WO1994005700A2 - Methods and reagents to detect and characterize norwalk and related viruses - Google Patents

Methods and reagents to detect and characterize norwalk and related viruses Download PDF

Info

Publication number
WO1994005700A2
WO1994005700A2 PCT/US1993/008447 US9308447W WO9405700A2 WO 1994005700 A2 WO1994005700 A2 WO 1994005700A2 US 9308447 W US9308447 W US 9308447W WO 9405700 A2 WO9405700 A2 WO 9405700A2
Authority
WO
WIPO (PCT)
Prior art keywords
norwalk
leu
protein
gly
ala
Prior art date
Application number
PCT/US1993/008447
Other languages
French (fr)
Other versions
WO1994005700A3 (en
Inventor
David O. Matson
Mary K. Estes
Xi Jiang
David Y. Graham
Original Assignee
Baylor College Of Medicine
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Baylor College Of Medicine filed Critical Baylor College Of Medicine
Priority to AU48514/93A priority Critical patent/AU4851493A/en
Priority to JP6507522A priority patent/JPH08500250A/en
Publication of WO1994005700A2 publication Critical patent/WO1994005700A2/en
Publication of WO1994005700A3 publication Critical patent/WO1994005700A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/127—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/16011—Caliciviridae
    • C12N2770/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates generally to synthesizing clones of Norwalk virus and calicivirus and to making probes to Norwalk and related viruses. It also relates to methods of detection and characterization of Norwalk and related viruses.
  • 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., Am. J. Hyg. 58:16-30 (1953); Kapikian and Chanock, "Norwalk group of viruses" in B.N. Fields' 2d ed. of Virology. Raven Press, New York, pp. 671-693 (1990)). Up to 42% of cases of adult viral gastroenteritis have been estimated to be caused by Norwalk or Norwalk-like viruses (Kaplan et al., Ann. Internal Med. 96(6):756-761 (1982)).
  • the concentration of the virus in stool is usually so low that virus detection with routine electron microscopy is not possible (Dolin et al., Proc. Soc. Exp. Med. and Biol. 140:578-583 (1972); Kapikian et al., J. Virol. 10:1075-1081 (1972); Thornhill et al., J. Infect. Dis. 132:28-34 (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.
  • RIAs radio immunoassays
  • ELISAs biotin-avidin enzyme linked immunoabsorbent assays
  • Viruses related to Norwalk virus include small round enteric viruses, such as viruses with typical calicivirus morphology and the astro viruses.
  • the classification scheme for the human small enteric viruses shown in Table 1 here is an updated version of a scheme outlined by Caul and Appleton in the Journal of Medical Virology, 9:257-265 (1982). This system is referred to in Cubitt et al., J. Infectious Diseases, 156:806-814 (1987); Table 1 of the article by Appleton entitled “Small round viruses: classification and role in food-borne infections", in the book Novel Diarrhoea Viruses. Ciba Foundation Symposium No. 128, pp. 108- 125 (John Wiley & Sons, N.Y. (1987)); and Table 1 of the chapter entitled “Norwalk group of viruses” by Kapikian and Chanock from the book Virology (B.N.Fields, 2d ed., Raven Press (1990)).
  • human small round structured enteric viruses include calicivirus and astro virus.
  • the recent sequencing of Norwalk virus indicates that Norwalk virus is a calicivirus and has a genome organization like that of other caliciviruses.
  • human small round enteric viruses are a large number of non-human small round viruses which have been classified as astroviruses, caliciviruses, and small round structured viruses based upon their morphology.
  • viruses examples include the primate calicivirus isolated from the pygmy chimpanzee, described in the journal Science 221:79-81 (1983), a porcine enteric calicivirus, described in the Journal of Clinical Microbiology 12:105-111 (1980), and bovine astroviruses described in VetPathol. 21:208-215 (1984).
  • Individual calicivirus types will at times exhibit host specificity and tissue tropisms, but as an overall group they cause gastroenteritis, hepatitis, abortion, skin lesions, pneumonia, myocarditis, and encephalitis.
  • the caliciviruses infecting humans fit in this context in that Norwalk-like viruses cause gastroenteritis, hepatitis E causes hepatitis, and San Miguel sea lion virus type 5 causes skin vesicles in humans as well as infections in seals, fish, pigs and cattle.
  • Yet 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 still further object of the invention is to express proteins to measure antibody responses.
  • a nucleotide sequence of the genome sense strand of the Norwalk virus cDNA clone intended to accomplish the foregoing objects includes the nucleotide sequence shown in Table 2. Within the Norwalk nucleotide sequence are regions which encode proteins. The nucleotide sequence of the Norwalk virus genome, its fragments and derivatives are used to make diagnostic products, vaccines and antivirals.
  • FIG. 2a Hybridization of stool samples with 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°C and 65°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.
  • 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 50ul 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.
  • Figure 4 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 also is shown.
  • FIG. 5 Schematic diagram of Norwalk cDNA clones. pUCNV-953 was the first positive cone identified. Overlapping clones were determined by restriction enzyme analyses and partial sequencing of the clones. AAA indicates the poly (a) tail at the 3' end of the viral genome.
  • 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 Figure 3 of Reyes eial, Science 247:1335-1339 (1990).
  • FIG. 8 This schematic shows the organization of Norwalk genome given in Table 2. The features shown here are based on analyses of the nucleotide sequence of the Norwalk virus genome and the deduced amino acid sequence of proteins encoded in the genome.
  • the genome contains 7753 nucleotides including 111 A's at the 3'-end. Translation of the sequence predicts that the genome encodes three open reading frames (shown by the open boxes in the second line). The first open reading frame is predicted to start from an initiation codon at nucleotide 146 and it extends to nucleotide 5359 (excluding the termination codon).
  • the second open reading frame is initiated at nucleotide 5346 and it extends to nucleotide 6935, and a third open reading frame exists between nucleotides 6938 and 7573.
  • the first open reading frame is a protein that is eventually cleaved to make at least three proteins. These three proteins include a picornavirus 2C-like protein, a 3C-like protease and a 3D-like RNA-dependent RNA polymerase.
  • the second open reading frame encodes the capsid protein, which contains sequence homology with the picornavirus VP3 protein.
  • Figure 9 Nucleotide and amino acid sequence of human calicivirus Sapporo cDNAs. The 551 nucleotide known sequence of human calicivirus
  • HuCV Sapporo HuCV Sapporo
  • HuCV Sapporo is presented in its entirety. Below the nucleotide sequence is the amino acid sequence for HuCV Sapporo. Above the HuCV Sapporo nucleotide sequence is the sequence of the cDNA from a Houston day care center outbreak (Day care). In the Day care sequence a ".” indicates the nucleotide is identical to the HuCV Sapporo nucleotide at that site. Where a nucleotide difference occurred in the Day care sequence, a new letter is indicated at that position. "N" indicates uncertainty of the nucleotide at that site.
  • FIG. 11 Strategy used to obtain nucleotide sequence of the Norwalk- related virus SRSV/KY/89 using primers from the Norwalk virus sequence.
  • This figure shows a partial schematic of the Norwalk virus genome and the predicted ORF1 showing the location of the 3D-like polymerase region, the second ORF showing the location of the VP3-like domain and the start of ORF 3.
  • the solid lines show regions of KY89 sequenced based on using primer sets (see numbers such as 36 and 35, etc) chosen from the sequence of the Norwalk virus genome.
  • FIG. 12 Comparison of the Norwalk virus nucleotide sequence with the Norwalk virus-related virus SRSV/KY/89 nucleotide sequence. Part of the nucleotide sequence of Norwalk-related virus SRSV/KY/89 was determined using primers from the Norwalk-virus (NV) genome. Primers from the NV genome used to obtain the sequence of this Norwalk-related virus are shown in Table 6. Some of these primers were modified based on the initial nucleotide sequence obtained from the SRSV/KY/89 to obtain the rest of the sequence of SRSV/KY/89. The primers shown here and in Table 6 are used by way of example only; other NV primers can be used.
  • NV Norwalk-virus
  • Figure 13 Comparison of deduced amino acid sequence of proteins of the Norwalk virus and the Norwalk-related virus SRSV/KY/89.
  • the protein sequence of SRSV/KY/89 was deduced from the nucleotide sequence shown in Figure 12.
  • Figure 13a shows a comparison of the deduced amino acid sequence of ORF2, the capsid, of SRSV/KY/89 with the same region encoded in the Norwalk virus genome.
  • Figure 13b shows a comparison of the deduced amino acid sequence of part of the polymerase protein of SRSV/KY/89 with that of Norwalk virus. Comparisons of similar sequences from other Norwalk-related viruses will permit discovery of conserved and divergent regions including antigenic regions.
  • the information will rapidly permit choices of broadly reactive primers to detect all Norwalk-related viruses and specific primer sets to detect individual Norwalk-related viruses. Similarily, fragments and peptides with common amino acid sequences or specific amino acid sequences can be selected for development of diagnostics, vaccines and antivirals.
  • Figure 14 Comparison of partial nucleotide sequences of Norwalk virus and six Norwalk-related viruses obtained using primers from the NV genome. Sequences from SRSV/CDC 6/91, SRSV/UT/88, SMA/78; SRSV/Ca bridge, UK/92, SRSV/CDC 32, Norwalk virus/68, SRSV-3/88, SRSV/KY89/89. Figures 14a and 14b show two different regions of the genome.
  • Figure 15 Expression of the Norwalk virus capsid protein.
  • Baculovirus recombinants C-6 and C-8 that contain a subgenomic piece of Norwalk virus DNA (from nucleotides 5337 to 7753) were selected and used to infect insect (Spodoptera fugiperda) cells at a multiplicity of infection of 10 PFU/cell. After 4 days of incubation at 27° C, the infected cells were harvested and the proteins were analyzed by electrophoresis on 12% polyacrylamide gels. The proteins were visualized after staining with Coomassie blue.
  • the Norwalk-expressed protein (highlighted by the arrowhead) is only seen in the recombinant-infected cells, but not in wild- type baculovirus (wt) or mock-infected (m) insect cells.
  • Figure 16 The Norwalk virus expressed protein shows immunoreactivity with sera from volunteers infected with Norwalk virus.
  • the expressed protein shown in Figure 11 was absorbed onto the wells of a 96-well ELISA plate and its reactivity was tested with dilutions of serum samples taken from volunteers before (pre) and three weeks after (post) infection with Norwalk virus.
  • Norwalk virus-like particles can be purified in gradients of CsCl.
  • Supernatants of insect cells infected with the baculovirus recombinant C-8 were processed by extraction with genetron and PEG precipitation and virus eluted from these PEG pellets was centrifuged in
  • Figure 19 Use of the expressed virus-like particles to measure the reactivity of pre- and post-serum samples from volunteers infected with Norwalk virus shows that most volunteers have an immune response. Volunteer 6 who did not show an immune response also did not become ill after being administered virus.
