| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 12.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 05.12.00 |
| PATENT TITLE |
Polyclonal and monoclonal antibodies to Norwalk virus and methods for making them |
| PATENT ABSTRACT |
Double-stranded cDNA was synthesized from nucleic acid extracted from Norwalk virus purified from stool specimens of volunteers. One clone was isolated from a cDNA library constructed in a pUC-13 vector after amplification of the cDNA. The specificity of this cDNA (pUCNV-953) was shown by hybridization assays. The cDNA reacted with post- (but not pre-) infection stool samples from Norwalk volunteers and with highly purified Norwalk virus, but not with other common enteric viruses such as hepatitis A virus and rotavirus. Finally, the probe detected virus in the same fractions of CsCl gradients in which viral antigen was detected using a specific Norwalk virus radioimmunoassay, and particles were detected by immune electron microscopy. Single-stranded RNA probes derived from the DNA clone after subcloning into an in vitro transcription vector were also used to show that the Norwalk virus contains a ssRNA genome of about 8 kb in size. The original clone was also used to detect additional cDNAs which represent at least 7 kb of nucleic acid of the Norwalk genome. The availability of a Norwalk-specific cDNA and the first partial genome sequence information allow rapid cloning of the entire genome and of establishment of sensitive diagnostic assays. Such assays can be based on detection of Norwalk virus nucleic acid or Norwalk viral antigen using polyclonal or monoclonal antibodies to proteins expressed from the cDNA or to synthetic peptides made based on the knowledge of the genome sequence. Vaccines made by recombinant DNA technology are now feasible. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 04.02.93 |
| PATENT REFERENCES CITED |
Greenberg et al, J. Virol. 37:994-999 Mar. 1981. Greenberg et al, J. Med Virol 2:97-108, 1978. Treanor et al, Proc. Natl. Acad. Sci., USA, 85: 3613-3617 May 1988. Walter, J. Immunol. Meth. 88: 149-161, 1986. Dresser, Ch 8 of "Handbook of Experimental Immunochemistry" Weir et al, Blackwell Publ v. 1:8.1-8.21 1986. Norrott, J. Med. Virology 44:280-286, 1994. Ploegh, PNAS 77:6081-6085, 1980. T. Tanaka et al.; High Yield Production of Monoclonal Antibodies to Recombinant Norwalk Virus-Like Particles by Oral Immunization; Presented at CDC meeting. Bruce Alberts et al.: Molecular Biology of The Cell, Second Addition; Chapter 5; Basic Genetic Mechanisms, 258-266 (1989). Chitra Mandal et al.; Production of Highly Specific Polyclonal and Monoclonal Antibodies Using Estradiol-3-O-Carboxymethyl Ether as Hapten; Steroids 52/5&6, 551-60, Nov. & Dec. 1988. Ed Harlow et al.; Antibodies: A Laboratory Manual; 115, 141, 156-57, 242 (1988). Bruce Alberts et al.; Molecular Biology of The Cell, Third Edition; The Functional Properties of Antibodies, pp. 1211-1227 (1994). Eli Benjamini et al.; Immunology: A Short Course, Second Edition; pp. 174-175 (1991). Evan M. Roitt et al.; Immunology; (1989). Charles A. Janeway et al.; Immuno Biology: The Immune System in Health and Disease, Fourth Edition; pp. 99-100. Xi Jiang et al.; Sequence and Genomic Organization of Norwalk Virus; Virology 195, 51-61 (1993). Xi Jiang et al.; Norwalk Virus Genome Cloning and Characterization; Science, 250:1580-83. Wang et al.; Sequence Diversity of Small, Round-Structured Viruses in the Norwalk Virus Group; Journal of Virology pp. 5982-5990 (1994). Xi Jiang et al.; Detection of Norwalk Virus in Stool by Polymerase Chain Reaction; Journal of Clinical Microbiology pp. 2529-2534 (1992). Appleton, H. "Small Round Viruses: Classification and Role in Food-Borne Infetions" in Novel Diarrhoea Viruses. (1987) pp. 108-125. Caul, E.O., et al., J.Medical Virology 9:257-265 (1982). Cubitt, W.D., et al., J. Infect.Diseases. 156(5):806-814 (1987). Dingle, J.H., et al., Am. J. Hyg., 58:16-30 (1953). Dolin, R., et al., Proc. Soc. Exp. Med. & Biol., 140:578-583 (1972). Dolin, R., et al., J. Infect. Dis., 123:307-312 (1971). Dupont, H.L., New England J. Med., 314(11):707-708 (1986). Enzymology, vol. 70, 1980, pp. 104-142. Geysen, et al., PNAS 8:3998-4002 (1984). Gill, O.N., et al., Br. Med. J., 287:1532-1534 (1983). Greenberg, H.B., et al., J. Med. Virol. 2(2):97-108 (1978). Greenberg, H.B., et al., J. Virol. 37(3):994-999 (1981). Gunn, R.A., et al., Am. J. Epidemiol., 115:348-351 (1982). Hayaski, Y., et al., J. Clin. Microbiol., 27:1728-1733 (1989). Jiang Xi, et al., J. Clin. Microbiol., 27:874-879 (1989). Jiang Xi, et al., App. Environ. Microbiol., 53:2487-2495 (1987). Jiang Xi, et al., App. Environ. Microbiol., 52:711-717 (1986). Kapikian, et al., "Norwalk Group of Viruses" in Field's Virology, (2nd ed. 1990) pp. 671-693. Kapikian, A.Z., et al., J. Virol., 10:1075-1081 (1972). Kaplan, J., et al., Ann. Internal Med., 96(6):756-761 (1982). Matsui, et al., J. Clin. Invest. 87:1456-1461 (1991). Morse, D.L. et al., New Engl. J. Med., 314:678-681 (1986). Murphy, A.N., et al., Med. J. Aust., 2:329-333 (1979). Peptide Protein and Gene Technology, No. 6, Cambridge Research Biochemicals, Wilmington, Del. Sekine, S., et al., Microbiol. Immunol., 33:207-217 (1989). Thornhill, T.S., et al., J. Infect. Dis., 132:28-34 (1975). Wilde, J., et al., J. Clin. Microbiol., 28(6):1300-1307 (1990). Wilson, R., et al., Am. J. Public Health, 72:72-74 (1982). |
| PATENT GOVERNMENT INTERESTS | This invention is supported in part through a grant or award from the Food and Drug Administration |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: __________________________________________________________________________ # SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 9 - - - - (2) INFORMATION FOR SEQ ID NO:1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7722 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown - - (ii) MOLECULE TYPE: cDNA - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Norwalk v - #irus (B) STRAIN: 8FIIa (C) INDIVIDUAL ISOLATE: - #8FIIa - - (vii) IMMEDIATE SOURCE: (B) CLONE: pUCNV-953 an - #d its derivatives - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - - GGCGTCAAAA GACGTCGTTC CTACTGCTGC TAGCAGTGAA AATGCTAACA AC - #AATAGTAG 60 - - TATTAAGTCT CGTCTATTGG CGAGACTCAA GGGTTCAGGT GGGGCTACGT CC - #CCACCCAA 120 - - CTCGATAAAG ATAACCAACC AAGATATGGC TCTGGGGCTG ATTGGACAGG TC - #CCAGCGCC 180 - - AAAGGCCACA TCCGTCGATG TCCCTAAACA ACAGAGGGAT AGACCACCAC GG - #ACTGTTGC 240 - - CGAAGTTCAA CAAAATTTGC GTTGGACTGA GAGACCACAA GACCAGAATG TT - #AAGACGTG 300 - - GGATGAGCTT GACCACACAA CAAAACAACA GATACTTGAT GAACACGCTG AG - #TGGTTTGA 360 - - TGCCGGTGGC TTAGGTCCAA GTACACTACC CACTAGTCAT GAACGGTACA CA - #CATGAGAA 420 - - TGATGAAGGC CACCAGGTAA AGTGGTCGGC TAGGGAAGGT GTAGACCTTG GC - #ATATCCGG 480 - - GCTCACGACG GTGTCTGGGC CTGAGTGGAA TATGTGCCCG CTACCACCAG TT - #GACCAAAG 540 - - GAGCACGACA CCTGCAACTG AGCCCACAAT TGGTGACATG ATCGAATTCT AT - #GAAGGGCA 600 - - CATCTATCAT TATGCTATAT ACATAGGTCA AGGCAAGACG GTGGGTGTAC AC - #TCCCCTCA 660 - - AGCAGCCTTC TCAATAACGA GGATCACCAT ACAGCCCATA TCAGCTTGGT GG - #CGAGTCTG 720 - - TTATGTCCCA CAACCAAAAC AGAGGCTCAC ATACGACCAA CTCAAAGAAT TA - #GAAAATGA 780 - - ACCATGGCCG TATGCCGCAG TCACGAACAA CTGCTTCGAA TTTTGTTGCC AG - #GTCATGTG 840 - - CTTGGAAGAT ACTTGGTTGC AAAGGAAGCT CATCTCCTCT GGCCGGTTTT AC - #CACCCGAC 900 - - CCAAGATTGG TCCCGAGACA CTCCAGAATT CCAACAAGAC AGCAAGTTAG AG - #ATGGTTAG 960 - - GGATGCAGTG CTAGCCGCTA TAAATGGGTT GGTGTCGCGG CCATTTAAAG AT - #CTTCTGGG 1020 - - TAAGCTCAAA CCCTTGAACG TGCTTAACTT ACTTTCAAAC TGTGATTGGA CG - #TTCATGGG 1080 - - GGTCGTGGAG ATGGTGGTCC TCCTTTTAGA ACTCTTTGGA ATCTTTTGGA AC - #CCACCTGA 1140 - - TGTTTCCAAC TTTATAGCTT CACTCCTGCC AGATTTCCAT CTACAGGGCC CC - #GAGGACCT 1200 - - TGCCAGGGAT CTCGTGCCAA TAGTATTGGG GGGGATCGGC TTAGCCATAG GA - #TTCACCAG 1260 - - AGACAAGGTA AGTAAGATGA TGAAGAATGC TGTTGATGGA CTTCGTGCGG CA - #ACCCAGCT 1320 - - CGGTCAATAT GGCCTAGAAA TATTCTCATT ACTAAAGAAG TACTTCTTCG GT - #GGTGATCA 1380 - - AACAGAGAAA ACCCTAAAAG ATATTGAGTC AGCAGTTATA GATATGGAAG TA - #CTATCATC 