 and NS1-53, also identified as critical for attenuation of LAV2 vaccine, were both conserved in VDV2 sequence.

[0036] Sequencing, at attenuation-specific loci, of virus recovered after transfection, did not reveal any mutation, compared to SEQ ID No.38. The biologically cloned VDV2 virus exhibits a homogenous plaque phenotype and a remarkable genetic stability with regard to its LAV2 parent as it can be deduced especially from the conservation of the attenuation genotype.

[0037] VDV2 (passage 11) strain was sequenced and compared with the serotype 2 Dengue Live Attenuated Virus (LAV2) strain sequence (SEQ ID No.38). A set of 10 nucleotide differences was identified, triggering six amino acid substitutions located in M and Env structural peptides, and also in non-structural peptides NS3 and NS5. None of these differences corresponds to any of the LAV2 attenuation positions.

TABLE-US-00002 TABLE 2 Sequence comparison between LAV2/PDK53 and VDV2 passage 11 strains. Nucleotides Amino Acids Position LAV2 VDV2 Region Position LAV2 VDV2 736 G C M 9 G R 1619 G A E 228 G E 1638 A G E 234 K K 2520 G A NS1 33 K K 4723 T A NS3 69 S T 5062 G C NS3 181 D H 9191 G A NS5 541 R K 9222 A G NS5 551 E E 10063 T A NS5 832 S T 10507 A G 3'nc -- -- -- Grey shading: differences in structural proteins; Bold characters: differences in non-structural proteins.

[0038] The invention thus provides for live attenuated dengue-2 virus strains that have been obtained from the wild type virus DEN-2 16681 attenuated by serial passages on PDK cells and then by sanitization on VERO cells. In particular the attenuated strains of the invention comprise at least the identified sequence mutations (non-silent and optionally silent) relative to the nucleotide sequence or polyprotein sequence of the wild-type DEN-2 16681 and LAV2/PDK53 strains.

[0039] Accordingly, the invention relates to an isolated live attenuated dengue-2 virus strain which comprises, or consists of, the sequence of LAV2/PDK53 strain (SEQ ID No. 38) wherein at least nucleotides at positions 736, 1619, 4723, 5062, 9191, 10063, and 10507, and optionally 1638, 2520, 9222, and 10361, are mutated, with the proviso that the following nucleotides are not mutated: 57, 524, 2055, 2579, 4018, 5547, 6599, and 8571. Preferably, the mutations are substitutions. Preferably, the nucleotide at position 736 is C, the nucleotide at position 1619 is A, the nucleotide at position 4723 is A, the nucleotide at position 5062 is A, the nucleotide at position 9191 is A, the nucleotide at position 10063 A, and the nucleotide at position 10507 is G.

The nucleotide at position 5270 may be A or T, preferably A.

[0040] Still preferably, the isolated strain according to the invention comprises the sequence SEQ ID No.38 wherein said sequence comprises at least the mutations 736 G>C, 1619 G>A, 4723 T>A, 5062 G>C, 9191 G>A, 10063 T>A, and 10507 A>G, and optionally the mutation 1638 A>G, 2520 G>A, and/or 9222 A>G.

[0041] Hence, a live attenuated dengue-2 virus strain according to the invention may comprise, or consist of, the sequence of wild-type dengue-2 strain 16681 (SEQ ID No.3) wherein said sequence comprises at least the mutations 57 C>T, 524 A>T, 736 G>C, 1619 G>A, 2055 C>T, 2579 G>A, 4018 C>T, 4723 T>A, 5062 G>C, 5547 T>C, 6599 G>C, 8571 C>T, 9191 G>A, 10063 T>A, and 10507 A>G. Preferably, a live attenuated strain according to the invention further comprises the mutation 1638 A>G, 2520 G>A, and/or 9222 A>G by reference to the nucleotide sequence of wild-type strain 16681 (SEQ ID No.3).

[0042] The live attenuated dengue-2 virus strains according to the invention may include variant strains that comprise a sequence SEQ ID No.38 mutated at positions 736, 1619, 4723, 5062, 9191, 10063, and 10507, as defined above, and that further comprise a substitution of one or more nucleotides in a given codon position that results in no alteration in the amino acid encoded at that position.

[0043] Advantageously, the live attenuated dengue-2 virus strain according to the invention comprises a sequence which differs by a limited number of mutations, e.g. no more than 5, still preferably no more than 2, from SEQ ID No.1.

[0044] Preferably, the genomic sequence of a dengue-2 virus strain according to the invention consists of the nucleotide sequence SEQ ID No.1.

[0045] The invention also relates to live attenuated dengue-2 strains that may be derived from the VDV2 strain of sequence SEQ ID No.1 by further passages on cells, in particular Vero cells.

[0046] The invention also relates to an isolated nucleic acid which comprises, or consists of, the DNA sequence SEQ ID No.1 or its equivalent RNA sequence.

[0047] A "nucleic acid molecule" refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix.

[0048] As used herein, by RNA sequence "equivalent" to SEQ ID No.1 is meant a sequence SEQ ID No.1 wherein deoxythymidines have been replaced by uridines. As SEQ ID No.1 constitutes VDV2 cDNA sequence, the equivalent RNA sequence thus corresponds to the positive strand RNA of VDV2.

[0049] The invention further relates to the polyprotein of sequence SEQ ID No.2 and to fragments thereof. SEQ ID No.2 is the sequence of the polyprotein encoded by SEQ ID No.1 A "fragment" of a reference protein is meant a polypeptide which sequence comprises a chain of consecutive amino acids of the reference protein. A fragment may be at least 8, at least 12, at least 20, amino acid long.

[0050] Said fragments of the polyprotein of sequence SEQ ID No.2 comprise at least an arginine at position 9 of M protein (position 214 of SEQ ID No.2), and/or a glutamic acid at position 228 of E protein (position 508 of SEQ ID No.2), and/or a threonine at position 69 of NS3 protein (position 1543 of SEQ ID No.2), and/or a histidine at position 181 of NS3 protein (position 1656 of SEQ ID No.2), and/or a lysine at position 541 of NS5 protein (position 1725 of SEQ ID No.2), and/or a threonine at position 832 of NS5 protein (position 3032 of SEQ ID No.2).

[0051] According to an embodiment the fragment of the polyprotein encoded by SEQ ID No.1 is or comprises M protein, and/or E protein, and/or NS3 protein and/or NS5 protein.

Immunogenic and Vaccine Compositions

[0052] The invention also relates to an immunogenic composition, suitable to be used as a vaccine, which comprises a VDV2 strain according to the invention.

[0053] The immunogenic compositions according to the invention elicit a specific humoral immune response toward the dengue virus comprising neutralizing antibodies.

[0054] Preferably, the immunogenic composition is a vaccine.

[0055] According to an embodiment, the immunogenic is a monovalent composition, i.e. it elicits en immune response and/or confers protection against Dengue-2 virus only.

[0056] According to another embodiment, the invention relates to a multivalent dengue immunogenic composition. Such a multivalent immunogenic composition or vaccine may be obtained by combining individual monovalent dengue vaccines. The immunogenic or vaccine composition may further comprise at least a live attenuated dengue virus of another serotype. In particular, the immunogenic or vaccine composition may comprise a VDV2 according to the invention in combination with at least a live attenuated dengue virus selected from the group consisting of serotype 1, serotype 3, and serotype 4.

[0057] Preferably, the immunogenic or vaccine composition may be a tetravalent dengue vaccine composition, i.e. a vaccine composition that comprises a VDV2 according to the invention in combination with a live attenuated dengue-1 virus strain, a live attenuated dengue-3 virus strain and a live attenuated dengue-4 virus strain.

[0058] Live attenuated dengue-1, dengue-3 and dengue-4 virus strains have been described previously. Reference may be made to the live-attenuated vaccines that were developed by Mahidol University by passaging dengue serotype 1 (strain 16007, passage 13; LAV1), and serotype 4 (strain 1036, passage 48, LAV4) viruses in Primary Dog Kidney (PDK) Cells, and for serotype 3 (strain 16562) in Primary Green Monkey Kidney (PGMK) cells (passage 30) and Fetal Rhesus Lung (FRhL) cells (passage 3) (LAV3). The nucleotide sequences of LAV1 (SEQ ID No.40), LAV3 (SEQ ID No.41), and LAV4 (SEQ ID No.42) are shown in the annexed sequence listing.

[0059] Advantageously, a live attenuated dengue-1 strain may correspond to a VDV1 strain which has been obtained from the LAV1 strain developed by Mahidol by the process of sanitization according to the invention. In particular a live attenuated dengue-1 strain (VDV1) may comprise, and advantageously consists of the sequence SEQ ID No.39.

[0060] Immunogenic compositions including vaccines may be prepared as injectables which can correspond to liquid solutions, suspensions or emulsions. The active immunogenic ingredients may be mixed with pharmaceutically acceptable excipients which are compatible therewith.

[0061] The immunogenic compositions or vaccines according to the present invention may be prepared using any conventional method known to those skilled in the art. Conventionally the antigens according to the invention are mixed with a pharmaceutically acceptable diluent or excipient, such as water or phosphate buffered saline solution, wetting agents, fillers, emulsifier stabilizer. The excipient or diluent will be selected as a function of the pharmaceutical form chosen, of the method and route of administration and also of pharmaceutical practice. Suitable excipients or diluents and also the requirements in terms of pharmaceutical formulation, are described in Remington's Pharmaceutical Sciences, which represents a reference book in this field.

[0062] Preferably, the immunogenic composition or vaccine corresponds to an injectable composition comprising an aqueous buffered solution to maintain e.g. a pH (as determined at RT with a pH meter) in the range of 6 to 9.

[0063] The composition according to the invention may further comprise an adjuvant, i.e. a substance which improves, or enhances, the immune response elicited by the VDV2 strain. Any pharmaceutically acceptable adjuvant or mixture of adjuvants conventionally used in the field of human vaccines may be used for this purpose.

[0064] The immunogenic compositions or vaccines according to the invention may be administered by any conventional route usually used in the field of human vaccines, such as the parenteral (e.g. intradermal, subcutaneous, intramuscular) route In the context of the present invention immunogenic compositions or vaccines are preferably injectable compositions administered subcutaneously in the deltoid region.

Method for Immunizing

[0065] The invention further provides for a method of immunizing a host in need thereof against a dengue infection which comprises administering the host with an immunoeffective amount of a vaccine composition according to the invention.

[0066] A "host in need thereof" denotes a person at risk for dengue infection, i.e. individuals travelling to regions where dengue virus infection is present, and also inhabitants of those regions.

[0067] The route of administration is any conventional route used in the vaccine field the choice of administration route depends on the formulation that is selected preferably, the immunogenic composition or vaccine corresponds to an injectable composition administered via subcutaneous route, advantageously in the deltoid region.

[0068] The amount of LAV or VDV, in particular VDV2, in the immunogenic compositions or vaccines may be conveniently expressed in viral plaque forming unit (PFU) unit or Cell Culture Infectious Dose 50% (CCID.sub.50) dosage form and prepared by using conventional pharmaceutical techniques. For instance, the composition according to the invention may be prepared in dosage form containing 10 to 10.sup.6 CCID.sub.50, or 10.sup.3 to 10.sup.5 CCID.sub.50 of LAV or VDV, for instance a dose of 4.+-.0.5 log.sub.10 CCID.sub..sub.50 of VDV2 strain for a monovalent composition. Where the composition is multivalent, to reduce the possibility of viral interference and thus to achieve a balanced immune response (i.e. an immune response against all the serotype contained in the composition), the amounts of each of the different dengue serotypes present in the administered vaccines may not be equal.

[0069] An "immunoeffective amount" is an amount which is capable of inducing a specific humoral immune response comprising neutralising antibodies in the serum of a vaccinee, as evaluated by the plaque reduction neutralization test as described in section 4.1.1.2.2; a serum being considered to be positive for the presence of neutralizing antibodies when the neutralizing antibody titer thus determined is at least superior or equal to 1:10.

[0070] The volume of administration may vary depending on the route of administration. Subcutaneous injections may range in volume from about 0.1 ml to 1.0 ml, preferably 0.5 ml.

[0071] The optimal time for administration of the composition is about one to three months before the initial exposure to the dengue virus. The vaccines of the invention can be administered as prophylactic agents in adults or children at risk of Dengue infection. The targeted population thus encompasses persons which are naive as well as well as non-naive with regards to dengue virus. The vaccines of the invention can be administered in a single dose or, optionally, administration can involve the use of a priming dose followed by a booster dose that is administered, e.g. 2-6 months later, as determined to be appropriate by those of skill in the art. The invention will be further described in view of the following figures and examples.

FIGURES

[0072] FIG. 1 is a summary of History of VDV2 seed.

[0073] FIG. 2 is a flow chart that summarises the developed manufacturing process that gives rise to the Filled Product (monovalent), "ready to use" doses.

[0074] FIG. 3 is a diagrammatic representation of VDV2 genome map. The above arrow is the polyprotein coding sequence. The below arrows represent mature peptides coding sequence. The vertical bars symbolize the nucleotidic variations between wild-type dengue 2 strain 16681 and LAV2 strain. The stars designate the nucleotidic variations between LAV2 and VDV2.

[0075] FIG. 4 shows plaque size analysis after 7 days of incubation at 37.degree. C. for dengue-1 viruses LAV2, VDV2, and strain 16681.

[0076] FIG. 5 is a graphic analysis showing plaque size distribution for dengue-2 viruses LAV2, VDV2, and strain 16681.

[0077] FIG. 6 is a summary of Trial Design for assessment of safety of VDV2 monovalent in healthy flavivirus-naive adults.

EXAMPLES

Example 1

Sanitization

[0078] 1.1 Viral RNA Purification

[0079] The RNA purification and transfection process was performed as follows. DEN-2/PDK50 suspension was resuspended in 0.5 ml of water and diluted in order to contain at least 3.times.10.sup.4 and up to 3.times.10.sup.7 TCID.sub.50 or PFU of virus per milliliter. One unit of benzonase diluted in 0.01 ml of William's medium was added to 0.5 ml of virus, in order to digest DNA or RNA molecules from cellular origin, and the solution was incubated for 2 hours at 4.degree. C. on an agitator. At the end of incubation step, 0.65 ml of a denaturing buffer containing guanidium chloride, detergent (SDS), and pmercaptoethanol (RTL-.beta.mercaptoethanol buffer, provided in the kit RNeasy Mini kit, Qiagen Ref. 74104) were added and proteins were extracted once with phenol/chloroform (1/1) vol/vol and once with chloroform vol/vol, followed by centrifugation for 5 min at 14,000 rpm at room temperature. After each extraction, the aqueous phase was collected, taking care not to collect material (white precipitate) at the interface, and transferred to a clean 1 ml-Eppendorf tube. The RNA solution was then applied onto a QIAgen column following the recommendations of the manufacturer (RNeasy minikit, QIAgen), in order to remove traces of solvent, and eluted with 0.06 ml of nuclease-free H.sub.2O water. The presence of viral RNA was confirmed by quantitative RT-PCR, using a reference curve established with known quantities of virus, in TCID.sub.50/ml.

[0080] 1.2 Transfection of Vero Cells with Purified RNA

[0081] Transfection was performed using lipofectamine (LF2000 Reagent, Life Technologies), a mixture of cationic lipids that associate to RNA through charge interactions and allows transfer of the complexes into the cytoplasm of the cells by fusion with the cell membrane. The optimal quantity of LF2000 reagent was determined in a preliminary experiment by incubating Vero cells, plated 16 to 24 hours before (0.3-0.5.times.10.sup..sup.6 cells per well in a 6 wells plate) with increasing doses (5 to 20 .mu.l) of lipofectamine. Cells were then incubating 4 to 5 hours at 32.degree. C., 5% CO.sub.2, before replacing the medium by fresh culture medium without FCS, and the incubation was continued overnight at 32.degree. C. Toxicity (round, refringent or floating cells, homogeneity of the cell monolayer) was checked regularly for 48 hours, under an inverted microscope. The highest dose of lipofectamine that was not toxic under these conditions was 10 .mu.l and was chosen for RNA transfection.

[0082] Four transfections were carried out in parallel, using 1/4 of the RNA preparation (about 2.times.10.sup.4 log eqTCID.sub.50, according to qRT-PCR). Twenty-five microliters of viral RNA solution were diluted in 500 .mu.l of OptiMEM medium (GIBCO) containing 15 .mu.l of LF2000 Reagent (a mixture of cationic lipids that associate to RNA through charge interactions, and allow transfer of the complexes into the cytoplasm of the cells by fusion with the cell membrane). 200 ng of yeast tRNA were added as carrier in 2 out of the 4 reactions.

[0083] The 4 transfection mixes were allowed to precipitate for 10 min at room temperature before addition to 6-wells plates of confluent Vero cells, and incubation at 36.degree. C. After four hours, transfection mix was removed and cells were rinsed once in PBS. Three milliliters of post-transfection medium (Williams, GIBCO) were added, and incubation was continued for 5 days at 32.degree. C. Culture medium was then replaced by 3 ml of Dengue infection medium (Williams supplemented with 10 mM MgSO.sub.4).

[0084] A focus of cells presenting typical cytopathic effects (round, refringent cells) was detected at day 8 post-transfection in 1 out of the 2 wells transfected in presence of tRNA. Release of virus in the supernatant of these cells was confirmed by qRT-PCR. Eleven days post-transfection, marked cytopathic effects were detected in this only well, while the supernatant of the three other transfected-wells remained negative.

[0085] The viral solution recovered after transfection was re-named TV100 (instead of 16681 PDK50Nero-2) and exhibited an infectious titer of 5.8 log TCID.sub.50/ml after dilution at 1/2 in an aqueous buffered solution comprising cryoprotective agents (pH=7.5).

[0086] 1.3 Characterization of Viruses Recovered after Transfection

[0087] Spot sequencing of specific loci important for attenuation was performed by R. Kinney (CDC, Fort Collins). Data are presented in Table 3.

TABLE-US-00003 TABLE 3 Sequencing of transfected virus at attenuation-specific positions 5'-NC-57 NS1-53 NS3-250 Virus Nt 57 Nt 2579 (aa) Nt 5270 (aa) DEN-2 16681 C G (Gly) A (Glu) DEN-2 PDK53 T A (Asp) T/A (Val/Glu) TV100 T A (Asp) A (Glu)

[0088] VDV2 has retained the important attenuating loci at 5'NC-57 and NS1-53, and the wild-type 16681 locus of the NS3-250-Glu variant in the PDK53 vaccine. The NS3-250-GluNal mix in the PDK53 vaccine was observed to be stable between passages PDK45 and PDK53 suggesting that selection has occurred in Vero cells. Previous analysis of DEN-2 vaccine isolated from serum of a vaccinee had demonstrated that this selection could also occur in humans.

[0089] Viral plaques diameters were measured in Vero cells. Briefly, Vero cells were plated at a density of 1.000.000 cells/cm.sup.2 in culture medium containing 4% of FBS. After overnight incubation, the medium was removed and cells were infected with serial twofold or fivefold dilutions of virus. After 1.5 hour at 37.degree. C. 5% CO.sub.2, the inoculum was removed and cells were incubated at 37.degree. C. 5% CO.sub.2 in Minimal Eagle Medium (MEM) containing 1.26% methylcellulose and 10% FBS. After 11 days of incubation, plates were fixed 20 minutes in cold acetone at -20.degree. C. and revealed by immuno-coloration with a flavivirus-specific mAb, diluted at 2.5 .mu.g/ml. Viral plaques were measured using an image analysis software (Saisam/Microvision). VDV2 was compared to LAV2 16681/PDK50 seed (Table 4) and exhibited similar homogeneous small plaques of 1-3 mm diameter.

TABLE-US-00004 TABLE 4 Plaques size of LAV2 16681/PDK50 and VDV2 Step Virus LP/MP SP Before LAV2 PDK50 0 319 transfection After transfection Uncloned VDV2 0 183 LP/MP: Number of Large/Medium Plaques in 6 wells SP: Number of Small Plaques in 6 wells

[0090] 1.4 Plaque-Purifications

[0091] Three additional amplification passages (P2 to P4) were performed on the virus recovered after transfection. Biological cloning by plaque-purification was performed on P3 and P4 passaged virus (named LST 003 and LST 007, respectively).

[0092] Briefly, Vero cells were plated in 6-well plates and infected with serial dilutions of virus, in order to get between 1 and 20 plaques by plate. After 1.5 hour at 37.degree. C. 5% CO.sub.2, the inoculum was removed and cells were incubated under 3 ml of solid medium composed of MEM-10% FCS pre-heated at 42.degree. C. and mixed extemporaneally with 2% of melted agarose equilibrated at 42.degree. C. The medium was allowed to solidify at room temperature for 30 min; under flow hood, and plates were incubated in inverted position for 10 days at 32.degree. C.-5% CO.sub.2. A second layer of the same medium supplemented with 0.01% of neutral red was then added and plates were incubated for an additional night at 32.degree. C. Six well-isolated small plaques were picked under sterile conditions using a micro-pipet equipped with an 0.1 ml tip, and transferred into sterile tubes containing 0.2 ml of MEM4% FCS: three from P3 passage (identified as clones 31, 32 and 33), and three from P4 passage (identified as clones 71, 72 and 73). The suspension was homogenised by vortexing, serially diluted in the same medium, and immediately used to infect 6-well plates of Vero cells. The protocol was repeated and a second picking of two SP was done on clones 32, 33, 71 and 72, and one SP on clone 31. Each picked plaque was diluted in 1 ml of medium, before amplification on Vero cells, in T25 cm.sup.2 flasks. Culture medium was collected at day 6 post-infection, diluted with the same volume of an aqueous buffered solution comprising cryoprotective agent (pH 7.5) and frozen at -70.degree. C. All these steps were performed at 32.degree. C.

[0093] Plaque purified virus were named 311, 321, 322, 331, 332, 341, 342, 351, 352, 711, 712, 721 and 722, respectively.

[0094] Infectious titers were determined on Vero cells at the end of the first amplification (see below)

TABLE-US-00005 Clone 311: 3.95 LogCCID.sub.50/ml Clone 321: 5.20 LogCCID.sub.50/ml Clone 322: 5.45 LogCCID.sub.50/ml Clone 331: 5.55 LogCCID.sub.50/ml Clone 332: 4.95 LogCCID.sub..50/ml Clone 341: 2.80 LogCCID.sub.50/ml Clone 342: 4.85 LogCCID.sub.50/ml Clone 351: 5.35 LogCCID.sub.50/ml Clone 352: 5.50 LogCCID.sub.50/ml Clone 711: 5.45 LogCCID.sub.50/ml Clone 712: 5.65 LogCCID.sub.50/ml Clone 721: 5.30 LogCCID.sub.50/ml Clone 722: 5.60 LogCCID.sub.50/ml

[0095] A second amplification on Vero cells was carried out for three clones: clones 331, 352, and 722. Culture supernatants were collected at day 8 post-infection, diluted at 1/2 with an aqueous buffered solution comprising cryoprotective agent (pH 7.5) and named TV331, TV352 and TV722.

[0096] 1.5 Characterization of Cloned Virus

[0097] After the 1.sup.st amplification, all amplified viruses exhibited same plaque size phenotype and titers equivalent to, or higher than 5 log CCID.sub.50/ml (except clones 311 and 341 which were significantly lower). Sequencing at attenuation-specific positions was performed on 6 clones from the 1.sup.st amplification (clones 321, 331, 351, 352, 711, 721) and the three clones from the 2.sup.nd amplification, and revealed no mutation.

[0098] In absence of any significant difference between the clones, TV722 was selected and amplified in VERO cells in order to generate a VDV2 vaccine candidate strain.

TABLE-US-00006 TABLE 5 Sequencing at attenuation-specific spots of DEN-2 viruses 5'-UTR prM E NS1 NS2a NS3 NS4A NS5 Step/cell Virus 57 524 2055 2579 4018 5270 5547 6599 8571 Wild-type/PGMK 16681 C A C G C A T G C Vaccine/PDK PDK53 T T T A T A/T C C T 2nd plaque- TV 321 T T T A T A C C T purification/VERO TV 331 T T T A T A C C T TV 342 T T T A T A C C T TV 352 T T T A T A C C T TV 711 T T T A T A C C T TV 722 T T T A T A C C T 2.sup.nd amplification/VERO TV722PM T T T A T A C C T

Nucleotides position are indicated below each gene and referred to published sequence of DEN-2 16681 strain.

[0099] In conclusion, a total number of 11 passages was necessary to obtain a biological clone of DEN-2 166681/PDK50 adapted on VERO cells.

[0100] Further characterizations have been performed then by determining VDV2 passage 11 complete sequence and phenotypic testing.

Example 2

Sequencing

[0101] The complete sequence of the virus was generated according to the following strategy. Viral genomic RNA was purified. The full genome was amplified by 16 overlapping RT-PCR reactions. Each PCR was designed so that sequencing tags were added on each DNA strand. This allowed simpler sequence reactions, all driven by a single pair of universal sequencing primers. Each PCR product was individually sequenced on both DNA strands. All results were reassembled to reconstruct the full VDV2 genome.

[0102] 2.1 Materials

[0103] 2.1.1 Viruses

[0104] The viruses to which it is referred here are DEN-2 16681; LAV-2/PDK53; VDV2, the sequences of which are given in the attached sequence listing.

[0105] The complete genome sequence of these viruses is 10723 nucleotides in length.

[0106] 2.1.2 Primers

[0107] All primers have been designed in Seqweb bioinformatics package (Accelrys), primer design module (Table 6).

TABLE-US-00007 TABLE 6 list of RT-PCT and sequencing primers Primer RT-PCR Over- Name Primers sequences NtStart NtEnd length length lap D2 01+ GTTTTCCCAGTCACGACacgtggaccgacaaagacag (SEQ ID No. 4) 13 32 37 978 -32 D2 01- AACAGCTATGACCATGttcctcctgaaaccccttcc (SEQ ID No. 5) 991 972 36 371 D2 02+ GTTTTCCCAGTCACGACatcacgtacaagtgtcccc (SEQ ID No. 6) 583 601 36 949 D2 02- AACAGCTATGACCATGagcaacaccatctcattgaag (SEQ ID No. 7) 1532 1512 37 163 D2 03+ GTTTTCCCAGTCACGACtgcaaccagaaaacttggaatacac (SEQ ID No. 8) 1325 1349 42 948 D2 03- AACAGCTATGACCATGgctccatagattgctccaaagac (SEQ ID No. 9) 2273 2251 39 203 D2 04+ GTTTTCCCAGTCACGACcccagtcaacatagaagcagaacc (SEQ ID No. 10) 2025 2048 41 878 D2 04- AACAGCTATGACCATGccaaagccatagtcttcaacttcc (SEQ ID No. 11) 2903 2880 40 155 D2 05+ GTTTTCCCAGTCACGACatcatgcaggcaggaaaac (SEQ ID No. 12) 2707 2725 36 949 D2 05- AACAGCTATGACCATGaccataaccatcactcttccc (SEQ ID No. 13) 3656 3636 37 240 D2 06+ AACAGCTATGACCATGaccataaccatcactcttccc (SEQ ID No. 14) 3368 3386 36 930 D2 06- AACAGCTATGACCATGgctctctccagttccaaatc (SEQ ID No. 15) 4298 4279 36 146 D2 07+ GTTTTCCCAGTCACGACaagaaccagcaagaaaaggag (SEQ ID No. 16) 4113 4133 38 868 D2 07- AACAGCTATGACCATGcaccattaccataaagacccac (SEQ ID No. 17) 4981 4960 38 226 D2 08+ GTTTTCCCAGTCACGACttgaaccatcatgggcggac (SEQ ID No. 18) 4715 4734 37 910 D2 08- AACAGCTATGACCATGtcctgcttttatacttggaacgaac (SEQ ID No. 19) 5625 5601 41 208 D2 09+ GTTTTCCCAGTCACGACaagcccatttcacagaccc (SEQ ID No. 20) 5375 5393 36 920 D2 09- AACAGCTATGACCATGtcaatttcttcctttccccttc (SEQ ID No. 21) 6295 6274 38 158 D2 10+ GTTTTCCCAGTCACGACgagaggagaagcaaggaaaac (SEQ ID No. 22) 6096 6116 38 923 D2 10- AACAGCTATGACCATGagggacacattcactgagg (SEQ ID No. 23) 7019 7001 35 233 D2 11+ GTTTTCCCAGTCACGACacagagaacaccccaagac (SEQ ID No. 24) 6750 6768 36 929 D2 11- AACAGCTATGACCATGtccacttcctggattccac (SEQ ID No. 25) 7679 7661 35 308 D2 12+ GTTTTCCCAGTCACGACacaagtaatgctcctagtcctc (SEQ ID No. 26) 7332 7353 39 935 D2 12- AACAGCTATGACCATGttcactgatgacactatgttcc (SEQ ID No. 27) 8267 8246 38 211 D2 13+ GTTTTCCCAGTCACGACgtcatcaccaaatcccacag (SEQ ID No. 28) 8016 8035 37 937 D2 13- AACAGCTATGACCATGgcttcttctctctttttcccatc (SEQ ID No. 29) 8953 8931 39 140 D2 14+ GTTTTCCCAGTCACGACaaggtgagaagcaatgcag (SEQ ID No. 30) 8773 8791 36 937 D2 14- AACAGCTATGACCATGtggaaatggtgtgaacagaag (SEQ ID No. 31) 9710 9690 37 209 D2 15+ GTTTTCCCAGTCACGACgcattcagcacctaacaatcac (SEQ ID No. 32) 9641 9482 39 9335 D2 15- AACAGCTATGACCATGggcatttatgatggcctgac (SEQ ID No. 33) 10396 10377 36 -- D2 16i+ ccatggaagctgtacgc (SEQ ID No. 34) 10480 10496 64 234 D2 16i- AACAGCTATGACCATGtgattcaacagcaccattcc (SEQ ID No. 35) 10714 10695 36 -28

[0108] 2.2 Methods

[0109] 2.2.1 Viral RNA Purification

[0110] From previous experience, a minimal of 1000 DICC.sub.50 is required to get a positive RT-PCR reaction in the next steps. This means that a minimum virus titer of 10.sup.4 DICC.sub.50/mL is necessary. Virus genomic RNA was purified using QIAamp viral RNA mini kit (Qiagen), according to the manufacturer's recommendations. Briefly, a volume of 140 .mu.l from a crude viral sample was incubated in the presence of the lysis solution, and loaded onto a kit column. After washing steps, the purified viral RNA was eluted by 60 .mu.l of sterile nuclease-free water containing 1 .mu.l (40 units) of RNAse inhibitor (RNAse Out, Sigma).