  • Figure 20 Partial sequence of the primate Pan paniscus cDNA atprcvw2.
  • fragment as used herein is defined as any portion of the Norwalk virus genome or a subgenomic clone of the Norwalk virus that is required to be expressed to produce or encodes a peptide which in turn is 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.
  • derivative as used herein is defined as larger pieces of
  • DNA or an additional cDNA which represents the Norwalk virus genome and which is detected by direct or sequential use of the original cDNA and any deduced amino acid sequences thereof.
  • Clone pUCNV-1011 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 affecting the reading or expression of the DNA or cDNA.
  • the terms Norwalk "related viruses” and “Norwalk-like viruses” as used herein are defined as human and non-human calicivirus, astro virus and small round structured viruses (SRSV).
  • Norwalk-related viruses As the genomic sequences of the viruses become known, those skilled in the art will be able to determine Norwalk-related viruses and Norwalk-like viruses based on nucleotide homologies.
  • the Norwalk group includes Snow Mountain Agent (SMA), Hawaii Agent, Taunton Agent, Amulree, Otofuke, and Montgomery County Agent.
  • SMA Snow Mountain Agent
  • the Norwalk group is characterized by small, round, structured viruses with an amorphous surface or ragged outline.
  • 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, MA 01730).
  • Sodium bicarbonate solution was taken by each person 2 minutes before and 5 minutes after virus administration.
  • the volunteer studies were approved by the Institutional Review Board for Human Research at Baylor College of Medicine, at the Cincinnati 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°C for later use.
  • a 10% solution of stool samples in TBS was clarified by low speed centrifugation at 3000 rpm for 15 minutes. The resulting supernate then was extracted two to three times with genetron in the presence of 0.5% Zwittergent 3-14 detergent (Calbiochem Corp., La Jolla, CA). 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, CA 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 hours 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 hour.
  • the purified virus was stored at about -70°C.
  • 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°C for about 30 minutes. 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 minutes in a microcentrifuge.
  • One method of synthesis and cloning included denaturing nucleic acids extracted from the purified Norwalk viruses with 10 mM CH 3 HgOH. Then cDNA was synthesized using the cDNA synthesis kit with the supplied random hexanucleotide primer (Amersham, Arlington Heights, IL 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, WI 53711-5305).
  • 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, CT). Replicate filter strips were prepared and hybridized with each labeled plasmid probe individually at 65°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.
  • Complementary DNA was prepared by reverse transcriptase-polymerase chain reaction (RT-PCR) using a primer pair (for example primers 36 and 35 shown in Table 6) derived from the sequence of Norwalk virus.
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • the resulting product was ligated into a plasmid vector and transfected into E. coli. Plasmids then were partially purified from the bacteria and the inserted PCR product was sequenced in the plasmid by dideoxy chain termination to examine the relation to Norwalk virus by nucleotide and predicted protein homology.
  • Norwalk virus and related viruses a cDNA library for Norwalk was derived from nucleic acid extracted from virions purified from stool samples. Norwalk virus was purified with methods used previously for hepatitis A and rotaviruses from stool samples with some modifications
  • Figure 1 shows an electron micrographs of purified Norwalk viruses isolated by the above procedure and Norwalk-related viruses used to produce cDNAs using RT-PCR.
  • Example 2 Initial cDNA synthesis, cloning and screening A cDNA library was generated from nucleic acids extracted from these purified viruses by proteinase K treatment of the samples followed by phenol-chloroform extraction and ethanol precipitation (Jiang et al., 1986; 1987). Because the nature of the viral genome was unknown, the extracted nucleic acids were denatured with methylmercuric hydroxide before cDNA synthesis. Random primed cDNA was synthesized with the Gubler-Hoffman method (cDNA synthesis system plus, Amersham) and a small amount of cDNA was obtained. Direct cloning of this small amount of cDNA was unsuccessful.
  • a step of amplification of the DNA was performed by synthesizing more copies of the DNA with random primers and the Klenow fragment of DNA polymerase before cloning.
  • the procedure involved cycles of denaturation, addition of random primers and the Klenow fragment of DNA polymerase, reannealing and elongation. With this procedure, a linear incorporation of labeled nucleotides into product was observed as the number of cycles of synthesis was increased. The number of cycles performed was limited ( ⁇ 10) to avoid the synthesis of an excess of smaller fragments.
  • Norwalk cDNA eight cycles of amplification were performed and approximately 2.5 ug of DNA were obtained, which was at least a 100-fold amplification of the starting template cDNA. This amplified cDNA was cloned into pUC-13 by blunt-end ligation and a positive clone (pUCNV-953) was isolated.
  • FIG. 2b shows a dot blot hybridization of the clone with stool samples collected at different times post-infection of the disease. Strong signals were observed only with stools from acute phase, but not before and after the illness. This result was consistent with previous RIA assays for viral antigen detection using convalescent sera from volunteers with Norwalk diarrhea and immune electron microscopy (IEM) studies of the samples for viral particle examination. This result also agrees with the patterns of virus shedding in stool in the course of the disease (Thornhill et al., 1975 ).
  • the pUCNV-953 cDNA was subcloned into the transcription vector pGEM-3Zf(+) and grown. ssRNA probes were then generated by in vitro transcription using SP6 and T7 polymerases (Bethesda Research
  • the hybridization signals were removed by treatment of the viral nucleic acid with RNAse (but not with DNAse) before loading them onto the filters, indicating the virus genome contains ssRNA.
  • RNAse but not with DNAse
  • a long open reading frame was found in one of the two strands of the inserted DNA by the computer analysis of the sequences of pUCNV-953.
  • the ssRNA probe with the same sequence as this coding strand does not react with the viral nucleic acid, but the complementary ssRNA probe does react in the hybridization tests. Therefore, Norwalk virus contains a positive sense single-stranded RNA genome.
  • the size of the genome of Norwalk virus was estimated to be about 8 kb based on comparisons of the migration rate of the purified viral RNA in agarose gels with molecular weight markers.
  • the pUCNV-953 cDNA was used to rescreen a second cDNA library made as follows.
  • a clone of the Norwalk or related virus was synthesized by isolating nucleic acid from purified Norwalk virus; cDNA was synthesized using reverse transcriptase and random primers; a second strand of DNA was synthesized from the cDNA; and at least one copy of DNA was inserted into a plasmid or a cloning and expression vector; and screening the library with the original puCNV-953 cDNA identified clones containing fragments of (or the complete) Norwalk or related genome.
  • a cloning and expression vector such as lambda ZAPII® (Stratagene Inc.)
  • the cDNA library was screened to identify recombinant phage containing fragments of or the complete Norwalk or related genome. Additional cDNAs were made and found with this method. Use of these additional cDNAs to made and found with this method. Use of these additional cDNAs to rescreen the library resulted in detection of new clones ( Figure 5).
  • Norwalk virus cDNA sequence can be used in assays to detect the genome of Norwalk and other related viruses.
  • the detection assays include labeled cDNA or ssRNA probes for direct detection of the Norwalk virus genome and measurement of the amount of probe binding.
  • primers or small oligonucleotide probes (10 nucleotides or greater) and polymerase chain reaction amplification are used to detect the Norwalk and Norwalk-related virus genomes.
  • Expression of the open reading frame in the cDNA is used to make hyperimmune or monoclonal antibodies for use in diagnostic products, vaccines and antivirals.
  • the nucleotide sequence in Table 2 was identified. Within that nucleotide sequence, the encoding regions for several proteins have been identified. In that sequence, the first protein is encoded by nucleotides 146 through 5339 and the amino acid sequence is shown in Table 3. This first protein is eventually cleaved to make at least three proteins including a picornavirus 2C-like protein, a 3C-like protease and an RNA-dependent RNA polymerase.
  • the RNA-dependent RNA polymerase The RNA-dependent
  • RNA polymerase is deduced from nucleotides 4543 to 4924 of the Norwalk virus genome as shown in Table 3. The fact that this portion of the genome contains an RNA polymerase is verified by comparisons with RNA polymerase in other positive sense RNA viruses ( Figure 6 SE ID NOS 38 through 50).
  • Hybridization assays are the assays of choice to detect Norwalk virus because small amounts of virus are present in clinical or contaminated water and food specimens. Previously, detection of Norwalk and related nucleic acids was not possible because the genome of Norwalk virus was not known and no sequence information was available. Probes made from the Norwalk virus cDNA or primers made from the Norwalk virus genome sequence allow methods to amplify the genome for diagnostic products to be established. Probes to identify Norwalk virus alone and to identify other Norwalk-related viruses enable development of either specific assays for Norwalk or general assays to detect sequences common to many or all of the Norwalk-related agents.
  • CTAB cetyltrimethylammonium bromide
  • T oligo d(T) cellulose
  • the resulting nucleic acid was further purified by adsorption onto and elution from oligo d(T) cellulose. This step removes unrelated nucleic acids that lack a poly(A) tail.
  • Norwalk virus was detected easily by PCR in very small amounts (400 ul of a 10% suspension) of stool sample.
  • RT-PCR active nucleic acids are extracted with CTAB and without oligo d(T) cellulose.
  • the inhibitor(s) can be removed from stool, it is also possible to detect and clone nucleic acids of other viruses (DNA viruses, non-poly(A) tailed RNA viruses) present in stool.
  • the CTAB and oligo d(T) cellulose technique of extraction followed by detection of viral RNA with RT-PCR was used on stool samples and could be used on water and food samples.
  • Stool sample was suspended in distilled water (about 10% wt/vol) and extracted once with genetron. Viruses in the supernatant were precipitated with polyethylene glycol at a final concentration of about 8%.
  • the viral pellets were treated with proteinase K (about 400 ug/ml) in the presence of SDS at about 37°C for about 30 minutes followed by one extraction with phenol chloroform and one with chloroform.
  • a solution of about 5% CTAB and about 0.4M NaCl was added at a ratio of sample:CTAB equal to about 5:2.
  • the nucleic acids including the viral RNA
  • the resultant pellets were suspended in about 1M NaCl and extracted twice with chloroform.
  • the viral RNA in the aqueous phase was used directly in RT-PCR reactions or further purified by adsorption/elution on oligo d(T) cellulose.
  • a batch method of adsorption/elution on oligo d(T) cellulose was used to purify poly(A) tailed RNA.
  • nucleic acids partially purified as described above or RNA extracted directly with phenol chloroform (without CTAB treatment) were mixed with oligo d(T) cellulose (about 2-4mg/sample) in a binding buffer (about 0.5M NaCl and lOmM Tris, pH 7.5). The mixture was incubated at about 4 ⁇ C for about 1 hr with gentle shaking and then centrifuged for about 2 minutes in a microcentrifuge.
  • the oligo d(T) cellulose pellet was washed 3-4 times with binding buffer and then the poly(A) tailed RNA was eluted with IX TE buffer (about lOmM Tris, ImM EDTA, pH 7.5). The supernate was collected following centrifugation to remove the oligo d(T) cellulose and the viral RNA in the supernate was precipitated with ethanol.