1440 - - TACATCAGTG ACTCAGCTCG TGAGGGACAA ACAGTCTGCA CGGGCTTATA TG - #GCCATCTT 1500 - - AGATAATGAA GAAGAAAAGG CAAGGAAATT ATCTGTCAGG AATGCCGACC CA - #CACGTAGT 1560 - - ATCCTCTACC AATGCTCTCA TATCCCGGAT CTCAATGGCT AGGGCTGCAT TG - #GCCAAGGC 1620 - - TCAAGCTGAA ATGACCAGCA GGATGCGTCC TGTGGTCATT ATGATGTGTG GG - #CCCCCTGG 1680 - - TATAGGTAAA ACCAAGGCAG CAGAACATCT GGCTAAACGC CTAGCCAATG AG - #ATACGGCC 1740 - - TGGTGGTAAG GTTGGGCTGG TCCCACGGGA GGCAGTGGAT CATTGGGATG GA - #TATCACGG 1800 - - AGAGGAAGTG ATGCTGTGGG ACGACTATGG AATGACAAAG ATACAGGAAG AC - #TGTAATAA 1860 - - ACTGCAAGCC ATAGCCGACT CAGCCCCCCT AACACTCAAT TGTGACCGAA TA - #GAAAACAA 1920 - - GGGAATGCAA TTTGTGTCTG ATGCTATAGT CATCACCACC AATGCTCCTG GC - #CCAGCCCC 1980 - - AGTGGACTTT GTCAACCTCG GGCCTGTTTG CCGAAGGGTG GACTTCCTTG TG - #TATTGCAC 2040 - - GGCACCTGAA GTTGAACACA CGAGGAAAGT CAGTCCTGGG GACACAACTG CA - #CTGAAAGA 2100 - - CTGCTTCAAG CCCGATTTCT CACATCTAAA AATGGAGTTG GCTCCCCAAG GG - #GGCTTTGA 2160 - - TAACCAAGGG AATACCCCGT TTGGTAAGGG TGTGATGAAG CCCACCACCA TA - #AACAGGCT 2220 - - GTTAATCCAG GCTGTAGCCT TGACGATGGA GAGACAGGAT GAGTTCCAAC TC - #CAGGGGCC 2280 - - TACGTATGAC TTTGATACTG ACAGAGTAGC TGCGTTCACG AGGATGGCCC GA - #GCCAACGG 2340 - - GTTGGGTCTC ATATCCATGG CCTCCCTAGG CAAAAAGCTA CGCAGTGTCA CC - #ACTATTGA 2400 - - AGGATTAAAG AATGCTCTAT CAGGCTATAA AATATCAAAA TGCAGTATAC AA - #TGGCAGTC 2460 - - AAGGGTGTAC ATTATAGAAT CAGATGGTGC CAGTGTACAA ATCAAAGAAG AC - #AAGCAAGC 2520 - - TTTGACCCCT CTGCAGCAGA CAATTAACAC GGCCTCACTT GCCATCACTC GA - #CTCAAAGC 2580 - - AGCTAGGGCT GTGGCATACG CTTCATGTTT CCAGTCCGCC ATAACTACCA TA - #CTACAAAT 2640 - - GGCGGGATCT GCGCTCGTTA TTAATCGAGC GGTCAAGCGT ATGTTTGGTA CC - #CGTACAGC 2700 - - AGCCATGGCA TTAGAAGGAC CTGGGAAAGA ACATAATTGC AGGGTCCATA AG - #GCTAAGGA 2760 - - AGCTGGAAAG GGGCCCATAG GTCATGATGA CATGGTAGAA AGGTTTGGCC TA - #TGTGAAAC 2820 - - TGAAGAGGAG GAGAGTGAGG ACCAAATTCA AATGGTACCA AGTGATGCCG TC - #CCAGAAGG 2880 - - AAAGAACAAA GGCAAGACCA AAAAGGGACG TGGTCGCAAA AATAACTATA AT - #GCATTCTC 2940 - - TCGCCGTGGT CTGAGTGATG AAGAATATGA AGAGTACAAA AAGATCAGAG AA - #GAAAAGAA 3000 - - TGGCAATTAT AGTATACAAG AATACTTGGA GGACCGCCAA CGATATGAGG AA - #GAATTAGC 3060 - - AGAGGTACAG GCAGGTGGTG ATGGTGGCAT AGGAGAAACT GAAATGGAAA TC - #CGTCACAG 3120 - - GGTCTTCTAT AAATCCAAGA GTAAGAAACA CCAACAAGAG CAACGGCGAC AA - #CTTGGTCT 3180 - - AGTGACTGGA TCAGACATCA GAAAACGTAA GCCCATTGAC TGGACCCCGC CA - #AAGAATGA 3240 - - ATGGGCAGAT GATGACAGAG AGGTGGATTA TAATGAAAAG ATCAATTTTG AA - #GCTCCCCC 3300 - - GACACTATGG AGCCGAGTCA CAAAGTTTGG ATCAGGATGG GGCTTTTGGG TC - #AGCCCGAC 3360 - - AGTGTTCATC ACAACCACAC ATGTAGTGCC AACTGGTGTG AAAGAATTCT TT - #GGTGAGCC 3420 - - CCTATCTAGT ATAGCAATCC ACCAAGCAGG TGAGTTCACA CAATTCAGGT TC - #TCAAAGAA 3480 - - AATGCGCCCT GACTTGACAG GTATGGTCCT TGAAGAAGGT TGCCCTGAAG GG - #ACAGTCTG 3540 - - CTCAGTCCTA ATTAAACGGG ATTCGGGTGA ACTACTTCCG CTAGCCGTCC GT - #ATGGGGGC 3600 - - TATTGCCTCC ATGAGGATAC AGGGTCGGCT TGTCCATGGC CAATCAGGGA TG - #TTACTGAC 3660 - - AGGGGCCAAT GCAAAGGGGA TGGATCTTGG CACTATACCA GGAGACTGCG GG - #GCACCATA 3720 - - CGTCCACAAG CGCGGGAATG ACTGGGTTGT GTGTGGAGTC CACGCTGCAG CC - #ACAAAGTC 3780 - - AGGCAACACC GTGGTCTGCG CTGTACAGGC TGGAGAGGGC GAAACCGCAC TA - #GAAGGTGG 3840 - - AGACAAGGGG CATTATGCCG GCCACGAGAT TGTGAGGTAT GGAAGTGGCC CA - #GCACTGTC 3900 - - AACTAAAACA AAATTCTGGA GGTCCTCCCC AGAACCACTG CCCCCCGGAG TA - #TATGAGCC 3960 - - AGCATACCTG GGGGGCAAGG ACCCCCGTGT ACAGAATGGC CCATCCCTAC AA - #CAGGTACT 4020 - - ACGTGACCAA CTGAAACCCT TTGCGGACCC CCGCGGCCGC ATGCCTGAGC CT - #GGCCTACT 4080 - - GGAGGCTGCG GTTGAGACTG TAACATCCAT GTTAGAACAG ACAATGGATA CC - #CCAAGCCC 4140 - - GTGGTCTTAC GCTGATGCCT GCCAATCTCT TGACAAAACT ACTAGTTCGG GG - #TACCCTCA 4200 - - CCATAAAAGG AAGAATGATG ATTGGAATGG CACCACCTTC GTTGGAGAGC TC - #GGTGAGCA 4260 - - AGCTGCACAC GCCAACAATA TGTATGAGAA TGCTAAACAT ATGAAACCCA TT - #TACACTGC 4320 - - AGCCTTAAAA GATGAACTAG TCAAGCCAGA AAAGATTTAT CAAAAAGTCA AG - #AAGCGTCT 4380 - - ACTATGGGGC GCCGATCTCG GAACAGTGGT CAGGGCCGCC CGGGCTTTTG GC - #CCATTTTG 4440 - - TGACGCTATA AAATCACATG TCATCAAATT GCCAATAAAA GTTGGCATGA AC - #ACAATAGA 4500 - - AGATGGCCCC CTCATCTATG CTGAGCATGC TAAATATAAG AATCATTTTG AT - #GCAGATTA 4560 - - TACAGCATGG GACTCAACAC AAAATAGACA AATTATGACA GAATCCTTCT CC - #ATTATGTC 4620 - - GCGCCTTACG GCCTCACCAG AATTGGCCGA GGTTGTGGCC CAAGATTTGC TA - #GCACCATC 4680 - - TGAGATGGAT GTAGGTGATT ATGTCATCAG GGTCAAAGAG GGGCTGCCAT CT - #GGATTCCC 4740 - - ATGTACTTCC CAGGTGAACA GCATAAATCA CTGGATAATT ACTCTCTGTG CA - #CTGTCTGA 4800 - - GGCCACTGGT TTATCACCTG ATGTGGTGCA ATCCATGTCA TATTTCTCAT TT - #TATGGTGA 4860 - - TGATGAGATT GTGTCAACTG ACATAGATTT TGACCCAGCC CGCCTCACTC AA - #ATTCTCAA 4920 - - GGAATATGGC CTCAAACCAA CAAGGCCTGA CAAAACAGAA GGACCAATAC AA - #GTGAGGAA 4980 - - AAATGTGGAT GGACTGGTCT TCTTGCGGCG CACCATTTCC CGTGATGCGG CA - #GGGTTCCA 5040 - - AGGCAGGTTA GATAGGGCTT CGATTGAACG CCAAATCTTC TGGACCCGCG GG - #CCCAATCA 5100 - - TTCAGATCCA TCAGAGACTC TAGTGCCACA CACTCAAAGA AAAATACAGT TG - #ATTTCACT 5160 - - TCTAGGGGAA GCTTCACTCC ATGGTGAGAA ATTTTACAGA AAGATTTCCA GC - #AAGGTCAT 5220 - - ACATGAAATC AAGACTGGTG GATTGGAAAT GTATGTCCCA GGATGGCAGG CC - #ATGTTCCG 5280 - - CTGGATGCGC TTCCATGACC TCGGATTGTG GACAGGAGAT CGCGATCTTC TG - #CCCGAATT 5340 - - CGTAAATGAT GATGCGTCTA AGGACGCTAC ATCAAGCGTG GATGGCGCTA GT - #GGCGCTGG 5400 - - TCAGTTGGTA CCGGAGGTTA ATGCTTCTGA CCCTCTTGCA ATGGATCCTG TA - #GCAGGTTC 5460 - - TTCGACAGCA GTCGCGACTG CTGGACAAGT TAATCCTATT GATCCCTGGA TA - #ATTAATAA 5520 - - TTTTGTGCAA GCCCCCCAAG GTGAATTTAC TATTTCCCCA AATAATACCC CC - #GGTGATGT 5580 - - TTTGTTTGAT TTGAGTTTGG GTCCCCATCT TAATCCTTTC TTGCTCCATC TA - #TCACAAAT 5640 - - GTATAATGGT TGGGTTGGTA ACATGAGAGT CAGGATTATG CTAGCTGGTA AT - #GCCTTTAC 5700 - - TGCGGGGAAG ATAATAGTTT CCTGCATACC CCCTGGTTTT GGTTCACATA AT - #CTTACTAT 5760 - - AGCACAAGCA ACTCTCTTTC CACATGTGAT TGCTGATGTT AGGACTCTAG AC - #CCCATTGA 5820 - - GGTGCCTTTG GAAGATGTTA GGAATGTTCT CTTTCATAAT AATGATAGAA AT - #CAACAAAC 5880 - - CATGCGCCTT GTGTGCATGC TGTACACCCC CCTCCGCACT GGTGGTGGTA CT - #GGTGATTC 5940 - - TTTTGTAGTT GCAGGGCGAG TTATGACTTG CCCCAGTCCT GATTTTAATT TC - #TTGTTTTT 6000 - - AGTCCCTCCT ACGGTGGAGC AGAAAACCAG GCCCTTCACA CTCCCAAATC TG - #CCATTGAG 6060 - - TTCTCTGTCT AACTCACGTG CCCCTCTCCC AATCAGTAGT ATGGGCATTT CC - #CCAGACAA 6120 - - TGTCCAGAGT GTGCAGTTCC AAAATGGTCG GTGTACTCTG GATGGCCGCC TG - #GTTGGCAC 6180 - - CACCCCAGTT TCATTGTCAC ATGTTGCCAA GATAAGAGGG ACCTCCAATG GC - #ACTGTAAT 6240 - - CAACCTTACT GAATTGGATG GCACACCCTT TCACCCTTTT GAGGGCCCTG CC - #CCCATTGG 6300 - - GTTTCCAGAC CTCGGTGGTT GTGATTGGCA TATCAATATG ACACAGTTTG GC - #CATTCTAG 6360 - - CCAGACCCAG TATGATGTAG ACACCACCCC TGACACTTTT GTCCCCCATC TT - #GGTTCAAT 6420 - - TCAGGCAAAT GGCATTGGCA GTGGTAATTA TGTTGGTGTT CTTAGCTGGA TT - #TCCCCCCC 6480 - - ATCACACCCG TCTGGCTCCC AAGTTGACCT TTGGAAGATC CCCAATTATG GG - #TCAAGTAT 6540 - - TACGGAGGCA ACACATCTAG CCCCTTCTGT ATACCCCCCT GGTTTCGGAG AG - #GTATTGGT 6600 - - CTTTTTCATG TCAAAAATGC CAGGTCCTGG TGCTTATAAT TTGCCCTGTC TA - #TTACCACA 6660 - - AGAGTACATT TCACATCTTG CTAGTGAACA AGCCCCTACT GTAGGTGAGG CT - #GCCCTGCT 6720 - - CCACTATGTT GACCCTGATA CCGGTCGGAA TCTTGGGGAA TTCAAAGCAT AC - #CCTGATGG 6780 - - TTTCCTCACT TGTGTCCCCA ATGGGGCTAG CTCGGGTCCA CAACAGCTGC CG - #ATCAATGG 6840 - - GGTCTTTGTC TTTGTTTCAT GGGTGTCCAG ATTTTATCAA TTAAAGCCTG TG - #GGAACTGC 6900 - - CAGCTCGGCA AGAGGTAGGC TTGGTCTGCG CCGATAATGG CCCAAGCCAT AA - #TTGGTGCA 6960 - - ATTGCTGCTT CCACAGCAGG TAGTGCTCTG GGAGCGGGCA TACAGGTTGG TG - #GCGACAGG 7020 - - CCCTCCAAAG CCAAAGGTAT CAACAAAATT TGCAACTGCA AGAAAATTCT TT - #TAAACATG 7080 - - ACAGGGAAAT GATTGGGTAT CAGGTTGAAG CTTCAAATCA ATTATTGGCT AA - #AAATTTGG 7140 - - CAACTAGATA TTCACTCCTC CGTGCTGGGG GTTTGACCAG TGCTGATGCA GC - #AAGATCTG 7200 - - TGGCAGGAGC TCCAGTCACC CGCATTGTAG ATTGGAATGG CGTGAGAGTG TC - #TGCTCCCG 7260 - - AGTCCTCTGC TACCACATTG AGATCCGGTG GCTTCATGTG AGTTCCCATA CC - #ATTTGCCT 7320 - - CTAAGCAAAA ACAGGTTCAA TCATCTGGTA TTAGTAATCC AAATTATTCC CC - #TTCATCCA 7380 - - TTTCTCGAAC CACTAGTTGG GTCGAGTCAC AAAACTCATC GAGATTTGGA AA - #TCTTTCTC 7440 - - CATACCACGC GGAGGCTCTC AATACAGTGT GGTTGACTCC ACCCGGTTCA AC - #AGCCTCTT 7500 - - CTACACTGTC TTCTGTGCCA CGTGGTTATT TCAATACAGA CAGGTTGCCA TT - #ATTCGCAA 7560 - - ATAATAGGCG ATGATGTTGT AATATGAAAT GTGGGCATCA TATTCATTTA AT - #TAGGTTTA 7620 - - ATTAGGTTTA ATTTGATGTT AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA - #AAAAAAAA 7680 - - AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA - # - #7722 - - - - (2) INFORMATION FOR SEQ ID NO:2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 381 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown - - (ii) MOLECULE TYPE: cDNA - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Norwalk v - #irus (B) STRAIN: 8FIIa (C) INDIVIDUAL ISOLATE: - #8FIIa - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: - - CATTTTGATG CAGATTATAC AGCATGGGAC TCAACACAAA ATAGACAAAT TA - #TGACAGAA 60 - - TCCTTCTCCA TTATGTCGCG CCTTACGGCC TCACCAGAAT TGGCCGAGGT TG - #TGGCCCAA 120 - - GATTTGCTAG CACCATCTGA GATGGATGTA GGTGATTATG TCATCAGGGT CA - #AAGAGGGG 180 - - CTGCCATCTG GATTCCCATG TACTTCCCAG GTGAACAGCA TAAATCACTG GA - #TAATTACT 240 - - CTCTGTGCAC TGTCTGAGGC CACTGGTTTA TCACCTGATG TGGTGCAATC CA - #TGTCATAT 300 - - TTCTCATTTT ATGGTGATGA TGAGATTGTG TCAACTGACA TAGATTTTGA CC - #CAGCCCGC 360 - - CTCACTCAAA TTCTCAAGGA A - # - # 381 - - - - (2) INFORMATION FOR SEQ ID NO:3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 127 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: peptide - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Norwalk - #virus - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: - - His Phe Asp Ala Asp Tyr Thr Ala Trp Asp Se - #r Thr Gln Asn Arg Gln 1 5 - # 10 - # 15 - - Ile Met Thr Glu Ser Phe Ser Thr Met Ser Ar - #g Leu Thr Ala Ser Pro 20 - # 25 - # 30 - - Glu Leu Ala Glu Val Val Ala Gln Asp Leu Le - #u Ala Pro Ser Glu Met 35 - # 40 - # 45 - - Asp Val Gly Asp Tyr Val Ile Arg Val Lys Gl - #u Gly Leu Pro Ser Gly 50 - # 55 - # 60 - - Phe Pro Cys Thr Ser Gln Val Asn Ser Ile As - #n His Trp Ile Ile Thr 65 - #70 - #75 - #80 - - Leu Cys Ala Leu Ser Glu Ala Thr Gly Leu Se - #r Pro Asp Val Val Gln 85 - # 90 - # 95 - - Ser Met Ser Tyr Phe Ser Phe Tyr Gly Asp As - #p Glu Ile Val Ser Thr 100 - # 105 - # 110 - - Asp Ile Asp Phe Asp Pro Ala Arg Leu Thr Gl - #n Ile Leu Lys Glu 115 - # 120 - # 125 - - - - (2) INFORMATION FOR SEQ ID NO:4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: - - CACGCGGAGG CTCTCAAT - # - # - # 18 - - - - (2) INFORMATION FOR SEQ ID NO:5: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: - - GGTGGCGACA GGCCCTCC - # - # - # 18 - - - - (2) INFORMATION FOR SEQ ID NO:6: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: - - TCAGCAGTTA TAGATATG - # - # - # 18 - - - - (2) INFORMATION FOR SEQ ID NO:7: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: - - ATGCTATATA CATAGGTC - # - # - # 18 - - - - (2) INFORMATION FOR SEQ ID NO:8: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: - - CAACAGGTAC TACGTGAC - # - # - # 18 - - - - (2) INFORMATION FOR SEQ ID NO:9: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: - - TGTGGCCCAA GATTTGCT - # - # - # 18 __________________________________________________________________________ 1. A hybridoma which produces a high affinity monoclonal antibody to a Norwalk virus protein or peptide or to a Norwalk-related virus protein or peptide specifically immunoreactive with a protein or peptide coded for by the coding sequence of a cDNA selected from the group consisting of a cDNA corresponding to SEQ.ID.NO. 1 or a fragment thereof, a cDNA obtained by screening a cDNA library of a Norwalk-related virus with the coding sequence of a cDNA of SEQ ID NO. 1 or a fragment thereof, and a cDNA obtained by screening a cDNA library of a Norwalk-related virus with a cDNA identified by screening a cDNA library of a Norwalk-related virus with a cDNA of SEQ. ID. NO. 1 or a fragment thereof; whereby said antibody produced specifically recognizes an antigen of a Norwalk virus protein or peptide or a Norwalk-related virus protein or peptide. 2. A high affinity monoclonal antibody produced by the hybridoma of claim 1. 3. A method of producing antibodies to a Norwalk virus protein or peptide or to a Norwalk-related virus protein or peptide comprising: synthesizing a synthetic protein or peptide of at least 15 amino acids in length and coded for by the coding sequence of a cDNA selected from the group consisting of a cDNA corresponding to SEQ.ID.NO. 1 or a fragment thereof, a cDNA obtained by screening a cDNA library of a Norwalk-related virus with the coding sequence of a cDNA of SEQ ID NO. 1 or a fragment thereof, and a cDNA obtained by screening a cDNA library of a Norwalk-related virus with a cDNA identified by screening a cDNA library of a Norwalk-related virus with a cDNA of SEQ. ID. NO. 1 or a fragment thereof; immunizing an animal with said synthetic protein or peptide; and recovering antibodies from said animal; whereby said antibodies specifically recognize an antigen of a Norwalk virus protein or peptide or a Norwalk-related virus protein or peptide. 4. A method of producing a hybridoma which produces a monoclonal antibody to a Norwalk virus protein or peptide or to a Norwalk-related virus protein or peptide comprising the steps of: synthesizing a protein or peptide of at least 15 amino acids in length and coded for by the coding sequence of a cDNA selected from the group consisting of a cDNA corresponding to SEQ.ID.NO. 1 or a fragment thereof a cDNA obtained by screening a cDNA library of a Norwalk-related virus with the coding sequence of a cDNA of SEQ ID NO. 1 or a fragment thereof, and a cDNA obtained by screening a cDNA library of a Norwalk-related virus with a cDNA identified by screening a cDNA library of a Norwalk-related virus with a cDNA of SEQ. ID. NO. 1 or a fragment thereof; immunizing an animal with said protein or peptide to produce an antigenic response; boosting said immunized animal with said protein or peptide to increase polyclonal antibody production in said animal; fusing splenocytes from said animal with myeloma cells; screening said fused cells for production of monoclonal antibodies immunoreactive with said protein or peptide; and recovering said monoclonal antibodies; whereby said monoclonal antibodies specifically recognize an antigen of a Norwalk virus protein or peptide or a Norwalk-related virus protein or peptide. 5. A polyclonal antibody to a Norwalk virus protein or peptide or to a Norwalk-related virus protein or peptide produced according to a process having the following steps: selecting a cDNA molecule from the group consisting of a cDNA corresponding to the coding sequence of SEQ.ID.NO. 1, or a fragment thereof, a cDNA obtained by screening a cDNA library of a Norwalk-related virus with the coding sequence of a cDNA of SEQ ID NO. 1 or a fragment thereof, and a cDNA obtained by screening a cDNA library of a Norwalk-related virus with a cDNA identified by screening a cDNA library of a Norwalk-related virus with a cDNA of SEQ. ID. NO. 1 or a fragment thereof, wherein said cDNA molecule codes for a protein or peptide of at least 15 amino acids in length; expressing said cDNA in an expression system to produce a protein or peptide; immunizing an animal with said protein or peptide; immunizing said animal with said protein or peptide a second time; and recovering antibodies from said animal; whereby said antibodies specifically recognize an antigen or a Norwalk virus protein or peptide or a Norwalk-related protein or peptide. 6. A hybridoma which produces a high affinity monoclonal antibody to a Norwalk virus protein or peptide or to a Norwalk-related virus protein or peptide produced according to a process having the following steps: selecting a cDNA molecule from the group consisting of a cDNA corresponding to the coding sequence of SEQ.ID.NO. 1, or a fragment thereof, a cDNA obtained by screening a cDNA library of a Norwalk-related virus with the coding sequence of a cDNA of SEQ ID NO. 1 or a fragment thereof, and a cDNA obtained by screening a cDNA library of a Norwalk-related virus with a cDNA identified by screening a cDNA library of a Norwalk-related virus with a cDNA of SEQ. ID. NO. 1 or a fragment thereof, wherein said cDNA molecule codes for a protein or peptide of at least 15 amino acids in length; expressing said cDNA in an expression system to produce a protein or peptide; immunizing an animal with said protein or peptide; immunizing said animal with said protein or peptide a second time; fusing splenocytes of said animal with myeloma cells; screening said fused cells for production of monoclonal antibodies immunoreactive with said protein or peptide; and recovering said monoclonal antibodies; whereby said monoclonal antibodies specifically recognize an antigen of a Norwalk virus protein or peptide or a Norwalk-related virus protein or peptide. 