[0111] 2.2.2 Reverse Transcription

[0112] Viral RNA was reverse transcribed into cDNA by a reverse transcriptase (reverse iT) from ABGene. Again, standard operating conditions were applied, using 10 .mu.l of purified RNA, in a final reaction volume of 20 .mu.l. The reaction was initiated by hybridization of the minus strand primers. One RT reaction per PCR was performed. The cDNA synthesis was obtained by 45 min incubation at 47.degree. C.

[0113] 2.2.3 PCR

[0114] All PCR were performed with Expand High Fidelity PCR system (Roche diagnostics), using all 16 pairs of primers (+) and (-) from Table 6. PCR conditions were the following ones:

TABLE-US-00008 PCR RT 2 .mu.l program 10x 2.5 .mu.l Denaturation 94.degree. C. 2 min buffer dNTP mix 2 .mu.l Denaturation 94.degree. C. 15 sec (10 mM) Primers 0.8 .mu.l each Hybrid- 55.degree. C. 30 sec 40 cycles ization H20 16.4 .mu.l Elongation 68.degree. C. 1 min Enzyme 0/5 .mu.l Elongation 68.degree. C. 5 min All PCR products were controlled by electrophoresis on agarose gel.

[0115] 2.2.4 Sequencing

[0116] The major part of the sequence reactions has been outsourced to Genome Express. Genome extremities, ambiguities, some inter-PCR junctions, and regions not sequenced by Genome Express for technical reasons were performed in-house. [0117] Sequencing at Genome Express: PCR products were shipped at +4.degree. C., and sequencing results were received as informatic sequence files. Text file, quality files and chromatograms are available for each individual sequence. After sequence alignment, all discrepancies were checked on the chromatogram, and corrected if identified as sequence algorithm errors. [0118] In-house sequencing: Sequence reactions were performed on thermocycler PTC-200 (MJ Research), with Sequitherm Excell II LC kit (Epicentre). Each PCR product was sequenced on both strands independently in a single reaction. Reactions were loaded onto a sequence electrophoresis gel. Run and analysis of sequence were performed on the automated sequencer Gene ReadIR 4200 (Li-Cor).

TABLE-US-00009 [0118] .cndot. Sequence reactioQn DNA up to 200/250 ng PCR program Reaction buffer 7.2 .mu.l Denaturation 92.degree. C. 2 min Primers (1-2 pM) 1.5 .mu.l each Denaturation 92.degree. C. 15 sec 30 cycles Enzyme 1 .mu.l Hybridization 50.degree. C. 30 sec H.sub.2O up to 20 .mu.l Elongation 70.degree. C. 1 min Elongation 70.degree. C. 10 sec

[0119] Addition of 3 .mu.l of denaturating/loading buffer.

[0120] Denaturation of samples 3 min at 95.degree. C. and ice cooling just before samples loading.

TABLE-US-00010 .cndot. Sequence Electrophoresis Electrophoresis parameters Gel parameters Voltage 1500 V Gel hight 41 cm Current 35 mA Gel thickness 0.2 mm Power 40 W Temperature 45.degree. C. Run time 9H00 Scan speed 3

[0121] 2.3 Results

[0122] All PCR fragments were sequenced from both ends using a common PCR added ail, i.e. a specific motif which has been added at 5' end of all primers:

TABLE-US-00011 (SEQ ID No. 36) 5' primers: M13SEQ-GTTTTCCCAGTCACGAC (SEQ ID No. 37) 3' primers: M13REV-AACAGCTATGACCATG

[0123] M13-SEQ and -REV sequences correspond to universal M13 primers motifs (New England Biolabs references).

[0124] For final contig assembly, a quick analysis was performed in Vector NTi, in ContigExpress module (Informax). The LAV2 reference sequence was compared with all individual sequencing results. In such conditions, all results could be aligned at the right place on the complete genome, even when some regions were still missing contig assembly, giving a quick visualization of the overall genome alignment.

[0125] 2.3.1 Complete VDV2 Sequence Assembly

[0126] The final sequence alignment was performed in Vector NTi, AlignX module (Informax). The classical multiple sequence alignment algorithm ClustalW (Thompson et al., 1994) was used by the software to build the global alignment. All the sequence results were aligned together with the LAV2 reference sequence, thus allowing for a better reconstruction of the genome. Any discrepancy in the sequence with regard to the reference required a confirmation on another independent sequence reaction. The complete sequence of VDV2 is shown in SEQ ID No.1.

[0127] Some ambiguities are often found in single sequences, especially near sequence extremities. This is inherent to the somewhat poor quality of the reaction at both ends of any PCR fragment. Such poor quality sequences were excluded from the alignment, until two other independent sequence reactions were available from other PCR products. Discrepancy towards the reference was not taken into account in the final alignment when not confirmed in at least two independent other PCR sequences matching the consensus. Conversely, any discrepancy confirmed on two independent sequences was kept in the final sequence.

[0128] Table 7 summarizes the characteristics of each individual sequence reaction, indicating start, end and length. Overlaps between adjacent PCR are also indicated, as well as differences with regard to the reference sequence in the last column.

TABLE-US-00012 TABLE 7 Dengue VDV2 individual sequences characteristics Name Start End Size Overlap Comments D2 01+ 33 365 332 0 2 sequences D2 01- 619 79 540 5 2 sequences D2 02+ 614 1334 720 736 G > C (M9-G > R) D2 02- 1488 654 834 127 736 G > C (M9-G > R) D2 03+ 1361 2135 774 1619 G > A (E228 G > E); 1638 A > G (E234K s) D2 03- 2227 1416 811 179 1619 G > A (E228 G > E); 1638 A > G (E234K s) D2 04+ 2048 2774 726 2520 G > A (NS1-33K s) D2 04- 2866 2210 656 133 2520 G > A (NS1-33K s) D2 05+ 2733 3495 762 D2 05- 3619 2819 800 251 D2 06+ 3393 4196 803 D2 06- 4257 3368 889 78 D2 07+ 4179 4830 651 4723 T > A (NS3-69 S > T) D2 07- 4851 4223 628 130 4723 T > A (NS3-69 S > T) D2 08+ 4742 5506 764 5062 G > C (NS3-181 DD > H) D2 08- 5582 4721 861 188 5062 G > C (NS3-181 DD > H) D2 09+ 5394 6100 706 D2 09- 6669 5979 690 545 D2 10+ 6124 6996 872 D2 10- 6983 6148 835 218 D2 11+ 6778 7567 789 D2 11- 7649 6781 868 317 D2 12+ 7365 8236 971 D2 12- 8241 7332 909 191 D2 13+ 8050 8797 747 D2 13- 8819 8147 672 22 D2 14+ 8707 9700 903 9191 G > A (NS5-541 R > K); 9222 A > G (NS5-551E s) D2 14- 9654 8804 850 199 9191 G > A (NS5-541 R > K); 9222 A > G (NS5-551E s) D2 15+ 9501 10285 784 10063 T > A (NS5-832 S > T) D2 15- 10347 9702 645 187 10063 T > A (NS5-832 S > T) D2 16i+ 10486 10687 201 10507 A > G D2 16i- 10694 10160 534 0 10507 A > G

[0129] The two extremities of the genome could not be sequenced from PCR amplification, because cDNA synthesis and PCR DNA reaction required oligonucleotides complementary to the ends of the genome. During the amplification step, these oligonucleotides are incorporated into the PCR fragment. The sequence result is that of the synthetic oligonucleotide, and not that of the virus itself. PCR from both ends of the virus genome did work properly, suggesting that the viral sequence was not significantly different from the oligonucleotide sequence (if it had been the case, PCR amplification should have failed or at least should have been of poor quality). We were not able to distinguish them from all other PCR amplifications. So, in the reconstructed genome, both genome ends were considered as identical to oligonucleotide sequences (and also identical to the reference). At 5' end, the sequence is that of nucleotides 1 to 32. At 3' end, the sequence is that of nucleotides 10695 to 10723.

[0130] 2.3.2 Sequence Comparison

[0131] Ten nucleotide differences have been detected with regard to the parent LAV2 genomic sequence. VDV2 vaccine strain is derived from LAV2, through virus sanitization and passage from dog to monkey cells.

[0132] Differences between LAV2 and VDV2 can have several origins. First, cloning steps can select a viral subpopulation that is not 100% identical to the major sequence previously detected in LAV2. Second, LAV2 has been produced on PDK cells, whereas VDV2 has been made on Vero cells. Such passage from dog to monkey cells is known to potentially induce virus changes that reflect adaptation to the new cell line. Third, as for all RNA viruses, the lower viral RNA polymerase fidelity triggers a higher genomic mutation rate than DNA polymerases do.

[0133] In term of sequences, all 9 nucleotide positions which have been linked to viral attenuation of LAV2 are conserved in VDV2 passage 11.

[0134] Furthermore, sequence comparison between VDV2 passage 9 and passage 11 showed the occurrence of two mutations between passages 9 and 11 which are linked to differences in phenotype, viremia and immunogenicity.

TABLE-US-00013 TABLE 8 Sequence comparison between LAV2/PDK53 strain and VDV2 passages 9 and 11 strains Nucleotides Amino acids VDV2 VDV2 Position LAV2 Passage 9 Passage 11 Region Position LAV2 Passage 9 Passage 11 736 G G C M 9 G G R 1619 G A A E 228 G E E 1638 A G G E 234 K K K 2520 G A A NS1 33 K K K 4723 T A A NS3 69 S T T 5062 G C C NS3 181 D H H 5270 A/T A A NS3 250 E/V V V 9191 G G A NS5 541 R R K 9222 A G G NS5 551 E E E 10063 T A A NS5 832 S T T 10507 A G G 3' nc -- -- -- -- Bold: sequence differences between VDV2 passage 9 and passage 11/

[0135] When performing sequence alignment between all available Genbank serotype 2 Dengue genomic sequences, it appears that only two positions are shared by other Dengue 2 strains (1638 and 2520), both silent at amino acid level. All other positions are specific to the VDV2 passage 11 strain, triggering an amino acid substitution (Table 8). Concerning amino acid changes, the four changes in non-structural peptides appear relatively conservative, from a biochemical point of view, whereas the two changes in M and in the envelope bring modification both in charge and hydrophobicity.

Example 3

Characterization

[0136] The objective of these studies was to assess whether changes in attenuation markers occurred through passages.

[0137] The flow chart shown on FIG. 2 summarises the developed manufacturing process that gives rise to the Filled Product (monovalent), "ready to use" doses

[0138] Briefly, after 2 successive passages on Vero cells of the VDV2 passage 8, the respective working seeds were obtained. The final virus cultivations are also conducted by infection of a Vero cell suspension. The viruses produced are then harvested. DesoxyRiboNucleic Acid (DNA) is digested according to an enzymatic treatment. Impurities are removed by ultrafiltration. Infectious titers are enhanced by a concentration step. An aqueous buffered solution comprising cryoprotective agents (pH=7.5) is added and this 0.22-.mu.m filtrated mixture is then diluted at the targeted dose within the same solution. The active substance is then filled into glass vials, freeze-dried, and stored before use.

[0139] 3.1 Phenotypic Markers

[0140] Table 9 presents data from three phenotypic assays performed on DEN-2 16681 wt strain, DEN-2 16681/PDK53 vaccine strain, VDV2 passage 9 and VDV2 passage 11 (last adaptation passage): temperature-sensitivity (Ts), growth curves on monkey (Vero) and mosquito (C6/36) cells and neurovirulence in Newborn mice (data obtained at CDC). Reduced mouse neurovirulence (reduced mortality and longer average survival time (AST)), restricted-growth at 39.degree. C. and restricted replication on C6/36 are currently accepted by the scientific community as attenuation criteria for Dengue viruses. Vero-adapted passages exhibit clear Ts profile, and are more restricted than DEN2/PDK53. Final adaptation passage is restricted by about 3 log in this assay. Temperature sensitivity was also confirmed by viral growth curves. On Vero cells, similar replication levels were observed with all tested viruses. On mosquito cells, viral growth of Vero-adapted viruses was clearly restricted (about 3 log) compared to wt DEN2, and slightly restricted (about 0.5 log) compared to DEN2-PDK53. Surprisingly, mouse neurovirulence of Vero-adapted viruses was close to neurovirulence of wt DEN2, and significantly higher than neurovirulence of DEN2/PDK53 vaccine. These data point out the low predictive value of this say, with regard to viral strain attenuation (cf clinical data).

[0141] Plaque size distribution of VDV2 passages 9 and 11, DEN2/PDK53 and wtDEN2 are compared to FIG. 5. Wt DEN2 exhibits heterogenous profile with 95% of plaques with a size homogeneous profile, with a major population (81%) of plaques<0.6 mm and a minor population (12%) of 1-2 mm plaques. This profile is close to, but distinct from DEN2-PDK53 profile. Noteworthy, the intermediate adaptation passage, VDV2 P9, exhibits a more heterogeneous profile, with a major population (70%) of 1-2 mm plaques, and a minor population (25%) of plaques<0.6 mm. These data demonstrate that VDV2 strain was not yet fully adapted at passage 9, and that the two additional passages were required for obtention of a homogeneous population replicating stably in Vero cells.

TABLE-US-00014 TABLE 9 Summary of DEN-2 viral phenotypes Growth curves (Peak log.sub.10 Neurovirulence in pfu/ml) newborn Swiss Temperature sensitivity Vero-LS10 Webster mice (Percent titer reduction at 39.degree. C.).sub.Fold-reduction at AST Virus Score Day 3 Day 4 Day 5 Day 6 Titer Day Mortality.sub.n (S.D.) D2- + n.d. 92.7.sub.13.7 n.d. 92.2.sub.12.8 7.5 8 100.0%.sub.16 12.2 16681 (1.5) D2- + n.d. 96.6.sub.29.4 n.d. 99.7.sub.333.3 7.3 8-10 43.75%.sub.16 16.0 PDK53 (2.4) VDV2 + n.d. 99.94.sub.1666.7 n.d. 99.97.sub.3333.3 7.5 8-10 100.0%.sub.16 10.9 P9 (0.7) VDV2 + n.d. 99.92.sub.1250.0 n.d. 99.88.sub.833 7.5 10 100.0%.sub.16 10.9 P11 (0.6) N: number of animals.

Example 4

Immunogenicity, Viremia, and Toxicology in Monkeys

[0142] The most solid and numerous data that can be obtained in monkeys concern immunogenicity and viremia. Viremia, in particular, has been identified as one of the factors associated with virulence and disease severity in humans, and then constitute an important parameter to consider. Obviously, immunogenicity is a key parameter when testing vaccines.

[0143] Inventors have established minimal/maximal values for viremia and immunogenicity.

TABLE-US-00015 TABLE 10 Minimal requirements for responses induced by Dengue vaccine candidates in monkeys, as measured in Vero or LLC-MK2 cells by plaque assay (these cells being considered equivalent in such an assay) Viremia mean duration Viremia mean peak titer Mean neutralizing titer (days) (log 10 pfu) Day 30 (all serotypes being (all serotypes being (for each serotype) considered) considered) PRNT 50 .ltoreq.3 days .ltoreq.1.5-2 .gtoreq.80 pfu: plaque forming unit PRNT 50: Plaque Reduction Neutralization Titer 50 (titre corresponding to a reduction of 50% of plaque number)

[0144] 4.1 Pre-Clinical Pharmacology, Pharmacokinetics, and Product Metabolism in Animals

[0145] 4.1.1 Material and methods

[0146] 4.1.1.1 Monkey Experiments

[0147] Monkey experiments were carried out according to European guidelines regarding animal experiments.

[0148] Immunizations were performed on cynomolgus monkeys (Macaca fascicularis) originating from Mauritius (CRP Le Vallon). Monkeys were quarantined for 6 weeks in the animal facility of Sanofi Pasteur before immunization.

[0149] Monkeys were immunized by subcutaneous (SC) route in the arm with vaccines in a volume of 0.5 ml (see each respective section). After light anesthesia with ketamine (Imalgene, Merial), blood was collected by puncture of the inguinal or saphene veins. At days 0 and 28, 5 ml of blood were sampled for evaluating antibody responses while between days 2 and 10 only 1 ml of blood was sampled for evaluating viremia. Blood was collected on ice and kept on ice until serum separation. To do so, blood was centrifuged for 20 minutes at 4.degree. C., and serum collected and stored at -80.degree. C. until testing in Rich Kinney's laboratory. Shipment to USA was performed in dry ice.

[0150] 4.1.1.2 Viremia and Neutralizing Antibody Responses (Plaque Reduction Neutralization Test, PRNT)

[0151] All analyses were performed in the laboratory of R. Kinney in CDC, Fort Collins, USA. Serum samples were shipped and stored at -80.degree. C. until the time of testing. At the time of first thawing, the samples were tested for viremia, and a 1:5 dilution of the serum was made. The 1:5 serum dilutions were inactivated for 30 min at 56.degree. C. before testing for neutralizing antibodies.

[0152] 4.1.1.2.1 Viremia

[0153] 0.125 ml of serum was added to 0.125 ml of diluent (RPMI medium) in the first well of 96-well plate and serial 10-fold dilution series were done, transferring 0.025 ml into 0.225 ml of diluent for each dilution. 0.2 ml of 10.sup.0.3-10.sup.5.3 dilution series was plated in 6-well plate of Vero cells (virus was adsorbed at 37.degree. C. for 1.5 hour, overlayed with 4 ml of agarose lacking neutral red, overlayed 6-7 days later with 2 ml of agarose containing neutral red, and plaques counted). The limit of virus detection was =10 PFU/ml. For controls stock DEN-16681 PDK-53 (LAV2) vaccine was plated.

[0154] 4.1.1.2.2 PRNT (Plaque Reduction Neutralization Test)

[0155] Neutralizing antibodies were quantified as described in Huang et al. (2000). Briefly, 0.2 ml of heat-inactivated, 1:5 dilution of serum was added to the first well of 96-well plate and serial 2-fold dilution series were made, transferring 0.1 ml into 0.1 ml of diluent (RPMI medium) for each dilution. This resulted in a 1:10-1:320 serum dilution series. 0.1 ml of DEN virus (60-160 PFU; parental DEN2 16681 virus) was added to each serum dilution well for a total of 0.2 ml of serum-virus mixture. 96-well plates were incubated overnight at 4.degree. C. 0.1 ml of serum-virus mixtures (containing 30-80 PFU of input virus) were plated in 6-well Vero plates (as indicated above in the Viremia section) and plaques were counted after staining with neutral red. Multiple back titrations of the input viruses at 2-fold, 1-fold, and 0.5-fold test concentrations provided direct experimental determination of the input PFU, which was the basis for determining 50% (PRNT.sub.50) and 70% (PRNT.sub.70) endpoint antibody titers. A negative serum result should have a neutralizing antibody titer of <1:10. Sera showing neutralization titers of 320 were retested at dilutions 1:80-1:2560 for determination of endpoint titer.

[0156] 4.1.2 Evaluation of Monovalent VDV2 Candidate at Passage 9 in Monkeys

[0157] Purification/selection of VDV2 candidate has been conducted as described in example 1. The selected clones (based on phenotypic markers and sequence) have been tested after 9 passages in cell culture in Sanofi Pasteur on male cynomolgus macaques (Macaca fascicularis, mean weight 3.1 kg) originating from CRP Le Vallon, Mauritius.

[0158] After immunization on DO, viremia was followed from D2 to D10, and immunogenicity measured at DO and D28. All viruses and vaccines, when in liquid form, were kept at -70.degree. C.

[0159] LAV2: titre: 10.sup.3,93DICC.sub.50/ml; lyophilized, resuspended in 0.5 ml of PBS (containing Ca.sup.2+ and Mg.sup.2+; CaCl.sub.2.2H.sub.2O 0.133 g/l; MgCl.sub..sub.2.6H.sub.2O, 0.1 g/l) and administered in totality.

[0160] Passage VDV2 DEN2-TV722 (2 plaque purifications+1 amplification): Titre: 10.sup.5,6 DICC.sub.50/ml; liquid, diluted at 10.sup.5,3 pfu/ml in PBS (containing Ca.sup.2+ and Mg.sup.2+; CaCl.sub.2.2H.sub.2O 0.133 g/l; MgCl..sub.2.6H.sub.2O, 0.1 g/l); 0.5 ml administered.

[0161] Injection was done by SC route in the arm with a 23G1 needle, at a 10.sup.5 DICC.sub.50 dose for VDV2.

[0162] The results are as presented in Table 11. Titration at day 28 were carried out in triplicate for both PRNT.sub.70 or and PRNT.sub.50.

[0163] The comparison between VDV2 and LAV2 showed clear differences in viremia, with high viremia of short duration for VDV2 in 3/4 monkeys compared to LAV2, and significant immunogenicity for both types (rather lower for VDV2). This viremia may be considered as too high for VDV2 at this pre-master level after only a few passages on Vero cells. However, wild type DEN-2 (and other types too) induce viremia of longer duration (6 to 7 days) and intensity (up to 5 logs plaque forming units [pfu]) (Monath et al., 2000; Bray et al., 1996).

TABLE-US-00016 TABLE 11 VDV2 passage 9 immunogenicity Neutralizing Antibody Titer Viremia (PFU/ml in Vero cells) Day (-15) Day 28 Day Day Day Day Day Day Day Day Day Day Serum Group PRNT.sub.70 PRNT.sub.50 PRNT.sub.70 PRNT.sub.50 -15 2 3 4 5 6 7 8 9 10 AD LAV <10 <10 80/80/160 320/160/320 0 0 0 0 0 0 5 0 50 20 097 DEN-2 AC <10 <10 160/80/320 320/160/640 0 0 0 0 0 5 5 0 0 0 170 AD <10 <10 1280/640/2560 2560/1280/2560 0 5 0 0 10 50 0 5 0 0 677 AC <10 <10 320/320/320 640/1280/1280 0 0 5 0 15 5 0 5 0 0 182 AC VDV <10 <10 160/160/160 320/160/640 0 550 35 0 0 0 0 0 0 0 658 DEN-2 AC <10 <10 160/80/160 160/160/160 0 1650 35 0 5 0 0 0 0 0 512 AD <10 <10 160/320/160 320/320/320 0 1700 60 100 0 0 0 0 0 0 608 AD <10 <10 80/80/80 80/160/160 0 70 10 0 50 10 100 0 0 0 132 Virus Exp#1 Exp#2 Exp#3 DEN-2 60PFU 54PFU 46PFU

[0164] 4.1.3 Evaluation of Monovalent VDV2 Candidate at Passage 11

[0165] As immunogenicity of the vaccines had been tested at the passage 9, a further experiment was designed to test the monovalent passage after two additional passages (passage 10).

[0166] Male Macaca fascicularis monkeys were used as before, originating from C.R.P. Le Vallon, Ile Maurice (24 monkeys, mean weight 3.4 kg).

[0167] Passage 11 VDV2: Batch: Titre: 8.07 log 10 g DICC.sub.50/ml

[0168] Placebo: PBS with Ca.sup.2+ and Mg.sup.2+

[0169] VDV3: VERO-Derived Vaccine Dengue serotype 3 strain, obtained by sanitization of LAV3 on Vero cells.

[0170] VDV4: VERO-Derived Vaccine Dengue serotype 4 strain, obtained by sanitization of LAV4 on Vero cells.

[0171] Vaccines were diluted at 10.sup.5.3 DICC.sub.50/ml in PBS (containing Ca.sup.2+ and Mg.sup.2+; CaCl.sub.2.2H.sub.2O 0.133 g/l; MgCl.sub.2.6H.sub.2O, 0.1 g/l); 0.5 ml administered by SC route in the arm with a 23G1 needle, corresponding to a dose of 10.sup..sup.5 DICC.sub.50.

[0172] Viremia and immunogenicity have been measured as usual in CDC by R Kinney. The results are shown in Table 12.

[0173] VDV2 passage 11 monovalent vaccine induced a significant immune response, while viremia was low or absent. The absent/low VDV2-induced viremia is to be considered in light of the previous experiment in which the passage 9 VDV2 induced high early viremia. Some evolution between passages 9 and 11 suppressed this high viremia while immunogenicity was maintained. VDV2 therefore constitutes an acceptable candidate.

[0174] It is to be noted that in the same experiment, 4 monkeys were vaccinated with a tetravalent formulation involving the same VDV2 passage 11 vaccine; no viremia was detected for VDV1 and VDV2 while VDV3 and VDV4 induced viremia.

[0175] Two other experiments involved the administration of VDV2, alone or in combination with the other serotypes.

[0176] In the first one (tetravalent study; 5-log of each serotype), no viremia was detected for VDV2, and VDV1, while high levels of viremia were detected for VDV3 and VDV4.

[0177] In the second experiment, VDV2 passage 11 was administered alone or within a tetravalent combination including VDV1. When administered alone, VDV2 passage 11 induced a low viremia (peak 40) in only 1 out of 4 monkeys while the 3 others were negative. When present within tetravalent formulations, VDV2 induced no or dramatically lower viremia than VDV3 and VDV4, even though VDV2 was administered at 4 log while VDV3 and VDV4 were administered at 2 log. This demonstrates the higher safety of VDV2, as far as viremia is concerned. Monovalent VDV2 thus fulfilled the success criteria initially defined in monkeys.

TABLE-US-00017 TABLE 12 passage 11 VDV2 immunogenicity and viremia Neutralizing Antibody Titer Viremia (PFU/ml in Vero cells) Day (-14) Day 29 Day Day Day Day Day Day Day Day Day Monkey Group PRNT.sub.50 PRNT.sub.70 PRNT.sub.50 PRNT.sub.50 -14 3 4 5 6 7 8 9 10 AE 971 VDV DEN-2 -- -- 180 80 0 0 0 0 0 0 0 0 0 AE 990 -- -- 160 50 0 15 0 0 5 0 0 0 0 AE 998 -- -- 905 508 0 0 0 0 0 0 0 0 0 AF 182 -- -- 285 101 0 0 0 0 0 0 0 0 0 Geo homologous -- -- 293 119 Mean AE 538 Placebo -/-/-/- -/-/-/- 2.5/-/2/2 -/-/-/- 0 0 0 0 0 0 0 0 0 AE 548 -/-/-/- -/-/-/- -/-/1/2 -/-/-/- 0 0 0 0 0 0 0 0 0 AE 556 -/-/1.5/2 -/-/-/- 1/-/-/- -/-/-/- 0 0 0 0 0 0 0 0 0 AE 572 -/-/1.5/5 -/-/1.5/2 5/-/-/2 -/-/-/- 0 0 0 0 0 0 0 0 0 Geo -/-/1.2/3 -/-/1/1 2/-/1.2/1.6 -/-/-/- Mean D1/D2/ D1/D2/ D1/D2/ D1/D2/ D3/D4 D3/D4 D3/D4 D3/D4

[0178] 4.2 Toxicology of VDV2

[0179] 4.2.1 Neurovirulence Tests in Monkeys

[0180] The objective of this test was to demonstrate the lack of neurotropic properties in monkeys (Ph. Eur. 2.6.18) of the attenuated 2 dengue virus seed produced by Sanofi Pasteur.

[0181] 10 cynomolgus monkeys from Mauritius were inoculated with VDV2 passage 9 by the intracerebral route (10.sup.710 CCID.sub.50/in the thalamus of each hemisphere). At the end of the test, the monkeys were sacrificed and perfused with formaline solution. Tissue samples were taken from the brain of each monkey (medulla oblongata, pons and cerebellum, midbrain, thalamus including the left and the right parts, the left and the right of the cerebral cortex). Sections were cut at a thickness of 8 .mu.m and stained by eosin and gallocyanin.

[0182] No histopathological signs of pathogenicity were observed in the monkey brains injected with serotype 2 primary virus seed.

[0183] 4.2.2 GLP Toxicity Study in the Cynomolgus Monkey after 1 Subcutaneous Administration of VDV2 Followed by a 28-Day Observation Period

[0184] The objective of this GLP study was to assess the interactions between VDV2 passage 9 and other Dengue vaccine candidates. The 1st step of the study was to assess the safety and immunogenicity of VDV2 prior to the administration of another vaccine candidate.

[0185] One human dose of VDV2 (approximately 10.sup.4 CCID.sub.50 per dose) was administered subcutaneously on Day 0 to cynomolgus monkeys (4 males and 4 females). A control group of 2 males and 2 females received the vehicle (4% NaCl).

[0186] Mortality, clinical condition, body weight, and food consumption were monitored throughout the study. Body temperature was taken once pre-test, daily from the day of each administration and during 2 days after. Blood samples were taken for clinical laboratory determinations once pre-test and on Days 8 and 27.

[0187] There were no effects on clinical signs, body weight, food consumption, dermal reactions, body temperature, haematology, clinical chemistry, or organ weights. No deaths were reported during the study.

[0188] In conclusion, the subcutaneous administration of VDV2 to the cynomolgus monkey (Macaca fascicularis) at the test doses did not adversely affect the health of the monkeys as assessed by in-life clinical observations and clinical pathology.

Example 5

Safety of Monovalent VDV2 in Healthy, Lavivirus-Naive Adults Aged 18 to 40 Years

[0189] The aim of this phase 1 trial is to document the safety, viremia, and immunogenicity profiles of monovalent VDV2 passage 11 at a virus concentration of 1 04 CCID.sub.50 compared to Stamaril.RTM. (used as control group) in flavivirus-naive adults. Single injections are given, with follow-up at 6 and 12 months. For safety precaution, sequential inclusions are performed in the study.

[0190] Enrollment and vaccinations are therefore staggered; a 1st cohort (n=4 per group, total n=12) have been vaccinated. The safety data collected up to Day 28 have been reviewed by an Independent Data Monitoring Committee (IDMC) and by the Royal Adelaide Hospital Investigational Drugs Subcommittee (IDSC) before deciding to proceed with the vaccination of the remaining subjects (n=8 per group, total n=16). A schematic representation of the trial design is provided in FIG. 6.

