cDNA sequence of Dengue virus serotype 1 (Singapore strain)

Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...

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4242181, 435 5, 435 691, 435 693, 435 697, 4352523, 43525233, 4352542, 435325, 435348, 4353201, 4352351, 536 2372, 530324, 530350, 5303879, C12N 1540, A61K 3912, C07K 1418, C07K 1610, C12Q 170

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060175354

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BRIEF SUMMARY
The present invention relates to Dengue Virus Type 1. Dengue virus infection may lead to dengue fever (DF) or its more severe dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). DHF is an important virus disease of global significance, especially in Southeast Asia. There are four serotypes of Dengue virus (DEN1, DEN2, DEN3 and DEN4) belonging to the family Flaviviradae.
The complete genomic sequence of DEN2 (Jamaica) has been published by Deubel et al; Virology 165, 234-244 (1988). The complete genomic sequence of DEN3 (HS87) has been published by Osatomi and Sumiyoshi; Virology 176, 643-647 (1990). The complete genomic sequence of DEN4 has been published by Zhao et al; Virology 155, 77-88. To date, only a partial sequence of any variant of DEN1, DEN1 (Nauru Island), has been determined; Mason et al, Virology 161, 262-267 (1987).
We have now identified a previously unknown strain of DEN1 and established its complete nucleotide sequence. The new strain, DEN1-S275/90, was deposited at the European Collection of Animal Cell Cultures (ECACC) Porton Down, GB under Budapest Treaty conditions on (Apr. 21, 1992) and given accession number V92042111. DEN1-S275/90 differs significantly from DEN2, DEN3 and DEN4 in terms of sequence homology. There are also a number of significant differences between DEN1-S275/90 and DEN1 (Nauru Island).
The present invention thus provides DEN1-S275/90 (ECACC V92042111). The invention further provides DEN1-S275/90 (ECACC V92042111) for use as a diagnostic reagent. The invention also provides DEN1-S275/90 in inactivated form for use as a diagnostic reagent or a vaccine.
The invention also provides the nucleic acid sequence of Seq. ID No. 1 and DNA sequences substantially corresponding to SEQ ID No. 1, e.g. degenerate variants thereof having one or more nucleotide changes but nevertheless capable of being translated to give the same protein sequence. The invention further provides fragments of such DNA polynucleotides, in particular the fragments encoding the C, C', PreM, M, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 genes of the genome of the virus. The start and end points of these preferred fragments in the nucleic acid sequence of Seq I.D. No. 1 are shown below in Table 1. Table 1 also shows the start and end points of the proteins encoded by these genes, using the numbering of Seq. ID Nos. 1 and 2.


TABLE 1 ______________________________________ Start and end points of the nucleic acid (n) numbers encoding the genes of S275/90. The table also shows the start and end points of the corresponding proteins (p) within the polyprotein encoded by S275/90. Gene Start(n) End(n) Start(p) End(p) ______________________________________ C 81 422 1 114 C' 123 422 15 114 PreM 423 695 115 205 M 696 920 206 280 E 921 2402 281 774 NS1 2403 3464 775 1128 NS2A 3465 4112 1129 1344 NS2B 4113 4499 1345 1474 NS3 4500 6359 1475 2093 NS4A 6360 6809 2094 2242 NS4B 6810 7556 2243 2492 NS5 7557 10268 2493 3396 ______________________________________
The nucleic acid sequences of the invention may be used as probes in an assay to determine the presence or absence of DEN1-S275/90, or they may be incorporated into a vector, eg. an expression vector.
Nucleic acid fragments according to the invention may be made by known methods of chemical synthesis or cloned from the virus itself using known recombinant techniques. Fragments according to the invention may also be produced by replication of DNA or RNA, by transcription from DNA to form RNA fragments or reverse transcription from RNA fragments to form DNA fragments. Such transcription may be in a cell free system or may be effected in cells for instance by cloning. Cell free systems include an appropriate replicase, transcriptase or reverse transcriptase, suitable nucleotide precursors and a nucleic acid template or appropriate sequence, together with buffers and any necessary or desirable cofactors.
The present invention also provides a polyprotein as set forth in Seq. ID No. 1 and Seq.