7. A high affinity monoclonal antibody to a Norwalk virus protein or peptide or to a Norwalk-related virus protein or peptide produced according to a process having the following steps: selecting a cDNA molecule from the group consisting of a cDNA corresponding to the coding sequence of SEQ.ID.NO. 1, or a fragment thereof, a cDNA obtained by screening a cDNA library of a Norwalk-related virus with the coding sequence of a cDNA of SEQ ID NO. 1 or a fragment thereof, and a cDNA obtained by screening a cDNA library of a Norwalk-related virus with a cDNA identified by screening a cDNA library of a Norwalk-related virus with a cDNA of SEQ. ID. NO. 1 or a fragment thereof, wherein said cDNA molecules codes for a protein or peptide of at least 15 amino acids in length; expressing said cDNA in an expression system to produce a protein or peptide; immunizing an animal with said protein or peptide; immunizing said animal with said protein or peptide a second time; fusing splenocytes from said animal with myeloma cells; screening said cells for the production of monoclonal antibodies immunoreactive with said protein or peptide; and recovering said monoclonal antibodies; whereby said monoclonal antibodies specifically recognize an antigen of a Norwalk virus protein or peptide or a Norwalk-related virus protein or peptide. 8. A polyclonal antibody to a Norwalk virus protein or peptide or to a Norwalk-related virus protein or peptide produced according to a process having the following steps: synthesizing a protein or peptide at least 15 amino acids in length and coded for by the coding sequence of a cDNA selected from the group consisting of a cDNA corresponding to SEQ.ID.NO. 1 or a fragment thereof, a cDNA obtained by screening a cDNA library of a Norwalk-related virus with the coding sequence of a cDNA of SEQ ID NO. 1 or a fragment thereof, and a cDNA obtained by screening a cDNA library of a Norwalk-related virus with a cDNA identified by screening a cDNA library of a Norwalk-related virus with a cDNA of SEQ. ID. NO. 1 or a fragment thereof; immunizing an animal with said protein or peptide; immunizing said animal with said protein or peptide a second time; and recovering antibodies from said animal; whereby said antibodies specifically recognize an antigen of a Norwalk virus protein or peptide or a Norwalk-related virus protein or peptide. 9. A hybridoma which produces a high affinity monoclonal antibody to a Norwalk virus protein or peptide or to a Norwalk-related virus protein or peptide produced according to a process having the following steps: synthesizing a protein or peptide at least 15 amino acids in length and coded for by the coding sequence of a cDNA selected from the group consisting of a cDNA corresponding to SEQ.ID.NO. 1 or a fragment thereof, a cDNA obtained by screening a cDNA library of a Norwalk-related virus with the coding sequence of a cDNA of SEQ ID NO. 1 or a fragment thereof, and a cDNA obtained by screening a cDNA library of a Norwalk-related virus with a cDNA identified by screening a cDNA library of a Norwalk-related virus with a cDNA of SEQ. ID. NO. 1 or a fragment thereof; immunizing an animal with said protein or peptide; immunizing said animal with said protein or peptide a second time; fusing splenocytes of said animal with myeloma cells; screening said fused cells for production of monoclonal antibodies immunoreactive with said protein or peptide; and recovering said monoclonal antibodies; whereby said monoclonal antibodies specifically recognize an antigen or a Norwalk virus protein or peptide or a Norwalk-related virus protein or peptide. 10. A high affinity monoclonal antibody to a Norwalk virus protein or peptide or to a Norwalk-related virus protein or peptide produced according to a process having the following steps: synthesizing a protein or peptide at least 15 amino acids in length and coded for by the coding sequence of a cDNA selected from the group consisting of a cDNA corresponding to SEQ.ID.NO. 1 or a fragment thereof, a cDNA obtained by screening a cDNA library of a Norwalk-related virus with the coding sequence of a cDNA of SEQ ID NO. 1 or a fragment thereof, and a cDNA obtained by screening a cDNA library of a Norwalk-related virus with a cDNA identified by screening a cDNA library of a Norwalk-related virus with a cDNA of SEQ. ID. NO. 1 or a fragment thereof; immunizing an animal with said protein or peptide; immunizing said animal with said protein or peptide a second time; fusing splenocytes from said animal with myeloma cells; screening said cells for the production of monoclonal antibodies immunoreactive with said protein or peptide; and recovering said monoclonal antibodies; whereby said monoclonal antibodies specifically recognize an antigen of a Norwalk virus protein or peptide or a Norwalk-related virus protein or peptide. 11. A method of producing antibodies to a Norwalk virus protein or peptide or to a Norwalk-related virus protein or peptide comprising immunizing an animal with a single protein or protein coded for by the coding sequence of a cDNA selected from the group consisting of a cDNA corresponding to SEQ.ID.NO. 1 or a fragment thereof, a cDNA obtained by screening a cDNA library of a Norwalk-related virus with the coding sequence of a cDNA of SEQ ID NO. 1 or a fragment thereof, and a cDNA obtained by screening a cDNA library of a Norwalk-related virus with a cDNA identified by screening a cDNa library of a Norwalk-related virus with a cDNA of SEQ. ID. NO. 1 or a fragment thereof, and recovering antibodies from said animal; whereby said antibodies specifically recognize an antigen of a Norwalk virus protein or peptide or a Norwalk-related virus protein or peptide. 12. A method of producing a hybridoma which produces a monoclonal antibody to a Norwalk virus protein or peptide or to a Norwalk-related virus protein or peptide comprising the steps of: immunizing an animal with a protein or peptide coded for by the coding sequence of a cDNA selected from the group consisting of a cDNA corresponding to SEQ ID NO. 1 or a fragment thereof, a cDNA obtained by screening a cDNA library of a Norwalk-related virus with the coding sequence of a cDNA of SEQ ID NO. 1 or a fragment thereof, and a cDNA obtained by screening a cDNA library of a Norwalk-related virus with a cDNA identified by screening a cDNA library of a Norwalk-related virus with a cDNA of SEQ. ID. NO. 1 or a fragment thereof; boosting said immunization with said protein or peptide to increase antibody production in said animal; fusing splenocytes from said animal with myeloma cells; screening said fused cells for production of monoclonal antibodies immunoreactive with said protein or peptide; and recovering said monoclonal antibodies; whereby said monoclonal antibodies specifically recognize an antigen of a Norwalk virus protein or peptide or a Norwalk-related virus protein or peptide. 