  • the RNA obtained at this stage was basically inhibitor-free and able to be used in RT-PCR.
  • Norwalk virus RNA was detected in less than 0.05g of stool samples using the CTAB technique.
  • a trace inhibitor activity was observed with RNA extracted with either CTAB or oligo d(T) alone, but this was easily removed by dilution (1:2) of the extracted nucleic acid before RT-PCR.
  • Combination of the CTAB and oligo d(T) techniques resulted in obtaining high quality, inhibitor free RNA which could be used directly for RT-PCR detection and for cloning of the viral genome.
  • this method to clone from small amounts of stool one skilled in the art will know that they can obtain cDNAs for the remainder of the genome including those representing the 5'-end of the genome.
  • primers based on the above nucleotide sequence of the genome were made by chemical methods. These primers include: Primer 1: CACGCGGAGGCTCTCAAT located at nucleotides 7448 to 7465; Primer 4: GGTGGCGAAGCGGCCCTC located at nucleotides 7010 to 7027; Primer 8: TCAGCAGTTATAGATATG located at nucleotides 1409 to 1426; Primer 9: ATGCTATATACATAGGTC located at nucleotides 612 to 629; Primer 16: CAACAGGTACTACGTGAC located at nucleotides 4010 to 4027; and Primer 17: TGTGGCCCAAGATTTGCT located at nucleotides 4654 to 4671 (SEQ ID NOS 51 through 56, respectively).
  • New, additional primer sets (Table 6 and SEQ ID NOS.: 15 to 37) are used as probes to detect the Norwalk-related viruses.
  • Table 7 shows the ability of newly selected primer sets 36-35, 69-39, 78-80 to detect many Norwalk-related viruses.
  • sequence of 2516 nucleotides of another small round structured virus (SRSV/KY/89 SEQ ID NO: 12) also was obtained by using a total of 8 additional sets of primers from the original Norwalk virus sequence (primers 56 and 23, 42 and 55, 58 and 59, 60 and 61, 72 and 63, 76 and 77, 64 and 75, and 74 and 3; Table 6).
  • Prokaryotic hosts may include Gram negative as well as Gram positive bacteria, such as E. coli. S. tvmphimurium. Serratia marcescens. and Bacillus subtilis.
  • Eukaryotic hosts may include yeast, insect or mammalian cells. Immunized animals may include mammals such as guinea pigs, mice, rabbits, cows, goats or horses or other non-mammalian or non-murine species such as chickens.
  • synthetic peptides of greater than 15 amino acids made to match the amino acid sequence deduced from the partial cDNA sequence (or from other sequences determined by sequencing additional cDNAs detected with the original or other clones) are linked to a carrier protein such as bovine serum albumin or lysozyme or cross-linked with treatment with glutaraldehyde and used to immunize animals to produce polyclonal antibodies for diagnostic tests.
  • a carrier protein such as bovine serum albumin or lysozyme or cross-linked with treatment with glutaraldehyde
  • the serum of animals immunized with either the expressed protein or with synthetic peptides are tested by immunologic assays such as immune electron microscopy, Western blots (immunoblots) and blocking ELISAs to demonstrate that antibodies to Norwalk and related viruses have been made.
  • Reactivities with the expressed protein or synthetic peptides show specificity of the polyclonal sera.
  • Reactivities with other viruses in the Norwalk group indicate production of a reagent which recognizes cross-reacting epitopes.
  • Balb ⁇ c mice injected with the immunogens as described above and shown to have produced polyclonal antibodies are boosted with immunogen and then sacrificed.
  • Hybridomas resulting from this fusion are screened for their reactivity with the expressed protein, the peptide and virus particles to select cells producing monoclonal antibodies to Norwalk virus. Screening of such hybridomas with Norwalk-related viruses permits identification of hybridomas secreting monoclonal antibodies to these viruses as well.
  • the Norwalk virus has the genetic organization shown in Figure 8. Expression of regions of this genome in cell-free translation systems and in the baculovirus expression system have shown that the 5 '-end of the genome encodes nonstructural proteins and the 3'- end of the genome encodes at least one structural protein. Based on this information, one can express the complete genome or subgenomic regions of the genome to produce diagnostic assays to detect viral antigens or immune responses to specific regions of the genome. This information can be used to detect the Norwalk virus, antigens or immune responses to Norwalk virus. This information also can be used to detect other similar currently uncharacterized viruses that cause gastroenteritis or possibly other diseases. Some of these viruses will be in the Caliciviridae or in the picornavirus superfamily. All of these viruses will have matching or similar genomic regions in their DNA sequences.
  • cDNA clones from viruses related to Norwalk virus enable the production of new antibodies and antisera for diagnostic assays for these related viruses.
  • availability of cDNA clones from caliciviruses which cannot be cultivated permits the expression of protein products of those clones.
  • the protein products are used to develop new antibodies and antisera.
  • genetic engineering is used to combine the cDNAs from viruses related to Norwalk virus with the cDNAs from Norwalk virus to produce chimeric proteins, such that part of the protein produced is derived from Norwalk virus genome sequence and another part of the protein is derived from the genome sequence of a virus related to Norwalk virus. These chimeric proteins are then used to produce diagnostic reagents, vaccines and antivirals. Examples of the diagnostic assays are shown in the specific examples and figures below.
  • Norwalk virus genome allows the prediction of specific regions of the gene sequence as regions where oligonucleotide primers or probes can be developed to detect Norwalk virus sequences and common sequences of other related or similar viruses. Some of these common genome sequences are found in viruses in the
  • Caliciviridae or in the picornavirus superfamily The detection can be done by standard PCR, hybridization or other gene amplification methods.
  • Norwalk virus genome SEQ ID NO: 16
  • These primers then were used to prepare a cDNA clone by reverse transcriptase-PCR from the nucleotide sequence of human calicivirus Sapporo strain (HuCV Sapporo), 1982 outbreak ( Figure 9, SEQ ID NO:5). The resulting sequence was compared to that of Norwalk virus and of feline and rabbit caliciviruses available from Genbank.
  • c-29_4-gel The first cDNA clone from Sapporo, named "c-29_4-gel", determined to contain calicivirus sequence was 488 nucleotides long, of which 40 nucleotides were contributed by primers 36 and 35, leaving 448 nucleotides unique to human calicivirus Sapporo.
  • the sequence of clone c-29_4-gel between primers 36 and 35 also is shown in Figure 9, SEQ ID NO:8.
  • the sequence exhibits strong homology with Norwalk virus, feline calicivirus, and the rabbit calicivirus at the nucleotide and amino acid levels. (See Figure 10 and Tables 7 and 8).
  • the sequence contains a continuous protein encoding region on the positive strand. In Norwalk, feline, and rabbit caliciviruses continuous protein encoding regions also are found in the region of homology.
  • the sequence contains the amino acid motif YGDD, which is a marker for RNA virus proteins which have RNA-dependent-RNA-polymerase activity. In c-29_4-gel, the YGDD motif is at the predicted distance from the ends of the sequence.
  • the same cDNA product was obtained from six different stool specimens. Fifth, no significant homologies were found for other sequences in the Genbank.
  • the nucleotide sequence of c-29_4-gel was used to synthesize an internal primer. This internal primer was used to prepare a second set of RT-PCR products from human calicivirus Sapporo RNA.
  • a number of new cDNA clones were obtained of which one, named "at23s2m31", contains overlapping sequence which is 5' on the virus genome from that contained in c-29_4-gel.
  • Sequence at23s2m31 is 149 nucleotides long (SEQ ID NO:7) and overlaps c-29_4-gel by 46 nucleotides. See Figure 9 for at23s2m31 sequence and area of overlap with c-29_4-gel.
  • the resulting combined sequence information of c-29_4-gel and at23s2m31 is 551 nucleotides in length, excluding the portion c-29_4-gel contributed by prime 35.
  • human calicivirus Sapporo sequence was generated from knowledge of the Norwalk virus sequence, the former is distinguishable in the same region (see Table 8 or Figure 9).
  • the known sequence of human calicivirus Sapporo indicates that this virus is more closely related to the animal caliciviruses than to Norwalk virus.
  • primer 36 used for the initial amplification of cDNA clone c-29_4-gel- Examination of the homology of known calicivirus sequences (Table 8 SEQ ID NOS 57 through 62) in that region indicated that a new 36 primer could be synthesized and used to amplify caliciviruses more closely related to human calicivirus Sapporo than Norwalk virus.
  • a new primer was synthesized and is called primer "new 36" (see Table 6, last line, and SEQ ID NO:37).
  • the new 36 primer was used with primer 35 to generate a cDNA clone from a calicivirus which caused a diarrhea outbreak in November, 1986, in a Houston day care center ("Day care").
  • the calicivirus strain causing this Day care outbreak was antigenically related to human calicivirus Sapporo but antigenically distinct from Norwalk virus by EIA.
  • the Day care cDNA product obtained from the RT-PCR reaction with primers new 36 and 35 is 445 nucleotides long, excluding the primers (see Figure 9 and SEQ ID NO: 9), and has close homology to human calicivirus Sapporo and a more distant, yet still significant homology with Norwalk virus, as shown in Figure 10.
  • Evidence that this Day care cDNA is correct is the same as that listed for c-29_4-gel above.
  • a calicivirus was isolated from the mouth of the pygmy chimpanzee, Pan paniscus. This calicivirus is antigenically distinct from the human calicivirus Sapporo strain by EIA.
  • a cDNA was produced from the primate calicivirus (PrCV) RNA using RT-PCR and primers 36 and 35. The complete nucleotide sequence of this cDNA is not yet available.
  • the cDNA called atprcvw2 ( Figure 20; SEQ. ID.
  • NOS 13 and 14 is of the predicted size and has significant nucleotide homology with human calicivirus Sapporo, feline calicivirus(es), and the rabbit calicivirus in the region of known sequence. No significant homology with Norwalk virus has been observed in the region of known sequence.
  • the known amino acid sequence contains the YGDD motif on the positive strand at the predicted distance from primer 35.
  • Norwalk virus genomic sequence to detect and characterize KY89.
  • Another small round virus associated with an outbreak of gastroenteritis The known sequence for Norwalk virus is used to obtain the sequence of other viruses such as SRSV/KY/89, an agent from a stool from an outbreak of gastroenteritis in Japan in 1989.
  • SRSV/KY/89 an agent from a stool from an outbreak of gastroenteritis in Japan in 1989.
  • cDNA products and sequence information were obtained using primer sets 36-35.
  • Norwalk-related viruses permits development of diagnostic assays to detect antibodies, antigens, viral genetic material or antivirals and to develop vaccines for specific Norwalk-related viruses in the same manner that Norwalk virus fragments and derivatives have been used.