[0191] After administration of the vaccine the patient are regularly submitted to various clinical examination and testing. A summary of this follow up is given in Table 13 below.

[0192] The enrolled population consists of adults aged 18 to 40 years (i.e. the day of the 18th birthday to the day before the 41st birthday) on day of inclusion who are flaviviruses-naive [persons presenting vaccination against flavivirus diseases (e.g. yellow fever, Japanese encephalitis, dengue fever); or history of flavivirus infection (confirmed either clinically, serologically or microbiologically) or previous residence in or travel to areas with high dengue infection endemicity (whatever the duration), or residence in or travel to North Queensland for 2 weeks or more) were excluded]

TABLE-US-00018 TABLE 13 Flow chart for follow up Visit Number V01 V02 V03 V04 V05 V06 V07 V08 V09 V10 V11 V12 Trial timelines.sup. D0 D2 D4 D6 D8 D10 D12 D14 D16 D28 D180 D365 Time Windows .+-.1 d .+-.1 d .+-.4 d .+-.15 d .+-.30 d Clinical Examination Vital signs (BP, pulse rate) Oral temperature Blood Sampling: Serology HBV/HCV/HIV Biological Safety Viremia Immunogenicity Cytokines in serum PBMCs for T cell (subset) immediate surveillance Local & systemic events V: visit D: day. Time intervals between visits will be calculated from the date of study vaccination which might differ from the date of visit (e.g. in case a temporary exclusion criterion is met). V06 and V07 must be done with at least 1-day interval.

[0193] The products tested are:

[0194] The vaccine evaluated is a lyophilised product in a vial that is reconstituted extemporaneously with the diluent provided separately:

[0195] Active ingredient: 4.+-.0.5 log.sub.10 CCID.sub.50 of monovalent Vero dengue virus serotype 2 (VDV2 passage 11) per 0.5 mL dose;

[0196] Diluent: Sterile NaCl.sup.4.Salinity. solution for vaccine reconstitution.

[0197] The reconstituted vaccine, i.e 0.5 mL of NaCl 4.Salinity. solution of monovalent VDV2, should be used immediately or be maintained until use +2.degree. C. and +8.degree. C.

[0198] The 0.5 mL vaccine dose is administered subcutaneously in the deltoid region.

[0199] The control vaccine Stamaril.RTM., is a yellow fever vaccine produced by Aventis Pasteur. Stamaril.RTM. is presented as a lyophilised, avian-leukosis-free, stabilised product to be reconstituted with a diluent immediately before use. (Active ingredient: Live attenuated yellow fever virus (17D strain): .gtoreq.1,000 mouse Lethal Dose 50% (LD.sub.50)/Diluent: Sterile NaCl 4.Salinity. solution).

[0200] The control vaccine is administered subcutaneously in the deltoid region.

[0201] No subject had clinically significant syndrome related to vaccination. One subject had a transient fever (<38.degree. C.). One subject had a local reaction (induration). No serious adverse event related to vaccination was observed.

[0202] All subjects have antibodies response 28 days after vaccination against dengue 2 (titer between 1888 and 6393)

REFERENCES

[0203] The following references are incorporated herein by reference as if set forth in their entirety herein: [0204] Bhamarapravati, N and Yoksan S. (1997). Dengue and Dengue Hemorrhagic Fever. Live attenuated tetravalent dengue vaccines, CABI Publishing, 367-379. [0205] Burke D S and Monath T P. Flaviviruses (2001) In Knipe D M and Howley P M, eds. Fields Virology 4th ed. Vol 1, 1043-1125 [0206] DeFraites R F, Smoak B L, Trofa A F, Hoke C H, Kanesa-thasan N, King A, MacArthy P O, et al. Dengue fever among U.S. military personnel--Haiti, September-November, 1994. MMWR 1994; 43: 845-848. [0207] Dunnen and Antonarakis (2000) Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutation. 15:7-12; Erratum in: Hum Mutat 2002; 20(5):403 [0208] Gubler D J. Dengue. (1988) In: Epidemiology of arthropod-borne viral disease. Monath T P M, editor, Boca Raton (Fla.): CRC Press:223-60 [0209] Gubler D J, Kuno G. Dengue and Dengue Hemorrhagic Fever. CAB International Publishing 1997 [0210] Gubler D. Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century. (2002) TRENDS in Microbiology. 10:100-103 [0211] Halstead S B and Simasthien P (1970). Observations related to pathogenesis of Dengue haemorrhagic fever. II. Antigenic and biological properties of dengue viruses and their association with disease response in the host. Yale J. Biol. Med; 42: 261-275. [0212] Huang et al. (2000). J. Virol 74; 3020-3028. [0213] Jirakanjanakit N, Khin M M, Yoksan S, Bhamarapravati N. (1999) Dynamics of susceptibility and transmissibility of the live, attenuated, candidate vaccines dengue-1 PDK13, dengue-3 PGMK30F3, and dengue-4 PDK48 after oral infection in Aedes aegypti. Am J Trop Med Hyg., 61(4):672-676 [0214] Kautner I, Robinson M J, Kubnle U. (1997) Dengue Virus infection: Epidemiology, pathogenesis, clinical presentation, diagnosis, and prevention. J of Pediatrics; 131:516-524 [0215] Monath, T P. (1994) Dengue: the risk to developed and developing countries. Proc Natl Acad Sci; 91: 2395-2400. [0216] Monath T P, Levenbook I, Soike K, Zhang Z X, Ratterree M, Draper K et al. (2000) Chimeric yellow fever virus 17D-Japanese encephalitis virus vaccine: dose-response effectiveness and extended safety testing in rhesus monkeys. Journal of Virology; 74(4):1742-1751 [0217] Bray M, Men R, Lai C J. (1996) Monkeys immunized with intertypic chimeric dengue viruses are protected against wild-type virus challenge. J Virol; 70(6):4162-4166 [0218] Rigau-Perez J G, Clark G G, Gubler D J, Reiter P, Sanders E J, Vorndam A V. (1998) Dengue and dengue haemorrhagic fever. Lancet; 352: 971-977. [0219] Rothman A L, Ennis F A. (1999) Immunopathogenesis of dengue hemorrhagic fever. Virology; 257: 1-6 [0220] Sabin A B. (1952) Research on dengue during World War II. Am J Trop Med Hyg; 1: 30-50 [0221] Shirtcliffe P, Cameron E, Nicholson K G, Wiselka M J. (1998) Don't forget dengue! Clinical features of dengue fever in returning travelers. J Roy Coll Phys Lond.; 32: 235-237. [0222] Thompson J D, Higgins D G, and Gibson T J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids. Res., 22 (22), 4673-4680 [0223] Vaughn D W, Green S, Kalayanarooj S, Innis B L, Nimmannitya S, Suntayakorn S, Rothman A L, Ennis F A, Nisalak A. (1997) Dengue in the early febrile phase: viremia and antibody response. J Infect Dis; 176: 322-30. [0224] Vaughn D W, Green S, Kalayanarooj S, Innis B L, Nimmannitya S, Suntayakorn S, Endy T P, Raengsakulrach B, Rothman A L, Ennis F A, Nisalak A. (2000) Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Inf Dis; 181: 2-9. [0225] WHO Technical Guide, 1986. Dengue haemorrhagic fever: diagnosis, treatment and control, p 1-2. World Health Organization, Geneva, Switzerland [0226] Wu S, Grouard-Vogel G, Sun W, Mascola J, Brachtel E, Putvatana R. (2000) Human skin Langerhans cells are targets of dengue virus infection. Nature Med; 7:816-820 [0227] Khin M M, Jirakanjanakit N, Yoksan S, Bhamarapravati N. (1994) Infection, dissemination, transmission, and biological attributes of dengue-2 PDK53 candidate vaccine virus after oral infection in Aedes aegypti. Am J Trop Med Hyg., 51(6):864-869

Sequence CWU 1

1

42110723DNADengue virusmisc_feature(1)..(10723)VDV2 1agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60gttctaacag ttttttaatt agagagcaga tctctg atg aat aac caa cgg aaa 114 Met Asn Asn Gln Arg Lys 1 5 aag gcg aaa aac acg cct ttc aat atg ctg aaa cgc gag aga aac cgc 162Lys Ala Lys Asn Thr Pro Phe Asn Met Leu Lys Arg Glu Arg Asn Arg 10 15 20 gtg tcg act gtg caa cag ctg aca aag aga ttc tca ctt gga atg ctg 210Val Ser Thr Val Gln Gln Leu Thr Lys Arg Phe Ser Leu Gly Met Leu 25 30 35 cag gga cga gga cca tta aaa ctg ttc atg gcc ctg gtg gcg ttc ctt 258Gln Gly Arg Gly Pro Leu Lys Leu Phe Met Ala Leu Val Ala Phe Leu 40 45 50 cgt ttc cta aca atc cca cca aca gca ggg ata ttg aag aga tgg gga 306Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly Ile Leu Lys Arg Trp Gly 55 60 65 70 aca att aaa aaa tca aaa gct att aat gtt ttg aga ggg ttc agg aaa 354Thr Ile Lys Lys Ser Lys Ala Ile Asn Val Leu Arg Gly Phe Arg Lys 75 80 85 gag att gga agg atg ctg aac atc ttg aat agg aga cgc aga tct gca 402Glu Ile Gly Arg Met Leu Asn Ile Leu Asn Arg Arg Arg Arg Ser Ala 90 95 100 ggc atg atc att atg ctg att cca aca gtg atg gcg ttc cat tta acc 450Gly Met Ile Ile Met Leu Ile Pro Thr Val Met Ala Phe His Leu Thr 105 110 115 aca cgt aac gga gaa cca cac atg atc gtc agc aga caa gag aaa ggg 498Thr Arg Asn Gly Glu Pro His Met Ile Val Ser Arg Gln Glu Lys Gly 120 125 130 aaa agt ctt ctg ttt aaa aca gag gtt ggc gtg aac atg tgt acc ctc 546Lys Ser Leu Leu Phe Lys Thr Glu Val Gly Val Asn Met Cys Thr Leu 135 140 145 150 atg gcc atg gac ctt ggt gaa ttg tgt gaa gac aca atc acg tac aag 594Met Ala Met Asp Leu Gly Glu Leu Cys Glu Asp Thr Ile Thr Tyr Lys 155 160 165 tgt ccc ctt ctc agg cag aat gag cca gaa gac ata gac tgt tgg tgc 642Cys Pro Leu Leu Arg Gln Asn Glu Pro Glu Asp Ile Asp Cys Trp Cys 170 175 180 aac tct acg tcc acg tgg gta act tat ggg acg tgt acc acc atg gga 690Asn Ser Thr Ser Thr Trp Val Thr Tyr Gly Thr Cys Thr Thr Met Gly 185 190 195 gaa cat aga aga gaa aaa aga tca gtg gca ctc gtt cca cat gtg cga 738Glu His Arg Arg Glu Lys Arg Ser Val Ala Leu Val Pro His Val Arg 200 205 210 atg gga ctg gag aca cga act gaa aca tgg atg tca tca gaa ggg gcc 786Met Gly Leu Glu Thr Arg Thr Glu Thr Trp Met Ser Ser Glu Gly Ala 215 220 225 230 tgg aaa cat gtc cag aga att gaa act tgg atc ttg aga cat cca ggc 834Trp Lys His Val Gln Arg Ile Glu Thr Trp Ile Leu Arg His Pro Gly 235 240 245 ttc acc atg atg gca gca atc ctg gca tac acc ata gga acg aca cat 882Phe Thr Met Met Ala Ala Ile Leu Ala Tyr Thr Ile Gly Thr Thr His 250 255 260 ttc caa aga gcc ctg att ttc atc tta ctg aca gct gtc act cct tca 930Phe Gln Arg Ala Leu Ile Phe Ile Leu Leu Thr Ala Val Thr Pro Ser 265 270 275 atg aca atg cgt tgc ata gga atg tca aat aga gac ttt gtg gaa ggg 978Met Thr Met Arg Cys Ile Gly Met Ser Asn Arg Asp Phe Val Glu Gly 280 285 290 gtt tca gga gga agc tgg gtt gac ata gtc tta gaa cat gga agc tgt 1026Val Ser Gly Gly Ser Trp Val Asp Ile Val Leu Glu His Gly Ser Cys 295 300 305 310 gtg acg acg atg gca aaa aac aaa cca aca ttg gat ttt gaa ctg ata 1074Val Thr Thr Met Ala Lys Asn Lys Pro Thr Leu Asp Phe Glu Leu Ile 315 320 325 aaa aca gaa gcc aaa cag cct gcc acc cta agg aag tac tgt ata gag 1122Lys Thr Glu Ala Lys Gln Pro Ala Thr Leu Arg Lys Tyr Cys Ile Glu 330 335 340 gca aag cta acc aac aca aca aca gaa tct cgc tgc cca aca caa ggg 1170Ala Lys Leu Thr Asn Thr Thr Thr Glu Ser Arg Cys Pro Thr Gln Gly 345 350 355 gaa ccc agc cta aat gaa gag cag gac aaa agg ttc gtc tgc aaa cac 1218Glu Pro Ser Leu Asn Glu Glu Gln Asp Lys Arg Phe Val Cys Lys His 360 365 370 tcc atg gta gac aga gga tgg gga aat gga tgt gga cta ttt gga aag 1266Ser Met Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys 375 380 385 390 gga ggc att gtg acc tgt gct atg ttc aga tgc aaa aag aac atg gaa 1314Gly Gly Ile Val Thr Cys Ala Met Phe Arg Cys Lys Lys Asn Met Glu 395 400 405 gga aaa gtt gtg caa cca gaa aac ttg gaa tac acc att gtg ata aca 1362Gly Lys Val Val Gln Pro Glu Asn Leu Glu Tyr Thr Ile Val Ile Thr 410 415 420 cct cac tca ggg gaa gag cat gca gtc gga aat gac aca gga aaa cat 1410Pro His Ser Gly Glu Glu His Ala Val Gly Asn Asp Thr Gly Lys His 425 430 435 ggc aag gaa atc aaa ata aca cca cag agt tcc atc aca gaa gca gaa 1458Gly Lys Glu Ile Lys Ile Thr Pro Gln Ser Ser Ile Thr Glu Ala Glu 440 445 450 ttg aca ggt tat ggc act gtc aca atg gag tgc tct cca aga acg ggc 1506Leu Thr Gly Tyr Gly Thr Val Thr Met Glu Cys Ser Pro Arg Thr Gly 455 460 465 470 ctc gac ttc aat gag atg gtg ttg ctg cag atg gaa aat aaa gct tgg 1554Leu Asp Phe Asn Glu Met Val Leu Leu Gln Met Glu Asn Lys Ala Trp 475 480 485 ctg gtg cac agg caa tgg ttc cta gac ctg ccg tta cca tgg ttg ccc 1602Leu Val His Arg Gln Trp Phe Leu Asp Leu Pro Leu Pro Trp Leu Pro 490 495 500 gga gcg gac aca caa gag tca aat tgg ata cag aag gag aca ttg gtc 1650Gly Ala Asp Thr Gln Glu Ser Asn Trp Ile Gln Lys Glu Thr Leu Val 505 510 515 act ttc aaa aat ccc cat gcg aag aaa cag gat gtt gtt gtt tta gga 1698Thr Phe Lys Asn Pro His Ala Lys Lys Gln Asp Val Val Val Leu Gly 520 525 530 tcc caa gaa ggg gcc atg cac aca gca ctt aca ggg gcc aca gaa atc 1746Ser Gln Glu Gly Ala Met His Thr Ala Leu Thr Gly Ala Thr Glu Ile 535 540 545 550 caa atg tca tca gga aac tta ctc ttc aca gga cat ctc aag tgc agg 1794Gln Met Ser Ser Gly Asn Leu Leu Phe Thr Gly His Leu Lys Cys Arg 555 560 565 ctg aga atg gac aag cta cag ctc aaa gga atg tca tac tct atg tgc 1842Leu Arg Met Asp Lys Leu Gln Leu Lys Gly Met Ser Tyr Ser Met Cys 570 575 580 aca gga aag ttt aaa gtt gtg aag gaa ata gca gaa aca caa cat gga 1890Thr Gly Lys Phe Lys Val Val Lys Glu Ile Ala Glu Thr Gln His Gly 585 590 595 aca ata gtt atc aga gtg caa tat gaa ggg gac ggc tct cca tgc aag 1938Thr Ile Val Ile Arg Val Gln Tyr Glu Gly Asp Gly Ser Pro Cys Lys 600 605 610 atc cct ttt gag ata atg gat ttg gaa aaa aga cat gtc tta ggt cgc 1986Ile Pro Phe Glu Ile Met Asp Leu Glu Lys Arg His Val Leu Gly Arg 615 620 625 630 ctg att aca gtc aac cca att gtg aca gaa aaa gat agc cca gtc aac 2034Leu Ile Thr Val Asn Pro Ile Val Thr Glu Lys Asp Ser Pro Val Asn 635 640 645 ata gaa gca gaa cct cca ttt gga gac agc tac atc atc ata gga gta 2082Ile Glu Ala Glu Pro Pro Phe Gly Asp Ser Tyr Ile Ile Ile Gly Val 650 655 660 gag ccg gga caa ctg aag ctc aac tgg ttt aag aaa gga agt tct atc 2130Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe Lys Lys Gly Ser Ser Ile 665 670 675 ggc caa atg ttt gag aca aca atg agg ggg gcg aag aga atg gcc att 2178Gly Gln Met Phe Glu Thr Thr Met Arg Gly Ala Lys Arg Met Ala Ile 680 685 690 tta ggt gac aca gcc tgg gat ttt gga tcc ttg gga gga gtg ttt aca 2226Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Leu Gly Gly Val Phe Thr 695 700 705 710 tct ata gga aag gct ctc cac caa gtc ttt gga gca atc tat gga gct 2274Ser Ile Gly Lys Ala Leu His Gln Val Phe Gly Ala Ile Tyr Gly Ala 715 720 725 gcc ttc agt ggg gtt tca tgg act atg aaa atc ctc ata gga gtc att 2322Ala Phe Ser Gly Val Ser Trp Thr Met Lys Ile Leu Ile Gly Val Ile 730 735 740 atc aca tgg ata gga atg aat tca cgc agc acc tca ctg tct gtg aca 2370Ile Thr Trp Ile Gly Met Asn Ser Arg Ser Thr Ser Leu Ser Val Thr 745 750 755 cta gta ttg gtg gga att gtg aca ctg tat ttg gga gtc atg gtg cag 2418Leu Val Leu Val Gly Ile Val Thr Leu Tyr Leu Gly Val Met Val Gln 760 765 770 gcc gat agt ggt tgc gtt gtg agc tgg aaa aac aaa gaa ctg aaa tgt 2466Ala Asp Ser Gly Cys Val Val Ser Trp Lys Asn Lys Glu Leu Lys Cys 775 780 785 790 ggc agt ggg att ttc atc aca gac aac gtg cac aca tgg aca gaa caa 2514Gly Ser Gly Ile Phe Ile Thr Asp Asn Val His Thr Trp Thr Glu Gln 795 800 805 tac aaa ttc caa cca gaa tcc cct tca aaa cta gct tca gct atc cag 2562Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys Leu Ala Ser Ala Ile Gln 810 815 820 aaa gcc cat gaa gag gac att tgt gga atc cgc tca gta aca aga ctg 2610Lys Ala His Glu Glu Asp Ile Cys Gly Ile Arg Ser Val Thr Arg Leu 825 830 835 gag aat ctg atg tgg aaa caa ata aca cca gaa ttg aat cac att cta 2658Glu Asn Leu Met Trp Lys Gln Ile Thr Pro Glu Leu Asn His Ile Leu 840 845 850 tca gaa aat gag gtg aag tta act att atg aca gga gac atc aaa gga 2706Ser Glu Asn Glu Val Lys Leu Thr Ile Met Thr Gly Asp Ile Lys Gly 855 860 865 870 atc atg cag gca gga aaa cga tct ctg cgg cct cag ccc act gag ctg 2754Ile Met Gln Ala Gly Lys Arg Ser Leu Arg Pro Gln Pro Thr Glu Leu 875 880 885 aag tat tca tgg aaa aca tgg ggc aaa gca aaa atg ctc tct aca gag 2802Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala Lys Met Leu Ser Thr Glu 890 895 900 tct cat aac cag acc ttt ctc att gat ggc ccc gaa aca gca gaa tgc 2850Ser His Asn Gln Thr Phe Leu Ile Asp Gly Pro Glu Thr Ala Glu Cys 905 910 915 ccc aac aca aat aga gct tgg aat tcg ttg gaa gtt gaa gac tat ggc 2898Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu Glu Val Glu Asp Tyr Gly 920 925 930 ttt gga gta ttc acc acc aat ata tgg cta aaa ttg aaa gaa aaa cag 2946Phe Gly Val Phe Thr Thr Asn Ile Trp Leu Lys Leu Lys Glu Lys Gln 935 940 945 950 gat gta ttc tgc gac tca aaa ctc atg tca gcg gcc ata aaa gac aac 2994Asp Val Phe Cys Asp Ser Lys Leu Met Ser Ala Ala Ile Lys Asp Asn 955 960 965 aga gcc gtc cat gcc gat atg ggt tat tgg ata gaa agt gca ctc aat 3042Arg Ala Val His Ala Asp Met Gly Tyr Trp Ile Glu Ser Ala Leu Asn 970 975 980 gac aca tgg aag ata gag aaa gcc tct ttc att gaa gtt aaa aac tgc 3090Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe Ile Glu Val Lys Asn Cys 985 990 995 cac tgg cca aaa tca cac acc ctc tgg agc aat gga gtg cta gaa 3135His Trp Pro Lys Ser His Thr Leu Trp Ser Asn Gly Val Leu Glu 1000 1005 1010 agt gag atg ata att cca aag aat ctc gct gga cca gtg tct caa 3180Ser Glu Met Ile Ile Pro Lys Asn Leu Ala Gly Pro Val Ser Gln 1015 1020 1025 cac aac tat aga cca ggc tac cat aca caa ata aca gga cca tgg 3225His Asn Tyr Arg Pro Gly Tyr His Thr Gln Ile Thr Gly Pro Trp 1030 1035 1040 cat cta ggt aag ctt gag atg gac ttt gat ttc tgt gat gga aca 3270His Leu Gly Lys Leu Glu Met Asp Phe Asp Phe Cys Asp Gly Thr 1045 1050 1055 aca gtg gta gtg act gag gac tgc gga aat aga gga ccc tct ttg 3315Thr Val Val Val Thr Glu Asp Cys Gly Asn Arg Gly Pro Ser Leu 1060 1065 1070 aga aca acc act gcc tct gga aaa ctc ata aca gaa tgg tgc tgc 3360Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile Thr Glu Trp Cys Cys 1075 1080 1085 cga tct tgc aca tta cca ccg cta aga tac aga ggt gag gat ggg 3405Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr Arg Gly Glu Asp Gly 1090 1095 1100 tgc tgg tac ggg atg gaa atc aga cca ttg aag gag aaa gaa gag 3450Cys Trp Tyr Gly Met Glu Ile Arg Pro Leu Lys Glu Lys Glu Glu 1105 1110 1115 aat ttg gtc aac tcc ttg gtc aca gct gga cat ggg cag gtc gac 3495Asn Leu Val Asn Ser Leu Val Thr Ala Gly His Gly Gln Val Asp 1120 1125 1130 aac ttt tca cta gga gtc ttg gga atg gca ttg ttc ctg gag gaa 3540Asn Phe Ser Leu Gly Val Leu Gly Met Ala Leu Phe Leu Glu Glu 1135 1140 1145 atg ctt agg acc cga gta gga acg aaa cat gca ata cta cta gtt 3585Met Leu Arg Thr Arg Val Gly Thr Lys His Ala Ile Leu Leu Val 1150 1155 1160 gca gtt tct ttt gtg aca ttg atc aca ggg aac atg tcc ttt aga 3630Ala Val Ser Phe Val Thr Leu Ile Thr Gly Asn Met Ser Phe Arg 1165 1170 1175 gac ctg gga aga gtg atg gtt atg gta ggc gcc act atg acg gat 3675Asp Leu Gly Arg Val Met Val Met Val Gly Ala Thr Met Thr Asp 1180 1185 1190 gac ata ggt atg ggc gtg act tat ctt gcc cta cta gca gcc ttc 3720Asp Ile Gly Met Gly Val Thr Tyr Leu Ala Leu Leu Ala Ala Phe 1195 1200 1205 aaa gtc aga cca act ttt gca gct gga cta ctc ttg aga aag ctg 3765Lys Val Arg Pro Thr Phe Ala Ala Gly Leu Leu Leu Arg Lys Leu 1210 1215 1220 acc tcc aag gaa ttg atg atg act act ata gga att gta ctc ctc 3810Thr Ser Lys Glu Leu Met Met Thr Thr Ile Gly Ile Val Leu Leu 1225 1230 1235 tcc cag agc acc ata cca gag acc att ctt gag ttg act gat gcg 3855Ser Gln Ser Thr Ile Pro Glu Thr Ile Leu Glu Leu Thr Asp Ala 1240 1245 1250 tta gcc tta ggc atg atg gtc ctc aaa atg gtg aga aat atg gaa 3900Leu Ala Leu Gly Met Met Val Leu Lys Met Val Arg Asn Met Glu 1255 1260 1265 aag tat caa ttg gca gtg act atc atg gct atc ttg tgc gtc cca 3945Lys Tyr Gln Leu Ala Val Thr Ile Met Ala Ile Leu Cys Val Pro 1270 1275 1280 aac gca gtg ata tta caa aac gca tgg aaa gtg agt tgc aca ata 3990Asn Ala Val Ile Leu Gln Asn Ala Trp Lys Val Ser Cys Thr Ile 1285 1290 1295 ttg gca gtg gtg tcc gtt tcc cca ctg ttc tta aca tcc tca