13. A method of obtaining monoclonal antibodies to a Norwalk virus protein or peptide or to a Norwalk-related virus protein or peptide comprising: screening hybridomas with a recombinant or synthetic peptide or protein, said peptide or protein being coded for by the coding sequence of a cDNA selected from the group consisting of a cDNA corresponding to SEQ ID NO. 1 or a fragment thereof, a cDNA obtained by screening a cDNA library of a Norwalk-related virus with the coding sequence of a cDNA of SEQ ID NO. 1 or a fragment thereof, and a cDNA obtained by screening a cDNA library of a Norwalk-related virus with a cDNA identified by screening a cDNA library of a Norwalk-related virus with a cDNA of SEQ. ID. NO. 1 or a fragment thereof; wherein said hybridomas are the fusion product of a myeloma cell and a splenocyte from an animal exposed to a Norwalk virus protein or peptide or a Norwalk related virus protein or peptide and harvesting monoclonal antibodies from the hybridomas which produces antibodies which react with said recombinant or synthetic peptide or protein; whereby said monoclonal antibodies specifically recognize an antigen of a Norwalk virus protein or peptide or a Norwalk-related virus protein or peptide. 14. A method of obtaining monoclonal antibodies to a Norwalk virus protein or peptide or to a Norwalk-related virus protein or peptide comprising: harvesting monoclonal antibodies from a hybridoma which produces a monoclonal antibody to a Norwalk virus protein or peptide or to a Norwalk-related virus protein or peptide specifically immunoreactive with a protein or peptide coded for by the coding sequence of a cDNA selected from the group consisting of a cDNA corresponding to SEQ.ID.NO. 1 or a fragment thereof, a cDNA obtained by screening a cDNA library of a Norwalk-related virus with the coding sequence of a cDNA of SEQ ID NO. 1 or a fragment thereof, and a cDNA obtained by screening a cDNA library of a Norwalk-related virus with a cDNA identified by screening a cDNA library of a Norwalk-related virus with a cDNA of SEQ. ID. NO. 1 or a fragment thereof. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention relates generally to synthesizing clones of Norwalk virus and to making probes to Norwalk and related viruses. It also relates to methods of detection and characterization of Norwalk and related viruses. BACKGROUND OF THE INVENTION Norwalk virus is one of the most important viral pathogens causing acute gastroenteritis, the second most common illness in the United States (Dingle et al., 1953; Kapikian and Chanock, 1985). Up to 42% of cases of viral gastroenteritis have been estimated to be caused by Norwalk or Norwalk-like viruses (Kaplan et al., 1982). Both water and foodborne transmission of Norwalk virus has been documented, and particularly large epidemic outbreaks of illness have occurred following consumption of contaminated shellfish including clams, cockles, and oysters (Murphy et al., 1979; Gunn et al., 1982; Wilson et al., 1982; Gill et al., 1983; DuPont 1986; Morse et al., 1986; Sekine et al., 1989). An increase in fish and shellfish-related food poisonings has recently been noted and attributed to increased recognition of these entities by clinicians as well as to increased consumption of seafood (Eastaugh and Shepherd, 1989). Norwalk virus was discovered in 1973. However, knowledge about the virus has remained limited because it has failed to grow in cell cultures and no suitable animal models have been found for virus cultivation. Human stool samples obtained from outbreaks and from human volunteer studies, therefore, are the only source of the virus. Still the concentration of the virus in stool is usually so low that virus detection with routine electron microscopy is not possible (Dolin et al., 1972; Kapikian et al., 1972; Thornhill et al., 1975). Current methods of Norwalk virus detection include immune electron microscopy and other immunologic methods such as radio immunoassays (RIAs) or a biotin-avidin enzyme linked immunoabsorbent assays (ELISAs) which utilize acute and convalescent phase serum from humans. To date, no hyperimmune serum from animals has been successfully prepared due either to insufficient quantities or unusual properties of the viral antigen. Preliminary biophysical characterization of virions has indicated particles contain one polypeptide (Greenberg et al., 1981), but efforts to characterize the viral genome have failed. Therefore, these viruses have remained unclassified. CITED AND RELEVANT INFORMATION 1. Dingle J, Badger G, Feller A et al. 1953. A study of illness in a group of Cleveland families: 1. Plan of study and certain general observations. Am. J. Hyg. 58:16-30. 2. Dolin R, Blacklow N R, DuPont H, Buscho R F, Wyatt R G, Kasel J A, Hornick R, and Chanock R M. 1972. Biological properties of Norwalk agent of acute infectious nonbacterial gastroenteritis. Proc. Soc. Exp. Med. and Biol. 140:578-583. 3. Dolin R, Blacklow N R, DuPont H, Formal S, Buscho R F, Kasel J A, Chames R P, Hornick R, and Chanock R M. 1971. Transmission of acute infectious nonbacterial gastroenteritis to volunteers by oral administration of stool filtrates. J. Infect. Dis. 123:307-312. 4. DuPont H L. 1986. Consumption of raw shellfish--is the risk now unacceptable? New Engl. J. Med. 314:707-708. 5. Eastaugh J, Shepherd S. 1989. Infectious and toxic syndromes from fish and shellfish consumption. Arch. Intern. Med. 149:1735-1740. 6. Gill O N, Cubitt W D, McSwiggan D A, Watney B M and Bartlett C L R. 1983. Epidemic of gastroenteritis caused by oysters contaminated with small round structured viruses. Br. Med. J. 287:1532-1534. 7. Greenberg H B, Valdesuso J R, Kalica A R, Wyatt R G, McAuliffe V J, Kapikian A Z and Chanock R M. 1981. Proteins of Norwalk virus. J. Virol. 37: 994-999. 8. Gunn R A, Janowski H T, Lieb S, Prather E C, and Greenberg H B. 1982. Norwalk virus gastroenteritis following raw oyster consumption. Am. J. Epidemiol. 115:348-351. 9. Jiang X, Estes M K, and Metcalf T G. 1989. In situ hybridization for quantitative assay of infectious hepatitis A virus. J. Clin. Microbiol. 27:874-879. 10. Jiang X, Estes M K, and Metcalf T G. 1987. Detection of hepatitis A virus by hybridization with single-stranded RNA probes. Appl. Environ. Microbiol. 53:2487-2495. 11. Jiang X, Estes M K, Metcalf T G, and Melnick J L. 1986. Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes. Appl. Environ. Microbiol. 52:711-717. 12. Kapikian A Z and Chanock R M. 1990. Norwalk group of viruses. In: BN Fields (ed.) Virology, Raven Press, New York, pp. 671-693. 13. Kapikian A Z, Wyatt R G, Dolin R, Thornhill T S, Kalica A R, and Chanock R M. 1972. Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis. J. Virol. 10:1075-1081. 14. Kaplan J, Feldman R, Campbell D et al. 1982. Epidemiology of Norwalk Gastroenteritis and the Role of Norwalk Virus in Outbreaks of Acute Nonbacterial Gastroenteritis. Ann. Internal Med. 96(6): 756-761. 15. Morse D L, Guzewich J J, Hanrahan J P, Stricof R, Shayegani M, Deibel R, Grabau J C, Nowak N A, Herrmann J E, Cukor G, and Blacklow N R. 1986. Widespread outbreaks of clam- and oyster-associated gastroenteritis: role of Norwalk virus. New Engl. J. Med. 314:678-681. 16. Murphy A M, Grohmann G S, Christopher P J, Lopez W A, Davey G R, and Millsom R H. 1979. An Australia-wide outbreak of gastroenteritis from oysters caused by Norwalk virus. Med. J. Aust. 2:329-333. 17. Sekine S, Okada S, Hayashi Y, Ando T, Terayama T, Yabuuchi K, Miki T, and Ohashi M. 1989. Prevalence of small round structured virus infections in acute gastroenteritis outbreaks in Tokyo. Microbiol. Immunol. 33:207-217. 18. Thornhill T S, Kalica A R, Wyatt R G, Kapikian A Z, and Chanock R M. 1975. Pattern of shedding of the Norwalk particle in stools during experimentally induced gastroenteritis in volunteers as determined by immune electron microscopy. J. Infect. Dis. 132:28-34. 19. Wilson R, Anderson L J, Holman R C, Gary G W, and Greenberg H B. 1982. Waterborne gastroenteritis due to the Norwalk agent: clinical and epidemiologic investigation. Am. J. Public Health 72:72-74. 20. Hayashi Y, Ando T, Utagawa E, Sekine S, Okada S, Yabuuchi K, Miki T, and Ohashi M. 1989. Western Blot (Immunoblot) Assay, Round-Structured Virus Associated with an Acute Gastroenteritis Outbreak in Tokyo. J. Clin. Microbiol. 27:1728-1733. 21. U.S. Pat. No. 4,358,535, issued Nov. 9, 1982, to Fahkow S and Moseley S L. Specific DNA Probes in Diagnostic Microbiology. 