  • Protein(s) encoded in the Norwalk virus genome or fragments or derivatives thereof is produced in a prokaryotic or eukaryotic expression system and used as antigens in diagnostic assays to detect immune responses following virus infections.
  • Prokaryotic hosts may include Gram negative as well as Gram positive bacteria, such as Escherichia coli, Salmonella tymphimurium, Serratia marcescens, Bacillus subtilis, Staphylococcus aureus and Streptococcus sanguinis.
  • Eukaryotic hosts may include yeast, insect or mammalian cells. Diagnostic assays may include many formats such as enzyme-linked immunosorbent assays, radioimmunoassays, immunoblots or other assays.
  • Figure 15 shows data for a capsid protein encoded from the 3 '-end of the Norwalk virus genome. It is expressed by nucleotides 5337 through 7753 of the DNA sequence shown in Table 2 and Figure 8. This protein has an approximate molecular weight of 58,500 and is hereinafter referred to as the 58,500 mwt protein. It was produced in insect cells infected with baculovirus recombinants (C-6 and C-8). A band (see arrow in Figure 15) representing the 58,500 mwt protein in C-6 and C-8 infected cells is not seen in insect cells infected with wild-type (WT) baculovirus or in mock infected cells.
  • WT wild-type
  • Figure 16 shows data using the 58,500 mwt protein produced using the baculovirus expression system to detect immune responses before and after infection of volunteers with Norwalk virus inoculum. Antigen was put on ELISA plates and pre- and post-infection human sera were added. The data show that when an individual has had the infection, the post- serum reacts strongly to the antigen. Other proteins encoded in the Norwalk virus cDNA or fragments or derivatives thereof are similarly used to detect immune responses following Norwalk virus infection.
  • Proteins have the intrinsic property of being able to form particles.
  • the 58,500 mwt protein discussed above has that property.
  • Particles formed from proteins are expressed in any expression system and used to produce diagnostic assays based on detection of antibody responses or immune responses.
  • Figure 17 shows an electron micrograph of particles produced using the baculovirus expression system from recombinants containing the 3'-end of the Norwalk genome. These particles are similar in size to the native virus particles. They are antigenic, immunoreactive and immunogenic. They differ from most of the virus particles resulting from natural infection in that many of the expressed particles lack nucleic acids.
  • the rNV particles are highly immunogenic when given parenterally to mice, rabbits and guinea pigs and when given orally to mice.
  • Figure 18 shows data on the properties of rNV particles following centrifugation in gradients of CsCl.
  • the density of the particles (symbolized by closed boxes) is 1.31 g/cc which is distinct from the 1.38 g/cc density of particles purified from the original infectious Norwalk inoculum given to volunteers.
  • the gradients were fractionated. Each fraction was put on an ELISA plate and human serum was then introduced.
  • the open boxes show that there was no ELISA activity with the pre-infection serum.
  • the closed diamonds show there was reactivity with the post-infection serum.
  • Other particles made from other proteins encoded in the Norwalk virus cDNA or fragments or derivatives thereof are similarly used to detect immune responses following Norwalk virus infection.
  • Figure 19 shows data using purified particles formed by the 58,500 mwt protein to detect immune responses in post-inoculation (but not pre- inoculation) serum samples of 9 volunteers infected with Norwalk virus.
  • Purified, expressed particles were put on ELISA plates and one pre- and one post-infection serum samples from each volunteer was added to the particles. The amount of antibody binding to the particles in each pre- and post-infection sample was measured.
  • the data in Figure 19 show that the expressed proteins form particles that are immunoreactive and antigenic.
  • Other proteins encoded in the Norwalk virus cDNA or fragments or derivatives thereof are similarly used to detect immunoreactive and antigenic activity.
  • ELISA assays were made based on utilizing the Norwalk virus capsid protein that was engineered to be synthesized from a cDNA fragment that was deduced from the Norwalk virus cDNA sequence and then produced using the baculovirus expression system. This expressed Norwalk virus capsid protein self-assembled into recombinant Norwalk virus particles (rNV). Two new ELISA assays were established using this rNV antigen. One assay detects antiviral antibody and the other detects viral antigen. Both the ELISAs are very sensitive when compared to the previous assays (based on reagents from human volunteers) available to detect such agents.
  • the antigen ELISA detects immune responses following human infections with Norwalk virus and a subset of human infections with viruses in the Norwalk group such as Snow Mountain and Hawaii agents.
  • the antigen ELISA is based on use of hyperimmune serum made to the baculovirus expressed recombinant Norwalk virus particles (rNV). This antigen ELISA has been found to be very specific in that is recognizes the prototype Norwalk virus (8FIIa) and a subset of closely related agents, but not all other viruses in the Norwalk group such as the Snow Mountain agent and Hawaii agent (See Tables 1 and 7).
  • the antigen ELISA does not detect other viruses in the Norwalk group such as the small round structured viruses or caliciviruses, these and other Norwalk-related viruses have been able to be detected using primers selected from the nucleotide sequence of Norwalk virus (See Table 7).
  • primers selected from the nucleotide sequence of Norwalk virus See Table 7.
  • ELISAs based on using other fragments of the Norwalk virus genome were developed.
  • the new diagnostic assays are based on detection of antibody responses or of antigens deduced from fragments of the Norwalk virus genome other than the capsid region. An example and data of this approach is the following.
  • One Norwalk virus nonstructural protein is predicted to be encoded in the first ORF of Norwalk viral genome.
  • This ORF is located at the 5 end of the viral genome and it has a predicated molecular weight of 190,000 (190K). Whether this ORF 1 is useful in diagnostic assays first was evaluated by expressing the protein encoded in the full length viral RNA, and then synthesizing and testing the immunoreactivity of the encoded protein using a cell-free system. This was accomplished by in vitro transcription of a full length cDNA (pGNV-F) of the Norwalk viral genome cDNAs. This full-length cDNA was constructed by ligation of subgenomic derivatives of the original Norwalk virus cDNAs shown in the physical map in Figure 5.
  • NV mRNAs next were examined for their ability to direct the synthesis of a Norwalk virus specific protein by cell-free translation in rabbit reticulocyte lysates in the presence of ⁇ S methionine to produce a radiolabeled protein.
  • the expressed proteins were analyzed by polyacrylamide gel electrophoresis (PAGE). A clear band of approximate molecular weight of 130,000 was observed in the sample containing the viral RNA but not in the negative control (without viral RNA).
  • the immunoreactivity of this protein was examined by reactivity with pre- and post-infection sera from volunteers given Norwalk virus.
  • the 130K protein was precipitated by a convalescent serum of a volunteer infected with Norwalk virus, but not by serum collected before infection, indicating this protein was virus-specific. This showed this 130K protein contains some immunoreactive epitopes.
  • the apparent smaller size of the protein made in this translation system suggested that either the protein migrates aberrantly on gels, or an internal initiation codon was used to begin translation or some type of post translational modification may have occurred after the protein was translated.
  • the 2C region of the Norwalk viral genome was expressed using the baculovirus expression system. This region was selected for initial expression because it is located at the 5'-end of the non-structural protein and a high level of conservation was found between the sequence of the predicted Norwalk virus protein, and new sequence published for related caliciviruses and picornavirus.
  • a 5 '-end cDNA fragment of the viral genome was subcloned into the baculovirus transfer vector pVL 1393. After co-transfection of insect Sf9 cells with wild-type baculovirus DNA, recombinants containing the Norwalk viral gene were identified and selected.
  • radiolabeled lysates of recombinant-infected insect cells were prepared, and the radiolabeled proteins were analyzed by PAGE.
  • the results showed that a protein of apparent molecular weight of 57,000 (57K) was made in recombinant-infected but not in uninfected cells.
  • the size of the protein suggested that the internal AUG initiation codon located at nucleotide 953 was used for making this protein.
  • This 57K protein also was precipitated by convalescent serum (but not by pre- infection serum) from a volunteer who was infected with Norwalk virus. This protein mainly remained cell-associated.
  • rNV Norwalk virus particles
  • mice guinea pigs and rabbits following parenteral immunization
  • mice given rNV orally also have developed serum antibodies.
  • Hybridomas from mice immunized with rNV also have been obtain following fusion with myeloma cells.
  • Use of these antibodies in a capture ELISA has shown NV antigen can be detected. This antigen ELISA based on the antiserum made to the rNV particles is quite specific and it detects only a subset of Norwalk-related viruses (See Table 7).
  • capsid antigens from other Norwalk-related viruses must be expressed to produce a more broadly reactive ELISA for capsid antigen.
  • the ELISA is only one format that can be used to detect virus antigen. Other formats could include immunofluorescence or immunocytochemistry, or immune electron microscopy.
  • the comparison of the capsid sequences of Norwalk virus and Norwalk-related viruses permits the identification of conserved regions of the capsid protein and use of fragments of such sequences to immunize animals and can result in the production of antisera with more broad reactivity to Norwalk-related viruses.
  • sequential immunization of animals with expressed proteins of Norwalk and Norwalk-related viruses will result in antiserum with the desired broad reactivity.
  • Antigen detection assays that are specific to one of a few strains of Norwalk and Norwalk-related viruses and additional assays that are more broadly reactive each will have use. Expression of fragments of proteins encoded in other regions of the genome can be used to produce antiserum to other proteins for use in ELISAs to detect viral antigens.
  • the expression of the first ORF that represents a polyprotein encoded in the 5'-end of the genome and fragment 2C of the polyprotein has shown that each of these nonstructural proteins in immunoreactive and antiserum made to these can be used to develop diagnostic assays to detect these viral proteins.
  • These assays can be broadly reactive and detect many other Norwalk- related viruses because of sequence conservation.
  • Example 10 Development of a vaccine using Norwalk virus expressed antigens Vaccines for Norwalk virus, the Norwalk group of viruses or other small round viruses are made from an expressed Norwalk virus protein. That expressed protein can be a Norwalk virus capsid protein expressed alone or in combination with one or more other Norwalk virus proteins or self-forming particles. For example, the particles shown in Figure 17 were produced using the baculovirus expression system. They are used as a vaccine when expressed alone or in combination with one or more other Norwalk virus proteins.
  • the other proteins encoded in the Norwalk virus cDNA or fragments or derivatives thereof are used as a vaccine when expressed alone or in combination with one or more Norwalk virus proteins.
  • Individuals are vaccinated orally, parenterally or by a combination of both methods.
  • parenteral vaccination the expressed protein is mixed with an adjuvant and administered in one or more doses in amounts and at intervals that give maximum immune response and protective immunity.
  • Oral vaccination parallels natural infection by Norwalk virus inoculum, i.e. the individual ingests the vaccine with dechlorinated water or buffer.
  • Oral vaccination may follow sodium bicarbonate treatment to neutralize stomach activity. For example, sodium bicarbonate solution is taken by each person 2 minutes before and 5 minutes after vaccine administration.
  • the Norwalk virus capsid protein carrier is mixed with or covalently linked to one or more heterologous protein antigens or synthetic peptides containing heterologous epitopes.