cag 4035Leu Ala Val Val Ser Val Ser Pro Leu Phe Leu Thr Ser Ser Gln 1300 1305 1310 caa aaa aca gat tgg ata cca tta gca ttg acg atc aaa ggt ctc 4080Gln Lys Thr Asp Trp Ile Pro Leu Ala Leu Thr Ile Lys Gly Leu 1315 1320 1325 aat cca aca gct att ttt cta aca acc ctc tca aga acc agc aag 4125Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu Ser Arg Thr Ser Lys 1330 1335 1340 aaa agg agc tgg cca tta aat gag gct atc atg gca gtc ggg atg 4170Lys Arg Ser Trp Pro Leu Asn Glu Ala Ile Met Ala Val Gly Met 1345 1350 1355 gtg agc att tta gcc agt tct ctc cta aaa aat gat att ccc atg 4215Val Ser Ile Leu Ala Ser Ser Leu Leu Lys Asn Asp Ile Pro Met 1360 1365 1370 aca gga cca tta gtg gct gga ggg ctc ctc act gtg tgc tac gtg 4260Thr Gly Pro Leu Val Ala Gly Gly Leu Leu Thr Val Cys Tyr Val 1375 1380 1385 ctc act gga cga tcg gcc gat ttg gaa ctg gag aga gca gcc gat 4305Leu Thr Gly Arg Ser Ala Asp Leu Glu Leu Glu Arg Ala Ala Asp 1390 1395 1400 gtc aaa tgg gaa gac cag gca gag ata tca gga agc agt cca atc 4350Val Lys Trp Glu Asp Gln Ala Glu Ile Ser Gly Ser Ser Pro Ile 1405 1410 1415 ctg tca ata aca ata tca gaa gat ggt agc atg tcg ata aaa aat 4395Leu Ser Ile Thr Ile Ser Glu Asp Gly Ser Met Ser Ile Lys Asn 1420 1425 1430 gaa gag gaa gaa caa aca ctg acc ata ctc att aga aca gga ttg 4440Glu Glu Glu Glu Gln Thr Leu Thr Ile Leu Ile Arg Thr Gly Leu 1435 1440 1445 ctg gtg atc tca gga ctt ttt cct gta tca ata cca atc acg gca 4485Leu Val Ile Ser Gly Leu Phe Pro Val Ser Ile Pro Ile Thr Ala 1450 1455 1460 gca gca tgg tac ctg tgg gaa gtg aag aaa caa cgg gcc gga gta 4530Ala Ala Trp Tyr Leu Trp Glu Val Lys Lys Gln Arg Ala Gly Val 1465 1470 1475 ttg tgg gat gtt cct tca ccc cca ccc atg gga aag gct gaa ctg 4575Leu Trp Asp Val Pro Ser Pro Pro Pro Met Gly Lys Ala Glu Leu 1480 1485 1490 gaa gat gga gcc tat aga att aag caa aaa ggg att ctt gga tat 4620Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys Gly Ile Leu Gly Tyr 1495 1500 1505 tcc cag atc gga gcc gga gtt tac aaa gaa gga aca ttc cat aca 4665Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu Gly Thr Phe His Thr 1510 1515 1520 atg tgg cat gtc aca cgt ggc gct gtt cta atg cat aaa gga aag 4710Met Trp His Val Thr Arg Gly Ala Val Leu Met His Lys Gly Lys 1525 1530 1535 agg att gaa cca aca tgg gcg gac gtc aag aaa gac cta ata tca 4755Arg Ile Glu Pro Thr Trp Ala Asp Val Lys Lys Asp Leu Ile Ser 1540 1545 1550 tat gga gga ggc tgg aag tta gaa gga gaa tgg aag gaa gga gaa 4800Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu Trp Lys Glu Gly Glu 1555 1560 1565 gaa gtc cag gta ttg gca ctg gag cct gga aaa aat cca aga gcc 4845Glu Val Gln Val Leu Ala Leu Glu Pro Gly Lys Asn Pro Arg Ala 1570 1575 1580 gtc caa acg aaa cct ggt ctt ttc aaa acc aac gcc gga aca ata 4890Val Gln Thr Lys Pro Gly Leu Phe Lys Thr Asn Ala Gly Thr Ile 1585 1590 1595 ggt gct gta tct ctg gac ttt tct cct gga acg tca gga tct cca 4935Gly Ala Val Ser Leu Asp Phe Ser Pro Gly Thr Ser Gly Ser Pro 1600 1605 1610 att atc gac aaa aaa gga aaa gtt gtg ggt ctt tat ggt aat ggt 4980Ile Ile Asp Lys Lys Gly Lys Val Val Gly Leu Tyr Gly Asn Gly 1615 1620 1625 gtt gtt aca agg agt gga gca tat gtg agt gct ata gcc cag act 5025Val Val Thr Arg Ser Gly Ala Tyr Val Ser Ala Ile Ala Gln Thr 1630 1635 1640 gaa aaa agc att gaa gac aac cca gag atc gaa gat cac att ttc 5070Glu Lys Ser Ile Glu Asp Asn Pro Glu Ile Glu Asp His Ile Phe 1645 1650 1655 cga aag aga aga ctg acc atc atg gac ctc cac cca gga gcg gga 5115Arg Lys Arg Arg Leu Thr Ile Met Asp Leu His Pro Gly Ala Gly 1660 1665 1670 aag acg aag aga tac ctt ccg gcc ata gtc aga gaa gct ata aaa 5160Lys Thr Lys Arg Tyr Leu Pro Ala Ile Val Arg Glu Ala Ile Lys 1675 1680 1685 cgg ggt ttg aga aca tta atc ttg gcc ccc act aga gtt gtg gca 5205Arg Gly Leu Arg Thr Leu Ile Leu Ala Pro Thr Arg Val Val Ala 1690 1695 1700 gct gaa atg gag gaa gcc ctt aga gga ctt cca ata aga tac cag 5250Ala Glu Met Glu Glu Ala Leu Arg Gly Leu Pro Ile Arg Tyr Gln 1705 1710 1715 acc cca gcc atc aga gct gag cac acc ggg cgg gag att gtg gac 5295Thr Pro Ala Ile Arg Ala Glu His Thr Gly Arg Glu Ile Val Asp 1720 1725 1730 cta atg tgt cat gcc aca ttt acc atg agg ctg cta tca cca gtt 5340Leu Met Cys His Ala Thr Phe Thr Met Arg Leu Leu Ser Pro Val 1735 1740 1745 aga gtg cca aac tac aac ctg att atc atg gac gaa gcc cat ttc 5385Arg Val Pro Asn Tyr Asn Leu Ile Ile Met Asp Glu Ala His Phe 1750 1755 1760 aca gac cca gca agt ata gca gct aga gga tac atc tca act cga 5430Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly Tyr Ile Ser Thr Arg 1765 1770 1775 gtg gag atg ggt gag gca gct ggg att ttt atg aca gcc act ccc 5475Val Glu Met Gly Glu Ala Ala Gly Ile Phe Met Thr Ala Thr Pro 1780 1785 1790 ccg gga agc aga gac cca ttt cct cag agc aat gca cca atc ata 5520Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser Asn Ala Pro Ile Ile 1795 1800 1805 gat gaa gaa aga gaa atc cct gaa cgc tcg tgg aat tcc gga cat 5565Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser Trp Asn Ser Gly His 1810 1815 1820 gaa tgg gtc acg gat ttt aaa ggg aag act gtt tgg ttc gtt cca 5610Glu Trp Val Thr Asp Phe Lys Gly Lys Thr Val Trp Phe Val Pro 1825 1830 1835 agt ata aaa gca gga aat gat ata gca gct tgc ctg agg aaa aat 5655Ser Ile Lys Ala Gly Asn Asp Ile Ala Ala Cys Leu Arg Lys Asn 1840 1845 1850 gga aag aaa gtg ata caa ctc agt agg aag acc ttt gat tct gag 5700Gly Lys Lys Val Ile Gln Leu Ser Arg Lys Thr Phe Asp Ser Glu 1855 1860 1865 tat gtc aag act aga acc aat gat tgg gac ttc gtg gtt aca act 5745Tyr Val Lys Thr Arg Thr Asn Asp Trp Asp Phe Val Val Thr Thr 1870 1875 1880 gac att tca gaa atg ggt gcc aat ttc aag gct gag agg gtt ata 5790Asp Ile Ser Glu Met Gly Ala Asn Phe Lys Ala Glu Arg Val Ile 1885 1890 1895 gac ccc aga cgc tgc atg aaa cca gtc ata cta aca gat ggt gaa 5835Asp Pro Arg Arg Cys Met Lys Pro Val Ile Leu Thr Asp Gly Glu 1900 1905 1910 gag cgg gtg att ctg gca gga cct atg cca gtg acc cac tct agt 5880Glu Arg Val Ile Leu Ala Gly Pro Met Pro Val Thr His Ser Ser 1915 1920 1925 gca gca caa aga aga ggg aga ata gga aga aat cca aaa aat gag 5925Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn Pro Lys Asn Glu 1930 1935 1940 aat gac cag tac ata tac atg ggg gaa cct ctg gaa aat gat gaa 5970Asn Asp Gln Tyr Ile Tyr Met Gly Glu Pro Leu Glu Asn Asp Glu 1945 1950 1955 gac tgt gca cac tgg aaa gaa gct aaa atg ctc cta gat aac atc 6015Asp Cys Ala His Trp Lys Glu Ala Lys Met Leu Leu Asp Asn Ile 1960 1965 1970 aac acg cca gaa gga atc att cct agc atg ttc gaa cca gag cgt 6060Asn Thr Pro Glu Gly Ile Ile Pro Ser Met Phe Glu Pro Glu Arg 1975 1980 1985 gaa aag gtg gat gcc att gat ggc gaa tac cgc ttg aga gga gaa 6105Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr Arg Leu Arg Gly Glu 1990 1995 2000 gca agg aaa acc ttt gta gac tta atg aga aga gga gac cta cca 6150Ala Arg Lys Thr Phe Val Asp Leu Met Arg Arg Gly Asp Leu Pro 2005 2010 2015 gtc tgg ttg gcc tac aga gtg gca gct gaa ggc atc aac tac gca 6195Val Trp Leu Ala Tyr Arg Val Ala Ala Glu Gly Ile Asn Tyr Ala 2020 2025 2030 gac aga agg tgg tgt ttt gat gga gtc aag aac aac caa atc cta 6240Asp Arg Arg Trp Cys Phe Asp Gly Val Lys Asn Asn Gln Ile Leu 2035 2040 2045 gaa gaa aac gtg gaa gtt gaa atc tgg aca aaa gaa ggg gaa agg 6285Glu Glu Asn Val Glu Val Glu Ile Trp Thr Lys Glu Gly Glu Arg 2050 2055 2060 aag aaa ttg aaa ccc aga tgg ttg gat gct agg atc tat tct gac 6330Lys Lys Leu Lys Pro Arg Trp Leu Asp Ala Arg Ile Tyr Ser Asp 2065 2070 2075 cca ctg gcg cta aaa gaa ttt aag gaa ttt gca gcc gga aga aag 6375Pro Leu Ala Leu Lys Glu Phe Lys Glu Phe Ala Ala Gly Arg Lys 2080 2085 2090 tct ctg acc ctg aac cta atc aca gaa atg ggt agg ctc cca acc 6420Ser Leu Thr Leu Asn Leu Ile Thr Glu Met Gly Arg Leu Pro Thr 2095 2100 2105 ttc atg act cag aag gca aga gac gca ctg gac aac tta gca gtg 6465Phe Met Thr Gln Lys Ala Arg Asp Ala Leu Asp Asn Leu Ala Val 2110 2115 2120 ctg cac acg gct gag gca ggt gga agg gcg tac aac cat gct ctc 6510Leu His Thr Ala Glu Ala Gly Gly Arg Ala Tyr Asn His Ala Leu 2125 2130 2135 agt gaa ctg ccg gag acc ctg gag aca ttg ctt tta ctg aca ctt 6555Ser Glu Leu Pro Glu Thr Leu Glu Thr Leu Leu Leu Leu Thr Leu 2140 2145 2150 ctg gct aca gtc acg gga ggg atc ttt tta ttc ttg atg agc gca 6600Leu Ala Thr Val Thr Gly Gly Ile Phe Leu Phe Leu Met Ser Ala 2155 2160 2165 agg ggc ata ggg aag atg acc ctg gga atg tgc tgc ata atc acg 6645Arg Gly Ile Gly Lys Met Thr Leu Gly Met Cys Cys Ile Ile Thr 2170 2175 2180 gct agc atc ctc cta tgg tac gca caa ata cag cca cac tgg ata 6690Ala Ser Ile Leu Leu Trp Tyr Ala Gln Ile Gln Pro His Trp Ile 2185 2190 2195 gca gct tca ata ata ctg gag ttt ttt ctc ata gtt ttg ctt att 6735Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu Ile Val Leu Leu Ile 2200 2205 2210 cca gaa cct gaa aaa cag aga aca ccc caa gac aac caa ctg acc 6780Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp Asn Gln Leu Thr 2215 2220 2225 tac gtt gtc ata gcc atc ctc aca gtg gtg gcc gca acc atg gca 6825Tyr Val Val Ile Ala Ile Leu Thr Val Val Ala Ala Thr Met Ala 2230 2235 2240 aac gag atg ggt ttc cta gaa aaa acg aag aaa gat ctc gga ttg 6870Asn Glu Met Gly Phe Leu Glu Lys Thr Lys Lys Asp Leu Gly Leu 2245 2250 2255 gga agc att gca acc cag caa ccc gag agc aac atc ctg gac ata 6915Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser Asn Ile Leu Asp Ile 2260 2265 2270 gat cta cgt cct gca tca gca tgg acg ctg tat gcc gtg gcc aca 6960Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu Tyr Ala Val Ala Thr 2275 2280 2285 aca ttt gtt aca cca atg ttg aga cat agc att gaa aat tcc tca 7005Thr Phe Val Thr Pro Met Leu Arg His Ser Ile Glu Asn Ser Ser 2290 2295 2300 gtg aat gtg tcc cta aca gct ata gcc aac caa gcc aca gtg tta 7050Val Asn Val Ser Leu Thr Ala Ile Ala Asn Gln Ala Thr Val Leu 2305 2310 2315 atg ggt ctc ggg aaa gga tgg cca ttg tca aag atg gac atc gga 7095Met Gly Leu Gly Lys Gly Trp Pro Leu Ser Lys Met Asp Ile Gly 2320 2325 2330 gtt ccc ctt ctc gcc att gga tgc tac tca caa gtc aac ccc ata 7140Val Pro Leu Leu Ala Ile Gly Cys Tyr Ser Gln Val Asn Pro Ile 2335 2340 2345 act ctc aca gca gct ctt ttc tta ttg gta gca cat tat gcc atc 7185Thr Leu Thr Ala Ala Leu Phe Leu Leu Val Ala His Tyr Ala Ile 2350 2355 2360 ata ggg cca gga ctc caa gca aaa gca acc aga gaa gct cag aaa 7230Ile Gly Pro Gly Leu Gln Ala Lys Ala Thr Arg Glu Ala Gln Lys 2365 2370 2375 aga gca gcg gcg ggc atc atg aaa aac cca act gtc gat gga ata 7275Arg Ala Ala Ala Gly Ile Met Lys Asn Pro Thr Val Asp Gly Ile 2380 2385 2390 aca gtg att gac cta gat cca ata cct tat gat cca aag ttt gaa 7320Thr Val Ile Asp Leu Asp Pro Ile Pro Tyr Asp Pro Lys Phe Glu 2395 2400 2405 aag cag ttg gga caa gta atg ctc cta gtc ctc tgc gtg act caa 7365Lys Gln Leu Gly Gln Val Met Leu Leu Val Leu Cys Val Thr Gln 2410 2415 2420 gta ttg atg atg agg act aca tgg gct ctg tgt gag gct tta acc 7410Val Leu Met Met Arg Thr Thr Trp Ala Leu Cys Glu Ala Leu Thr 2425 2430 2435 tta gct acc ggg ccc atc tcc aca ttg tgg gaa gga aat cca ggg 7455Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp Glu Gly Asn Pro Gly 2440 2445 2450 agg ttt tgg aac act acc att gcg gtg tca atg gct aac att ttt 7500Arg Phe Trp Asn Thr Thr Ile Ala Val Ser Met Ala Asn Ile Phe 2455 2460 2465 aga ggg agt tac ttg gcc gga gct gga ctt ctc ttt tct att atg 7545Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Leu Phe Ser Ile Met 2470 2475 2480 aag aac aca acc aac aca aga agg gga act ggc aac ata gga gag 7590Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr Gly Asn Ile Gly Glu 2485 2490 2495 acg ctt gga gag aaa tgg aaa agc cga ttg aac gca ttg gga aaa 7635Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu Asn Ala Leu Gly Lys 2500 2505 2510 agt gaa ttc cag atc tac aag aaa agt gga atc cag gaa gtg gat 7680Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly Ile Gln Glu Val Asp 2515 2520 2525 aga acc tta gca aaa gaa ggc att aaa aga gga gaa acg gac cat 7725Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg Gly Glu Thr Asp His 2530 2535 2540 cac gct gtg tcg cga ggc tca gca aaa ctg aga tgg ttc gtt gag 7770His Ala Val Ser Arg Gly Ser Ala Lys Leu Arg Trp Phe Val Glu 2545 2550 2555

aga aac atg gtc aca cca gaa ggg aaa gta gtg gac ctc ggt tgt 7815Arg Asn Met Val Thr Pro Glu Gly Lys Val Val Asp Leu Gly Cys 2560 2565 2570 ggc aga gga ggc tgg tca tac tat tgt gga gga cta aag aat gta 7860Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Gly Gly Leu Lys Asn Val 2575 2580 2585 aga gaa gtc aaa ggc cta aca aaa gga gga cca gga cac gaa gaa 7905Arg Glu Val Lys Gly Leu Thr Lys Gly Gly Pro Gly His Glu Glu 2590 2595 2600 ccc atc ccc atg tca aca tat ggg tgg aat cta gtg cgt ctt caa 7950Pro Ile Pro Met Ser Thr Tyr Gly Trp Asn Leu Val Arg Leu Gln 2605 2610 2615 agt gga gtt gac gtt ttc ttc atc ccg cca gaa aag tgt gac aca 7995Ser Gly Val Asp Val Phe Phe Ile Pro Pro Glu Lys Cys Asp Thr 2620 2625 2630 tta ttg tgt gac ata ggg gag tca tca cca aat ccc aca gtg gaa 8040Leu Leu Cys Asp Ile Gly Glu Ser Ser Pro Asn Pro Thr Val Glu 2635 2640 2645 gca gga cga aca ctc aga gtc ctt aac tta gta gaa aat tgg ttg 8085Ala Gly Arg Thr Leu Arg Val Leu Asn Leu Val Glu Asn Trp Leu 2650 2655 2660 aac aac aac act caa ttt tgc ata aag gtt ctc aac cca tat atg 8130Asn Asn Asn Thr Gln Phe Cys Ile Lys Val Leu Asn Pro Tyr Met 2665 2670 2675 ccc tca gtc ata gaa aaa atg gaa gca cta caa agg aaa tat gga 8175Pro Ser Val Ile Glu Lys Met Glu Ala Leu Gln Arg Lys Tyr Gly 2680 2685 2690 gga gcc tta gtg agg aat cca ctc tca cga aac tcc aca cat gag 8220Gly Ala Leu Val Arg Asn Pro Leu Ser Arg Asn Ser Thr His Glu 2695 2700 2705 atg tac tgg gta tcc aat gct tcc ggg aac ata gtg tca tca gtg 8265Met Tyr Trp Val Ser Asn Ala Ser Gly Asn Ile Val Ser Ser Val 2710 2715 2720 aac atg att tca agg atg ttg atc aac aga ttt aca atg aga tac 8310Asn Met Ile Ser Arg Met Leu Ile Asn Arg Phe Thr Met Arg Tyr 2725 2730 2735 aag aaa gcc act tac gag ccg gat gtt gac ctc gga agc gga acc 8355Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp Leu Gly Ser Gly Thr 2740 2745 2750 cgt aac atc ggg att gaa agt gag ata cca aac cta gat ata att 8400Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro Asn Leu Asp Ile Ile 2755 2760 2765 ggg aaa aga ata gaa aaa ata aag caa gag cat gaa aca tca tgg 8445Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu His Glu Thr Ser Trp 2770 2775 2780 cac tat gac caa gac cac cca tac aaa acg tgg gca tac cat ggt 8490His Tyr Asp Gln Asp His Pro Tyr Lys Thr Trp Ala Tyr His Gly 2785 2790 2795 agc tat gaa aca aaa cag act gga tca gca tca tcc atg gtc aac 8535Ser Tyr Glu Thr Lys Gln Thr Gly Ser Ala Ser Ser Met Val Asn 2800 2805 2810 gga gtg gtc agg ctg ctg aca aaa cct tgg gac gtt gtc ccc atg 8580Gly Val Val Arg Leu Leu Thr Lys Pro Trp Asp Val Val Pro Met 2815 2820 2825 gtg aca cag atg gca atg aca gac acg act cca ttt gga caa cag 8625Val Thr Gln Met Ala Met Thr Asp Thr Thr Pro Phe Gly Gln Gln 2830 2835 2840 cgc gtt ttt aaa gag aaa gtg gac acg aga acc caa gaa ccg aaa 8670Arg Val Phe Lys Glu Lys Val Asp Thr Arg Thr Gln Glu Pro Lys 2845 2850 2855 gaa ggc acg aag aaa cta atg aaa ata aca gca gag tgg ctt tgg 8715Glu Gly Thr Lys Lys Leu Met Lys Ile Thr Ala Glu Trp Leu Trp 2860 2865 2870 aaa gaa tta ggg aag aaa aag aca ccc agg atg tgc acc aga gaa 8760Lys Glu Leu Gly Lys Lys Lys Thr Pro Arg Met Cys Thr Arg Glu 2875 2880 2885 gaa ttc aca aga aag gtg aga agc aat gca gcc ttg ggg gcc ata 8805Glu Phe Thr Arg Lys Val Arg Ser Asn Ala Ala Leu Gly Ala Ile 2890 2895 2900 ttc act gat gag aac aag tgg aag tcg gca cgt gag gct gtt gaa 8850Phe Thr Asp Glu Asn Lys Trp Lys Ser Ala Arg Glu Ala Val Glu 2905 2910 2915 gat agt agg ttt tgg gag ctg gtt gac aag gaa agg aat ctc cat 8895Asp Ser Arg Phe Trp Glu Leu Val Asp Lys Glu Arg Asn Leu His 2920 2925 2930 ctt gaa gga aag tgt gaa aca tgt gtg tac aac atg atg gga aaa 8940Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr Asn Met Met Gly Lys 2935 2940 2945 aga gag aag aag cta ggg gaa ttc ggc aag gca aaa ggc agc aga 8985Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys Ala Lys Gly Ser Arg 2950 2955 2960 gcc ata tgg tac atg tgg ctt gga gca cgc ttc tta gag ttt gaa 9030Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Phe Leu Glu Phe Glu 2965 2970 2975 gcc cta gga ttc tta aat gaa gat cac tgg ttc tcc aga gag aac 9075Ala Leu Gly Phe Leu Asn Glu Asp His Trp Phe Ser Arg Glu Asn 2980 2985 2990 tcc ctg agt gga gtg gaa gga gaa ggg ctg cac aag cta ggt tac 9120Ser Leu Ser Gly Val Glu Gly Glu Gly Leu His Lys Leu Gly Tyr 2995 3000 3005 att cta aga gac gtg agc aag aaa gag gga gga gca atg tat gcc 9165Ile Leu Arg Asp Val Ser Lys Lys Glu Gly Gly Ala Met Tyr Ala 3010 3015 3020 gat gac acc gca gga tgg gat aca aaa atc aca cta gaa gac cta 9210Asp Asp Thr Ala Gly Trp Asp Thr Lys Ile Thr Leu Glu Asp Leu 3025 3030 3035 aaa aat gaa gag atg gta aca aac cac atg gaa gga gaa cac aag 9255Lys Asn Glu Glu Met Val Thr Asn His Met Glu Gly Glu His Lys 3040 3045 3050 aaa cta gcc gag gcc att ttc aaa cta acg tac caa aac aag gtg 9300Lys Leu Ala Glu Ala Ile Phe Lys Leu Thr Tyr Gln Asn Lys Val 3055 3060 3065 gtg cgt gtg caa aga cca aca cca aga ggc aca gta atg gac atc 9345Val Arg Val Gln Arg Pro Thr Pro Arg Gly Thr Val Met Asp Ile 3070 3075 3080 ata tcg aga aga gac caa aga ggt agt gga caa gtt ggc acc tat 9390Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly Gln Val Gly Thr Tyr 3085 3090 3095 gga ctc aat act ttc acc aat atg gaa gcc caa cta atc aga cag 9435Gly Leu Asn Thr Phe Thr Asn Met Glu Ala Gln Leu Ile Arg Gln 3100 3105 3110 atg gag gga gaa gga gtc ttt aaa agc att cag cac cta aca atc 9480Met Glu Gly Glu Gly Val Phe Lys Ser Ile Gln His Leu Thr Ile 3115 3120 3125 aca gaa gaa atc gct gtg caa aac tgg tta gca aga gtg ggg cgc 9525Thr Glu Glu Ile Ala Val Gln Asn Trp Leu Ala Arg Val Gly Arg 3130 3135 3140 gaa agg tta tca aga atg gcc atc agt gga gat gat tgt gtt gtg 9570Glu Arg Leu Ser Arg Met Ala Ile Ser Gly Asp Asp Cys Val Val 3145 3150 3155 aaa cct tta gat gac agg ttc gca agc gct tta aca gct cta aat 9615Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala Leu Thr Ala Leu Asn 3160 3165 3170 gac atg gga aag att agg aaa gac ata caa caa tgg gaa cct tca 9660Asp Met Gly Lys Ile Arg Lys Asp Ile Gln Gln Trp Glu Pro Ser 3175 3180 3185 aga gga tgg aat gat tgg aca caa gtg ccc ttc tgt tca cac cat 9705Arg Gly Trp Asn Asp Trp Thr Gln Val Pro Phe Cys Ser His His 3190 3195 3200 ttc cat gag tta atc atg aaa gac ggt cgc gta ctc gtt gtt cca 9750Phe His Glu Leu Ile Met Lys Asp Gly Arg Val Leu Val Val Pro 3205 3210 3215 tgt aga aac caa gat gaa ctg att ggc aga gcc cga atc tcc caa 9795Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg Ala Arg Ile Ser Gln 3220 3225 3230 gga gca ggg tgg tct ttg cgg gag acg gcc tgt ttg ggg aag tct 9840Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys Ser 3235 3240 3245 tac gcc caa atg tgg agc ttg atg tac ttc cac aga cgc gac ctc 9885Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe His Arg Arg Asp Leu 3250 3255 3260 agg ctg gcg gca aat gct att tgc tcg gca gta cca tca cat tgg 9930Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala Val Pro Ser His Trp 3265 3270 3275 gtt cca aca agt cga aca acc tgg tcc ata cat gct aaa cat gaa 9975Val Pro Thr Ser Arg Thr Thr Trp Ser Ile His Ala Lys His Glu 3280 3285 3290 tgg atg aca acg gaa gac atg ctg aca gtc tgg aac agg gtg tgg 10020Trp Met Thr Thr Glu Asp Met Leu Thr Val Trp Asn Arg Val Trp 3295 3300 3305 att caa gaa aac cca tgg atg gaa gac aaa act cca gtg gaa aca 10065Ile Gln Glu Asn Pro Trp Met Glu Asp Lys Thr Pro Val Glu Thr 3310 3315 3320 tgg gag gaa atc cca tac ttg ggg aaa aga gaa gac caa tgg tgc 10110Trp Glu Glu Ile Pro Tyr Leu Gly Lys Arg Glu Asp Gln Trp Cys 3325 3330 3335 ggc tca ttg att ggg tta aca agc agg gcc acc tgg gca aag aac 10155Gly Ser Leu Ile Gly Leu Thr Ser Arg Ala Thr Trp Ala Lys Asn 3340 3345 3350 atc caa gca gca ata aat caa gtt aga tcc ctt ata ggc aat gaa 10200Ile Gln Ala Ala Ile Asn Gln Val Arg Ser Leu Ile Gly Asn Glu 3355 3360 3365 gaa tac aca gat tac atg cca tcc atg aaa aga ttc aga aga gaa 10245Glu Tyr Thr Asp Tyr Met Pro Ser Met Lys Arg Phe Arg Arg Glu 3370 3375 3380 gag gaa gaa gca gga gtt ctg tgg tag aaagcaaaac taacatgaaa 10292Glu Glu Glu Ala Gly Val Leu Trp 3385 3390 caaggctaga agtcaggtcg gattaagcca tagtacggaa aaaactatgc tacctgtgag 10352ccccgtccaa ggacgttaaa agaagtcagg ccatcataaa tgccatagct tgagtaaact 10412atgcagcctg tagctccacc tgagaaggtg taaaaaatcc gggaggccac aaaccatgga 10472agctgtacgc atggcgtagt ggactagcgg ttaggggaga cccctccctt acaaatcgca 10532gcaacaatgg gggcccaagg cgagatgaag ctgtagtctc gctggaagga ctagaggtta 10592gaggagaccc ccccgaaaca aaaaacagca tattgacgct gggaaagacc agagatcctg 10652ctgtctcctc agcatcattc caggcacaga acgccagaaa atggaatggt gctgttgaat 10712caacaggttc t 1072323391PRTDengue virus 2Met Asn Asn Gln Arg Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu 1 5 10 15 Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg 20 25 30 Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met 35 40 45 Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly 50 55 60 Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val 65 70 75 80 Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn 85 90 95 Arg Arg Arg Arg Ser Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val 100 105 110 Met Ala Phe His Leu Thr Thr Arg Asn Gly Glu Pro His Met Ile Val 115 120 125 Ser Arg Gln Glu Lys Gly Lys Ser Leu Leu Phe Lys Thr Glu Val Gly 130 135 140 Val Asn Met Cys Thr Leu Met Ala Met Asp Leu Gly Glu Leu Cys Glu 145 150 155 160 Asp Thr Ile Thr Tyr Lys Cys Pro Leu Leu Arg Gln Asn Glu Pro Glu 165 170 175 Asp Ile Asp Cys Trp Cys Asn Ser Thr Ser Thr Trp Val Thr Tyr Gly 180 185 190 Thr Cys Thr Thr Met Gly Glu His Arg Arg Glu Lys Arg Ser Val Ala 195 200 205 Leu Val Pro His Val Arg Met Gly Leu Glu Thr Arg Thr Glu Thr Trp 210 215 220 Met Ser Ser Glu Gly Ala Trp Lys His Val Gln Arg Ile Glu Thr Trp 225 230 235 240 Ile Leu Arg His Pro Gly Phe Thr Met Met Ala Ala Ile Leu Ala Tyr 245 250 255 Thr Ile Gly Thr Thr His Phe Gln Arg Ala Leu Ile Phe Ile Leu Leu 260 265 270 Thr Ala Val Thr Pro Ser Met Thr Met Arg Cys Ile Gly Met Ser Asn 275 280 285 Arg Asp Phe Val Glu Gly Val Ser Gly Gly Ser Trp Val Asp Ile Val 290 295 300 Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr 305 310 315 320 Leu Asp Phe Glu Leu Ile Lys Thr Glu Ala Lys Gln Pro Ala Thr Leu 325 330 335 Arg Lys Tyr Cys Ile Glu Ala Lys Leu Thr Asn Thr Thr Thr Glu Ser 340 345 350 Arg Cys Pro Thr Gln Gly Glu Pro Ser Leu Asn Glu Glu Gln Asp Lys 355 360 365 Arg Phe Val Cys Lys His Ser Met Val Asp Arg Gly Trp Gly Asn Gly 370 375 380 Cys Gly Leu Phe Gly Lys Gly Gly Ile Val Thr Cys Ala Met Phe Arg 385 390 395 400 Cys Lys Lys Asn Met Glu Gly Lys Val Val Gln Pro Glu Asn Leu Glu 405 410 415 Tyr Thr Ile Val Ile Thr Pro His Ser Gly Glu Glu His Ala Val Gly 420 425 430 Asn Asp Thr Gly Lys His Gly Lys Glu Ile Lys Ile Thr Pro Gln Ser 435 440 445 Ser Ile Thr Glu Ala Glu Leu Thr Gly Tyr Gly Thr Val Thr Met Glu 450 455 460 Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu Gln 465 470 475 480 Met Glu Asn Lys Ala Trp Leu Val His Arg Gln Trp Phe Leu Asp Leu 485 490 495 Pro Leu Pro Trp Leu Pro Gly Ala Asp Thr Gln Glu Ser Asn Trp Ile 500 505 510 Gln Lys Glu Thr Leu Val Thr Phe Lys Asn Pro His Ala Lys Lys Gln 515 520 525 Asp Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu 530 535 540 Thr Gly Ala Thr Glu Ile Gln Met Ser Ser Gly Asn Leu Leu Phe Thr 545 550 555 560 Gly His Leu Lys Cys Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly 565 570 575 Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Val Val Lys Glu Ile 580 585 590 Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln Tyr Glu Gly 595 600 605 Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu Glu Lys 610 615 620 Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu 625 630 635 640 Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Asp Ser 645 650 655 Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe 660 665 670 Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Glu Thr Thr Met Arg