22. U.S. Pat. No. 4,751,080, issued Jun. 14, 1988, to Wyatt R G, Kapikian A Z, Chanock R M, Midthum K, Flores J, Hoshino Y. Vaccine Against Rotavirus Diseases. 23. U.S. Pat. No. 4,814,268, issued Mar. 21, 1989, to Kreider J W and Howett M. K. Methods for Propagating Fastidious Human Viruses and for Producing Purified Suspensions Thereof. SUMMARY OF THE INVENTION It is therefore an object of the invention to characterize the Norwalk and related virus genomes by synthesizing and cloning a cDNA library. It is an associated object of the invention to deduce an amino acid sequence of cDNA. Another object of the invention is to develop a method of preparing polyclonal and monoclonal antibodies to the Norwalk and related viruses. Still another object of the invention is to develop a method of making probes to detect Norwalk and related viruses. A further object of the invention is to use the cDNA or fragments or derivatives thereof in assays to detect Norwalk and related viruses in samples suspected of containing the viruses. A nucleotide sequence of the genome sense strand of a fragment of the Norwalk virus cDNA clone according to the presently preferred embodiment of the invention intended to accomplish the foregoing objects includes: GGCGTCAAAA GACGTCGTTC CTACTGCTGC TAGCAGTGAA 40 - AATGCTAACA ACAATAGTAG TATTAAGTCT CGTCTATTGG 80 - CGAGACTCAA GGGTTCAGGT GGGGCTACGT CCCCACCCAA 120 - CTCGATAAAG ATAACCAACC AAGATATGGC TCTGGGGCTG 160 - ATTGGACAGG TCCCAGCGCC AAAGGCCACA TCCGTCGATG 200 - TCCCTAAACA ACAGAGGGAT AGACCACCAC GGACTGTTGC 240 - CGAAGTTCAA CAAAATTTGC GTTGGACTGA GAGACCACAA 280 - GACCAGAATG TTAAGACGTG GGATGAGCTT GACCACACAA 320 - CAAAACAACA GATACTTGAT GAACACGCTG AGTGGTTTGA 360 - TGCCGGTGGC TTAGGTCCAA GTACACTACC CACTAGTCAT 400 - GAACGGTACA CACATGAGAA TGATGAAGGC CACCAGGTAA 440 - AGTGGTCGGC TAGGGAAGGT GTAGACCTTG GCATATCCGG 480 - GCTCACGACG GTGTCTGGGC CTGAGTGGAA TATGTGCCCG 520 - CTACCACCAG TTGACCAAAG GAGCACGACA CCTGCAACTG 560 - AGCCCACAAT TGGTGACATG ATCGAATTCT ATGAAGGGCA 600 - CATCTATCAT TATGCTATAT ACATAGGTCA AGGCAAGACG 640 - GTGGGTGTAC ACTCCCCTCA AGCAGCCTTC TCAATAACGA 680 - GGATCACCAT ACAGCCCATA TCAGCTTGGT GGCGAGTCTG 720 - TTATGTCCCA CAACCAAAAC AGAGGCTCAC ATACGACCAA 760 - CTCAAAGAAT TAGAAAATGA ACCATGGCCG TATGCCGCAG 800 - TCACGAACAA CTGCTTCGAA TTTTGTTGCC AGGTCATGTG 840 - CTTGGAAGAT ACTTGGTTGC AAAGGAAGCT CATCTCCTCT 880 - GGCCGGTTTT ACCACCCGAC CCAAGATTGG TCCCGAGACA 920 - CTCCAGAATT CCAACAAGAC AGCAAGTTAG AGATGGTTAG 960 - GGATGCAGTG CTAGCCGCTA TAAATGGGTT GGTGTCGCGG 1000 - CCATTTAAAG ATCTTCTGGG TAAGCTCAAA CCCTTGAACG 1040 - TGCTTAACTT ACTTTCAAAC TGTGATTGGA CGTTCATGGG 1080 - GGTCGTGGAG ATGGTGGTCC TCCTTTTAGA ACTCTTTGGA 1120 - ATCTTTTGGA ACCCACCTGA TGTTTCCAAC TTTATAGCTT 1160 - CACTCCTGCC AGATTTCCAT CTACAGGGCC CCGAGGACCT 1200 - TGCCAGGGAT CTCGTGCCAA TAGTATTGGG GGGGATCCGC 1240 - TTAGCCATAG GATTCACCAG AGACAAGGTA AGTAAGATGA 1280 - TGAAGAATGC TGTTGATGGA CTTCGTGCGG CAACCCAGCT 1320 - CGGTCAATAT GGCCTAGAAA TATTCTCATT ACTAAAGAAG 1360 - TACTTCTTCG GTGGTGATCA AACAGAGAAA ACCCTAAAAG 1400 - ATATTGAGTC AGCAGTTATA GATATGGAAG TACTATCATC 1440 - TACATCAGTG ACTCAGCTCG TGAGGGACAA ACAGTCTGCA 1480 - CGGGCTTATA TGGCCATCTT AGATAATGAA GAAGAAAAGG 1520 - CAAGGAAATT ATCTGTCAGG AATGCCGACC CACACGTAGT 1560 - ATCCTCTACC AATGCTCTCA TATCCCGGAT CTCAATGGCT 1600 - AGGGCTGCAT TGGCCAAGGC TCAAGCTGAA ATGACCAGCA 1640 - GGATGCGTCC TGTGGTCATT ATGATGTGTG GGCCCCCTGG 1680 - TATAGGTAAA ACCAAGGCAG CAGAACATCT GGCTAAACGC 1720 - CTAGCCAATG AGATACGGCC TGGTGGTAAG GTTGGGCTGG 1760 - TCCCACGGGA GGCAGTGGAT CATTGGGATG GATATCACGG 1800 - AGAGGAAGTG ATGCTGTGGG ACGACTATGG AATGACAAAG 1840 - ATACAGGAAG ACTGTAATAA ACTGCAAGCC ATAGCCGACT 1880 - CAGCCCCCCT AACACTCAAT TGTGACCGAA TAGAAAACAA 1920 - GGGAATGCAA TTTGTGTCTG ATGCTATAGT CATCACCACC 1960 - AATGCTCCTG GCCCAGCCCC AGTGGACTTT GTCAACCTCG 2000 - GGCCTGTTTG CCGAAGGGTG GACTTCCTTG TGTATTGCAC 2040 - GGCACCTGAA GTTGAACACA CGAGGAAAGT CAGTCCTGGG 2080 - GACACAACTG CACTGAAAGA CTGCTTCAAG CCCGATTTCT 2120 - CACATCTAAA AATGGAGTTG GCTCCCCAAG GGGGCTTTGA 2160 - TAACCAAGGG AATACCCCGT TTGGTAAGGG TGTGATGAAG 2200 - CCCACCACCA TAAACAGGCT GTTAATCCAG GCTGTAGCCT 2240 - TGACGATGGA GAGACAGGAT GAGTTCCAAC TCCAGGGGCC 2280 - TACGTATGAC TTTGATACTG ACAGAGTAGC TGCGTTCACG 2320 - AGGATGGCCC GAGCCAACGG GTTGGGTCTC ATATCCATGG 2360 - CCTCCCTAGG CAAAAAGCTA CGCAGTGTCA CCACTATTGA 2400 - AGGATTAAAG AATGCTCTAT CAGGCTATAA AATATCAAAA 2440 - TGCAGTATAC AATGGCAGTC AAGGGTGTAC ATTATAGAAT 2480 - CAGATGGTGC CAGTGTACAA ATCAAAGAAG ACAAGCAAGC 2520 - TTTGACCCCT CTGCAGCAGA CAATTAACAC GGCCTCACTT 2560 - GCCATCACTC GACTCAAAGC AGCTAGGGCT GTGGCATACG 2600 - CTTCATGTTT CCAGTCCGCC ATAACTACCA TACTACAAAT 2640 - GGCGGGATCT GCGCTCGTTA TTAATCGAGC GGTCAAGCGT 2680 - ATGTTTGGTA CCCGTACAGC AGCCATGGCA TTAGAAGGAC 2720 - CTGGGAAAGA ACATAATTGC AGGGTCCATA AGGCTAAGGA 2760 - AGCTGGAAAG GGGCCCATAG GTCATGATGA CATGGTAGAA 2800 - AGGTTTGGCC TATGTGAAAC TGAAGAGGAG GAGAGTGAGG 2840 - ACCAAATTCA AATGGTACCA AGTGATGCCG TCCCAGAAGG 2880 - AAAGAACAAA GGCAAGACCA AAAAGGGACG TGGTCGCAAA 2920 - AATAACTATA ATGCATTCTC TCGCCGTGGT CTGAGTGATG 2960 - AAGAATATGA AGAGTACAAA AAGATCAGAG AAGAAAAGAA 3000 - TGGCAATTAT AGTATACAAG AATACTTGGA GGACCGCCAA 3040 - CGATATGAGG AAGAATTAGC AGAGGTACAG GCAGGTGGTG 3080 - ATGGTGGCAT AGGAGAAACT GAAATGGAAA TCCGTCACAG 3120 - GGTCTTCTAT AAATCCAAGA GTAAGAAACA CCAACAAGAG 3160 - CAACGGCGAC AACTTGGTCT AGTGACTGGA TCAGACATCA 3200 - GAAAACGTAA GCCCATTGAC TGGACCCCGC CAAAGAATGA 3240 - ATGGGCAGAT GATGACAGAG AGGTGGATTA TAATGAAAAG 3280 - ATCAATTTTG AAGCTCCCCC GACACTATGG AGCCGAGTCA 3320 - CAAAGTTTGG ATCAGGATGG GGCTTTTGGG TCAGCCCGAC 3360 - AGTGTTCATC ACAACCACAC ATGTAGTGCC AACTGGTGTG 3400 - AAAGAATTCT TTGGTGAGCC CCTATCTAGT ATAGCAATCC 3440 - ACCAAGCAGG TGAGTTCACA CAATTCAGGT TCTCAAAGAA 3480 - AATGCGCCCT GACTTGACAG GTATGGTCCT TGAAGAAGGT 3520 - TGCCCTGAAG GGACAGTCTG CTCAGTCCTA ATTAAACGGG 3560 - ATTCGGGTGA ACTACTTCCG CTAGCCGTCC GTATGGGGGC 3600 - TATTGCCTCC ATGAGGATAC AGGGTCGGCT TGTCCATGGC 3640 - CAATCAGGGA TGTTACTGAC AGGGGCCAAT GCAAAGGGGA 3680 - TGGATCTTGG CACTATACCA GGAGACTGCG GGGCACCATA 3720 - CGTCCACAAG CGCGGGAATG ACTGGGTTGT GTGTGGAGTC 3760 - CACGCTGCAG CCACAAAGTC AGGCAACACC GTGGTCTGCG 3800 - CTGTACAGGC TGGAGAGGGC GAAACCGCAC TAGAAGGTGG 3840 - AGACAAGGGG CATTATGCCG GCCACGAGAT TGTGAGGTAT 3880 - GGAAGTGGCC CAGCACTGTC AACTAAAACA AAATTCTGGA 3920 - GGTCCTCCCC AGAACCACTG CCCCCCGGAG TATATGAGCC 3960 - AGCATACCTG GGGGGCAAGG ACCCCCGTGT ACAGAATGGC 4000 - CCATCCCTAC AACAGGTACT ACGTGACCAA CTGAAACCCT 4040 - TTGCGGACCC CCGCGGCCGC ATGCCTGAGC CTGGCCTACT 4080 - GGAGGCTGCG GTTGAGACTG TAACATCCAT GTTAGAACAG 4120 - ACAATGGATA CCCCAAGCCC GTGGTCTTAC GCTGATGCCT 4160 - GCCAATCTCT TGACAAAACT ACTAGTTCGG GGTACCCTCA 4200 - CCATAAAAGG AAGAATGATG ATTGGAATGG CACCACCTTC 4240 - GTTGGAGAGC TCGGTGAGCA AGCTGCACAC GCCAACAATA 4280 - TGTATGAGAA TGCTAAACAT ATGAAACCCA TTTACACTGC 4320 - AGCCTTAAAA GATGAACTAG TCAAGCCAGA AAAGATTTAT 4360 - CAAAAAGTCA AGAAGCGTCT ACTATGGGGC GCCGATCTCG 4400 - GAACAGTGGT CAGGGCCGCC CGGGCTTTTG GCCCATTTTG 4440 - TGACGCTATA AAATCACATG TCATCAAATT GCCAATAAAA 4480 - GTTGGCATGA ACACAATAGA AGATGGCCCC CTCATCTATG 4520 - CTGAGCATGC TAAATATAAG AATCATTTTG ATGCAGATTA 4560 - TACAGCATGG GACTCAACAC AAAATAGACA AATTATGACA 4600 - GAATCCTTCT CCATTATGTC GCGCCTTACG GCCTCACCAG 4640 - AATTGGCCGA GGTTGTGGCC CAAGATTTGC TAGCACCATC 4680 - TGAGATGGAT GTAGGTGATT ATGTCATCAG GGTCAAAGAG 4720 - GGGCTGCCAT CTGGATTCCC ATGTACTTCC CAGGTGAACA 4760 - GCATAAATCA CTGGATAATT ACTCTCTGTG CACTGTCTGA 4800 - GGCCACTGGT TTATCACCTG ATGTGGTGCA ATCCATGTCA 4840 - TATTTCTCAT TTTATGGTGA TGATGAGATT GTGTCAACTG 4880 - ACATAGATTT TGACCCAGCC CGCCTCACTC AAATTCTCAA 4920 - GGAATATGGC CTCAAACCAA CAAGGCCTGA CAAAACAGAA 4960 - GGACCAATAC AAGTGAGGAA AAATGTGGAT GGACTGGTCT 5000 - TCTTGCGGCG CACCATTTCC CGTGATGCGG CAGGGTTCCA 5040 - AGGCAGGTTA GATAGGGCTT CGATTGAACG CCAAATCTTC 5080 - TGGACCCGCG GGCCCAATCA TTCAGATCCA TCAGAGACTC 5120 - TAGTGCCACA CACTCAAAGA AAAATACAGT TGATTTCACT 5160 - TCTAGGGGAA GCTTCACTCC ATGGTGAGAA ATTTTACAGA 5200 - AAGATTTCCA GCAAGGTCAT ACATGAAATC AAGACTGGTG 5240 - GATTGGAAAT GTATGTCCCA GGATGGCAGG CCATGTTCCG 5280 - CTGGATGCGC TTCCATGACC TCGGATTGTG GACAGGAGAT 5320 - CGCGATCTTC TGCCCGAATT CGTAAATGAT GATGCGTCTA 5360 ** - AGGACGCTAC ATCAAGCGTG GATGGCGCTA GTGGCGCTGG 5400 - TCAGTTGGTA CCGGAGGTTA ATGCTTCTGA