REFERENCES:
Fu et al, "Full-Length cDNA Sequence of Dengue Type 1 Virus (Singapore Strain S275/90)", Virology 188:953-958 (1992).
Rico-Hesse, "Molecular Evolution and Distribution of Dengue Viruses Type 1 and 2 in Nature", Virology 174:479-493 (1990).
Lal et al, "Cloning Full-Length DNA Sequences of the Dneue Virus Genome for Use in Elucidating Pathogenesis and Development of Immunoprophylaxis", Vaccines, pp. 393-399 (1988).
Deubel et al, "Nucleotide Sequence and Deduced Amino Acid Sequence of the Nonstructural Proteins of Dengue Type 2 Virus, Jamaica Genotype: Comparative Analysis of the Full-Length Genome", Virology 165:234-244 (1988).
Osatomi et al, "Complete Nucleotide Sequence of Dengue Type 3 Virus Genome RNA", Virology 176:643-647 (1990).
Zhao et al, "Cloning Full-Length Dengue Type 4 Viral DNA Sequences: Analysis of Genes Coding for Structural Proteins", Virology 155:77-88 (1986).
Mason et al, "Sequence of the Dengue-1 Virus Genome in the REgion Encoding the Three Structural Proteins and the Major Nonstructural Protein NS1", Virology 161:262-267 (1987).
Westaway et al, "Flaviviridae", Intervirology 24:183-192 (1985).
Rosen, "The Pathogenesis of denuge haemorrhagic fever", Supplement to S. Afr. J. Med. 11:40-42 (1986).
Halstead, "Pathogenesis of Dengue: Challenges to Molecular Biology", Science 239:476-481 (1988).
Brinton and Dispoto, "Sequence and Secondary Structure Analysis of the 5'-Terminal Region of Flavivirus Genome RNA", Virology 162:290-299 (1988).
Irie et al, "Sequence analysis of cloned dengue virus type 2 genome (New Guinea-C strain)", Gene 75:197-211 (1989).
Wilbur and Lipman, "Rapid similarity searches of nucleic acid and protein data banks", Proc. Natl. Acad. Sci. USA 80:726-730 (1983).
Rice et al, "Nucleotide Sequence of Yellow Fever Virus: Implications for Flavivirus Gene Expression and Evolution", Science 229:726-733 (1985).
Chu et al, "Genetic Relatedness among Structural Protein Genes of Dengue 1 Virus Strains", J. Gen. Virol. 70:1701-1712 (1989).
Guan and Dixon, "Eukaryotic Proteins Expressed in Escherichia coli: An Improved Thrombin Cleavaged and Purification Procedure of Fusion Proteins with Glutathione S-Transferase", Anal. Biochem. 192:262-267 (1991).
Ford et al, "Fusion Tails for the Recovery and Purification of Recombinant Protiens", Prot. Exp. Pur. 2:96-107 (1991).
Maina et al, "An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein", Gene 74:365-373 (1988).
di Guan et al, "Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein", Gene 67:21-30 (1991).
Zhang et al., Journal of Virology 62:3027-3031 "Immunization of mice with dengue structural proteins and nonstructural protein NS1 expressed by baculovirus recombinant induces resistance to dengue virus encephalitis", 1988.
Henchal et al., Journal of General Virology 69:2101-2107 "Synergistic interactions of anti-NS1 monoclonal antibodies protect passively immunized mice from lethal challenge with dengue 2 virus", 1988.
Hayes et al., Pediatric Infectious Disease Journal 11:311-317, "Dengue and dengue hemorrhagic fever", (Abstract Only), 1992.
Suggs et al. "Use of Synthetic oligonucleotides as hybridization probes: Isolation of cloned cDNA for human beta-2 microglobulin". PNAS. vol. 87, No. 11, pp. 6613-6617, Nov. 1981.
Doraisingham, S. "Dengue Virus Infection". Singapore Medical Journal. No. 30:523-524, 1989.

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