  • This mixture is antigenic, induces antibodies, and protects against Norwalk virus and its antigens, and against the heterologous epitopes or antigens.
  • Example 12 Kit Kits for detecting immune responses to Norwalk virus are prepared by supplying in a container a protein deduced from the Norwalk virus genome shown in Table 2 or fragments or derivatives thereof. Similar proteins are prepared from Norwalk-related viruses to detect immune responses to the Norwalk-related viruses.
  • the protein encoded by Norwalk virus nucleotides 1 through 7753 the protein encoded by Norwalk virus nucleotides 146 through 5359, the protein encoded by Norwalk virus nucleotides 5337 through 7573, the protein encoded by Norwalk virus nucleotides 5346 through 6935, the protein encoded by Norwalk virus nucleotides 6938 through 7573 and any combinations thereof may be used in such kits.
  • the kit can also include controls for false positive and false negatives, reagents and sample collection devices. The kit can be equipped to detect one sample or multiple samples.
  • Kits for detecting Norwalk viruses and Norwalk-related viruses are prepared by supplying in a container at least one antiserum made from a protein expressed from the deduced amino acid sequence of the Norwalk virus genome shown in Tables 3, 4, or 5 or from a fragment or derivative the deduced amino acid sequence. Similar antiserum are made from proteins encoded by Norwalk-related viruse genomes.
  • kits for example, an antiserum made to the protein encoded by Norwalk virus nucleotides 1 through 7753, the protein encoded by Norwalk virus nucleotides 146 through 5359, the protein encoded by Norwalk virus nucleotides 5337 through 7573, the protein encoded by Norwalk virus nucleotides 5346 through 6935, the protein encoded by Norwalk virus nucleotides 6938 through 7573 and any combination thereof may be used in such kits.
  • the kit can also include controls for false positives and false negatives, reagents and sample collection devices.
  • the kit can be equipped to detect one sample or multiple samples.
  • Virus Morphology Physical features Examples Astrovirus 5—6-pointed surface RNA Lamb x star BD 1.36-1.38 g/cm 3 Human
  • GGT GGC TTA GGT CCA AGT ACA CTA CCC ACT AGT CAT GAA CGG TAC ACA 412 Gly Gly Leu Gly Pro Ser Thr Leu Pro Thr Ser His Glu Arg Tyr Thr 75 80 85
  • CAC CCG ACC CAA GAT TGG TCC CGA GAC ACT CCA GAA TTC CAA GAC 940 His Pro Thr Gin Asp Trp Ser Arg Asp Thr Pro Glu Phe Gin Gin Asp 250 255 260 265
  • AGC AAG TTA GAG ATG GTT AGG GAT GCA GTG CTA GCC GCT ATA AAT GGG 988 Ser Lys Leu Glu Met Val Arg Asp Ala Val Leu Ala Ala He Asn Gly 270 275 280
  • AAA CAG TCT GCA CGG GCT TAT ATG GCC ATC TTA GAT AAT GAA GAA GAA 1516 Lys Gin Ser Ala Arg Ala Tyr Met Ala He Leu Asp Asn Glu Glu Glu 445 450 455
  • AAG GCA AGG AAA TTA TCT GTC AGG AAT GCC GAC CCA CAC GTA GTA TCC 1564 Lys Ala Arg Lys Leu Ser Val Arg Asn Ala Asp Pro His Val Val Ser 460 465 470
  • GCT CCC CAA GGG GGC TTT GAT AAC CAA GGG AAT ACC CCG TTT GGT AAG 2188 Ala Pro Gin Gly Gly Phe Asp Asn Gin Gly Asn Thr Pro Phe Gly Lys 670 675 680
  • GGA CCT GGG AAA GAA CAT AAT TGC AGG
  • GCT 2764 Gly Pro Gly Lys Glu His Asn Cys Arg Val His Lys Ala Lys Glu Ala 860 865 870
  • GGT AAT TAT GTT GGT GTT CTT AGC TGG ATT TCC CCC CCA TCA CAC CCG 6491 Gly Asn Tyr Val Gly Val Leu Ser Trp He Ser Pro Pro Ser His Pro 370 375 380
  • N/A Not available Internal primers were used to amplify this agent.
  • the primate CV 35-36 PCR product sequence is not yet complete. Similarity information is based upon the partial sequence.
  • Primer 36 extends across the first 19 nucleotides in the Norwalk sequence above. Primer "new 36" is the first 19 - nucleotides of the HuCV Sapporo sequence,
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM human calicivirus Sapporo
  • GATTCCACAC AAAACCCTGC AGTGACAGCA GCCTCCCTGG CAATATTGGA GAGATTTGCT 240
  • GAGCCCCATC CAATTGTGTC TTGTGCCATT GAGGCTCTTT CCTCCCCTGC AGAGGGCTAT 300
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM human calicivirus Saporro (Day care)
  • MOLECULE TYPE DNA (genomic)
  • GGCCATGTTA TAGTGGTGTT CACATGAAAG ATGGCGACAA GATGTTGATA GATGCCAATC 60
  • MOLECULE TYPE DNA (genomic)
  • TTGTTCTTGG TTCCTCCCAC AGTGGAACAG AAGACTAGGC
  • ACAGTAATCA ATCTCACCGA ATTGGATGGC
  • ACCCCCTTCC ACCCTTTTGA AGGCCCTGCC 1800
  • GGAACTGCCA GTACGGCAAG AGGTAGGCTT GGTTTGCGCC
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM primate calcicvirus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM primate calcicvirus
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:15: CTTGTTGGTT TGAGGCCATA T 21
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:16: ATAAAAGTTG GCATGAACA 19
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:17: GTTGACACAA TCTCATCATC 20
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:18: GGCCTGCCAT CTGGATTGCC 20
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:19: GGGCCCCCTG GTATAGGTAA 20
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:20: TGGTGATGAC TATAGCATCA GACACAAA 28
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:22: GTTCTGACCA CCTAACCT 18
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:27: ATTATAGTTT CTTGCATA 18
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:29: CATTGGGTTT CCAGACCTA 19
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:30: ATAATTGGGG ATCTTCCAAA 20
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:31: TAGTGGCATG GGTATTTC 18
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:32: TATGCCAATC ACAGCCAC 18
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:33: GTCTGGCTCC CAAGTTGACC 20
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:35: TGAGGCTGCC CTGCTCCA 18
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:37: GTTGCTGTTG GCATTAACA 19
  • ORGANISM hepatitis C virus
  • ORGANISM hepatitis A virus

Abstract

Double-stranded cDNA was synthesized from nucleic acid extracted from Norwalk virus purified from stool specimens of volunteers. 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 an ssRNA genome of about 8 kb in size. The availability of a Norwalk-specific cDNA and the genome sequence information allow rapid cloning of the entire genome and establishment of sensitive diagnostic assays. Such assays can be based on detection of Norwalk and Norwalk-related virus nucleic acids or Norwalk and Norwalk-related viral antigens using probes or primers and polyclonal or monoclonal antibodies to proteins expressed from the cDNA or to synthetic peptides made based on the knowledge of the genome sequence. Assays using proteins deduced from the Norwalk virus genome and produced in expression systems can measure antibody responses. Vaccines for Norwalk and related viruses are made from an expressed Norwalk virus protein.

Description

Methods and Reagents To Detect and Characterize Norwalk and Related Viruses
This application is a Continuation-in-Part of Applicant's Co- Pending U.S. Application Serial No. 07/443,492 filed November 8, 1989, U.S. Application Serial No. 07/515,993, now abandoned, filed April 27, 1990, U.S. Application Serial No. 07/573,509 filed August 27, 1990, and U. S. Application Serial No. 07/696,454 filed May 6, 1991, all entitled "Methods and Reagents To Detect and Characterize Norwalk and Related Viruses." This invention is supported in part through grants or awards from the Food and Drug Administration and the National Institute of Health. The United States Government may have certain rights to this invention.
Field of the Invention The present invention relates generally to synthesizing clones of Norwalk virus and calicivirus 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., Am. J. Hyg. 58:16-30 (1953); Kapikian and Chanock, "Norwalk group of viruses" in B.N. Fields' 2d ed. of Virology. Raven Press, New York, pp. 671-693 (1990)). Up to 42% of cases of adult viral gastroenteritis have been estimated to be caused by Norwalk or Norwalk-like viruses (Kaplan et al., Ann. Internal Med. 96(6):756-761 (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, Med. J. Aust. 2:329-333 (1979); Gunn et al., Am. J. Epidemiol. 115:348-351 (1982); Wilson et al., Am. J. Public Health 72:72-74 (1982); Gill et al., Br. Med. J. 287:1532-1534 (1983); DuPont Veu Engl. J. Med. 314:707-708 (1986); Morse et al., New Engl. J. Med. 314:678-681 (1986); Sekine et al., Microbiol. Immunol. 33:207-217 (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, Arch. Intern. Med. 149:1735-1740 (1989)). Norwalk virus was discovered in 1973. Until recently, 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., Proc. Soc. Exp. Med. and Biol. 140:578-583 (1972); Kapikian et al., J. Virol. 10:1075-1081 (1972); Thornhill et al., J. Infect. Dis. 132:28-34 (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 antiserum 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., J. Virol. 37: 994-999 (1981)), but efforts to characterize the viral genome have failed.
Viruses related to Norwalk virus include small round enteric viruses, such as viruses with typical calicivirus morphology and the astro viruses. The classification scheme for the human small enteric viruses shown in Table 1 here is an updated version of a scheme outlined by Caul and Appleton in the Journal of Medical Virology, 9:257-265 (1982). This system is referred to in Cubitt et al., J. Infectious Diseases, 156:806-814 (1987); Table 1 of the article by Appleton entitled "Small round viruses: classification and role in food-borne infections", in the book Novel Diarrhoea Viruses. Ciba Foundation Symposium No. 128, pp. 108- 125 (John Wiley & Sons, N.Y. (1987)); and Table 1 of the chapter entitled "Norwalk group of viruses" by Kapikian and Chanock from the book Virology (B.N.Fields, 2d ed., Raven Press (1990)).