CCCTCTTGCA 5440 - ATGGATCCTG TAGCAGGTTC TTCGACAGCA GTCGCGACTG 5480 - CTGGACAAGT TAATCCTATT GATCCCTGGA TAATTAATAA 5520 - TTTTGTGCAA GCCCCCCAAG GTGAATTTAC TATTTCCCCA 5560 - AATAATACCC CCGGTGATGT TTTGTTTGAT TTGAGTTTGG 5600 - GTCCCCATCT TAATCCTTTC TTGCTCCATC TATCACAAAT 5640 - GTATAATGGT TGGGTTGGTA ACATGAGAGT CAGGATTATG 5680 - CTAGCTGGTA ATGCCTTTAC TGCGGGGAAG ATAATAGTTT 5720 - CCTGCATACC CCCTGGTTTT GGTTCACATA ATCTTACTAT 5760 - AGCACAAGCA ACTCTCTTTC CACATGTGAT TGCTGATGTT 5800 - AGGACTCTAG ACCCCATTGA GGTGCCTTTG GAAGATGTTA 5840 - GGAATGTTCT CTTTCATAAT AATGATAGAA ATCAACAAAC 5880 - CATGCGCCTT GTGTGCATGC TGTACACCCC CCTCCGCACT 5920 - GGTGGTGGTA CTGGTGATTC TTTTGTAGTT GCAGGGCGAG 5960 - TTATGACTTG CCCCAGTCCT GATTTTAATT TCTTGTTTTT 6000 - AGTCCCTCCT ACGGTGGAGC AGAAAACCAG GCCCTTCACA 6040 - CTCCCAAATC TGCCATTGAG TTCTCTGTCT AACTCACGTG 6080 - CCCCTCTCCC AATCAGTAGT ATGGGCATTT CCCCAGACAA 6120 - TGTCCAGAGT GTGCAGTTCC AAAATGGTCG GTGTACTCTG 6160 - GATGGCCGCC TGGTTGGCAC CACCCCAGTT TCATTGTCAC 6200 - ATGTTGCCAA GATAAGAGGG ACCTCCAATG GCACTGTAAT 6240 - CAACCTTACT GAATTGGATG GCACACCCTT TCACCCTTTT 6280 - GAGGGCCCTG CCCCCATTGG GTTTCCAGAC CTCGGTGGTT 6320 - GTGATTGGCA TATCAATATG ACACAGTTTG GCCATTCTAG 6360 - CCAGACCCAG TATGATGTAG ACACCACCCC TGACACTTTT 6400 - GTCCCCCATC TTGGTTCAAT TCAGGCAAAT GGCATTGGCA 6440 - GTGGTAATTA TGTTGGTGTT CTTAGCTGGA TTTCCCCCCC 6480 - ATCACACCCG TCTGGCTCCC AAGTTGACCT TTGGAAGATC 6520 - CCCAATTATG GGTCAAGTAT TACGGAGGCA ACACATCTAG 6560 - CCCCTTCTGT ATACCCCCCT GGTTTCGGAG AGGTATTGGT 6600 - CTTTTTCATG TCAAAAATGC CAGGTCCTGG TGCTTATAAT 6640 - TTGCCCTGTC TATTACCACA AGAGTACATT TCACATCTTG 6680 - CTAGTGAACA AGCCCCTACT GTAGGTGAGG CTGCCCTGCT 6720 - CCACTATGTT GACCCTGATA CCGGTCGGAA TCTTGGGGAA 6760 - TTCAAAGCAT ACCCTGATGG TTTCCTCACT TGTGTCCCCA 6800 - ATGGGGCTAG CTCGGGTCCA CAACAGCTGC CGATCAATGG 6840 - GGTCTTTGTC TTTGTTTCAT GGGTGTCCAG ATTTTATCAA 6880 - TTAAAGCCTG TGGGAACTGC CAGCTCGGCA AGAGGTAGGC 6920 - TTGGTCTGCG CCGATAATGG CCCAAGCCAT AATTGGTGCA 6960 - ATTGCTGCTT CCACAGCAGG TAGTGCTCTG GGAGCGGGCA 7000 - TACAGGTTGG TGGCGACAGG CCCTCCAAAG CCAAAGGTAT 7040 - CAACAAAATT TGCAACTGCA AGAAAATTCT TTTAAACATG 7080 - ACAGGGAAAT GATTGGGTAT CAGGTTGAAG CTTCAAATCA 7120 - ATTATTGGCT AAAAATTTGG CAACTAGATA TTCACTCCTC 7160 - CGTGCTGGGG GTTTGACCAG TGCTGATGCA GCAAGATCTG 7200 - TGGCAGGAGC TCCAGTCACC CGCATTGTAG ATTGGAATGG 7240 - CGTGAGAGTG TCTGCTCCCG AGTCCTCTGC TACCACATTG 7280 - AGATCCGGTG GCTTCATGTG AGTTCCCATA CCATTTGCCT 7320 - CTAAGCAAAA ACAGGTTCAA TCATCTGGTA TTAGTAATCC 7360 - AAATTATTCC CCTTCATCCA TTTCTCGAAC CACTAGTTGG 7400 - GTCGAGTCAC AAAACTCATC GAGATTTGGA AATCTTTCTC 7440 - CATACCACGC GGAGGCTCTC AATACAGTGT GGTTGACTCC 7480 - ACCCGGTTCA ACAGCCTCTT CTACACTGTC TTCTGTGCCA 7520 - CGTGGTTATT TCAATACAGA CAGGTTGCCA TTATTCGCAA 7560 - ATAATAGGCG ATGATGTTGT AATATGAAAT GTGGGCATCA 7600 - TATTCATTTA ATTAGGTTTA ATTAGGTTTA ATTTGATGTT 7640 - AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 7680 - AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 7720 - AA 7722 Within the above nucleotide sequence is a region where a RNA-dependent RNA polymerase is found. The RNA-dependent RNA polymerase is between bases 4543 and 4924 of the nucleotide sequence of the genome. The RNA polymerase and the corresponding oligopeptide include: CAT TTT GAT GCA GAT TAT ACA GCA TGG GAC TCA ACA CAA AAT HIS PHE ASP ALA ASP TYR THR ALA TRP ASP SER THR GLN ASN 5 10 - AGA CAA ATT ATG ACA GAA TCC TTC TCC ATT ATG TCG CGC CTT ARG GLN ILE MET THR GLU SER PHE SER THR MET SER ARG LEU 15 20 25 - ACG GCC TCA CCA GAA TTG GCC GAG GTT GTG GCC CAA GAT TTG THR ALA SER PRO GLU LEU ALA GLU VAL VAL ALA GLN ASP LEU 30 35 40 - CTA GCA CCA TCT GAG ATG GAT GTA GGT GAT TAT GTC ATC AGG LEU ALA PRO SER GLU MET ASP VAL GLY ASP TYR VAL ILE ARG 50 55 60 - GTC AAA GAG GGG CTG CCA TCT GGA TTC CCA TGT ACT TCC CAG VAL LYS GLU GLY LEU PRO SER GLY PHE PRO CYS THR SER GLN 65 70 75 - GTG AAC AGC ATA AAT CAC TGG ATA ATT ACT CTC TGT GCA CTG VAL ASN SER ILE ASN HIS TRP ILE ILE THR LEU CYS ALA LEU 80 85 - TCT GAG GCC ACT GGT TTA TCA CCT GAT GTG GTG CAA TCC ATG SER GLU ALA THR GLY LEU SER PRO ASP VAL VAL GLN SER MET 90 95 100 - TCA TAT TTC TCA TTT TAT GGT GAT GAT GAG ATT GTG TCA ACT SER TYR PHE SER PHE TYR GLY ASP ASP GLU ILE VAL SER THR 105 110 115 - GAC ATA GAT TTT GAC CCA GCC CGC CTC ACT CAA ATT CTC AAG GAA ASP ILE ASP PHE ASP PRO ALA ARG LEU THR GLN ILE LEU LYS GLU 120 125 130 This oligopeptide and other cDNAs representing the entire genome found with this cDNA, or other oligonucleotides or fragments of the entire cDNA, are used to make diagnostic products and vaccines. Other and still further objects, features and advantages of the present invention will be apparent from the following description of a presently preferred embodiment of the invention. BRIEF DESCRIPTION OF THE FIGURES FIG. 1. EM picture of Norwalk viruses after CsCl gradient purification. FIG. 2a. Hybridization of stool samples with .sup.32 P-labeled plasmid DNA for screening positive Norwalk cDNA clones. Nucleic acids from paired stools [before (b) and after (a) infection with Norwalk virus] from two volunteers (1 and 2) were dotted on Zetabind filters. Replicate strips were prepared and hybridized at 50.degree. C. and 65.degree. C. with each test clone (pUC-27, pUC-593, pUC-13 and pUCNV-953). One clone (pUCNV-953) which reacted only with stool samples after (but not before) Norwalk infection was considered as a potential positive clone and was chosen for further characterization. FIG. 2b. Dot blot hybridization of clone .sup.32 P-labeled pUCNV-953 with another 3 sets of stool samples collected at different times after infection (B=before acute phase of illness; A=acute phase of illness; P=post-acute phase of illness) of 3 volunteers. The nucleic acids were dotted directly or after treatment with RNAse or with DNAse before dotting. Double-stranded homologous cDNA (pUCNV-953) was dotted after the same treatments as the stool samples. FIG. 3. Dot blot hybridization of Norwalk viruses in a CsCl gradient with ssRNA probes made from pGEMNV-953. Aliquots of 50 ul from each fraction in a CsCl gradient were dotted onto a Zetabind filter. Duplicates of filters were made and hybridized with the two ssRNA probes respectively. The two strands were subsequently called cRNA (positive hybridization with the viral nucleic acid) and vRNA (no hybridization with the viral nucleic acid, data not shown). The graph shows EM counts of Norwalk viruses from each fraction of the same CsCl gradient for the dot blot hybridization. Five squares from each grid were counted and the average of the number of viral particles per square was calculated. FIG. 4. Hybridization of Norwalk viral RNA with .sup.32 P-labeled clone pUCNV-953. Nucleic acids extracted from partially purified viruses were electrophoresed in a native agarose gel as described previously (Jiang et al., 1989). The gel was then dried at 80.degree. C. for 1 h and hybridized with .sup.32 P-labeled pUCNV-953 insert. Lane 1, 23 S and 16 S rRNA from E. coli (Miles Laboratories Inc., Naperville, Ill. 60566), lanes 2 and 4, total nucleic acids from partially purified stool samples containing Norwalk virus, and lane 3, HAV RNA. FIG. 5. The nucleotide sequence of the genome sense strand of the first Norwalk virus cDNA clone. The deduced amino acid sequence of a long open reading frame in this cDNA is also shown. FIG. 6. Physical map of Norwalk virus specific clones isolated from the pUC-13 library. This map assumes the Norwalk genome is 8 kb and shows only a subset (the four largest) of .about.100 characterized clones. cDNAs which represent at least 7 kb of nucleic acid have been identified by hybridization with pre-and post infected stool samples, or by rescreening the library with 5'-end probes of the original (pUCNV-953) and subsequent positive clones. A poly(A) tail (.about.80 bases) is present at the 3'-end of clone pUCNV-4145. Clone pUCNV-1011 also hybridized specifically with post- (but not pre-) infection stools from volunteers (see FIG. 7). FIG. 7a. Dot blot hybridization of stool samples with .sup.32 P-labeled probes, derived from pUCNV-953, representing