Gly 675 680 685 Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser 690 695 700 Leu Gly Gly Val Phe Thr Ser Ile Gly Lys Ala Leu His Gln Val Phe 705 710 715 720 Gly Ala Ile Tyr Gly Ala Ala Phe Ser Gly Val Ser Trp Thr Met Lys 725 730 735 Ile Leu Ile Gly Val Ile Ile Thr Trp Ile Gly Met Asn Ser Arg Ser 740 745 750 Thr Ser Leu Ser Val Thr Leu Val Leu Val Gly Ile Val Thr Leu Tyr 755 760 765 Leu Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys 770 775 780 Asn Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val 785 790 795 800 His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys 805 810 815 Leu Ala Ser Ala Ile Gln Lys Ala His Glu Glu Asp Ile Cys Gly Ile 820 825 830 Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro 835 840 845 Glu Leu Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met 850 855 860 Thr Gly Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg 865 870 875 880 Pro Gln Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala 885 890 895 Lys Met Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly 900 905 910 Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu 915 920 925 Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu 930 935 940 Lys Leu Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser 945 950 955 960 Ala Ala Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp 965 970 975 Ile Glu Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe 980 985 990 Ile Glu Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser 995 1000 1005 Asn Gly Val Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Leu Ala 1010 1015 1020 Gly Pro Val Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln 1025 1030 1035 Ile Thr Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp 1040 1045 1050 Phe Cys Asp Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly Asn 1055 1060 1065 Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile 1070 1075 1080 Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr 1085 1090 1095 Arg Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro Leu 1100 1105 1110 Lys Glu Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala Gly 1115 1120 1125 His Gly Gln Val Asp Asn Phe Ser Leu Gly Val Leu Gly Met Ala 1130 1135 1140 Leu Phe Leu Glu Glu Met Leu Arg Thr Arg Val Gly Thr Lys His 1145 1150 1155 Ala Ile Leu Leu Val Ala Val Ser Phe Val Thr Leu Ile Thr Gly 1160 1165 1170 Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met Val Met Val Gly 1175 1180 1185 Ala Thr Met Thr Asp Asp Ile Gly Met Gly Val Thr Tyr Leu Ala 1190 1195 1200 Leu Leu Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala Gly Leu 1205 1210 1215 Leu Leu Arg Lys Leu Thr Ser Lys Glu Leu Met Met Thr Thr Ile 1220 1225 1230 Gly Ile Val Leu Leu Ser Gln Ser Thr Ile Pro Glu Thr Ile Leu 1235 1240 1245 Glu Leu Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met 1250 1255 1260 Val Arg Asn Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala 1265 1270 1275 Ile Leu Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys 1280 1285 1290 Val Ser Cys Thr Ile Leu Ala Val Val Ser Val Ser Pro Leu Phe 1295 1300 1305 Leu Thr Ser Ser Gln Gln Lys Thr Asp Trp Ile Pro Leu Ala Leu 1310 1315 1320 Thr Ile Lys Gly Leu Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu 1325 1330 1335 Ser Arg Thr Ser Lys Lys Arg Ser Trp Pro Leu Asn Glu Ala Ile 1340 1345 1350 Met Ala Val Gly Met Val Ser Ile Leu Ala Ser Ser Leu Leu Lys 1355 1360 1365 Asn Asp Ile Pro Met Thr Gly Pro Leu Val Ala Gly Gly Leu Leu 1370 1375 1380 Thr Val Cys Tyr Val Leu Thr Gly Arg Ser Ala Asp Leu Glu Leu 1385 1390 1395 Glu Arg Ala Ala Asp Val Lys Trp Glu Asp Gln Ala Glu Ile Ser 1400 1405 1410 Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser Glu Asp Gly Ser 1415 1420 1425 Met Ser Ile Lys Asn Glu Glu Glu Glu Gln Thr Leu Thr Ile Leu 1430 1435 1440 Ile Arg Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro Val Ser 1445 1450 1455 Ile Pro Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys Lys 1460 1465 1470 Gln Arg Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Pro Met 1475 1480 1485 Gly Lys Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys 1490 1495 1500 Gly Ile Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu 1505 1510 1515 Gly Thr Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu 1520 1525 1530 Met His Lys Gly Lys Arg Ile Glu Pro Thr Trp Ala Asp Val Lys 1535 1540 1545 Lys Asp Leu Ile Ser Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu 1550 1555 1560 Trp Lys Glu Gly Glu Glu Val Gln Val Leu Ala Leu Glu Pro Gly 1565 1570 1575 Lys Asn Pro Arg Ala Val Gln Thr Lys Pro Gly Leu Phe Lys Thr 1580 1585 1590 Asn Ala Gly Thr Ile Gly Ala Val Ser Leu Asp Phe Ser Pro Gly 1595 1600 1605 Thr Ser Gly Ser Pro Ile Ile Asp Lys Lys Gly Lys Val Val Gly 1610 1615 1620 Leu Tyr Gly Asn Gly Val Val Thr Arg Ser Gly Ala Tyr Val Ser 1625 1630 1635 Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu Asp Asn Pro Glu Ile 1640 1645 1650 Glu Asp His Ile Phe Arg Lys Arg Arg Leu Thr Ile Met Asp Leu 1655 1660 1665 His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro Ala Ile Val 1670 1675 1680 Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu Ala Pro 1685 1690 1695 Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu 1700 1705 1710 Pro Ile Arg Tyr Gln Thr Pro Ala Ile Arg Ala Glu His Thr Gly 1715 1720 1725 Arg Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg 1730 1735 1740 Leu Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met 1745 1750 1755 Asp Glu Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly 1760 1765 1770 Tyr Ile Ser Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe 1775 1780 1785 Met Thr Ala Thr Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser 1790 1795 1800 Asn Ala Pro Ile Ile Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser 1805 1810 1815 Trp Asn Ser Gly His Glu Trp Val Thr Asp Phe Lys Gly Lys Thr 1820 1825 1830 Val Trp Phe Val Pro Ser Ile Lys Ala Gly Asn Asp Ile Ala Ala 1835 1840 1845 Cys Leu Arg Lys Asn Gly Lys Lys Val Ile Gln Leu Ser Arg Lys 1850 1855 1860 Thr Phe Asp Ser Glu Tyr Val Lys Thr Arg Thr Asn Asp Trp Asp 1865 1870 1875 Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly Ala Asn Phe Lys 1880 1885 1890 Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met Lys Pro Val Ile 1895 1900 1905 Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly Pro Met Pro 1910 1915 1920 Val Thr His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg 1925 1930 1935 Asn Pro Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu Pro 1940 1945 1950 Leu Glu Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met 1955 1960 1965 Leu Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met 1970 1975 1980 Phe Glu Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr 1985 1990 1995 Arg Leu Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg 2000 2005 2010 Arg Gly Asp Leu Pro Val Trp Leu Ala Tyr Arg Val Ala Ala Glu 2015 2020 2025 Gly Ile Asn Tyr Ala Asp Arg Arg Trp Cys Phe Asp Gly Val Lys 2030 2035 2040 Asn Asn Gln Ile Leu Glu Glu Asn Val Glu Val Glu Ile Trp Thr 2045 2050 2055 Lys Glu Gly Glu Arg Lys Lys Leu Lys Pro Arg Trp Leu Asp Ala 2060 2065 2070 Arg Ile Tyr Ser Asp Pro Leu Ala Leu Lys Glu Phe Lys Glu Phe 2075 2080 2085 Ala Ala Gly Arg Lys Ser Leu Thr Leu Asn Leu Ile Thr Glu Met 2090 2095 2100 Gly Arg Leu Pro Thr Phe Met Thr Gln Lys Ala Arg Asp Ala Leu 2105 2110 2115 Asp Asn Leu Ala Val Leu His Thr Ala Glu Ala Gly Gly Arg Ala 2120 2125 2130 Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr Leu Glu Thr Leu 2135 2140 2145 Leu Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly Ile Phe Leu 2150 2155 2160 Phe Leu Met Ser Ala Arg Gly Ile Gly Lys Met Thr Leu Gly Met 2165 2170 2175 Cys Cys Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln Ile 2180 2185 2190 Gln Pro His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu 2195 2200 2205 Ile Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln 2210 2215 2220 Asp Asn Gln Leu Thr Tyr Val Val Ile Ala Ile Leu Thr Val Val 2225 2230 2235 Ala Ala Thr Met Ala Asn Glu Met Gly Phe Leu Glu Lys Thr Lys 2240 2245 2250 Lys Asp Leu Gly Leu Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser 2255 2260 2265 Asn Ile Leu Asp Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu 2270 2275 2280 Tyr Ala Val Ala Thr Thr Phe Val Thr Pro Met Leu Arg His Ser 2285 2290 2295 Ile Glu Asn Ser Ser Val Asn Val Ser Leu Thr Ala Ile Ala Asn 2300 2305 2310 Gln Ala Thr Val Leu Met Gly Leu Gly Lys Gly Trp Pro Leu Ser 2315 2320 2325 Lys Met Asp Ile Gly Val Pro Leu Leu Ala Ile Gly Cys Tyr Ser 2330 2335 2340 Gln Val Asn Pro Ile Thr Leu Thr Ala Ala Leu Phe Leu Leu Val 2345 2350 2355 Ala His Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala Thr 2360 2365 2370 Arg Glu Ala Gln Lys Arg Ala Ala Ala Gly Ile Met Lys Asn Pro 2375 2380 2385 Thr Val Asp Gly Ile Thr Val Ile Asp Leu Asp Pro Ile Pro Tyr 2390 2395 2400 Asp Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu Leu Val 2405 2410 2415 Leu Cys Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala Leu 2420 2425 2430 Cys Glu Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp 2435 2440 2445 Glu Gly Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser 2450 2455 2460 Met Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu 2465 2470 2475 Leu Phe Ser Ile Met Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr 2480 2485 2490 Gly Asn Ile Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu 2495 2500 2505 Asn Ala Leu Gly Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly 2510 2515 2520 Ile Gln Glu Val Asp Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg 2525 2530 2535 Gly Glu Thr Asp His His Ala Val Ser Arg Gly Ser Ala Lys Leu 2540 2545 2550 Arg Trp Phe Val Glu Arg Asn Met Val Thr Pro Glu Gly Lys Val 2555 2560 2565 Val Asp Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Gly 2570 2575 2580 Gly Leu Lys Asn Val Arg Glu Val Lys Gly Leu Thr Lys Gly Gly 2585 2590 2595 Pro Gly His Glu Glu Pro Ile Pro Met Ser Thr Tyr Gly Trp Asn 2600 2605 2610 Leu Val Arg Leu Gln Ser Gly Val Asp Val Phe Phe Ile Pro Pro 2615 2620 2625 Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Pro 2630 2635 2640 Asn Pro Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu Asn Leu 2645 2650 2655 Val Glu Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys Val 2660 2665 2670 Leu Asn Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu 2675 2680 2685 Gln Arg Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg 2690 2695 2700 Asn Ser Thr His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn 2705 2710 2715 Ile Val Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg 2720 2725 2730 Phe Thr Met Arg Tyr Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp 2735 2740 2745 Leu Gly Ser Gly Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro 2750 2755 2760 Asn Leu Asp Ile Ile Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu 2765 2770 2775 His Glu Thr Ser Trp His Tyr Asp Gln Asp His Pro Tyr Lys Thr 2780 2785 2790 Trp Ala Tyr His Gly Ser Tyr Glu Thr Lys Gln Thr Gly Ser Ala 2795 2800 2805 Ser Ser Met Val Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp 2810 2815 2820 Asp Val Val Pro Met Val Thr Gln Met Ala Met Thr Asp Thr Thr 2825 2830 2835 Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg 2840 2845 2850 Thr Gln Glu Pro Lys Glu Gly Thr Lys Lys Leu Met Lys Ile Thr 2855 2860 2865 Ala Glu Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Thr Pro Arg 2870 2875 2880 Met Cys Thr Arg Glu Glu Phe Thr Arg Lys Val Arg

Ser Asn Ala 2885 2890 2895 Ala Leu Gly Ala Ile Phe Thr Asp Glu Asn Lys Trp Lys Ser Ala 2900 2905 2910 Arg Glu Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys 2915 2920 2925 Glu Arg Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr 2930 2935 2940 Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys 2945 2950 2955 Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg 2960 2965 2970 Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp 2975 2980 2985 Phe Ser Arg Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu 2990 2995 3000 His Lys Leu Gly Tyr Ile Leu Arg Asp Val Ser Lys Lys Glu Gly 3005 3010 3015 Gly Ala Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Lys Ile 3020 3025 3030 Thr Leu Glu Asp Leu Lys Asn Glu Glu Met Val Thr Asn His Met 3035 3040 3045 Glu Gly Glu His Lys Lys Leu Ala Glu Ala Ile Phe Lys Leu Thr 3050 3055 3060 Tyr Gln Asn Lys Val Val Arg Val Gln Arg Pro Thr Pro Arg Gly 3065 3070 3075 Thr Val Met Asp Ile Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly 3080 3085 3090 Gln Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Ala 3095 3100 3105 Gln Leu Ile Arg Gln Met Glu Gly Glu Gly Val Phe Lys Ser Ile 3110 3115 3120 Gln His Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn Trp Leu 3125 3130 3135 Ala Arg Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser Gly 3140 3145 3150 Asp Asp Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala 3155 3160 3165 Leu Thr Ala Leu Asn Asp Met Gly Lys Ile Arg Lys Asp Ile Gln 3170 3175 3180 Gln Trp Glu Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro 3185 3190 3195 Phe Cys Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg 3200 3205 3210 Val Leu Val Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg 3215 3220 3225 Ala Arg Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala 3230 3235 3240 Cys Leu Gly Lys Ser Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe 3245 3250 3255 His Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala 3260 3265 3270 Val Pro Ser His Trp Val Pro Thr Ser Arg Thr Thr Trp Ser Ile 3275 3280 3285 His Ala Lys His Glu Trp Met Thr Thr Glu Asp Met Leu Thr Val 3290 3295 3300 Trp Asn Arg Val Trp Ile Gln Glu Asn Pro Trp Met Glu Asp Lys 3305 3310 3315 Thr Pro Val Glu Thr Trp Glu Glu Ile Pro Tyr Leu Gly Lys Arg 3320 3325 3330 Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu Thr Ser Arg Ala 3335 3340 3345 Thr Trp Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln Val Arg Ser 3350 3355 3360 Leu Ile Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser Met Lys 3365 3370 3375 Arg Phe Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp 3380 3385 3390 310723DNADengue virusmisc_feature(1)..(10723)Wild-type DEN2 strain 16681 3agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaacgta 60gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360ggaaggatgc tgaacatctt gaataggaga cgcagatctg caggcatgat cattatgctg 420attccaacag tgatggcgtt ccatttaacc acacgtaacg gagaaccaca catgatcgtc 480agcagacaag agaaagggaa aagtcttctg tttaaaacag aggatggcgt gaacatgtgt 540accctcatgg ccatggacct tggtgaattg tgtgaagaca caatcacgta caagtgtccc 600cttctcaggc agaatgagcc agaagacata gactgttggt gcaactctac gtccacgtgg 660gtaacttatg ggacgtgtac caccatggga gaacatagaa gagaaaaaag atcagtggca 720ctcgttccac atgtgggaat gggactggag acacgaactg aaacatggat gtcatcagaa 780ggggcctgga aacatgtcca gagaattgaa acttggatct tgagacatcc aggcttcacc 840atgatggcag caatcctggc atacaccata ggaacgacac atttccaaag agccctgatt 900ttcatcttac tgacagctgt cactccttca atgacaatgc gttgcatagg aatgtcaaat 960agagactttg tggaaggggt ttcaggagga agctgggttg acatagtctt agaacatgga 1020agctgtgtga cgacgatggc aaaaaacaaa ccaacattgg attttgaact gataaaaaca 1080gaagccaaac agcctgccac cctaaggaag tactgtatag aggcaaagct aaccaacaca 1140acaacagaat ctcgctgccc aacacaaggg gaacccagcc taaatgaaga gcaggacaaa 1200aggttcgtct gcaaacactc catggtagac agaggatggg gaaatggatg tggactattt 1260ggaaagggag gcattgtgac ctgtgctatg ttcagatgca aaaagaacat ggaaggaaaa 1320gttgtgcaac cagaaaactt ggaatacacc attgtgataa cacctcactc aggggaagag 1380catgcagtcg gaaatgacac aggaaaacat ggcaaggaaa tcaaaataac accacagagt 1440tccatcacag aagcagaatt gacaggttat ggcactgtca caatggagtg ctctccaaga 1500acgggcctcg acttcaatga gatggtgttg ctgcagatgg aaaataaagc ttggctggtg 1560cacaggcaat ggttcctaga cctgccgtta ccatggttgc ccggagcgga cacacaaggg 1620tcaaattgga tacagaaaga gacattggtc actttcaaaa atccccatgc gaagaaacag 1680gatgttgttg ttttaggatc ccaagaaggg gccatgcaca cagcacttac aggggccaca 1740gaaatccaaa tgtcatcagg aaacttactc ttcacaggac atctcaagtg caggctgaga 1800atggacaagc tacagctcaa aggaatgtca tactctatgt gcacaggaaa gtttaaagtt 1860gtgaaggaaa tagcagaaac acaacatgga acaatagtta tcagagtgca atatgaaggg 1920gacggctctc catgcaagat cccttttgag ataatggatt tggaaaaaag acatgtctta 1980ggtcgcctga ttacagtcaa cccaattgtg acagaaaaag atagcccagt caacatagaa 2040gcagaacctc cattcggaga cagctacatc atcataggag tagagccggg acaactgaag 2100ctcaactggt ttaagaaagg aagttctatc ggccaaatgt ttgagacaac aatgaggggg 2160gcgaagagaa tggccatttt aggtgacaca gcctgggatt ttggatcctt gggaggagtg 2220tttacatcta taggaaaggc tctccaccaa gtctttggag caatctatgg agctgccttc 2280agtggggttt catggactat gaaaatcctc ataggagtca ttatcacatg gataggaatg 2340aattcacgca gcacctcact gtctgtgaca ctagtattgg tgggaattgt gacactgtat 2400ttgggagtca tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460aaatgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag 2520ttccaaccag aatccccttc aaaactagct tcagctatcc agaaagccca tgaagagggc 2580atttgtggaa tccgctcagt aacaagactg gagaatctga tgtggaaaca aataacacca 2640gaattgaatc acattctatc agaaaatgag gtgaagttaa ctattatgac aggagacatc 2700aaaggaatca tgcaggcagg aaaacgatct ctgcggcctc agcccactga gctgaagtat 2760tcatggaaaa catggggcaa agcaaaaatg ctctctacag agtctcataa ccagaccttt 2820ctcattgatg gccccgaaac agcagaatgc cccaacacaa atagagcttg gaattcgttg 2880gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa attgaaagaa 2940aaacaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060aaagcctctt tcattgaagt taaaaactgc cactggccaa aatcacacac cctctggagc 3120aatggagtgc tagaaagtga gatgataatt ccaaagaatc tcgctggacc agtgtctcaa 3180cacaactata gaccaggcta ccatacacaa ataacaggac catggcatct aggtaagctt 3240gagatggact ttgatttctg tgatggaaca acagtggtag tgactgagga ctgcggaaat 3300agaggaccct ctttgagaac aaccactgcc tctggaaaac tcataacaga atggtgctgc 3360cgatcttgca cattaccacc gctaagatac agaggtgagg atgggtgctg gtacgggatg 3420gaaatcagac cattgaagga gaaagaagag aatttggtca actccttggt cacagctgga 3480catgggcagg tcgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaggaa 3540atgcttagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtaggc 3660gccactatga cggatgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720aaagtcagac caacttttgc agctggacta ctcttgagaa agctgacctc caaggaattg 3780atgatgacta ctataggaat tgtactcctc tcccagagca ccataccaga gaccattctt 3840gagttgactg atgcgttagc cttaggcatg atggtcctca aaatggtgag aaatatggaa 3900aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaacgc agtgatatta 3960caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgctc 4020ttaacatcct cacagcaaaa aacagattgg ataccattag cattgacgat caaaggtctc 4080aatccaacag ctatttttct aacaaccctc tcaagaacca gcaagaaaag gagctggcca 4140ttaaatgagg ctatcatggc agtcgggatg gtgagcattt tagccagttc tctcctaaaa 4200aatgatattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260ctcactggac gatcggccga tttggaactg gagagagcag ccgatgtcaa atgggaagac 4320caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga agatggtagc 4380atgtcgataa aaaatgaaga ggaagaacaa acactgacca tactcattag aacaggattg 4440ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500tgggaagtga agaaacaacg ggccggagta ttgtgggatg ttccttcacc cccacccatg 4560ggaaaggctg aactggaaga tggagcctat agaattaagc aaaaagggat tcttggatat 4620tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680cgtggcgctg ttctaatgca taaaggaaag aggattgaac catcatgggc ggacgtcaag 4740aaagacctaa tatcatatgg aggaggctgg aagttagaag gagaatggaa ggaaggagaa 4800gaagtccagg tattggcact ggagcctgga aaaaatccaa gagccgtcca aacgaaacct 4860ggtcttttca aaaccaacgc cggaacaata ggtgctgtat ctctggactt ttctcctgga 4920acgtcaggat ctccaattat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagcattgaa 5040gacaacccag agatcgaaga tgacattttc cgaaagagaa gactgaccat catggacctc 5100cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga agctataaaa 5160cggggtttga gaacattaat cttggccccc actagagttg tggcagctga aatggaggaa 5220gcccttagag gacttccaat aagataccag accccagcca tcagagctga gcacaccggg 5280cgggagattg tggacctaat gtgtcatgcc acatttacca tgaggctgct atcaccagtt 5340agagtgccaa actacaacct gattatcatg gacgaagccc atttcacaga cccagcaagt 5400atagcagcta gaggatacat ctcaactcga gtggagatgg gtgaggcagc tgggattttt 5460atgacagcca ctcccccggg aagcagagac ccatttcctc agagcaatgc accaatcata 5520gatgaagaaa gagaaatccc tgaacgttcg tggaattccg gacatgaatg ggtcacggat 5580tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640tgcctgagga aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700tatgtcaaga ctagaaccaa tgattgggac ttcgtggtta caactgacat ttcagaaatg 5760ggtgccaatt tcaaggctga gagggttata gaccccagac gctgcatgaa accagtcata 5820ctaacagatg gtgaagagcg ggtgattctg gcaggaccta tgccagtgac ccactctagt 5880gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgagaatga ccagtacata 5940tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000ctcctagata acatcaacac gccagaagga atcattccta gcatgttcga accagagcgt 6060gaaaaggtgg atgccattga tggcgaatac cgcttgagag gagaagcaag gaaaaccttt 6120gtagacttaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180ggcatcaact acgcagacag aaggtggtgt tttgatggag tcaagaacaa ccaaatccta 6240gaagaaaacg tggaagttga aatctggaca aaagaagggg aaaggaagaa attgaaaccc 6300agatggttgg atgctaggat ctattctgac ccactggcgc taaaagaatt taaggaattt 6360gcagccggaa gaaagtctct gaccctgaac ctaatcacag aaatgggtag gctcccaacc 6420ttcatgactc agaaggcaag agacgcactg gacaacttag cagtgctgca cacggctgag 6480gcaggtggaa gggcgtacaa ccatgctctc agtgaactgc cggagaccct ggagacattg 6540cttttactga cacttctggc tacagtcacg ggagggatct ttttattctt gatgagcgga 6600aggggcatag ggaagatgac cctgggaatg tgctgcataa tcacggctag catcctccta 6660tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720atagttttgc ttattccaga acctgaaaaa cagagaacac cccaagacaa ccaactgacc 6780tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840ctagaaaaaa cgaagaaaga tctcggattg ggaagcattg caacccagca acccgagagc 6900aacatcctgg acatagatct acgtcctgca tcagcatgga cgctgtatgc cgtggccaca 6960acatttgtta caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtgtcccta 7020acagctatag ccaaccaagc cacagtgtta atgggtctcg ggaaaggatg gccattgtca 7080aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140actctcacag cagctctttt cttattggta gcacattatg ccatcatagg gccaggactc 7200caagcaaaag caaccagaga agctcagaaa agagcagcgg cgggcatcat gaaaaaccca 7260actgtcgatg gaataacagt gattgaccta gatccaatac cttatgatcc aaagtttgaa 7320aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtatt gatgatgagg 7380actacatggg ctctgtgtga ggctttaacc ttagctaccg ggcccatctc cacattgtgg 7440gaaggaaatc cagggaggtt ttggaacact accattgcgg tgtcaatggc taacattttt 7500agagggagtt acttggccgg agctggactt ctcttttcta ttatgaagaa cacaaccaac 7560acaagaaggg gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg 7620aacgcattgg gaaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740ggctcagcaa aactgagatg gttcgttgag agaaacatgg tcacaccaga agggaaagta 7800gtggacctcg gttgtggcag aggaggctgg tcatactatt gtggaggact aaagaatgta 7860agagaagtca aaggcctaac aaaaggagga ccaggacacg aagaacccat ccccatgtca 7920acatatgggt ggaatctagt gcgtcttcaa agtggagttg acgttttctt catcccgcca 7980gaaaagtgtg acacattatt gtgtgacata ggggagtcat caccaaatcc cacagtggaa 8040gcaggacgaa cactcagagt ccttaactta gtagaaaatt ggttgaacaa caacactcaa 8100ttttgcataa aggttctcaa cccatatatg ccctcagtca tagaaaaaat ggaagcacta 8160caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220atgtactggg tatccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaagg 8280atgttgatca acagatttac aatgagatac aagaaagcca cttacgagcc ggatgttgac 8340ctcggaagcg gaacccgtaa catcgggatt gaaagtgaga taccaaacct agatataatt 8400gggaaaagaa tagaaaaaat aaagcaagag catgaaacat catggcacta tgaccaagac 8460cacccataca aaacgtgggc ataccatggt agctatgaaa caaaacagac tggatcagca 8520tcatccatgg tcaacggagt ggtcaggctg ctgacaaaac cttgggacgt cgtccccatg 8580gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagag 8640aaagtggaca cgagaaccca agaaccgaaa gaaggcacga agaaactaat gaaaataaca 8700gcagagtggc tttggaaaga attagggaag aaaaagacac ccaggatgtg caccagagaa 8760gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccatattcac tgatgagaac 8820aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940agagagaaga agctagggga attcggcaag gcaaaaggca gcagagccat atggtacatg 9000tggcttggag cacgcttctt agagtttgaa gccctaggat tcttaaatga agatcactgg 9060ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120attctaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180tgggatacaa gaatcacact agaagaccta aaaaatgaag aaatggtaac aaaccacatg 9240gaaggagaac acaagaaact agccgaggcc attttcaaac taacgtacca aaacaaggtg 9300gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360caaagaggta gtggacaagt tggcacctat ggactcaata ctttcaccaa tatggaagcc 9420caactaatca gacagatgga gggagaagga gtctttaaaa gcattcagca cctaacaatc 9480acagaagaaa tcgctgtgca aaactggtta gcaagagtgg ggcgcgaaag gttatcaaga 9540atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600ttaacagctc taaatgacat gggaaagatt aggaaagaca tacaacaatg ggaaccttca 9660agaggatgga atgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720atgaaagacg gtcgcgtact cgttgttcca tgtagaaacc aagatgaact gattggcaga 9780gcccgaatct cccaaggagc agggtggtct ttgcgggaga cggcctgttt ggggaagtct 9840tacgcccaaa tgtggagctt gatgtacttc cacagacgcg acctcaggct ggcggcaaat 9900gctatttgct cggcagtacc atcacattgg gttccaacaa gtcgaacaac ctggtccata 9960catgctaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggttaac aagcagggcc 10140acctgggcaa agaacatcca agcagcaata aatcaagtta gatcccttat aggcaatgaa 10200gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaagcagga 10260gttctgtggt agaaagcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc 10320catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380ggccatcata aatgccatag cttgagtaaa ctatgcagcc tgtagctcca cctgagaagg 10440tgtaaaaaat ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500ggttagagga gacccctccc ttacaaatcg cagcaacaat gggggcccaa ggcgagatga 10560agctgtagtc tcgctggaag gactagaggt tagaggagac ccccccgaaa caaaaaacag 10620catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca 10680gaacgccaga aaatggaatg gtgctgttga atcaacaggt tct 10723437DNAArtificial sequenceprimer 4gttttcccag tcacgacacg tggaccgaca aagacag 37536DNAArtificial sequenceprimer 5aacagctatg accatgttcc tcctgaaacc ccttcc 36636DNAArtificial sequenceprimer 6gttttcccag tcacgacatc acgtacaagt gtcccc 36737DNAArtificial sequenceprimer 7aacagctatg accatgagca acaccatctc attgaag 37842DNAArtificialprimer 8gttttcccag tcacgactgc aaccagaaaa cttggaatac ac 42939DNAArtificial sequenceprimer 9aacagctatg accatggctc catagattgc tccaaagac 391041DNAArtificial sequenceprimer 10gttttcccag tcacgacccc agtcaacata gaagcagaac c 411140DNAArtificial sequenceprimer 11aacagctatg accatgccaa agccatagtc ttcaacttcc 401236DNAArtificial sequenceprimer 12gttttcccag tcacgacatc atgcaggcag