As shown in Table 1, human small round structured enteric viruses include calicivirus and astro virus. The recent sequencing of Norwalk virus indicates that Norwalk virus is a calicivirus and has a genome organization like that of other caliciviruses. In addition to the human small round enteric viruses are a large number of non-human small round viruses which have been classified as astroviruses, caliciviruses, and small round structured viruses based upon their morphology. Examples of these viruses are the primate calicivirus isolated from the pygmy chimpanzee, described in the journal Science 221:79-81 (1983), a porcine enteric calicivirus, described in the Journal of Clinical Microbiology 12:105-111 (1980), and bovine astroviruses described in VetPathol. 21:208-215 (1984). Individual calicivirus types will at times exhibit host specificity and tissue tropisms, but as an overall group they cause gastroenteritis, hepatitis, abortion, skin lesions, pneumonia, myocarditis, and encephalitis. The caliciviruses infecting humans fit in this context in that Norwalk-like viruses cause gastroenteritis, hepatitis E causes hepatitis, and San Miguel sea lion virus type 5 causes skin vesicles in humans as well as infections in seals, fish, pigs and cattle. (D. O. Matson "Calicivirus Infections" in Textbook of Pediatric Infectious Disease, 3d ed., R. D. Feigin and J. D. Cherry, eds., W. B. Sanders, Philadelphia, (in press)). Summary of the Invention It is therefore an object of the invention to detect and characterize the Norwalk and related virus genomes by synthesizing and cloning a cDNA library. It is an associated object of the invention to deduce a ino acid sequences from Norwalk and related viral cDNA.
Another object of the invention is to develop probes or primers to confirm the genetic relationship between the Norwalk virus and the Norwalk-related viruses. Still another object of the invention is to develop a method of preparing polyclonal and monoclonal antibodies to the Norwalk and related viruses.
Yet 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 still further object of the invention is to express proteins to measure antibody responses. A nucleotide sequence of the genome sense strand of the Norwalk virus cDNA clone intended to accomplish the foregoing objects includes the nucleotide sequence shown in Table 2. Within the Norwalk nucleotide sequence are regions which encode proteins. The nucleotide sequence of the Norwalk virus genome, its fragments and derivatives are used to make diagnostic products, vaccines and antivirals.
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 Figure 1. EM picture of Norwalk and related viruses. Norwalk virus (A), human Calicivirus (B), small round structured virus (C), and human astrovirus (D). The var is 0.1 μm.
Figure 2a. Hybridization of stool samples with 32P-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°C and 65°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.
Figure 2b. Dot blot hybridization of clone 32P-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.
Figure 3. Dot blot hybridization of Norwalk viruses in a CsCl gradient with ssRNA probes made from pGEMNV-953. Aliquots of 50ul 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. Figure 4. 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 also is shown.
Figure 5. Schematic diagram of Norwalk cDNA clones. pUCNV-953 was the first positive cone identified. Overlapping clones were determined by restriction enzyme analyses and partial sequencing of the clones. AAA indicates the poly (a) tail at the 3' end of the viral genome.
Figure 6. 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 Figure 3 of Reyes eial, Science 247:1335-1339 (1990).
Figure 7. Three pairs of initial primers used to amplify the Norwalk virus genome. RNA was extracted from a stool sample (sample 543-11) by the CTAB technique and amplified by RT-PCR. Lanes 1 and 5, 1-kb markers from Bethesda Research Laboratories (the markers that migrated as 1.6, 1.0 and 0.5 kb are labeled); lane 2, PCR with Norwalk virus primers 8 and 9; lane 3, PCR with Norwalk primers 16 and 17; lane 4, PCR with Norwalk primers 1 and 4. The amplified products were separated on the agarose gel and visualized with UV light after staining with ethidium bromide. The small product seen in lane 3 was made in variable amount in different experiments. The positions of the three primer pairs used in this study are given above the autoradiograph. The numbers below the map indicate the size (in base pairs) of the RT-PCR product. Figure 8. This schematic shows the organization of Norwalk genome given in Table 2. The features shown here are based on analyses of the nucleotide sequence of the Norwalk virus genome and the deduced amino acid sequence of proteins encoded in the genome. The genome contains 7753 nucleotides including 111 A's at the 3'-end. Translation of the sequence predicts that the genome encodes three open reading frames (shown by the open boxes in the second line). The first open reading frame is predicted to start from an initiation codon at nucleotide 146 and it extends to nucleotide 5359 (excluding the termination codon). The second open reading frame is initiated at nucleotide 5346 and it extends to nucleotide 6935, and a third open reading frame exists between nucleotides 6938 and 7573. Based on comparisons of these predicted proteins with other proteins in the protein databank, the first open reading frame is a protein that is eventually cleaved to make at least three proteins. These three proteins include a picornavirus 2C-like protein, a 3C-like protease and a 3D-like RNA-dependent RNA polymerase. The second open reading frame encodes the capsid protein, which contains sequence homology with the picornavirus VP3 protein.
Figure 9. Nucleotide and amino acid sequence of human calicivirus Sapporo cDNAs. The 551 nucleotide known sequence of human calicivirus
Sapporo (HuCV Sapporo) is presented in its entirety. Below the nucleotide sequence is the amino acid sequence for HuCV Sapporo. Above the HuCV Sapporo nucleotide sequence is the sequence of the cDNA from a Houston day care center outbreak (Day care). In the Day care sequence a "." indicates the nucleotide is identical to the HuCV Sapporo nucleotide at that site. Where a nucleotide difference occurred in the Day care sequence, a new letter is indicated at that position. "N" indicates uncertainty of the nucleotide at that site. Below the HuCV Sapporo amino acid sequence are arrows, indicating the extent of cDNAs at23s2m31 and c-29_4-gel (which together contribute to the 551 nucleotides of the known sequence) and the new 36 primer (see Table 6). Figure 10. Nucleotide homologies between calicivirus cDNAs and calicivirus strains with known sequences. All comparisons are in reference to the sequence of human calicivirus Sapporo. The length of the baseline indicates the known sequence region. The boxes indicate areas of nucleotide sequence homology between HuCV Sapporo and the indicated strain. The length of the box indicates the part of the indicated strain where homology exists and the height of the box indicates the strength of the homology. SD = standard deviation. SD 3 or greater is significant. The numbers under the Norwalk homology box indicate the region of the Norwalk virus genome where homology was observed.
Figure 11. Strategy used to obtain nucleotide sequence of the Norwalk- related virus SRSV/KY/89 using primers from the Norwalk virus sequence. This figure shows a partial schematic of the Norwalk virus genome and the predicted ORF1 showing the location of the 3D-like polymerase region, the second ORF showing the location of the VP3-like domain and the start of ORF 3. On the bottom, the solid lines show regions of KY89 sequenced based on using primer sets (see numbers such as 36 and 35, etc) chosen from the sequence of the Norwalk virus genome.
Figure 12. Comparison of the Norwalk virus nucleotide sequence with the Norwalk virus-related virus SRSV/KY/89 nucleotide sequence. Part of the nucleotide sequence of Norwalk-related virus SRSV/KY/89 was determined using primers from the Norwalk-virus (NV) genome. Primers from the NV genome used to obtain the sequence of this Norwalk-related virus are shown in Table 6. Some of these primers were modified based on the initial nucleotide sequence obtained from the SRSV/KY/89 to obtain the rest of the sequence of SRSV/KY/89. The primers shown here and in Table 6 are used by way of example only; other NV primers can be used.
Figure 13. Comparison of deduced amino acid sequence of proteins of the Norwalk virus and the Norwalk-related virus SRSV/KY/89. The protein sequence of SRSV/KY/89 was deduced from the nucleotide sequence shown in Figure 12. Figure 13a shows a comparison of the deduced amino acid sequence of ORF2, the capsid, of SRSV/KY/89 with the same region encoded in the Norwalk virus genome. Figure 13b shows a comparison of the deduced amino acid sequence of part of the polymerase protein of SRSV/KY/89 with that of Norwalk virus. Comparisons of similar sequences from other Norwalk-related viruses will permit discovery of conserved and divergent regions including antigenic regions. The information will rapidly permit choices of broadly reactive primers to detect all Norwalk-related viruses and specific primer sets to detect individual Norwalk-related viruses. Similarily, fragments and peptides with common amino acid sequences or specific amino acid sequences can be selected for development of diagnostics, vaccines and antivirals.
Figure 14. Comparison of partial nucleotide sequences of Norwalk virus and six Norwalk-related viruses obtained using primers from the NV genome. Sequences from SRSV/CDC 6/91, SRSV/UT/88, SMA/78; SRSV/Ca bridge, UK/92, SRSV/CDC 32, Norwalk virus/68, SRSV-3/88, SRSV/KY89/89. Figures 14a and 14b show two different regions of the genome.
Figure 15. Expression of the Norwalk virus capsid protein. Baculovirus recombinants (C-6 and C-8) that contain a subgenomic piece of Norwalk virus DNA (from nucleotides 5337 to 7753) were selected and used to infect insect (Spodoptera fugiperda) cells at a multiplicity of infection of 10 PFU/cell. After 4 days of incubation at 27° C, the infected cells were harvested and the proteins were analyzed by electrophoresis on 12% polyacrylamide gels. The proteins were visualized after staining with Coomassie blue. The Norwalk-expressed protein (highlighted by the arrowhead) is only seen in the recombinant-infected cells, but not in wild- type baculovirus (wt) or mock-infected (m) insect cells. Figure 16. The Norwalk virus expressed protein shows immunoreactivity with sera from volunteers infected with Norwalk virus. The expressed protein shown in Figure 11 was absorbed onto the wells of a 96-well ELISA plate and its reactivity was tested with dilutions of serum samples taken from volunteers before (pre) and three weeks after (post) infection with Norwalk virus. After an incubation at 37° C for 2 hours, a peroxidase-conjugated goat-anti-human IgG, IgM and IgA serum was added and reactivity was subsequently observed by reading the optical density at 414nm after addition of the substrate. The data show that post-infection sera reacted strongly with the expressed antigen at serum dilutions of 1:100 and 1:1000, and some sera were still specifically reactive at a dilution of 1:10,000.
Figure 17. Baculovirus recombinants containing the 3 '-end of the Norwalk genome produce virus-like particles in insect cells. Lysates from insect cells infected with baculovirus recombinant C-8 (see Figure 11) were analyzed by electron microscopy and shown to contain numerous virus-like particles. These particles are the same size as virus particles obtained from the stools of volunteers infected with Norwalk virus. Bar = 50 nm.
Figure 18. Norwalk virus-like particles can be purified in gradients of CsCl. Supernatants of insect cells infected with the baculovirus recombinant C-8 were processed by extraction with genetron and PEG precipitation and virus eluted from these PEG pellets was centrifuged in
CsCl gradient in a SW50.1 rotor for 24 hours at 4°C. The gradient was fractionated and material in each fraction was adsorbed onto two wells of an ELISA plate. Duplicate wells were then treated either with pre- or post-infection serum, peroxidase-conjugated goat anti-human serum and substrate and the reactions were monitored by reading the OD4l4nm. A peak was observed in the gradient at a density of 1.31 g/cm3 and this peak was shown to contain virus-like particles by electron microscopy. This peak also contained a major protein of an approximate molecular weight of 58,500 that co-migrated with the protein expressed in the insect cells from the same baculovirus recombinant.
Figure 19. Use of the expressed virus-like particles to measure the reactivity of pre- and post-serum samples from volunteers infected with Norwalk virus shows that most volunteers have an immune response. Volunteer 6 who did not show an immune response also did not become ill after being administered virus.