the 3'-end of the Norwalk viral genome. Stool samples were collected from 5 volunteers at different times (a-e) after infection with Norwalk virus. Samples in column (a) were collected in the first 24 h post-infection, before symptoms appeared. The rest of the stool samples were collected from day 2 to day 5 post-infection. Nucleic acids were extracted and duplicate dots were immobilized on a Zetabind filter. FIG. 7b. Dot blot hybridization of stool samples with .sup.32 P-labeled probes, derived from pUCNV-1011, representing the 5'-end of the Norwalk viral genome. Stool samples were collected from 5 volunteers at different times (a-e) after infection with Norwalk virus. Samples in column (a) were collected in the first 24 h post-infection, before symptoms appeared. The rest of the stool samples were collected from day 2 to day 5 post-infection. Nucleic acids were extracted and duplicate dots were immobilized on a Zetabind filter. FIG. 8. Norwalk virus encodes an RNA-directed RNA polymerase sequence motif. The deduced amino acid sequence of a portion of Norwalk virus pUCNV-4095 (NV) is compared with consensus amino acid residues thought to encode putative RNA-directed RNA polymerases of hepatitis E virus (HEV), hepatitis C virus (HCV), hepatitis A virus (HAV), Japanese encephalitis virus (JE), poliovirus (polio), foot-and-mouth disease virus (FMD), encephalomyocarditis virus (EMC), Sindbis virus (SNBV), tobacco mosaic virus (TMV), alfalfa mosaic virus (AMV), brome mosaic virus (BMV), and cowpea mosaic virus (CpMV). Sequences for viruses other than NV are from FIG. 3 of Reyes et al., Science 247:1335-1339. FIG. 9. Three sets of primers used to amplify the Norwalk virus genome. DETAILED DESCRIPTION OF THE INVENTION It is readily apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The term "fragment" as used herein is defined as a fragment of a genome or a subgenomic clone that is required to be expressed to produce a peptide fragment which might be able to induce a polyclonal or monoclonal antibody. It is possible a peptide of only 5 amino acids could be immunogenic but usually peptides of 15 amino acids or longer are required. This depends on the properties of the peptide and it cannot be predicted in advance. The term "derivative" as used herein is defined as larger pieces of DNA or an additional cDNA which represents the Norwalk genome and which is detected by direct or sequential use of the original cDNA and any deduced amino acid sequences thereof. Clone pUCNV-1011, therefore, is a derivative, although it does not overlap or share sequences with the original clone. Also included within the definition of derivative are RNA counterparts of DNA fragments and DNA or cDNA fragments in which one or more bases have been substituted or to which labels and end structures have been added without effecting the reading or expression of the DNA or cDNA. Production of Norwalk Virus for Molecular Cloning Norwalk virus was produced by administration of safety tested Norwalk virus (8FIIa) to adult volunteers. The virus inoculum used in the volunteer study, was kindly supplied by Dr. Albert Kapikian (Laboratory of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md.). This virus originated from an outbreak of acute gastroenteritis in Norwalk, Ohio (Dolin et al., 1971). Two ml of a 1 to 100 dilution of 8FIIa in TBS was administered orally to each individual with 80 ml of milli-Q water (Millipore, Bedford, Mass. 01730). Sodium bicarbonate solution was taken by each person 2 min before and 5 min after virus administration. The volunteer studies were approved by the Institutional Review Board for Human Research at Baylor College of Medicine, at the Methodist Hospital and at the General Clinical Research Center. The virus was administered to the volunteers in the General Clinical Research Center where the volunteers were hospitalized and under extensive medical care for 4 days. All stools were collected and kept at -70.degree. C. for later use. Purification of Norwalk Viruses from Stool Samples A 10% solution of stool samples in TBS was clarified by low speed centrifugation at 3000 rpm for 15 min. The resultant supernate were then extracted two to three times with genetron in the presence of 0.5% Zwittergent 3-14 detergent (Calbiochem Corp., La Jolla, Calif.). Viruses in the aqueous phase were concentrated by pelleting at 36,000 rpm for 90 minutes through a 40% sucrose cushion in a 50.2 Ti rotor (Beckman Instruments, Inc., Palo Alto, Calif. 94304). The pellets were suspended in TBS and mixed with CsCl solution (refractive index 1.368) and centrifuged at about 35,000 rpm for about 24 h in a SW50.1 rotor (Beckman). The CsCl gradient was fractionated by bottom puncture and each fraction was monitored for virus by EM examination. The peak fractions containing Norwalk virus were pooled and CsCl in the samples was diluted with TBS and removed by pelleting the viruses at about 35,000 rpm for 1 h. The purified virus was stored at about -70.degree. C. Extraction of Nucleic Acids from Purified Virus One method of extraction involved treating purified Norwalk virus from CsCl gradients with proteinase K (400 ug/ml) in proteinase K buffer (0.1 M Tris--Cl pH 7.5, 12.5 mM EDTA, 0.15 M NaCl, 1% w/v SDS) at about 37.degree. C. for about 30 min. The samples were then extracted once with phenol-chloroform and once with chloroform. Nucleic acids in the aqueous phase were concentrated by precipitation with 2.5 volumes of ethanol in the presence of 0.2 M NaOAc followed by pelleting for 15 min in a microcentrifuge. cDNA Synthesis and Cloning of Amplified of cDNA One method of synthesis and cloning included denaturing nucleic acids extracted from the purified Norwalk viruses with 10 mM CH.sub.3 HgOH. Then cDNA was synthesized using the cDNA synthesis kit with the supplied random hexanucleotide primer (Amersham, Arlington Heights, Ill. 60005). After the second strand synthesis, the reaction mixture was extracted once with phenol-chloroform and once with chloroform followed by ethanol precipitation. Amplification of DNA was performed using the random prime kit for DNA labeling (Promega Corp., Madison, Wis. 53711-5305). Eight cycles of denaturation (100.degree. C. for 2 min), reannealing (2 min cooling to room temperature) and elongation (room temperature for 30 min) were performed after addition of Klenow fragment (Promega Corp.). A DNA library was constructed in pUC-13 with blunt-end ligation into the Sma I site. Screening of the Library for Positive Clones As one method of screening, white colonies from transformed DH5 alpha bacterial cells (BRL) were picked and both a master plate and minipreps of plasmid DNA were prepared for each clone. Clones containing inserts were identified after electrophoresis of the plasmid DNA in an agarose gel. The insert DNA in the agarose gel was cut out and labeled with .sup.32 P using random primers and Klenow DNA polymerase such as in the prime-a-gene.RTM. labeling system (Promega Corp.). Other isotopic or biochemical labels, such as enzymes, and fluorescent, chemiluminescent or bioluminescent substrates can also be used. Nucleic acids extracted from paired stool samples (before and after Norwalk infection) from two volunteers (543 and 544) were dotted onto Zetabind filters (AFM, Cuno, Meriden, Conn.). Replicate filter strips were prepared and hybridized with each labeled plasmid probe individually at 65.degree. C. without formamide. Potential positive clones were judged by their different reactions with the pre- and post-infection stools. Clones which reacted with post- (but not pre-) infection stools of volunteers were considered positive and these clones on the master plates were characterized further. Once one Norwalk clone was identified, it was used to rescreen the cDNA library to identify additional overlapping clones. Rescreening the cDNA library with these additional clones can ultimately identify clones representing the entire Norwalk virus genome. |
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