gaaaac 361337DNAArtificial sequenceprimer 13aacagctatg accatgacca taaccatcac tcttccc 371437DNAArtificial sequenceprimer 14aacagctatg accatgacca taaccatcac tcttccc 371536DNAArtificial sequenceprimer 15aacagctatg accatggctc tctccagttc caaatc 361638DNAArtificial sequencepriemr 16gttttcccag tcacgacaag aaccagcaag aaaaggag 381738DNAArtificial sequenceprimer 17aacagctatg accatgcacc attaccataa agacccac 381837DNAArtificial sequenceprimer 18gttttcccag tcacgacttg aaccatcatg ggcggac 371941DNAArtificial sequenceprimer 19aacagctatg accatgtcct gcttttatac ttggaacgaa c 412036DNAArtificial sequenceprimer 20gttttcccag tcacgacaag cccatttcac agaccc 362138DNAArtificial sequenceprimer 21aacagctatg accatgtcaa tttcttcctt tccccttc 382238DNAArtificial sequenceprimer 22gttttcccag tcacgacgag aggagaagca aggaaaac 382335DNAArtificial sequenceprimer 23aacagctatg accatgaggg acacattcac tgagg 352436DNAArtificial sequenceprimer 24gttttcccag tcacgacaca gagaacaccc caagac 362535DNAArtificial sequenceprimer 25aacagctatg accatgtcca cttcctggat tccac 352639DNAArtificial sequenceprimer 26gttttcccag tcacgacaca agtaatgctc ctagtcctc 392738DNAArtificial sequenceprimer 27aacagctatg accatgttca ctgatgacac tatgttcc 382837DNAArtificial sequenceprimer 28gttttcccag tcacgacgtc atcaccaaat cccacag 372939DNAArtificial sequenceprimer 29aacagctatg accatggctt cttctctctt tttcccatc 393036DNAArtificial sequenceprimer 30gttttcccag tcacgacaag gtgagaagca atgcag 363137DNAArtificial sequenceprimer 31aacagctatg accatgtgga aatggtgtga acagaag 373239DNAArtificial sequenceprimer 32gttttcccag tcacgacgca ttcagcacct aacaatcac 393335DNAArtificial sequenceprimer 33aacagctatg accatgggca tttatgatgg cctga 353417DNAArtificial sequenceprimer 34ccatggaagc tgtacgc 173536DNAArtificial sequenceprimer 35aacagctatg accatgtgat tcaacagcac cattcc 363617DNAArtificial sequenceprimer tail 36gttttcccag tcacgac 173716DNAArtificial sequenceprimer tail 37aacagctatg accatg 163810723DNADengue virusmisc_feature(1)..(10723)LAV2 38agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360ggaaggatgc tgaacatctt gaataggaga cgcagatctg caggcatgat cattatgctg 420attccaacag tgatggcgtt ccatttaacc acacgtaacg gagaaccaca catgatcgtc 480agcagacaag agaaagggaa aagtcttctg tttaaaacag aggttggcgt gaacatgtgt 540accctcatgg ccatggacct tggtgaattg tgtgaagaca caatcacgta caagtgtccc 600cttctcaggc agaatgagcc agaagacata gactgttggt gcaactctac gtccacgtgg 660gtaacttatg ggacgtgtac caccatggga gaacatagaa gagaaaaaag atcagtggca 720ctcgttccac atgtgggaat gggactggag acacgaactg aaacatggat gtcatcagaa 780ggggcctgga aacatgtcca gagaattgaa acttggatct tgagacatcc aggcttcacc 840atgatggcag caatcctggc atacaccata ggaacgacac atttccaaag agccctgatt 900ttcatcttac tgacagctgt cactccttca atgacaatgc gttgcatagg aatgtcaaat 960agagactttg tggaaggggt ttcaggagga agctgggttg acatagtctt agaacatgga 1020agctgtgtga cgacgatggc aaaaaacaaa ccaacattgg attttgaact gataaaaaca 1080gaagccaaac agcctgccac cctaaggaag tactgtatag aggcaaagct aaccaacaca 1140acaacagaat ctcgctgccc aacacaaggg gaacccagcc taaatgaaga gcaggacaaa 1200aggttcgtct gcaaacactc catggtagac agaggatggg gaaatggatg tggactattt 1260ggaaagggag gcattgtgac ctgtgctatg ttcagatgca aaaagaacat ggaaggaaaa 1320gttgtgcaac cagaaaactt ggaatacacc attgtgataa cacctcactc aggggaagag 1380catgcagtcg gaaatgacac aggaaaacat ggcaaggaaa tcaaaataac accacagagt 1440tccatcacag aagcagaatt gacaggttat ggcactgtca caatggagtg ctctccaaga 1500acgggcctcg acttcaatga gatggtgttg ctgcagatgg aaaataaagc ttggctggtg 1560cacaggcaat ggttcctaga cctgccgtta ccatggttgc ccggagcgga cacacaaggg 1620tcaaattgga tacagaaaga gacattggtc actttcaaaa atccccatgc gaagaaacag 1680gatgttgttg ttttaggatc ccaagaaggg gccatgcaca cagcacttac aggggccaca 1740gaaatccaaa tgtcatcagg aaacttactc ttcacaggac atctcaagtg caggctgaga 1800atggacaagc tacagctcaa aggaatgtca tactctatgt gcacaggaaa gtttaaagtt 1860gtgaaggaaa tagcagaaac acaacatgga acaatagtta tcagagtgca atatgaaggg 1920gacggctctc catgcaagat cccttttgag ataatggatt tggaaaaaag acatgtctta 1980ggtcgcctga ttacagtcaa cccaattgtg acagaaaaag atagcccagt caacatagaa 2040gcagaacctc catttggaga cagctacatc atcataggag tagagccggg acaactgaag 2100ctcaactggt ttaagaaagg aagttctatc ggccaaatgt ttgagacaac aatgaggggg 2160gcgaagagaa tggccatttt aggtgacaca gcctgggatt ttggatcctt gggaggagtg 2220tttacatcta taggaaaggc tctccaccaa gtctttggag caatctatgg agctgccttc 2280agtggggttt catggactat gaaaatcctc ataggagtca ttatcacatg gataggaatg 2340aattcacgca gcacctcact gtctgtgaca ctagtattgg tgggaattgt gacactgtat 2400ttgggagtca tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460aaatgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag 2520ttccaaccag aatccccttc aaaactagct tcagctatcc agaaagccca tgaagaggac 2580atttgtggaa tccgctcagt aacaagactg gagaatctga tgtggaaaca aataacacca 2640gaattgaatc acattctatc agaaaatgag gtgaagttaa ctattatgac aggagacatc 2700aaaggaatca tgcaggcagg aaaacgatct ctgcggcctc agcccactga gctgaagtat 2760tcatggaaaa catggggcaa agcaaaaatg ctctctacag agtctcataa ccagaccttt 2820ctcattgatg gccccgaaac agcagaatgc cccaacacaa atagagcttg gaattcgttg 2880gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa attgaaagaa 2940aaacaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060aaagcctctt tcattgaagt taaaaactgc cactggccaa aatcacacac cctctggagc 3120aatggagtgc tagaaagtga gatgataatt ccaaagaatc tcgctggacc agtgtctcaa 3180cacaactata gaccaggcta ccatacacaa ataacaggac catggcatct aggtaagctt 3240gagatggact ttgatttctg tgatggaaca acagtggtag tgactgagga ctgcggaaat 3300agaggaccct ctttgagaac aaccactgcc tctggaaaac tcataacaga atggtgctgc 3360cgatcttgca cattaccacc gctaagatac agaggtgagg atgggtgctg gtacgggatg 3420gaaatcagac cattgaagga gaaagaagag aatttggtca actccttggt cacagctgga 3480catgggcagg tcgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaggaa 3540atgcttagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtaggc 3660gccactatga cggatgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720aaagtcagac caacttttgc agctggacta ctcttgagaa agctgacctc caaggaattg 3780atgatgacta ctataggaat tgtactcctc tcccagagca ccataccaga gaccattctt 3840gagttgactg atgcgttagc cttaggcatg atggtcctca aaatggtgag aaatatggaa 3900aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaacgc agtgatatta 3960caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgttc 4020ttaacatcct cacagcaaaa aacagattgg ataccattag cattgacgat caaaggtctc 4080aatccaacag ctatttttct aacaaccctc tcaagaacca gcaagaaaag gagctggcca 4140ttaaatgagg ctatcatggc agtcgggatg gtgagcattt tagccagttc tctcctaaaa 4200aatgatattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260ctcactggac gatcggccga tttggaactg gagagagcag ccgatgtcaa atgggaagac 4320caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga agatggtagc 4380atgtcgataa aaaatgaaga ggaagaacaa acactgacca tactcattag aacaggattg 4440ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500tgggaagtga agaaacaacg ggccggagta ttgtgggatg ttccttcacc cccacccatg 4560ggaaaggctg aactggaaga tggagcctat agaattaagc aaaaagggat tcttggatat 4620tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680cgtggcgctg ttctaatgca taaaggaaag aggattgaac catcatgggc ggacgtcaag 4740aaagacctaa tatcatatgg aggaggctgg aagttagaag gagaatggaa ggaaggagaa 4800gaagtccagg tattggcact ggagcctgga aaaaatccaa gagccgtcca aacgaaacct 4860ggtcttttca aaaccaacgc cggaacaata ggtgctgtat ctctggactt ttctcctgga 4920acgtcaggat ctccaattat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagcattgaa 5040gacaacccag agatcgaaga tgacattttc cgaaagagaa gactgaccat catggacctc 5100cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga agctataaaa 5160cggggtttga gaacattaat cttggccccc actagagttg tggcagctga aatggaggaa 5220gcccttagag gacttccaat aagataccag accccagcca tcagagctgw gcacaccggg 5280cgggagattg tggacctaat gtgtcatgcc acatttacca tgaggctgct atcaccagtt 5340agagtgccaa actacaacct gattatcatg gacgaagccc atttcacaga cccagcaagt 5400atagcagcta gaggatacat ctcaactcga gtggagatgg gtgaggcagc tgggattttt 5460atgacagcca ctcccccggg aagcagagac ccatttcctc agagcaatgc accaatcata 5520gatgaagaaa gagaaatccc tgaacgctcg tggaattccg gacatgaatg ggtcacggat 5580tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640tgcctgagga aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700tatgtcaaga ctagaaccaa tgattgggac ttcgtggtta caactgacat ttcagaaatg 5760ggtgccaatt tcaaggctga gagggttata gaccccagac gctgcatgaa accagtcata 5820ctaacagatg gtgaagagcg ggtgattctg gcaggaccta tgccagtgac ccactctagt 5880gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgagaatga ccagtacata 5940tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000ctcctagata acatcaacac gccagaagga atcattccta gcatgttcga accagagcgt 6060gaaaaggtgg atgccattga tggcgaatac cgcttgagag gagaagcaag gaaaaccttt 6120gtagacttaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180ggcatcaact acgcagacag aaggtggtgt tttgatggag tcaagaacaa ccaaatccta 6240gaagaaaacg tggaagttga aatctggaca aaagaagggg aaaggaagaa attgaaaccc 6300agatggttgg atgctaggat ctattctgac ccactggcgc taaaagaatt taaggaattt 6360gcagccggaa gaaagtctct gaccctgaac ctaatcacag aaatgggtag gctcccaacc 6420ttcatgactc agaaggcaag agacgcactg gacaacttag cagtgctgca cacggctgag 6480gcaggtggaa gggcgtacaa ccatgctctc agtgaactgc cggagaccct ggagacattg 6540cttttactga cacttctggc tacagtcacg ggagggatct ttttattctt gatgagcgca 6600aggggcatag ggaagatgac cctgggaatg tgctgcataa tcacggctag catcctccta 6660tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720atagttttgc ttattccaga acctgaaaaa cagagaacac cccaagacaa ccaactgacc 6780tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840ctagaaaaaa cgaagaaaga tctcggattg ggaagcattg caacccagca acccgagagc 6900aacatcctgg acatagatct acgtcctgca tcagcatgga cgctgtatgc cgtggccaca 6960acatttgtta caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtgtcccta 7020acagctatag ccaaccaagc cacagtgtta atgggtctcg ggaaaggatg gccattgtca 7080aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140actctcacag cagctctttt cttattggta gcacattatg ccatcatagg gccaggactc 7200caagcaaaag caaccagaga agctcagaaa agagcagcgg cgggcatcat gaaaaaccca 7260actgtcgatg gaataacagt gattgaccta gatccaatac cttatgatcc aaagtttgaa 7320aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtatt gatgatgagg 7380actacatggg ctctgtgtga ggctttaacc ttagctaccg ggcccatctc cacattgtgg 7440gaaggaaatc cagggaggtt ttggaacact accattgcgg tgtcaatggc taacattttt 7500agagggagtt acttggccgg agctggactt ctcttttcta ttatgaagaa cacaaccaac 7560acaagaaggg gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg 7620aacgcattgg gaaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740ggctcagcaa aactgagatg gttcgttgag agaaacatgg tcacaccaga agggaaagta 7800gtggacctcg gttgtggcag aggaggctgg tcatactatt gtggaggact aaagaatgta 7860agagaagtca aaggcctaac aaaaggagga ccaggacacg aagaacccat ccccatgtca 7920acatatgggt ggaatctagt gcgtcttcaa agtggagttg acgttttctt catcccgcca 7980gaaaagtgtg acacattatt gtgtgacata ggggagtcat caccaaatcc cacagtggaa 8040gcaggacgaa cactcagagt ccttaactta gtagaaaatt ggttgaacaa caacactcaa 8100ttttgcataa aggttctcaa cccatatatg ccctcagtca tagaaaaaat ggaagcacta 8160caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220atgtactggg tatccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaagg 8280atgttgatca acagatttac aatgagatac aagaaagcca cttacgagcc ggatgttgac 8340ctcggaagcg gaacccgtaa catcgggatt gaaagtgaga taccaaacct agatataatt 8400gggaaaagaa tagaaaaaat aaagcaagag catgaaacat catggcacta tgaccaagac 8460cacccataca aaacgtgggc ataccatggt agctatgaaa caaaacagac tggatcagca 8520tcatccatgg tcaacggagt ggtcaggctg ctgacaaaac cttgggacgt tgtccccatg 8580gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagag 8640aaagtggaca cgagaaccca agaaccgaaa gaaggcacga agaaactaat gaaaataaca 8700gcagagtggc tttggaaaga attagggaag aaaaagacac ccaggatgtg caccagagaa 8760gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccatattcac tgatgagaac 8820aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940agagagaaga agctagggga attcggcaag gcaaaaggca gcagagccat atggtacatg 9000tggcttggag cacgcttctt agagtttgaa gccctaggat tcttaaatga agatcactgg 9060ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120attctaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180tgggatacaa gaatcacact agaagaccta aaaaatgaag aaatggtaac aaaccacatg 9240gaaggagaac acaagaaact agccgaggcc attttcaaac taacgtacca aaacaaggtg 9300gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360caaagaggta gtggacaagt tggcacctat ggactcaata ctttcaccaa tatggaagcc 9420caactaatca gacagatgga gggagaagga gtctttaaaa gcattcagca cctaacaatc 9480acagaagaaa tcgctgtgca aaactggtta gcaagagtgg ggcgcgaaag gttatcaaga 9540atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600ttaacagctc taaatgacat gggaaagatt aggaaagaca tacaacaatg ggaaccttca 9660agaggatgga atgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720atgaaagacg gtcgcgtact cgttgttcca tgtagaaacc aagatgaact gattggcaga 9780gcccgaatct cccaaggagc agggtggtct ttgcgggaga cggcctgttt ggggaagtct 9840tacgcccaaa tgtggagctt gatgtacttc cacagacgcg acctcaggct ggcggcaaat 9900gctatttgct cggcagtacc atcacattgg gttccaacaa gtcgaacaac ctggtccata 9960catgctaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggttaac aagcagggcc 10140acctgggcaa agaacatcca agcagcaata aatcaagtta gatcccttat aggcaatgaa 10200gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaagcagga 10260gttctgtggt agaaagcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc 10320catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380ggccatcata aatgccatag cttgagtaaa ctatgcagcc tgtagctcca cctgagaagg 10440tgtaaaaaat ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500ggttagagga gacccctccc ttacaaatcg cagcaacaat gggggcccaa ggcgagatga 10560agctgtagtc tcgctggaag gactagaggt tagaggagac ccccccgaaa caaaaaacag 10620catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca 10680gaacgccaga aaatggaatg gtgctgttga atcaacaggt tct 107233910735DNADengue virusmisc_feature(1)..(10735)VDV1 39agttgttagt ctacgtggac cgacaagaac agtttcgaat cggaagcttg cttaacgtag 60ttctaacagt tttttattag agagcagatc tctgatgatc aaccaacgaa aaaagacggg 120tcgaccgtct ttcaatatgc tgaaacgcgc gagaaaccgc gtgtcaactg tttcacagtt 180ggcgaagaga ttctcaaaag gattgctctc aggccaagga cccatgaaat tggtgatggc 240tttcatagca ttcttaagat ttctagccat acccccaaca gcaggaattt tggctagatg 300gggctcattc aagaagaatg gagcgattaa agtgttacgg ggtttcaaga gagaaatctc 360aaacatgcta aacataatga acaggaggaa aagatccgtg accatgctcc ttatgctgct 420gcccacagcc ctggcgttcc atctgacgac acgaggggga gagccgcata tgatagttag 480caagcaggaa agaggaaagt cacttttgtt caagacctct gcaggtgtca acatgtgcac 540cctcattgcg atggatttgg gagagttgtg tgaggacacg atgacctaca aatgcccccg 600gatcactgag gcggaaccag atgacgttga ctgttggtgc aatgccacgg acacatgggt 660gacctatgga acgtgctctc aaactggcga acaccgacga gacaaacgtt ccgtcgcatt 720ggccccacac gtggggcttg gcctagaaac aagagccgaa acgtggatgt cctctgaagg 780tgcttggaaa cagatacaaa aagtagagac ttgggctctg agacatccag gattcacggt 840gatagccctt tttctagcac atgccatagg aacatccatc acccagaaag ggatcatttt 900cattttgctg atgctggtaa caccatctat ggccatgcga tgcgtgggaa taggcaacag 960agacttcgtg gaaggactgt caggagcaac atgggtggat gtggtactgg agcatggaag 1020ttgcgtcacc accatggcaa aaaacaaacc aacactggac attgaactct tgaagacgga 1080ggtcacaaac cctgcagttc tgcgtaaatt gtgcattgaa gctaaaatat caaacaccac 1140caccgattcg agatgtccaa cacaaggaga agccacactg gtggaagaac aagacgcgaa 1200ctttgtgtgc cgacgaacgt tcgtggacag aggctggggc aatggctgtg ggctattcgg 1260aaaaggtagt ctaataacgt gtgccaagtt taagtgtgtg acaaaactag aaggaaagat 1320agctcaatat gaaaacctaa aatattcagt gatagtcacc gtccacactg gagatcagca 1380ccaggtggga aatgagacta cagaacatgg aacaactgca accataacac ctcaagctcc 1440tacgtcggaa atacagctga ccgactacgg aacccttaca ttagattgtt cacctaggac 1500agggctagat tttaacgaga tggtgttgct gacaatgaaa aagaaatcat ggcttgtcca 1560caaacagtgg tttctagact taccactgcc ttggacctct ggggctttaa catcccaaga 1620gacttggaac agacaagatt tactggtcac atttaagaca gctcatgcaa agaagcagga 1680agtagtcgta ctaggatcac aagaaggagc aatgcacact gcgctgactg gagcgacaga 1740aatccaaacg tcaggaacga caacaatttt cgcaggacac ctaaaatgca gactaaaaat 1800ggacaaacta actttaaaag ggatgtcata tgtgatgtgc acaggctcat tcaagttaga 1860gaaagaagtg gctgagaccc agcatggaac tgttctggtg caggttaaat atgaaggaac 1920agacgcacca