Figure 20. Partial sequence of the primate Pan paniscus cDNA atprcvw2.
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 any portion of the Norwalk virus genome or a subgenomic clone of the Norwalk virus that is required to be expressed to produce or encodes a peptide which in turn is 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 virus 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 affecting the reading or expression of the DNA or cDNA. The terms Norwalk "related viruses" and "Norwalk-like viruses" as used herein are defined as human and non-human calicivirus, astro virus and small round structured viruses (SRSV). As the genomic sequences of most of these viruses are not known, this classification is based on morphology as described by Caul and Appleton in the Journal of Medical Virology, 9:257-265 (1982); by Appleton in the article entitled "Small round viruses: classification and role in food-borne infections", in the book Novel Diarrhoea Viruses. Ciba Foundation Symposium No. 128, pp. 108- 125 (John Wiley & Sons, N.Y. (1987)); and by Kapikian and Chanock in the chapter entitled "Norwalk group of viruses" from the book Virology (B.N.Fields, 2d ed., Raven Press (1990)). As the genomic sequences of the viruses become known, those skilled in the art will be able to determine Norwalk-related viruses and Norwalk-like viruses based on nucleotide homologies. Within the Norwalk-related viruses is a subgroup of viruses referred to herein as the SRSV's or the Norwalk group. The Norwalk group includes Snow Mountain Agent (SMA), Hawaii Agent, Taunton Agent, Amulree, Otofuke, and Montgomery County Agent. The Norwalk group is characterized by small, round, structured viruses with an amorphous surface or ragged outline.
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, MA 01730). Sodium bicarbonate solution was taken by each person 2 minutes before and 5 minutes 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°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 minutes. The resulting supernate then was extracted two to three times with genetron in the presence of 0.5% Zwittergent 3-14 detergent (Calbiochem Corp., La Jolla, CA). 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, CA 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 hours 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 hour. The purified virus was stored at about -70°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°C for about 30 minutes. 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 minutes 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 CH3HgOH. Then cDNA was synthesized using the cDNA synthesis kit with the supplied random hexanucleotide primer (Amersham, Arlington Heights, IL 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, WI 53711-5305). Eight cycles of denaturation (100°C for 2 minutes), reannealing (2 minutes cooling to room temperature) and elongation (room temperature for 30 minutes) 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 32P using random primers and Klenow DNA polymerase such as in the PRIME-A-GENE® 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, CT). Replicate filter strips were prepared and hybridized with each labeled plasmid probe individually at 65°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.
Reverse Transcriptase-Polvmerase Chain Reaction Production of cDNA Clones from Viruses Related to Norwalk Virus
One method for producing cDNA clones of viruses related to Norwalk virus using the knowledge of the Norwalk virus genome sequence is the reverse transcription-polymerase chain reaction method. In this procedure, RNA was extracted from 300 uL of specimen containing the related virus. Complementary DNA was prepared by reverse transcriptase-polymerase chain reaction (RT-PCR) using a primer pair (for example primers 36 and 35 shown in Table 6) derived from the sequence of Norwalk virus. The resulting product was ligated into a plasmid vector and transfected into E. coli. Plasmids then were partially purified from the bacteria and the inserted PCR product was sequenced in the plasmid by dideoxy chain termination to examine the relation to Norwalk virus by nucleotide and predicted protein homology.
The following examples are offered by way of illustration and are not intended to limit the invention in any manner.
Example 1
Electron micrograph confirmation
To permit better diagnosis and molecular characterization of
Norwalk virus and related viruses, a cDNA library for Norwalk was derived from nucleic acid extracted from virions purified from stool samples. Norwalk virus was purified with methods used previously for hepatitis A and rotaviruses from stool samples with some modifications
(Jiang et al., 1986). Basically, stool samples obtained from volunteers administered Norwalk virus were treated with genetron to remove lipid and water insoluble materials. Virus in the aqueous phase was then pelleted through a 40% sucrose cushion. The resulting pellets were resuspended, sonicated and loaded in a CsCl gradient for isopycnic centrifugation.
Figure 1 shows an electron micrographs of purified Norwalk viruses isolated by the above procedure and Norwalk-related viruses used to produce cDNAs using RT-PCR.
Example 2 Initial cDNA synthesis, cloning and screening A cDNA library was generated from nucleic acids extracted from these purified viruses by proteinase K treatment of the samples followed by phenol-chloroform extraction and ethanol precipitation (Jiang et al., 1986; 1987). Because the nature of the viral genome was unknown, the extracted nucleic acids were denatured with methylmercuric hydroxide before cDNA synthesis. Random primed cDNA was synthesized with the Gubler-Hoffman method (cDNA synthesis system plus, Amersham) and a small amount of cDNA was obtained. Direct cloning of this small amount of cDNA was unsuccessful. Therefore, a step of amplification of the DNA was performed by synthesizing more copies of the DNA with random primers and the Klenow fragment of DNA polymerase before cloning. The procedure involved cycles of denaturation, addition of random primers and the Klenow fragment of DNA polymerase, reannealing and elongation. With this procedure, a linear incorporation of labeled nucleotides into product was observed as the number of cycles of synthesis was increased. The number of cycles performed was limited (<10) to avoid the synthesis of an excess of smaller fragments. In the case of Norwalk cDNA, eight cycles of amplification were performed and approximately 2.5 ug of DNA were obtained, which was at least a 100-fold amplification of the starting template cDNA. This amplified cDNA was cloned into pUC-13 by blunt-end ligation and a positive clone (pUCNV-953) was isolated.
To obtain the positive Norwalk virus clone, minipreparations of the plasmid DNAs containing potential inserts were screened by agarose gel electrophoresis. Inserts of the larger clones in the gel were cut out and probes were made with the DNA in the gel using the PRIME-A-GENE® labeling system (Promega Corp.). These probes were hybridized individually with paired stool samples (before and after Norwalk infection) from two volunteers (Figure 2a). One clone (pUCNV-953) reacted with post- but not pre-infection stool samples from both volunteers.
Example 3
Confirmation of viral origin of the clone pUCNV-953 To further confirm the viral origin of the clone pUCNV-953, six more paired stool samples were tested and the same results were obtained. Figure 2b shows a dot blot hybridization of the clone with stool samples collected at different times post-infection of the disease. Strong signals were observed only with stools from acute phase, but not before and after the illness. This result was consistent with previous RIA assays for viral antigen detection using convalescent sera from volunteers with Norwalk diarrhea and immune electron microscopy (IEM) studies of the samples for viral particle examination. This result also agrees with the patterns of virus shedding in stool in the course of the disease (Thornhill et al., 1975 ). When the pUCNV-953 clone was hybridized with fractions of a CsCl gradient from the Norwalk virus purification scheme, an excellent correlation between hybridization and EM viral particle counts was observed (Figure 3). The peaks of the hybridization signals and viral particle counts both were at fractions with a density of 1.38 g/cm3, which agrees with previous reports of the biophysical properties of Norwalk virus. Finally, the clone was tested by hybridization with highly purified Norwalk virus electrophoresed on an agarose gel. A single hybridization band was observed with Norwalk virus but not with HAV and rotavirus. Sequence analysis of the pUCNV-953 cDNA showed this clone is 511 bp (Figure 4). This partial genomic cDNA encodes a potential open reading frame for which the amino acid sequence has been deduced (Figure 4). No significant nucleotide or deduced amino acid sequence homology was found by comparison with other sequences in the Gen Bank (Molecular Biology Information Resource, Eugene Software, Baylor College of Medicine). Example 4
Use of Norwalk virus cDNA to characterize the viral genome
The pUCNV-953 cDNA was subcloned into the transcription vector pGEM-3Zf(+) and grown. ssRNA probes were then generated by in vitro transcription using SP6 and T7 polymerases (Bethesda Research
Laboratory). When two opposite sense ssRNA probes were hybridized with the viral nucleic acid separately, only one strand reacted with the virus, indicating the viral genome is single-stranded. As shown in Figure
2b, the hybridization signals were removed by treatment of the viral nucleic acid with RNAse (but not with DNAse) before loading them onto the filters, indicating the virus genome contains ssRNA. A long open reading frame was found in one of the two strands of the inserted DNA by the computer analysis of the sequences of pUCNV-953. The ssRNA probe with the same sequence as this coding strand does not react with the viral nucleic acid, but the complementary ssRNA probe does react in the hybridization tests. Therefore, Norwalk virus contains a positive sense single-stranded RNA genome. The size of the genome of Norwalk virus was estimated to be about 8 kb based on comparisons of the migration rate of the purified viral RNA in agarose gels with molecular weight markers.
The pUCNV-953 cDNA was used to rescreen a second cDNA library made as follows. A clone of the Norwalk or related virus was synthesized by isolating nucleic acid from purified Norwalk virus; cDNA was synthesized using reverse transcriptase and random primers; a second strand of DNA was synthesized from the cDNA; and at least one copy of DNA was inserted into a plasmid or a cloning and expression vector; and screening the library with the original puCNV-953 cDNA identified clones containing fragments of (or the complete) Norwalk or related genome. Alternatively at least one copy of DNA was inserted in a cloning and expression vector, such as lambda ZAPII® (Stratagene Inc.), and the cDNA library was screened to identify recombinant phage containing fragments of or the complete Norwalk or related genome. Additional cDNAs were made and found with this method. Use of these additional cDNAs to made and found with this method. Use of these additional cDNAs to rescreen the library resulted in detection of new clones (Figure 5).
Thus, those skilled in the art will recognize that entire Norwalk virus cDNA sequence, or fragments or derivatives thereof, can be used in assays to detect the genome of Norwalk and other related viruses. The detection assays include labeled cDNA or ssRNA probes for direct detection of the Norwalk virus genome and measurement of the amount of probe binding. Alternatively, primers or small oligonucleotide probes (10 nucleotides or greater) and polymerase chain reaction amplification are used to detect the Norwalk and Norwalk-related virus genomes. Expression of the open reading frame in the cDNA is used to make hyperimmune or monoclonal antibodies for use in diagnostic products, vaccines and antivirals.
Using the above methodology, the nucleotide sequence in Table 2 was identified. Within that nucleotide sequence, the encoding regions for several proteins have been identified. In that sequence, the first protein is encoded by nucleotides 146 through 5339 and the amino acid sequence is shown in Table 3. This first protein is eventually cleaved to make at least three proteins including a picornavirus 2C-like protein, a 3C-like protease and an RNA-dependent RNA polymerase. The RNA-dependent
RNA polymerase is deduced from nucleotides 4543 to 4924 of the Norwalk virus genome as shown in Table 3. The fact that this portion of the genome contains an RNA polymerase is verified by comparisons with RNA polymerase in other positive sense RNA viruses (Figure 6 SE ID NOS 38 through 50).