tgcaagattc ccttttcgac ccaagatgag aaaggagcaa cccagaatgg 1980gagattaata acagccaacc ccatagtcac tgacaaagaa aaaccagtca atattgaggc 2040agaaccaccc tttggtgaga gctacatcgt ggtaggagca ggtgaaaaag ctttgaaact 2100aagctggttc aagaaaggaa gcagcatagg gaaaatgttt gaagcaactg cccgaggagc 2160acgaaggatg gccattctgg gagacaccgc atgggacttc ggttctatag gaggagtgtt 2220cacgtctatg ggaaaactgg tacaccaggt ttttggaact gcatatggag ttttgtttag 2280cggagtttct tggaccatga aaataggaat agggattctg ctgacatggc taggattaaa 2340ttcaaggaac acgtcccttt cggtgatgtg catcgcagtt ggcatggtca cactgtacct 2400aggagtcatg gttcaggcag attcgggatg tgtaatcaac tggaaaggca gagaacttaa 2460atgtggaagc ggcatttttg tcactaatga agttcacact tggacagagc aatacaaatt 2520ccaggctgac tcccccaaga gactatcagc agccattggg aaggcatggg aggagggtgt 2580gtgtggaatc cgatcagcca ctcgtctcga gaacatcatg tggaaacaaa tatcaaatga 2640attgaaccac atcctacttg aaaatgacat gaaatttaca gtggtcgtgg gagacgttag 2700tggaatcttg gcccaaggaa aaaaaatgat taggccacaa cccatggaac acaaatactc 2760gtggaaaagc tggggaaaag ctaaaatcat aggagcggat gtacagaaca ccaccttcat 2820catcgacggc ccaaacaccc cagaatgccc tgacaatcaa agagcatgga atatttggga 2880agtagaggac tatggatttg ggattttcac gacaaacata tggttgaaat tgcgtgactc 2940ctacacccaa gtatgtgacc accggctgat gtcagctgcc attaaggaca gcaaggcagt 3000ccatgctgac atggggtact ggatagaaag tgaaaagaac gagacatgga agttggcgag 3060agcctccttt atagaagtta agacatgcat ctggccaaaa tcccacactc tatggagcaa 3120tggagttctg gaaagtgaaa tgataattcc aaagatatat ggaggaccaa tatctcagca 3180caactacaga ccaggatatt tcacacaaac agcagggccg tggcacctag gcaagttgga 3240actagatttc gatttttgtg aaggtaccac agttgttgtg gatgaacatt gtggaaatcg 3300aggaccatct ctcagaacca caacagtcac aggaaagata atccatgaat ggtgctgcag 3360atcttgtacg ctaccccccc tacgtttcaa aggggaagac gggtgttggt acggcatgga 3420aatcagacca gtgaaggaca aggaagagaa cctggtcaag tcaatggtct ctgcagggtc 3480aggagaagtg gacagctttt cactaggact gctatgcata tcaataatga ttgaagaagt 3540gatgagatcc agatggagca aaaaaatgct gatgactgga acactggctg tgttcctcct 3600tcttataatg ggacaattga catggagtga tctgatcagg ttatgtatta tggttggagc 3660caacgcttca gacaagatgg ggatgggaac aacgtaccta gctttaatgg ccactttcaa 3720aatgagacca atgttcgccg tcgggctatt atttcgcaga ctaacatcta gagaagttct 3780tcttcttaca attggcttga gcctggtggc atccgtggag ctaccaagtt ccctagagga 3840gctgggggat ggacttgcaa taggcatcat gatgttgaaa ttattgactg attttcagtc 3900acaccagcta tgggctactc tgctatcctt gacatttatt aaaacaactt tttcattgca 3960ctatgcatgg aagacaatgg ctatggtact gtcaattgta tctctcttcc ctttatgcct 4020gtccacgacc tctcaaaaaa caacatggct tccggtgctg ttgggatctc ttggatgcaa 4080accactaccc atgtttctta taacagaaaa caaaatctgg ggaaggaaga gttggcccct 4140caatgaagga attatggctg ttggaatagt tagtattcta ctaagttcac ttttaaaaaa 4200tgatgtgccg ctagccggcc cattaatagc tggaggcatg ctaatagcat gttatgtcat 4260atccggaagc tcagctgatt tatcactgga gaaagcggct gaggtctcct gggaggaaga 4320agcagaacac tcaggcgcct cacacaacat actagtagag gttcaagatg atggaaccat 4380gaagataaaa gatgaagaga gagatgacac gctcaccatt ctccttaaag caactctgct 4440ggcagtctca ggggtgtacc caatgtcaat accagcgacc ctttttgtgt ggtatttttg 4500gcagaaaaag aaacagagat caggagtgct atgggacaca cccagccccc cagaagtgga 4560aagagcagtt cttgatgatg gcatctatag aattttgcaa agaggactgt tgggcaggtc 4620ccaagtagga gtaggagttt tccaagaagg cgtgttccac acaatgtggc acgtcactag 4680gggagctgtc ctcatgtatc aaggaaaaag gctggaacca agctgggcca gtgtcaaaaa 4740agacttgatc tcatatggag gaggttggag gtttcaagga tcctggaaca cgggagaaga 4800agtacaggtg attgctgttg aaccgggaaa aaaccccaaa aatgtacaaa caacgccggg 4860taccttcaag acccctgaag gcgaagttgg agccatagcc ttagacttta aacctggcac 4920atctggatct cccatcgtaa acagagaggg aaaaatagta ggtctttatg gaaatggagt 4980ggtgacaaca agcggaactt acgttagtgc catagctcaa gctaaggcat cacaagaagg 5040gcctctacca gagattgagg acaaggtgtt taggaaaaga aacttaacaa taatggacct 5100acatccagga tcgggaaaaa caagaagata ccttccagcc atagtccgtg aggccataaa 5160aaggaagctg cgcacgctaa tcctagctcc cacaagagtt gtcgcttctg aaatggcaga 5220ggcactcaag ggagtgccaa taaggtatca gacaacagca gtgaagagtg aacacacagg 5280aaaggagata gttgacctta tgtgccacgc cactttcacc atgcgcctcc tgtctcccgt 5340gagagttccc aattataaca tgattatcat ggatgaagca cacttcaccg atccagccag 5400catagcagcc agagggtaca tctcaacccg agtgggtatg ggtgaagcag ctgcgatctt 5460tatgacagcc actcccccag gatcggtgga ggcctttcca cagagcaatg caattatcca 5520agatgaggaa agagacattc ctgagagatc atggaactca ggctatgact ggatcactga 5580ttttccaggt aaaacagtct ggtttgttcc aagcatcaaa tcaggaaatg acattgccaa 5640ctgtttaaga aaaaacggga aacgggtgat ccaattgagc agaaaaacct ttgacactga 5700gtaccagaaa acaaaaaaca acgactggga ctatgtcgtc acaacagaca tttccgaaat 5760gggagcaaat ttccgggccg acagggtaat agacccaagg cggtgtctga aaccggtaat 5820actaaaagat ggtccagagc gcgtcattct agccggaccg atgccagtga ctgtggccag 5880tgccgcccag aggagaggaa gaattggaag gaaccaaaac aaggaaggtg atcagtatat 5940ttacatggga cagcctttaa aaaatgatga ggaccacgct cattggacag aagcaaagat 6000gctccttgac aatataaaca caccagaagg gattatccca gccctctttg agccggagag 6060agaaaagagt gcagctatag acggggaata cagactgcgg ggtgaagcaa ggaaaacgtt 6120cgtggagctc atgagaagag gggatctacc agtctggcta tcctacaaag ttgcctcaga 6180aggcttccag tactccgaca gaaggtggtg cttcgatggg gaaaggaaca accaggtgtt 6240ggaggagaac atggacgtgg agatctggac aaaagaagga gaaagaaaga aactacgacc 6300tcgctggttg gacgccagaa catactctga cccactggct ctgcgcgagt ttaaagagtt 6360tgcagcagga agaagaagcg tctcaggtga cctaatatta gaaataggga aacttccaca 6420acatttgacg caaagggccc agaatgcttt ggacaacttg gtcatgttgc acaattccga 6480acaaggagga aaagcctata gacatgctat ggaagaactg ccagacacaa tagaaacgtt 6540gatgctccta gccttgatag ctgtgttgac tggtggagtg acgctgttct tcctatcagg 6600aagaggtcta ggaaaaacat ctatcggctt actctgcgtg atggcctcaa gcgcactgtt 6660atggatggcc agtgtggagc cccattggat agcggcctcc atcatactgg agttctttct 6720gatggtactg cttattccag agccagacag acagcgcact ccacaggaca accagctagc 6780atatgtggtg ataggtctgt tattcgtgat attgacagtg gcagccaatg agatgggatt 6840attggaaacc acaaagaaag acctggggat tggccatgta gctgctgaaa accaccacca 6900tgctacaatg ctggacgtag acctacatcc agcttcagcc tggaccctct atgcagtggc 6960cacaacaatc atcactccta tgatgagaca cacaattgaa aacacaacgg caaatatttc 7020cctgacagcc atcgcaaacc aagcagctat attgatggga cttgacaagg gatggccaat 7080atcgaagatg gacataggag ttccacttct cgccttgggg tgctattccc aagtgaatcc 7140gctgacactg atagcggcag tattgatgct agtagctcat tacgccataa ttggacctgg 7200actgcaagca aaagctacta gagaagctca aaaaagaaca gcggctggaa taatgaaaaa 7260tccaactgtc gacgggattg ttgcaataga cttagatccc gtggtttacg atgcaaaatt 7320tgaaaaacag ctaggccaaa taatgttgtt gatactttgc acatcacaga ttcttttgat 7380gcggactaca tgggccttgt gtgaatccat cacattggct actggacctc tgaccactct 7440ttgggaggga tctccaggaa aattctggaa caccacaata gcggtatcca tggcaaacat 7500tttcaggggg agttatctag caggagcagg tctggccttc tcattaatga aatctctagg 7560aggaggtagg agaggcacgg gagcccaagg ggaaacactg ggagaaaaat ggaaaagaca 7620actaaaccaa ctgagcaagt cagaattcaa tacttacaag aggagtggga ttatggaggt 7680ggatagatcc gaagccaaag agggactgaa aagaggagaa acaaccaaac acgcagtatc 7740gagaggaacg gccaaactga ggtggttcgt ggagaggaac cttgtgaaac cagaagggaa 7800agtcatagac ctcggttgtg gaagaggtgg ctggtcatat tattgcgctg ggctgaagaa 7860agtcacagaa gtgaaaggat acacaaaagg aggacctgga catgaggaac caatcccaat 7920ggcgacctat ggatggaacc tagtaaggct gcactccgga aaagatgtat tttttatacc 7980acctgagaaa tgtgacaccc ttttgtgtga tattggtgag tcctctccga acccaactat 8040agaggaagga agaacgttac gtgttctgaa aatggtggaa ccatggctca gaggaaacca 8100attttgcata aaaattctaa atccctatat gccgagcgtg gtagaaactc tggaacaaat 8160gcaaagaaaa catggaggaa tgctagtgcg aaacccactc tcaagaaatt ccacccatga 8220aatgtactgg gtttcatgtg gaacaggaaa cattgtgtca gcagtaaaca tgacatctag 8280aatgttgcta aatcggttca caatggctca caggaagcca acatatgaaa gagacgtgga 8340cttaggcgct ggaacaagac atgtggcagt agaaccagag gtagccaacc tagatatcat 8400tggccagagg atagagaata taaaaaatga acataagtca acatggcatt atgatgagga 8460caatccatac aaaacatggg cctatcatgg atcatatgag gttaagccat caggatcggc 8520ctcatccatg gtcaatggcg tggtgagatt gctcaccaaa ccatgggatg ttatccccat 8580ggtcacacaa atagccatga ctgataccac accctttgga caacagaggg tgtttaaaga 8640gaaagttgac acgcgcacac caaaagcaaa acgtggcaca gcacaaatta tggaagtgac 8700agccaggtgg ttatggggtt tcctttctag aaacaaaaaa cccagaattt gcacaagaga 8760ggagtttaca agaaaagtta ggtcaaacgc agctattgga gcagtgttcg ttgatgaaaa 8820tcaatggaac tcggcaaaag aagcagtgga agacgaacgg ttctgggaac ttgtccacag 8880agagagggag cttcataaac aggggaaatg tgccacgtgt gtctacaata tgatggggaa 8940gagagagaaa aaattaggag agttcggaaa ggcaaaagga agtcgtgcaa tatggtacat 9000gtggttggga gcacgcttcc tagagtttga agcccttggt ttcatgaatg aagatcactg 9060gttcagtaga gagaattcac tcagtggagt ggaaggagaa ggactccaca aacttggata 9120catactcaga gacatatcaa ggattccagg ggggaacatg tatgcagatg acacagccgg 9180atgggacaca agaataacag aggatgatct ccagaatgag gctaaaatca ctgacatcat 9240ggagcccgaa catgccctgc tggctacgtc aatctttaag ctgacctacc aaaataaggt 9300ggtaagggtg cagagaccag caaaaaatgg aaccgtgatg gatgttatat ccagacgtga 9360ccagagaggc agtggacagg ttggaactta tggcttaaac actttcacca acatggaggc 9420ccaactgata agacaaatgg agtctgaggg aatcttttta cccagcgaat tggaaacccc 9480aaatctagcc ggaagagttc tcgactggtt ggaaaaatat ggtgtcgaaa ggctgaaaag 9540aatggcaatc agcggagatg actgtgtggt gaaaccaatt gatgacaggt tcgcaacagc 9600cttaacagct ttgaatgaca tgggaaaagt aagaaaagac ataccacaat gggaaccttc 9660aaaaggatgg aatgattggc aacaagtgcc tttctgttca caccacttcc accagctaat 9720tatgaaggat gggagggaga tagtggtgcc atgccgcaac caagatgaac ttgtggggag 9780ggccagagta tcacaaggcg ccggatggag cctgagagaa accgcatgcc taggcaagtc 9840atatgcacaa atgtggcagc tgatgtattt ccacaggaga gacctgagac tggcggctaa 9900cgctatttgt tcagccgttc cagttgattg ggtcccaacc agccgcacca cctggtcgat 9960ccatgcccat caccaatgga tgacaacaga agacatgtta tcagtatgga atagggtctg 10020gatagaggaa aacccatgga tggaggataa gactcatgtg tccagttggg aagaagttcc 10080atacctagga aagagggaag atcagtggtg tggatccctg ataggcttaa cagcaagggc 10140cacctgggcc actaatatac aagtggccat aaaccaagtg agaaggctca ttgggaatga 10200gaattatcta gattacatga catcaatgaa gagattcaag aatgagagtg atcccgaagg 10260ggcactctgg taagtcaaca cattcacaaa ataaaggaaa ataaaaaatc aaatgaggca 10320agaagtcagg ccagattaag ccatagtacg gtaagagcta tgctgcctgt gagccccgtc 10380caaggacgta aaatgaagtc aggccgaaag ccacggtttg agcaagccgt gctgcctgtg 10440gctccatcgt ggggatgtaa aaacccggga ggctgcaacc catggaagct gtacgcatgg 10500ggtagcagac tagtggttag aggagacccc tcccaagaca caacgcagca gcggggccca 10560acaccagggg aagctgtacc ctggtggtaa ggactagagg ttagaggaga ccccccgcgt 10620aacaataaac agcatattga cgctgggaga gaccagagat cctgctgtct ctacagcatc 10680attccaggca cagaacgcca gaaaatggaa tggtgctgtt gaatcaacag gttct 107354010735DNADengue virusmisc_feature(1)..(10735)LAV1/PDK13 40agttgttagt ctacgtggac cgacaagaac agtttcgaat cggaagcttg cttaacgtag 60ttctaacagt tttttattag agagcagatc tctgatgatc aaccaacgaa aaaagacggg 120tcgaccgtct ttcaatatgc tgaaacgcgc gagaaaccgc gtgtcaactg tttcacagtt 180ggcgaagaga ttctcaaaag gattgctctc aggccaagga cccatgaaat tggtgatggc 240tttcatagca ttcttaagat ttctagccat acccccaaca gcaggaattt tggctagatg 300gggctcattc aagaagaatg gagcgattaa agtgttacgg ggtttcaaga gagaaatctc 360aaacatgcta aacataatga acaggaggaa aagatccgtg accatgctcc ttatgctgct 420gcccacagcc ctggcgttcc atctgacgac acgaggggga gagccgcata tgatagttag 480caagcaggaa agaggaaagt cacttttgtt caagacctct gcaggtgtca acatgtgcac 540cctcattgcg atggatttgg gagagttgtg tgaggacacg atgacctaca aatgcccccg 600gatcactgag gcggaaccag atgacgttga ctgttggtgc aatgccacgg acacatgggt 660gacctatgga acgtgctctc aaactggcga acaccgacga gacaaacgtt ccgtcgcatt 720ggccccacac gtggggcttg gcctagaaac aagagccgaa acgtggatgt cctctgaagg 780tgcttggaaa cagatacaaa aagtagagac ttgggctctg agacatccag gattcacggt 840gatagccctt tttctagcac atgccatagg aacatccatc acccagaaag ggatcatttt 900cattttgctg atgctggtaa caccatctat ggccatgcga tgcgtgggaa taggcaacag 960agacttcgtg gaaggactgt caggagcaac atgggtggat gtggtactgg agcatggaag 1020ttgcgtcacc accatggcaa aaaacaaacc aacactggac attgaactct tgaagacgga 1080ggtcacaaac cctgcagttc tgcgtaaatt gtgcattgaa gctaaaatat caaacaccac 1140caccgattcg agatgtccaa cacaaggaga agccacactg gtggaagaac aagacgcgaa 1200ctttgtgtgc cgacgaacgt tcgtggacag aggctggggc aatggctgtg ggctattcgg 1260aaaaggtagt ctaataacgt gtgccaagtt taagtgtgtg acaaaactag aaggaaagat 1320agctcaatat gaaaacctaa aatattcagt gatagtcacc gtccacactg gagatcagca 1380ccaggtggga aatgagacta cagaacatgg aacaactgca accataacac ctcaagctcc 1440tacgtcggaa atacagctga ccgactacgg aacccttaca ttagattgtt cacctaggac 1500agggctagat tttaacgaga tggtgttgct gacaatgaaa aagaaatcat ggcttgtcca 1560caaacagtgg tttctagact taccactgcc ttggacctct ggggctttaa catcccaaga 1620gacttggaac agacaagatt tactggtcac atttaagaca gctcatgcaa agaagcagga 1680agtagtcgta ctaggatcac aagaaggagc aatgcacact gcgctgactg gagcgacaga 1740aatccaaacg tcaggaacga caacaatttt cgcaggacac ctaaaatgca gactaaaaat 1800ggacaaacta actttaaaag ggatgtcata tgtgatgtgc acaggctcat tcaagttaga 1860gaaagaagtg gctgagaccc agcatggaac tgttctggtg caggttaaat atgaaggaac 1920agacgcacca tgcaagattc ccttttcgac ccaagatgag aaaggagcaa cccagaatgg 1980gagattaata acagccaacc ccatagtcac tgacaaagaa aaaccagtca atattgaggc 2040agaaccaccc tttggtgaga gctacatcgt ggtaggagca ggtgaaaaag ctttgaaact 2100aagctggttc aagaaaggaa gcagcatagg gaaaatgttt gaagcaactg cccgaggagc 2160acgaaggatg gccattctgg gagacaccgc atgggacttc ggttctatag gaggagtgtt 2220cacgtctatg ggaaaactgg tacaccaggt ttttggaact gcatatggag ttttgtttag 2280cggagtttct tggaccatga aaataggaat agggattctg ctgacatggc taggattaaa 2340ttcaaggaac acgtcccttt cggtgatgtg catcgcagtt ggcatggtca cactgtacct 2400aggagtcatg gttcaggcag attcgggatg tgtaatcaac tggaaaggca gagaacttaa 2460atgtggaagc ggcatttttg tcactaatga agttcacact tggacagagc aatacaaatt 2520ccaggctgac tcccccaaga gactatcagc agccattggg aaggcatggg aggagggtgt 2580gtgtggaatc cgatcagcca ctcgtctcga gaacatcatg tggaaacaaa tatcaaatga 2640attgaaccac atcctacttg aaaatgacat gaaatttaca gtggtcgtgg gagacgttag 2700tggaatcttg gcccaaggga aaaaaatgat taggccacaa cccatggaac acaaatactc 2760gtggaaaagc tggggaaaag ctaaaatcat aggagcggat gtacagaaca ccaccttcat 2820catcgacggc ccaaacaccc cagaatgccc tgacaatcaa agagcatgga atatttggga 2880agtagaggac tatggatttg ggattttcac gacaaacata tggttgaaat tgcgtgactc 2940ctacacccaa gtatgtgacc accggctgat gtcagctgcc attaaggaca gcaaggcagt 3000ccatgctgac atggggtact ggatagaaag tgaaaagaac gagacatgga agttggcgag 3060agcctccttt atagaagtta agacatgcat ctggccaaaa tcccacactc tatggagcaa 3120tggagttctg gaaagtgaaa tgataattcc aaagatatat ggaggaccaa tatctcagca 3180caactacaga ccaggatatt tcacacaaac agcagggccg tggcacctag gcaagttgga 3240actagatttc gatttttgtg aaggtaccac agttgttgtg gatgaacatt gtggaaatcg 3300aggaccatct ctcagaacca caacagtcac aggaaagata atccatgaat ggtgctgcag 3360atcttgtacg ctaccccccc tacgtttcaa aggggaagac gggtgttggt acggcatgga 3420aatcagacca gtgaaggaca aggaagagaa cctggtcaag tcaatggtct ctgcagggtc 3480aggagaagtg gacagctttt cactaggact gctatgcata tcaataatga ttgaagaagt 3540gatgagatcc agatggagca aaaaaatgct gatgactgga acactggctg tgttcctcct 3600tcttataatg ggacaattga catggagtga tctgatcagg ttatgtatta tggttggagc 3660caacgcttca gacaagatgg ggatgggaac aacgtaccta gctttaatgg ccactttcaa 3720aatgagacca atgttcgccg tcgggctatt atttcgcaga ctaacatcta gagaagttct 3780tcttcttaca attggcttga gcctggtggc atccgtggag ctaccaagtt ccctagagga 3840gctgggggat ggacttgcaa taggcatcat gatgttgaaa ttattgactg attttcagtc 3900acaccagcta tgggctactc tgctatcctt gacatttatt aaaacaactt tttcattgca 3960ctatgcatgg aagacaatgg ctatggtact gtcaattgta tctctcttcc ctttatgcct 4020gtccacgacc tctcaaaaaa caacatggct tccggtgctg ttgggatctc ttggatgcaa 4080accactaccc atgtttctta taacagaaaa caaaatctgg ggaaggaaga gttggcccct 4140caatgaagga attatggctg ttggaatagt tagtattcta ctaagttcac ttttaaaaaa 4200tgatgtgccg ctagccggcc cattaatagc tggaggcatg ctaatagcat gttatgtcat 4260atccggaagc tcagctgatt tatcactgga gaaagcggct gaggtctcct gggaggaaga 4320agcagaacac tcaggcgcct cacacaacat actagtagag gttcaagatg atggaaccat 4380gaagataaaa gatgaagaga gagatgacac gctcaccatt ctccttaaag caactctgct 4440ggcagtctca ggggtgtacc caatgtcaat accagcgacc ctttttgtgt ggtatttttg 4500gcagaaaaag aaacagagat caggagtgct atgggacaca cccagccccc cagaagtgga 4560aagagcagtt cttgatgatg gcatctatag aattttgcaa agaggactgt tgggcaggtc 4620ccaagtagga gtaggagttt tccaagaagg cgtgttccac acaatgtggc acgtcactag 4680gggagctgtc ctcatgtatc aaggaaaaag gctggaacca agctgggcca gtgtcaaaaa 4740agacttgatc tcatatggag gaggttggag gtttcaagga tcctggaaca cgggagaaga 4800agtacaggtg attgctgttg aaccgggaaa aaaccccaaa aatgtacaaa caacgccggg 4860taccttcaag acccctgaag gcgaagttgg agccatagcc ttagacttta aacctggcac 4920atctggatct cccatcgtaa acagagaggg aaaaatagta ggtctttatg gaaatggagt 4980ggtgacaaca agcggaactt acgttagtgc catagctcaa gctaaggcat cacaagaagg 5040gcctctacca gagattgagg acaaggtgtt taggaaaaga aacttaacaa taatggacct 5100acatccagga tcgggaaaaa caagaagata ccttccagcc atagtccgtg aggccataaa 5160aaggaagctg cgcacgctaa tcctagctcc cacaagagtt gtcgcttctg aaatggcaga 5220ggcactcaag ggagtgccaa taaggtatca gacaacagca gtgaagagtg aacacacagg 5280aaaggagata gttgacctta tgtgccacgc cactttcacc atgcgcctcc tgtctcccgt 5340gagagttccc aattataaca tgattatcat ggatgaagca cacttcaccg atccagccag 5400catagcagcc agagggtaca tctcaacccg agtgggtatg ggtgaagcag ctgcgatctt 5460tatgacagcc actcccccag gatcggtgga ggcctttcca cagagcaatg caattatcca 5520agatgaggaa agagacattc ctgagagatc atggaactca ggctatgact ggatcactga 5580ttttccaggt aaaacagtct ggtttgttcc aagcatcaaa tcaggaaatg acattgccaa 5640ctgtttaaga aaaaacggga aacgggtgat ccaattgagc agaaaaacct ttgacactga 5700gtaccagaaa acaaaaaaca acgactggga ctatgtcgtc acaacagaca tttccgaaat 5760gggagcaaat ttccgggccg acagggtaat agacccaagg cggtgtctga aaccggtaat 5820actaaaagat ggtccagagc gcgtcattct agccggaccg atgccagtga ctgtggccag 5880tgccgcccag aggagaggaa gaattggaag gaaccaaaac aaggaaggtg atcagtatat 5940ttacatggga cagcctttaa acaatgatga ggaccacgct cattggacag aagcaaagat 6000gctccttgac aatataaaca caccagaagg gattatccca gccctctttg agccggagag 6060agaaaagagt gcagctatag acggggaata cagactgcgg ggtgaagcaa ggaaaacgtt 6120cgtggagctc atgagaagag gggatctacc agtctggcta tcctacaaag ttgcctcaga

6180aggcttccag tactccgaca gaaggtggtg cttcgatggg gaaaggaaca accaggtgtt 6240ggaggagaac atggacgtgg agatctggac aaaagaagga gaaagaaaga aactacgacc 6300tcgctggttg gacgccagaa catactctga cccactggct ctgcgcgagt ttaaagagtt 6360tgcagcagga agaagaagcg tctcaggtga cctaatatta gaaataggga aacttccaca 6420acatttgacg caaagggccc agaatgcttt ggacaacttg gtcatgttgc acaattccga 6480acaaggagga aaagcctata gacatgctat ggaagaactg ccagacacaa tagaaacgtt 6540gatgctccta gccttgatag ctgtgttgac tggtggagtg acgctgttct tcctatcagg 6600aagaggtcta ggaaaaacat ctatcggctt actctgcgtg atggcctcaa gcgcactgtt 6660atggatggcc agtgtggagc cccattggat agcggcctcc atcatactgg agttctttct 6720gatggtactg cttattccag agccagacag acagcgcact ccacaggaca accagctagc 6780atatgtggtg ataggtctgt tattcgtgat attgacagtg gcagccaatg agatgggatt 6840attggaaacc acaaagaaag acctggggat tggccatgta gctgctgaaa accaccacca 6900tgctacaatg ctggacgtag acctacatcc agcttcagcc tggaccctct atgcagtggc 6960cacaacaatc atcactccta tgatgagaca cacaattgaa aacacaacgg caaatatttc 7020cctgacagcc atcgcaaacc aagcagctat attgatggga cttgacaagg gatggccaat 7080atcgaagatg gacataggag ttccacttct cgccttgggg tgctattccc aagtgaatcc 7140gctgacactg atagcggcag tattgatgct agtagctcat tacgccataa ttggacctgg 7200actgcaagca aaagctacta gagaagctca aaaaagaaca gcggctggaa taatgaaaaa 7260tccaactgtc gacgggattg ttgcaataga cttagatccc gtggtttacg atgcaaaatt 7320tgaaaaacag ctaggccaaa taatgttgtt gatactttgc acatcacaga ttcttttgat 7380gcggactaca tgggccttgt gtgaatccat cacattggct actggacctc tgaccactct 7440ttgggaggga tctccaggaa aattctggaa caccacaata gcggtatcca tggcaaacat 7500tttcaggggg agttatctag caggagcagg tctggccttc tcattaatga aatctctagg 7560aggaggtagg agaggcacgg gagcccaagg ggaaacactg ggagaaaaat ggaaaagaca 7620actaaaccaa ctgagcaagt cagaattcaa tacttacaag aggagtggga ttatggaggt 7680ggatagatcc gaagccaaag agggactgaa aagaggagaa acaaccaaac acgcagtatc 7740gagaggaacg gccaaactga ggtggttcgt ggagaggaac cttgtgaaac cagaagggaa 7800agtcatagac ctcggttgtg gaagaggtgg ctggtcatat tattgcgctg ggctgaagaa 7860agtcacagaa gtgaaaggat acacaaaagg aggacctgga catgaggaac caatcccaat 7920ggcgacctat ggatggaacc tagtaaagct gcactccgga aaagatgtat tttttatacc 7980acctgagaaa tgtgacaccc ttttgtgtga tattggtgag tcctctccga acccaactat 8040agaggaagga agaacgttac gtgttctgaa aatggtggaa ccatggctca gaggaaacca 8100attttgcata aaaattctaa atccctatat gccgagcgtg gtagaaactc tggaacaaat 8160gcaaagaaaa catggaggaa tgctagtgcg aaacccactc tcaagaaatt ccacccatga 8220aatgtactgg gtttcatgtg gaacaggaaa cattgtgtca gcagtaaaca tgacatctag 8280aatgttgcta aatcggttca caatggctca caggaagcca acatatgaaa gagacgtgga 8340cttaggcgct ggaacaagac atgtggcagt agaaccagag gtagccaacc tagatatcat 8400tggccagagg atagagaata taaaaaatga acataagtca acatggcatt atgatgagga 8460caatccatac aaaacatggg cctatcatgg atcatatgag gttaagccat caggatcggc 8520ctcatccatg gtcaatggcg tggtgagatt gctcaccaaa ccatgggatg ttatccccat 8580ggtcacacaa atagccatga ctgataccac accctttgga caacagaggg tgtttaaaga 8640gaaagttgac acgcgcacac caaaagcaaa acgtggcaca gcacaaatta tggaagtgac 8700agccaggtgg ttatggggtt tcctttctag aaacaaaaaa cccagaattt gcacaagaga 8760ggagtttaca agaaaagtta ggtcaaacgc agctattgga gcagtgttcg ttgatgaaaa 8820tcaatggaac tcggcaaaag aagcagtgga agacgaacgg ttctgggaac ttgtccacag 8880agagagggag cttcataaac aggggaaatg tgccacgtgt gtctacaata tgatggggaa 8940gagagagaaa aaattaggag agttcggaaa ggcaaaagga agtcgtgcaa tatggtacat 9000gtggttggga gcacgcttcc tagagtttga agcccttggt ttcatgaatg aagatcactg 9060gttcagtaga gagaattcac tcagtggagt ggaaggagaa ggactccaca aacttggata 9120catactcaga gacatatcaa ggattccagg ggggaacatg tatgcagatg acacagccgg 9180atgggacaca agaataacag aggatgatct ccagaatgag gctaaaatca ctgacatcat 9240ggagcccgaa catgccctgc tggctacgtc aatctttaag ctgacctacc aaaataaggt 9300ggtaagggtg cagagaccag caaaaaatgg aaccgtgatg gatgttatat ccagacgtga 9360ccagagaggc agtggacagg ttggaactta tggcttaaac actttcacca acatggaggc 9420ccaactgata agacaaatgg agtctgaggg aatcttttta cccagcgaat tggaaacccc 9480aaatctagcc ggaagagttc tcgactggtt ggaaaaatat ggtgtcgaaa ggctgaaaag 9540aatggcaatc agcggagatg actgtgtggt gaaaccaatt gatgacaggt tcgcaacagc 9600cttaacagct ttgaatgaca tgggaaaagt aagaaaagac ataccacaat gggaaccttc 9660aaaaggatgg aatgattggc aacaagtgcc tttctgttca caccacttcc accagctaat 9720tatgaaggat gggagggaga tagtggtgcc atgccgcaac caagatgaac ttgtggggag 9780ggccagagta tcacaaggcg ccggatggag cctgagagaa accgcatgcc taggcaagtc 9840atatgcacaa atgtggcagc tgatgtattt ccacaggaga gacctgagac tggcggctaa 9900cgctatttgt tcagccgttc cagttgattg ggtcccaacc agccgcacca cctggtcgat 9960ccatgcccat caccaatgga tgacaacaga agacatgtta tcagtatgga atagggtctg 10020gatagaggaa aacccatgga tggaggataa gactcatgtg tccagttggg aagaagttcc 10080atacctagga aagagggaag atcagtggtg tggatccctg ataggcttaa cagcaagggc 10140cacctgggcc actaatatac aagtggccat aaaccaagtg agaaggctca ttgggaatga 10200gaattatcta gattacatga catcaatgaa gagattcaag aatgagagtg atcccgaagg 10260ggcactctgg taagtcaaca cattcacaaa ataaaggaaa ataaaaaatc aaatgaggca 10320agaagtcagg ccagattaag ccatagtacg gtaagagcta tgctgcctgt gagccccgtc 10380caaggacgta aaatgaagtc aggccgaaag ccacggtttg agcaagccgt gctgcctgtg 10440gctccatcgt ggggatgtaa aaacccggga ggctgcaacc catggaagct gtacgcatgg 10500ggtagcagac tagtggttag aggagacccc tcccaagaca caacgcagca gcggggccca 10560acaccagggg aagctgtacc ctggtggtaa ggactagagg ttagaggaga ccccccgcgt 10620aacaataaac agcatattga cgctgggaga gaccagagat cctgctgtct ctacagcatc 10680attccaggca cagaacgcca gaaaatggaa tggtgctgtt gaatcaacag gttct 107354110699DNADengue virusmisc_feature(1)..(10699)LAV3 41agttgttagt ctacgtggac cgacaagaac agtttcgact cggaagcttg cttaacgtag 60tgctgacagt tttttattag agagcagatc tctgatgaac aaccaacgga aaaagacggg 120aaaaccgtct atcaatatgc tgaaacgcgt gagaaaccgt gtgtcaactg gatcacagtt 180ggcgaagaga ttctcaagag gattgctgaa cggccaagga ccaatgaaat tggttatggc 240atttatagct ttcctcagat ttctagccat tccaccgaca gcaggagtct tggctagatg 300gggtaccttt aagaagtcgg gggctattaa ggtcttaaaa ggcttcaaga aggagatctc 360aaacatgctg agcattatca acaaacggaa aaagacatcg ctctgtctca tgatgatgtt 420accagcaaca cttgctttcc acttaacttc acgagatgga gagccgcgca tgattgtggg 480gaagaatgaa agaggaaaat ccctactttt caagacagcc tctggaatca acatgtgcac 540actcatagct atggatctgg gagagatgtg tgatgacacg gtcacttaca aatgccccca 600cattaccgaa gtggagcctg aagacattga ctgctggtgc aaccttacat cgacatgggt 660gacttatgga acatgcaatc aagctggaga gcatagacgc gataagagat cagtggcgtt 720agctccccat gttggcatgg gactggacac acgcactcaa acctggatgt cggctgaagg 780agcttggaga caagtcgaga aggtagagac atgggccctt aggcacccag ggtttaccat 840actagcccta tttcttgccc attacatagg cacttccttg acccagaaag tggttatttt 900tatactatta atgctggtta ccccatccat gacaatgaga tgtgtaggag taggaaacag 960agattttgtg gaaggcctat cgggagctac gtgggttgac gtggtgctcg agcacggtgg 1020gtgtgtgact accatggcta agaacaagcc cacgctggac atagagcttc agaagaccga 1080ggccacccaa ctggcgaccc taaggaagct atgcattgag ggaaaaatta ccaacataac 1140aaccgactca agatgtccca cccaagggga agcgatttta cctgaggagc aggaccagaa 1200ctacgtgtgt aagcatacat acgtggacag aggctgggga aacggttgtg gtttgtttgg 1260caagggaagc ttggtgacat gcgcgaaatt tcaatgttta gaatcaatag agggaaaagt 1320ggtgcaacat gagaacctca aatacaccgt catcatcaca gtgcacacag gagaccaaca 1380ccaggtggga aatgaaacgc agggagtcac ggctgagata acaccccagg catcaaccgc 1440tgaagccatt ttacctgaat atggaaccct cgggctagaa tgctcaccac ggacaggttt 1500ggatttcaat gaaatgatct yattgacaat gaagaacaaa gcatggatgg tacatagaca 1560atggttcttt gacttacccc taccatggac atcaggagct acagcagaaa caccaacttg 1620gaacaggaaa gagcttcttg tgacatttaa aaatgcacat gcaaaaaagc aagaagtagt 1680tgttcttgga tcacaagagg gagcaatgca tacagcactg acaggagcta cagagatcca 1740aacctcagga ggcacaagta tctttgcggg gcacttaaaa tgtagactca agatggacaa 1800attggaactc aaagggatga gctatgcaat gtgcttgggt agctttgtgt tgaagaaaga 1860agtctccgaa acgcagcatg ggacaatact cattaaggtt gagtacaaag ggaaagatgc 1920accctgcaag attcctttct ccacggagga tggacaagga aaagctcaca atggcagact 1980gatcacagcc aatccagtgg tgaccaagaa ggaggagcct gtcaacattg aggctgaacc 2040tccttttgga gaaagtaaca tagtaattgg aattggagac aaagccctga aaatcaactg 2100gtacaagaag ggaagctcga ttgggaagat gttcgaggct actgccagag gtgcaaggcg 2160catggccatc ttgggagaca cagcctggga ctttggatca gtgggtggtg ttttgaattc 2220attagggaaa atggtccacc aaatatttgg gagtgcttac acagccctat ttggtggagt 2280ctcctggatg atgaaaattg gaataggtgt cctcttaacc tggatagggt tgaactcaaa 2340aaatacttct atgtcatttt catgcatcgc gataggaatc attacactct atctgggagc 2400cgtggtgcaa gctgacatgg ggtgtgtcat aaactggaaa ggcaaagaac tcaaatgtgg 2460aagtggaatt ttcgtcacta atgaggtcca cacctggaca gagcaataca aatttcaagc 2520agactccccc aagagactgg caacagccat tgcaggcgct tgggaaaatg gagtgtgcgg 2580aattaggtca acaaccagaa tggagaacct cttgtggaag caaatagcca atgaactgaa 2640ttacatatta tgggaaaaca acattaaatt aacggtagtt gtaggcgaca taactggggt 2700cttagagcaa gggaaaagaa cactaacacc acaacccatg gagctaaaat attcttggaa 2760aacatgggga aaggcaaaaa tagtgacagc tgaaacacaa aattcctctt tcataataga 2820tgggccaagc acaccggagt gtccaagtgc ctcaagagca tggaatgtgt gggaggtgga 2880ggattacggg ttcggagttt tcacaaccaa catatggctg aaactccgag aggtgtacac 2940ccaactatgt gaccataggc taatgtcggc agccgtcaag gatgagaggg ctgtacatgc 3000cgacatgggc tattggatag aaagccaaaa gaatgggagt tggaagctag aaaaagcatc 3060cttcatagag gtgaaaacct gcacatggcc aaaatcacac actctctgga gcaatggtgt 3120gctagagagt gacatgatta tcccaaagag tctagctggt cccatttcgc aacacaacca 3180caggcccggg taccacaccc aaacggcagg accctggcac ttaggaaaat tggagctgga 3240cttcaactat tgtgaaggaa caacagttgt catctcagaa aactgtggga caagaggccc 3300atcattgaga acaacaacgg tgtcagggaa gttgatacac gaatggtgct gccgctcgtg 3360cacacttcct cccctacgat acatgggaga agacggctgc tggtatggca tggaaatcag 3420acccattaat gagaaagaag agaatatggt aaagtctcta gcctcagcag ggagtggaaa 3480ggtggacaac ttcacaatgg gtgtcttgtg tttggcaatc ctctttgaag aggtgatgag 3540aggaaaattt gggaaaaaac acatgattgc aggggttctc ttcacgtttg tgctcctcct 3600ctcagggcaa ataacatgga gagacatggc gcacacactc ataatgattg ggtccaacgc 3660ctctgacaga atggggatgg gcgtcactta cctagctcta attgcaacat ttaaaattca 3720gccactcctg gctttgggat tcttcctgag gaaactgaca tctagagaaa atttattgct 3780gggagttggg ttggccatgg cagcaacgtt acgactgcca gaggacattg aacagatggc 3840gaatggaatt gctttggggc tcatggctct taaactgata acacaatttg aaacatacca 3900actatggacg gcattagttt ccctaacgtg ttcaaataca attttcacgt tgactgttgc 3960ctggagaaca gccactctga ttttagccgg aatttcgctt ttgccagtgt gccagtcttc 4020gagcatgagg aaaacagatt ggctcccaat gactgtggca gctatgggag ctcaacccct 4080accacttttt attttcagtc tgaaagatac actcaaaagg agaagctggc cactgaatga 4140gggggtgatg gcagttggac ttgtgagcat tctagctagt tctctcctta ggaatgatgt 4200gcctatggct ggaccattag tggctggggg cttgctgata gcgtgctacg tcataactgg 4260cacgtcagca gacctcactg tagaaaaagc agcagatgta acatgggagg aagaggccga 4320gcaaacagga gtgtcccaca atttaatggt cacagttgat gatgatggaa caatgagaat 4380aaaagatgac gagactgaga acatcttaac agtgctttta aaaacagcac tactaatagt 4440atcaggcatc tttccatact ccatacccgc aacactgttg gtctggcata cttggcaaaa 4500gcaaacccaa agatccggcg tcctatggga cgtacccagc cccccagaga cacagaaagc 4560ggaactggaa gaaggggtct ataggatcaa acagcaagga atttttggga aaacccaagt 4620gggggttgga gtacagaaag aaggagtttt ccacaccatg tggcatgtca caagaggggc 4680agtgttgaca cacaatggga aaagactgga accaaactgg gctagcgtga aaaaagatct 4740gatttcatac ggaggaggat ggagattgag tgcacaatgg aaaaaggggg aggaggtgca 4800ggttattgcc gtagagcctg ggaagaaccc aaagaacttt caaaccatgc caggcatttt 4860tcagacaaca acaggggaaa taggagcaat tgcactggat ttcaagcctg gaacttcagg 4920atctcccatc ataaacagag agggaaaggt agtgggactg tatggcaatg gagtggttac 4980aaagaatgga ggctatgtca gtggaatagc gcaaacaaat gcagaaccag atggaccgac 5040accagagttg gaagaagaga tgttcaaaaa gcgaaatcta accataatgg atctccatcc 5100tgggtcagga aagacgcgga aatatcttcc agctattgtt agagaggcaa tcaagagacg 5160cttaaggact ctaattttgg caccaacaag ggtagttgca gctgagatgg aagaagcatt 5220gaaagggctc ccaataaggt atcaaacaac tgcaacaaaa tctgaacaca caggaagaga 5280gattgttgat ctaatgtgtc acgcaacgtt cacaatgcgc ttgctgtcac cagtcagggt 5340tccaaactac aacttgataa taatggatga ggcccatttc acagacccag ccagtatagc 5400ggctagaggg tacatatcaa ctcgtgtagg aatgggagag gcagccgcaa ttttcatgac 5460agcaacaccc cctggaacag ctgatgcctt tcctcagagc aacgctccaa ttcaagatga 5520agagagagac ataccggaac gctcatggaa ttcaggcaat gaatggatta ctgactttgt 5580tgggaagaca gtgtggtttg tccctagcat caaagccgga aatgacatag caaactgctt 5640gcggaaaaat ggaaaaaagg ttattcaact cagcaggaag acctttgaca cagaatatca 5700aaagaccaaa ctgaatgatt gggactttgt ggtgacaaca gacatttcag aaatgggagc 5760caatttcaaa gcagatagag tgatcgaccc aagaagatgt ctcaagccgg tgattttgac 5820agatggaccc gagcgggtga tcctggctgg accaatgcca gtcaccgtag cgagcgctgc 5880gcaaaggaga gggagagttg gcaggaaccc acaaaaagaa aatgaccagt acatattcat 5940gggccagcct ctcaacaatg atgaagacca tgctcactgg acagaagcaa aaatgctgct 6000ggacaacatc aacacaccag aagggattat accagctctc tttgaaccag aaagggagaa 6060gtcagccgcc atagacggcg aataccgcct gaagggtgag tccaggaaga ctttcgtgga 6120actcatgagg aggggtgacc tcccagtttg gctagcccat aaagtagcat cagaagggat 6180caaatataca gatagaaaat ggtgctttga tggagaacgt aataatcaaa ttttagagga 6240gaatatggat gtggaaatct ggacaaagga aggagaaaag aaaaaactga gacctaggtg 6300gcttgatgcc cgcacttatt cagatccttt agcactcaaa gaattcaagg attttgcagc 6360tggcagaaag tcaatcgccc ttgatcttgt gacagaaata ggaagagtgc cttcacactt 6420agcccacaga acgagaaacg ccctggataa tttggtgatg ctgcacacgt cagaacatgg 6480cggtagggcc tacaggcatg cagtggagga actaccagaa acgatggaaa cactcttact 6540cctgggactg atgatcttgt taacaggtgg agcaatgctc ttcttgatat caggtaaagg 6600gattggaaag acttcaatag gactcatttg tgtaattgct tccagcggca tgttatggat 6660ggctgatgtc ccactccaat ggatcgcatc ggctatagtc ctggagtttt ttatgatggt 6720gttgctcata ccagaaccag aaaagcagag aactccccaa gacaaccaac tcgcatatgt 6780cgtgataggc atacttacat tggctgcaat agtagcggcc aatgaaatgg gactgttgga 6840aactacaaag agagatttag gaatgtctaa agaaccaggt gttgtttctc caaccagcta 6900tttggatgtg gacttgcacc cagcatcagc ctggacattg tacgccgtgg ccacaacagt 6960aataacacca atgttgagac acaccataga gaattccaca gcaaatgtgt ctctggcagc 7020catagctaac caggcagtgg tcctgatggg tttagacaaa ggatggccga tatcgaaaat 7080ggacttgggc gtaccactat tggcactggg ttgctattca caagtgaacc cactaactct 7140tgcagcggca gtacttttgc tagtcacaca ttatgcaatt ataggtccag gattgcaggc 7200aaaagccacc cgtgaagctc agaaaaggac agctgctgga ataatgaaga atccaacggt 7260ggatggaata atgacaatag acctagatcc tgtaatatat gattcaaaat ttgaaaagca 7320actaggacag gtcatgctcc tggttctgtg tgcagtccaa cttttattga tgagaacatc 7380atgggccttg tgtgaagttc taaccctagc cacaggacca ataacaacac tctgggaagg 7440atcacctggg aagttctgga acaccacgat agctgtttcc atggcgaaca tctttagagg 7500gagctattta gcaggagctg ggcttgcttt ttctatcatg aaatcagttg gaacaggaaa 7560gagaggaaca gggtcacaag gtgaaacctt aggagaaaag tggaaaaaga aattaaatca 7620gttatcccgg aaagagtttg acctttacaa gaaatccgga atcaccgaag tggatagaac 7680agaagccaaa gaagggttaa aaagaggaga aataacacac catgccgtgt ccagaggcag 7740cgcaaaactt caatggttcg tggagagaaa catggtcatt cctgaaggaa gagtcataga 7800cctaggctgt ggaagaggag gctggtcata ttactgtgca ggactgaaaa aagttacaga 7860agtgcgagga tacacaaaag gcggcccagg acacgaagaa ccagtaccta tgtctacata 7920cggatggaac atagtcaagt taatgagtgg aaaggatgtt ttttatctgc cacctgaaaa 7980gtgtgatacc ctattgtgtg acattggaga atcttcacca agcccaacag tggaagaaag 8040cagaaccata agagttttga agatggttga accatggcta aagaacaacc agttttgcat 8100taaagtattg aacccataca tgccaactgt gattgagcac ttagaaagac tacaaaggaa 8160acatggagga atgcttgtga gaaatccact ctcacgaaac tccacgcacg aaatgtattg 8220gatatccaat ggtacaggca atatcgtctc ttcagtcaac atggtatcca gattgctact 8280gaacagattc acaatgacac acaggagacc caccatagag aaagatgtgg atctaggagc 8340aggaacccga catgtcaatg cggaaccaga aacacccaac atggatgtca ttggggaaag 8400aataaaaagg atcaaagagg agcatagttc aacatggcac tatgatgatg aaaatcctta 8460caaaacgtgg gcttaccatg gatcctatga agtaaaagcc acaggctcag cctcctccat 8520gataaatgga gtcgtgaaac tcctcacaaa accatgggat gtggtgccca tggtgacaca 8580gatggcaatg acagatacaa ctccattcgg ccagcaaaga gtttttaaag agaaagtgga 8640caccaggaca cctaggccca tgccaggaac aagaaaggtt atggagatca cagcggagtg 8700gctttggagg accctgggaa ggaacaaaag acccagatta tgcacaaggg aggaattcac 8760aaagaaggtc agaaccaacg cagctatggg cgctgtcttc acagaagaga accaatggga 8820cagtgcgaga gctgctgttg aggacgaaga attttggaaa cttgtggaca gagaacgtga 8880actccacaaa ctgggcaagt gtggaagctg cgtttacaac atgatgggca agagagagaa 8940aaaacttgga gagtttggta aagcaaaagg cagtagggct atatggtaca tgtggttggg 9000agccaggtac cttgagttcg aggcgctcgg attcctcaat gaagaccact ggttctcgcg 9060tgaaaactct tacagtggag tagaaggaga aggactgcac aagctgggat acatcttgag 9120agatatttcc aagatacccg gaggagccat gtatgctgat gacacagccg gttgggacac 9180aagaataaca gaagatgacc tgcacaatga ggaaaaaatc acacagcaga tggaccctga 9240acacaggcag ctagcgaacg ctatattcaa gctcacatac caaaacaaag tggtcaaagt 9300ccaacgacca actccaaagg gcacggtaat ggacatcata tctaggaaag accaaagagg 9360cagtggacag gtgggaactt atggtctgaa cacattcacc aacatggaag cccagctaat 9420cagacaaatg gaaggagaag gcgtgttgtc aaaggcagac ctcgagaacc cccatccgct 9480agagaagaaa attacacaat ggttggaaac taaaggagtg gaaaggttaa aaagaatggc 9540catcagcggg gatgattgcg ttgtgaaacc aatcgacgac agattcgcca atgccctgct 9600tgccctgaac gatatgggaa aggttagaaa ggacatacct caatggcagc catcaaaggg 9660atggcatgat tggcaacagg tccccttctg ctcccaccac tttcatgaat tgatcatgaa 9720agatggaaga aagttggtag ttccctgcag accccaggac gaactaatag gaagagcgag 9780aatctcccaa ggagcaggat ggagccttag agaaactgca tgtctaggga aagcctacgc 9840tcaaatgtgg gctctcatgt attttcacag aagagatctt agactagcat ccaacgccat 9900atgttcagca gtaccagtcc actgggtccc cacgagcaga acgacatggt ctattcatgc 9960tcaccatcag tggatgacta cagaagacat gcttactgtc tggaacaggg tgtggataga 10020ggacaatcca tggatggaag acaaaactcc agtcacaacg tgggaagatg ttccatatct 10080agggaagaga gaagaccaat ggtgcggatc actcataggt ctcacttcca gagcaacctg 10140ggcccagaac atactcacag caatccaaca ggtgagaagc ctcataggca atgaagagtt 10200tctggactac atgccttcga tgaagagatt caggaaggag gaggagtcag agggagccat 10260ttggtaaaag caggaggtaa actgtcaggc cacattaagc cacagtacgg aagaagctgt 10320gcagcctgtg agccccgtcc aaggacgtta aaagaagaag tcaggcccaa aagccacggt 10380ttgagcaaac cgtgctgcct gtagctccgt cgtggggacg taaagcctgg gaggctgcaa 10440accgtggaag

ctgtacgcac ggtgtagcag actagtggtt agaggagacc cctcccatga 10500cacaacgcag cagcggggcc cgagcactga gggaagctgt acctccttgc aaaggactag 10560aggttagagg agaccccccg caaacaaaaa cagcatattg acgctgggag agaccagaga 10620tcctgctgtc tcctcagcat cattccaggc acagaacgcc agaaaatgga atggtgctgt 10680tgaatcaaca ggttctagt 106994210648DNADengue virusmisc_feature(1)..(10648)LAV4 42agttgttagt ctgtgtggac cgacaaggac agttccaaat cggaagcttg cttaacacag 60ttctaacagt ttgtttgaat agagagcaga tctctggaaa aatgaaccaa cgaaaaaagg 120tggttagacc acctttcaat atgctgaaac gcgagagaaa ccgcgtatca acccctcaag 180ggttggtgaa gagattctca accggacttt tttctgggaa aggaccctta cggatggtgc 240tagcattcat cacgtttttg cgagtccttt ccatcccacc aacagcaggg attctgaaaa 300gatggggaca gttgaagaaa aataaggcca tcaggatact gattggattc aggaaggaga 360taggccgcat gctgaacatc ttgaacggga gaaaaaggtc aacgataaca ttgctgtgct 420tgattcccac cgtaatggcg tttcacttgt caacaagaga tggcgaaccc ctcatgatag 480tggcaaaaca tgaaaggggg agacctctct tgtttaagac aacagagggg atcaacaaat 540gcactctcat tgccatggac ttgggtgaaa tgtgtgagga cactgtcacg tataaatgcc 600ccttactggt caataccgaa cctgaagaca ttgattgctg gtgcaatctc acgtctacct 660gggtcatgta tgggacatgc acccagagcg gagaacggag acgagagaag cgctcagtag 720ctttaacacc acattcagga atgggattgg aaacaagagc tgagacatgg atgtcatcgg 780aaggggcttg gaagcatgct cagagagtag agagctggat actcagaaac ccaggattcg 840cgctcttggc aggatttatg gcttatatga ttgggcaaac aggaatccag cgaactgtct 900tctttgtcct aatgatgctg gtcgccccat cctacggaat gcgatgcgta ggagtaggaa 960acagagactt tgtggaagga gtctcaggtg gagcatgggt cgatctggtg ctagaacatg 1020gaggatgcgt cacaaccatg gcccagggaa aaccaacctt ggattttgaa ctgactaaga 1080caacagccaa ggaagtggct ctgttaagaa cctattgcat tgaagcctca atatcaaaca 1140taaccacggc aacaagatgt ccaacgcaag gagagcctta tctaaaagag gaacaagacc 1200aacagtacat ctgccggaga gatgtggtag acagagggtg gggcaatggc tgtggcttgt 1260ttggaaaagg aggagttgtg acatgtgcga agttttcatg ttcggggaag ataacaggca 1320atttggtcca aattgagaac cttgaataca cagtggttgt aacagtccac aatggagaca 1380cccatgcagt aggaaatgac acatccaatc atggagttac agccacgata actcccaggt 1440caccatcggt ggaagtcaaa ttgccggact atggagaact aacactcgat tgtgaaccca 1500ggtctggaat tgactttaat gagatgattc tgatgaaaat gaaaaagaaa acatggcttg 1560tgcataagca atggtttttg gatctacctc taccatggac agcaggagca gacacatcag 1620aggttcactg gaattacaaa gagagaatgg tgacatttaa ggttcctcat gccaagagac 1680aggatgtgac agtgctggga tctcaggaag gagccatgca ttctgccctc gctggagcca 1740cagaagtgga ctccggtgat ggaaatcaca tgtttgcagg acatctcaag tgcaaagtcc 1800gtatggagaa attgagaatc aagggaatgt catacacgat gtgttcagga aagttctcaa 1860ttgacaaaga gatggcagaa acacagcatg ggacaacagt ggtgaaagtc aagtatgaag 1920gtgctggagc tccgtgtaaa gtccccatag agataagaga tgtgaacaag aaaaaagtgg 1980ttgggcgtat catctcatcc acccctttgg ctgagaatac caacagtgca accaacatag 2040agttagaacc cccctttggg gacagctaca tagtgatagg tgttggaaac agtgcattaa 2100cactccattg gttcaggaaa gggagttcca ttggcaagat gtttgagtcc acatacagag 2160gtgcaaaacg aatggccatt ctaggtgaaa cagcttggga ttttggttcc gttggtggac 2220tgttcacatc attgggaaag gctgtgcacc aggtttttgg aagtgtgtat acaaccatgt 2280ttggaggagt ctcatggatg attagaatcc taattgggtt cctagtgttg tggattggca 2340cgaactcaag gaacacttca atggctatga cgtgcatagc tgttggagga atcactctgt 2400ttctgggctt cacagttcaa gcagacatgg gttgtgtggt gtcatggagt gggaaagaat 2460tgaagtgtgg aagcggaatt tttgtggttg acaacgtgca cacttggaca gaacagtaca 2520aatttcaacc ggagtcccca gcgagactag cgtctgcaat attgaatgcc cacaaagatg 2580gggtctgtgg aattagatca accacgaggc tggaaaatgt catgtggaag caaataacca 2640acgagctaaa ttatgttctc tgggaaggag gacatgacct cactgtagtg gctggggatg 2700tgaagggggt gttgaccaaa ggcaagagag cactcacacc cccagtgaat gatctgaaat 2760attcatggaa gacatgggga aaagcaaaaa tcttcacccc agaagcaaga aatagcacat 2820ttttaataga cggaccagac acctccgaat gccccaatga acgaagagca tggaactttc 2880ttgaggtgga agactatgga tttggcatgt tcacgaccaa catatggatg aaattccgag 2940aaggaagttc agaagtgtgt gaccacaggt taatgtcagc ggcaattaaa gatcagaaag 3000ctgtgcatgc tgacatgggt tattggatag agagctcaaa aaaccagacc tggcagatag 3060agaaagcatc tcttattgaa gtgaaaacat gtctgtggcc caagacccac acattgtgga 3120gcaatggagt gctggaaagc cagatgctca ttccaaaatc atatgcgggc cctttttcac 3180accacaatta ccgccagggc tatgccacgc aaaccgtggg cccatggcac ttaggcaaat 3240tagagataga ctttggagaa tgccccggaa caacagtcgc aattcaggag gattgtgacc 3300atagaggccc atctttgagg accaccactg catctggaaa actagtcacg caatggtgct 3360gccgctcctg cacgatgcct cccttaaggt tcttgggaga agatgggtgc tggtatggga 3420tggagattag gcccttgagt gaaaaagaag agaacatggt caaatcacag gtaacggccg 3480gacagggcac atcagaaact ttttctatgg gtctgttgtg cctgaccttg tttgtggaag 3540aatgcttgag gagaagagtc actaggaaac acatgatatt ggttgtggtg atcactcttt 3600gtgccatcat cctaggaggc ctcacatgga tggacttact acgagccctc atcatgttgg 3660gggacactat gtctggtaga ataggaggac agatccacct agccatcatg gcagtgttca 3720agatgtcacc aggatacgtg ctgggtgtgt ttttaaggaa actcacttca agagagacag 3780cactaatggt aataggaatg gccatgacaa cggtgctttc aattccacat gaccttatgg 3840aactcattga tggaatatca ctggggctaa ttttgctaaa aatagtgaca cattttgaca 3900acacccaagt gggaacctta gccctttcct tgaccttcat aagatcaaca atgccattgg 3960tcatggcttg gaggaccatt atggctgtgt tgtttgtggt cacactcatt cctttgtgca 4020ggacaagctg tcttcaaaaa cagtctcatt gggtagaaat aacagcactc atcctaggag 4080cccaagctct gccagtgtac ctaatgactc ttatgaaagg agcctcaaga agatcttggc 4140ctcttaacga gggcataatg gctgtgggtt tggttagtct cttaggaagc gctcttttaa 4200agaatgatgt ccctttagct ggcccaatgg tggcaggagg cttacttctg gcggcttacg 4260tgatgagtgg tagctcagca gatctgtcac tagagaaggc cgccaatgtg cagtgggatg 4320aaatggcaga cataacaggc tcaagcccaa tcatagaagt gaagcaggat gaagatggct 4380ctttctccat acgggacgtc gaggaaacca atatgataac ccttttggtg aaactggcac 4440tgataacagt gtcaggtctc taccccttgg caattccagt cacaatgacc ttatggtaca 4500tgtggcaagt gaaaacacaa agatcaggag ccctgtggga cgtcccctca cccgctgcca 4560ctcaaaaagc cgcactgtct gaaggagtgt acaggatcat gcaaagaggg ttatttggga 4620aaactcaggt tggagtaggg atacacatgg aaggtgtatt tcacacaatg tggcatgtaa 4680caagaggatc agtgatctgc catgagactg ggagattgga gccatcttgg gctgacgtca 4740ggaatgacat gatatcatac ggtgggggat ggagacttgg agacaaatgg gacaaagaag 4800aagatgttca ggtcctcgcc atagaaccag gaaaaaatcc taaacatgtc caaacgaaac 4860ccggcctttt caagacccta actggagaaa ttggagcagt aacattagat ttcaaacccg 4920gaacgtctgg ttctcccatc atcaacagga aaggaaaagt catcggactc tatggaaatg 4980gagtagttac caaatcaggt gattacgtca gtgccataac gcaagccgaa agaattggag 5040agccagatta tgaagtggat gaggacattt ttcgaaagaa aagattaact ataatggact 5100tacaccccgg agctggaaag acaaaaagaa ttcttccatc aatagtgaga gaagccttaa 5160aaaggaggct gcgaaccttg attttggctc ccacgagagt ggtggcggcc gagatggaag 5220aggccctacg tggactgcca atccgttatc agaccccagc tgtgaaatca gaacacacag 5280gaagagagat tgtagacctc atgtgtcatg caaccttcac aacaagactt ttgtcatcaa 5340ccagagttcc aaattacaac ctcatagtga tggatgaagc acatttcacc gatccttcta 5400gtgtcgcggc tagaggatac atctcgacca gggtggaaat gggagaggca gcagccatct 5460tcatgaccgc aacccctccc ggagcgacag atccctttcc ccagagcaac agcccaatag 5520aagacatcga gagggaaatt ccggaaaggt catggaacac agggttcgac tggataacag 5580actaccaagg gaaaactgtg tggtttgttc ccagcataaa agctggaaat gacattgcaa 5640attgtttgag aaagtcggga aagaaagtta tccagttgag taggaaaacc tttgatacag 5700agtatccaaa aacgaaactc acggactggg attttgtggt cactacagac atatctgaaa 5760tgggggccaa ttttagagct gggagagtga tagaccctag gagatgcctc aagccagtta 5820tcctaacaga tgggccagag agagtcattt tagcaggtcc tattccagtg actccagcaa 5880gcgctgctca gagaagaggg cgaataggaa ggaacccagc acaagaagac gaccaatacg 5940ttttctccgg agacccacta aaaaatgatg aagatcatgc ccactggaca gaagcaaaga 6000tgctgcttga caatatctac accccagaag ggatcattcc aacattgttt ggtccggaaa 6060gggaaaaaac ccaagccatt gatggagagt ttcgcctcag aggggaacaa aggaagactt 6120ttgtggaatt aatgaggaga ggagaccttc cggtgtggct gagctataag gtagcttctg 6180ctggcatttc ttacaaagat cgggaatggt gcttcacagg ggaaaggaat aaccaaattt 6240tagaagaaaa catggaggtt gaaatttgga ctagagaggg agaaaagaaa aagctaaggc 6300caagatggtt agatgcacgt gtatacgctg accccatggc tttgaaggat tttaaggagt 6360ttgctagtgg aaggaagagc ataactctcg acatcctaac agagattgcc agtttgccaa 6420cttacctttc ctctagggcc aagctcgccc ttgataacat agtcatgctc cacacaacag 6480aaagaggagg gagggcctac caacacgccc tgaacgaact cccggagtca ctggaaacac 6540ttatgcttgt agctttacta ggtgctatga cagcaggtat cttcctgttt ttcatgcaag 6600ggaaaggaat agggaaattg tcaatgggtt tgataaccat tgcggtggct agtggcttgt 6660tctgggtagc agaaattcaa ccccagtgga tagcggcctc aatcatacta gagttttttc 6720tcatggtact gttgataccg gaaccagaaa aacaaaggac cccacaagac aatcaattga 6780tctacgtcat attgaccatt ctcaccatta ttggtctcat agcagccaac gagatggggc 6840tgattgaaaa aacaaaaacg gattttgggt tttaccaggt aaaaacagaa accaccatcc 6900tcgatgtgga cttgagacca gcttcagcat ggacgctcta tgcagtagcc accacawttc 6960tgactcccat gctgagacac accatagaaa acacgtcggc caacctatct ctagcagcca 7020ttgccaacca ggcggccgtc ctaatggggc ttggaaaagg atggccgctc cacagaatgg 7080acctcggtgt gccgctgtta gcaatgggat gctattctca agtgaaccca acaactttga 7140cagcatcctt agtcatgctt tcagtccatt atgcaataat aggtccagga ttgcaggcaa 7200aagccacaag agaggcccag aaaaggacag ctgctgggat catgaaaaac cccacggtgg 7260acgggataac agtaatagat ctagaaccaa tatcctatga cccaaaattt gaaaagcaat 7320tagggcaggt catgctactc gtcttgtgtg ctggacaact actcttgatg agaacaacat 7380gggctttctg tgaagtcttg actttggcca caggaccaat cttgaccttg tgggagggca 7440acccgggaag gttttggaac acgaccatag ccgtatccac cgccaacatt ttcaggggaa 7500gttacctggc gggagctgga ctggcttttt cactcataaa gaatgyacaa acccctagga 7560ggggaactgg gaccacagga gagacactgg gagagaagtg gaagagacag ctaaactcat 7620takacagaaa agagtttgaa gagtataaaa gaagtggaat actagaagtg gacaggactg 7680aagccaagtc tgccctgaaa gatgggtcta aaatcaagca tgcagtatct agagggtcca 7740gtaagattag atggattgtt gagagaggga tggtaaagcc aaaagggaaa gttgtagatc 7800ttggctgtgg gagaggagga tggtcttatt acatggcgac gctcaagaac gtgactgaag 7860tgaaagggta tacaaaagga ggtccaggac atgaagaacc gattcccatg gctacttatg 7920gctggaattt ggtcaaactc cattcagggg ttgacgtgtt ctacaaaccc acagagcaag 7980tggacaccct gctctgtgat attggggagt catcttctaa tccaacaata gaggaaggaa 8040gaacattaag agttttgaag atggtggagc catggctctc ttcaaaacct gaattctgca 8100tcaaagtcct taacccctac atgccaacag tcatagaaga gctggagaaa ctgcagagaa 8160aacatggtgg gaaccttgtc agatgcccgc tgtccaggaa ctccacccat gagatgtatt 8220gggtgtcagg agcgtcggga aacattgtga gctctgtgaa cacaacatca aagatgttgt 8280tgaacaggtt cacaacaagg cataggaaac ccacttatga gaaggacgta gatcttgggg 8340caggaacgag aagtgtctcc actgaaacag aaaaaccaga catgacaatt attgggagaa 8400ggcttcagcg attgcaagag gagcacaaag aaacctggca ttatgatcag gaaaacccat 8460acagaacctg ggcgtatcat ggaagctatg aagctccttc gacaggctct gcatcctcca 8520tggtgaacgg ggtagtaaaa ctgctaacaa aaccttggga tgtggttcca atggtgaccc 8580agttagccat gacagacaca accccttttg ggcaacaaag agtgttcaaa gagaaggtgg 8640ataccagaac accacaacca aaacccggta cacgaatggt tatgaccacg acagccaatt 8700ggctgtgggc cctccttggg aagaagaaaa atcccagact gtgcacaagg gaagagttca 8760tctcaaaagt tagatcaaac gcagccatag gcgcagtctt tcaggaagaa cagggatgga 8820catcagccag tgaagctgtg aatgacagcc ggttttggga actggttgac aaagaaaggg 8880ccctacacca ggaagggaaa tgtgaatcgt gtgtctacaa catgatggga aaacgtgaga 8940aaaagttagg agagtttggc agagccaagg gaagccgagc aatctggtac atgtggctgg 9000gagcgcggtt tctggaattt gaagccctgg gttttttgaa tgaagatcac tggtttggca 9060gagaaaattc atggagtgga gtggaagggg aaggtctgca cagattggga tatatcctgg 9120aggagataga caagaaggat ggagacctaa tgtatgctga tgacacagca ggctgggaca 9180caagaatcac tgaggatgac cttcaaaatg aagaactgat cacggaacag atggcccccc 9240accacaagat cctagccaaa gccattttca aactaaccta tcaaaacaaa gtggtgaaag 9300tcctcagacc cacaccgaga ggagcggtga tggatatcat atccaggaaa gaccaaagag 9360gtagtggaca agttggaaca tatggtttga acacattcac caacatggaa gttcaactca 9420tccgccaaat ggaagctgaa ggagtcatca cacaagatga catgcagaac ccaaaagggt 9480tgaaagaaag agttgagaaa tggctgaaag agtgtggtgt cgacaggtta aagaggatgg 9540caatcagtgg agacgattgc gtggtgaagc ccctggatga gaggtttggc acttccctcc 9600tcttcttgaa cgacatggga aaggtgagga aagacattcc gcagtgggaa ccatctaagg 9660gatggaaaaa ctggcaagag gttccttttt gctcccacca ctttcacaag atcttcatga 9720aggatggccg ctcactagtt gttccatgta gaaaccagga tgaactgata gggagagcca 9780gaatctcgca gggggctgga tggagcttaa gagaaacagc ctgcctgggc aaagcttacg 9840cccagatgtg gtcgctcatg tacttccaca gaagggatct gcgtttagcc tccatggcca 9900tatgctcagc agttccaacg gaatggtttc caacaagcag aacaacatgg tcaatccacg 9960ctcatcatca gtggatgacc actgaagata tgctcaaagt gtggaacaga gtgtggatag 10020aagacaaccc taatatgact gacaagactc cagtccattc gtgggaagat ataccttacc 10080tagggaaaag agaggatttg tggtgtggat ccctgattgg actttcttcc agagccacct 10140gggcgaagaa cattcacacg gccataaccc aggtcagaaa cctgatcgga aaagaggaat 10200acgtggatta catgccagta atgaaaagat acagcgctcc ttcagagagt gaaggagttc 10260tgtaattacc aacaacaaac accaaaggct attgaagtca ggccacttgt gccacggctt 10320gagcaaaccg tgctgcctgt agctccgcca ataatgggag gcgtgaaatc cctagggagg 10380ccatgcgcca cggaagctgt acgcgtggca tattggacta gcggttagag gagacccctc 10440ccatcactga caaaacgcag caaaaggggg cccgaagcca ggaggaagct gtactcctgg 10500tggaaggact agaggttaga ggagaccccc ccaacacaaa aacagcatat tgacgctggg 10560aaagaccaga gatcctgctg tctctgcaac atcaatccag gcacagagcg aagcaagatg 10620gattggtgtt gttgatccaa caggttct 10648

Claims

1. An isolated polyprotein encoded by SEQ ID No.1, or fragments thereof that comprise at least an arginine at position 9 of M protein, and/or a glutamic acid at position 228 of E protein, and/or a threonine at position 69 of NS3 protein, and/or a histidine at position 181 of NS3 protein, and/or a lysine at position 541 of NS5 protein, and/or a threonine at position 832 of NS5 protein.

2. The isolated polyprotein or fragments thereof according to claim 1, wherein the polyprotein is the polyprotein of sequence SEQ ID No. 2.

3. A fragment of the polyprotein according to claim 1 which is at least 20 amino acids long.

4. A fragment of the polyprotein according to claim 1 which comprises M protein, and/or E protein, and/or NS3 protein, and/or NS5 protein.