Also in the sequence in Table 2, two other protein encoding regions were found. They are encoded by nucleotides 5346 through 6935 and nucleotides 6938 through 7573. The amino acid sequences for these two proteins are shown in Tables 4 and 5, respectively. Example 5
Diagnostic assays based on detection of the sequences of the Norwalk virus genome
Hybridization assays are the assays of choice to detect Norwalk virus because small amounts of virus are present in clinical or contaminated water and food specimens. Previously, detection of Norwalk and related nucleic acids was not possible because the genome of Norwalk virus was not known and no sequence information was available. Probes made from the Norwalk virus cDNA or primers made from the Norwalk virus genome sequence allow methods to amplify the genome for diagnostic products to be established. Probes to identify Norwalk virus alone and to identify other Norwalk-related viruses enable development of either specific assays for Norwalk or general assays to detect sequences common to many or all of the Norwalk-related agents. In the past, one major difficulty encountered in RT-PCR detection of viral RNA in stool samples was that uncharacterized factor(s) are present in stools which inhibit the enzymatic activity of both reverse transcriptase and Taq polymerase (Wilde et al., J. Clin. Microbiol. 28:1300-1307, 1990). These factor(s) were difficult to remove by routine methods of nucleic acid extraction. Techniques were developed using cetyltrimethylammonium bromide (CTAB) and oligo d(T) cellulose specifically to separate viral RNA from the inhibitory factor (s). These techniques were based on the unique properties of CTAB which selectively precipitates nucleic acid while leaving acid insoluble polysaccharide in the supernatant. The resulting nucleic acid was further purified by adsorption onto and elution from oligo d(T) cellulose. This step removes unrelated nucleic acids that lack a poly(A) tail. With this technique, Norwalk virus was detected easily by PCR in very small amounts (400 ul of a 10% suspension) of stool sample. For example, one skilled in the art will recognize that it is now possible to clone the genome of RNA viruses present in low concentrations in small amounts of stool after RT-PCR and a step of amplification of the viral RNA by RT-PCR using random primers. In some cases, RT-PCR active nucleic acids are extracted with CTAB and without oligo d(T) cellulose. In addition, now that the inhibitor(s) can be removed from stool, it is also possible to detect and clone nucleic acids of other viruses (DNA viruses, non-poly(A) tailed RNA viruses) present in stool. The CTAB and oligo d(T) cellulose technique of extraction followed by detection of viral RNA with RT-PCR was used on stool samples and could be used on water and food samples. Stool sample was suspended in distilled water (about 10% wt/vol) and extracted once with genetron. Viruses in the supernatant were precipitated with polyethylene glycol at a final concentration of about 8%. The viral pellets were treated with proteinase K (about 400 ug/ml) in the presence of SDS at about 37°C for about 30 minutes followed by one extraction with phenol chloroform and one with chloroform. A solution of about 5% CTAB and about 0.4M NaCl was added at a ratio of sample:CTAB equal to about 5:2. After incubation at about room temperature for about 15 minutes and at about 45©C for about 5 minutes, the nucleic acids (including the viral RNA) were collected by centrifugation in a microcentrifuge for about 30 minutes. The resultant pellets were suspended in about 1M NaCl and extracted twice with chloroform. The viral RNA in the aqueous phase was used directly in RT-PCR reactions or further purified by adsorption/elution on oligo d(T) cellulose.
A batch method of adsorption/elution on oligo d(T) cellulose was used to purify poly(A) tailed RNA. In this procedure, nucleic acids partially purified as described above or RNA extracted directly with phenol chloroform (without CTAB treatment) were mixed with oligo d(T) cellulose (about 2-4mg/sample) in a binding buffer (about 0.5M NaCl and lOmM Tris, pH 7.5). The mixture was incubated at about 4©C for about 1 hr with gentle shaking and then centrifuged for about 2 minutes in a microcentrifuge. The oligo d(T) cellulose pellet was washed 3-4 times with binding buffer and then the poly(A) tailed RNA was eluted with IX TE buffer (about lOmM Tris, ImM EDTA, pH 7.5). The supernate was collected following centrifugation to remove the oligo d(T) cellulose and the viral RNA in the supernate was precipitated with ethanol. The RNA obtained at this stage was basically inhibitor-free and able to be used in RT-PCR.
In preliminary experiments, Norwalk virus RNA was detected in less than 0.05g of stool samples using the CTAB technique. A trace inhibitor activity was observed with RNA extracted with either CTAB or oligo d(T) alone, but this was easily removed by dilution (1:2) of the extracted nucleic acid before RT-PCR. Combination of the CTAB and oligo d(T) techniques resulted in obtaining high quality, inhibitor free RNA which could be used directly for RT-PCR detection and for cloning of the viral genome. With development of this method to clone from small amounts of stool, one skilled in the art will know that they can obtain cDNAs for the remainder of the genome including those representing the 5'-end of the genome.
For detection with PCR, primers based on the above nucleotide sequence of the genome were made by chemical methods. These primers include: Primer 1: CACGCGGAGGCTCTCAAT located at nucleotides 7448 to 7465; Primer 4: GGTGGCGAAGCGGCCCTC located at nucleotides 7010 to 7027; Primer 8: TCAGCAGTTATAGATATG located at nucleotides 1409 to 1426; Primer 9: ATGCTATATACATAGGTC located at nucleotides 612 to 629; Primer 16: CAACAGGTACTACGTGAC located at nucleotides 4010 to 4027; and Primer 17: TGTGGCCCAAGATTTGCT located at nucleotides 4654 to 4671 (SEQ ID NOS 51 through 56, respectively). These primers have been shown to be useful to detect virus using reverse transcription and polymerase chain reaction methods (RT-PCR). Figure 7 shows data using these primers. In primer sets 1 and 4, 8 and 9, and 16 and 17, the reverse compliments for the sequences given above for primers 1, 8, and 17 were used.
New, additional primer sets (Table 6 and SEQ ID NOS.: 15 to 37) are used as probes to detect the Norwalk-related viruses. Table 7 shows the ability of newly selected primer sets 36-35, 69-39, 78-80 to detect many Norwalk-related viruses. These results are additional examples of the use of primer sets from the original Norwalk virus sequence to detect Norwalk-related viruses. Nucleotide sequence data of many of these viruses indicates that there is a continuum of genetic relatedness within the RNA region described by primer sets 36-35 or 69-39 of these different viruses (from 87% to 0%), yet these different agents can b& detected using primers from the Norwalk virus genome sequence. The sequence of 2516 nucleotides of another small round structured virus (SRSV/KY/89 SEQ ID NO: 12) also was obtained by using a total of 8 additional sets of primers from the original Norwalk virus sequence (primers 56 and 23, 42 and 55, 58 and 59, 60 and 61, 72 and 63, 76 and 77, 64 and 75, and 74 and 3; Table 6).
Example 6
Preparation of polyclonal antibodies and monoclonal antibodies to Norwalk virus proteins Protein(s) encoded in the cDNA fragments or derivatives thereof, is produced in a prokaryotic or eukaryotic expression system and used to immunize animals to produce polyclonal antibodies for diagnostic assays. Prokaryotic hosts may include Gram negative as well as Gram positive bacteria, such as E. coli. S. tvmphimurium. Serratia marcescens. and Bacillus subtilis. Eukaryotic hosts may include yeast, insect or mammalian cells. Immunized animals may include mammals such as guinea pigs, mice, rabbits, cows, goats or horses or other non-mammalian or non-murine species such as chickens. Repeated immunization of these animals with the expressed protein mixed with an adjuvant such as Freund adjuvant to enhance stimulation of an immune response produces antibodies to the protein. Alternatively, synthetic peptides of greater than 15 amino acids made to match the amino acid sequence deduced from the partial cDNA sequence (or from other sequences determined by sequencing additional cDNAs detected with the original or other clones) are linked to a carrier protein such as bovine serum albumin or lysozyme or cross-linked with treatment with glutaraldehyde and used to immunize animals to produce polyclonal antibodies for diagnostic tests. The serum of animals immunized with either the expressed protein or with synthetic peptides are tested by immunologic assays such as immune electron microscopy, Western blots (immunoblots) and blocking ELISAs to demonstrate that antibodies to Norwalk and related viruses have been made. Reactivities with the expressed protein or synthetic peptides show specificity of the polyclonal sera. Reactivities with other viruses in the Norwalk group (Snow Mountain Agent, Hawaii Agent, Taunton Agent, etc.) indicate production of a reagent which recognizes cross-reacting epitopes. Balb\c mice injected with the immunogens as described above and shown to have produced polyclonal antibodies are boosted with immunogen and then sacrificed. Their spleens are removed for fusion of splenocytes with myeloma cells to produce hybridomas. Hybridomas resulting from this fusion are screened for their reactivity with the expressed protein, the peptide and virus particles to select cells producing monoclonal antibodies to Norwalk virus. Screening of such hybridomas with Norwalk-related viruses permits identification of hybridomas secreting monoclonal antibodies to these viruses as well.
Development of Diagnostic Assays Analysis of the deduced amino acid sequence of the Norwalk virus genome has shown that the Norwalk virus has the genetic organization shown in Figure 8. Expression of regions of this genome in cell-free translation systems and in the baculovirus expression system have shown that the 5 '-end of the genome encodes nonstructural proteins and the 3'- end of the genome encodes at least one structural protein. Based on this information, one can express the complete genome or subgenomic regions of the genome to produce diagnostic assays to detect viral antigens or immune responses to specific regions of the genome. This information can be used to detect the Norwalk virus, antigens or immune responses to Norwalk virus. This information also can be used to detect other similar currently uncharacterized viruses that cause gastroenteritis or possibly other diseases. Some of these viruses will be in the Caliciviridae or in the picornavirus superfamily. All of these viruses will have matching or similar genomic regions in their DNA sequences.
The availability of cDNA clones from viruses related to Norwalk virus enables the production of new antibodies and antisera for diagnostic assays for these related viruses. For example, availability of cDNA clones from caliciviruses which cannot be cultivated permits the expression of protein products of those clones. The protein products are used to develop new antibodies and antisera. In addition, genetic engineering is used to combine the cDNAs from viruses related to Norwalk virus with the cDNAs from Norwalk virus to produce chimeric proteins, such that part of the protein produced is derived from Norwalk virus genome sequence and another part of the protein is derived from the genome sequence of a virus related to Norwalk virus. These chimeric proteins are then used to produce diagnostic reagents, vaccines and antivirals. Examples of the diagnostic assays are shown in the specific examples and figures below.
Example 7
Development of diagnostic assays to detect nucleic acids of Norwalk virus or Norwalk-related viruses by detection of specific regions of the viral genomes based on an understanding of the Norwalk virus genome.
The genetic organization of the Norwalk virus genome allows the prediction of specific regions of the gene sequence as regions where oligonucleotide primers or probes can be developed to detect Norwalk virus sequences and common sequences of other related or similar viruses. Some of these common genome sequences are found in viruses in the
Caliciviridae or in the picornavirus superfamily. The detection can be done by standard PCR, hybridization or other gene amplification methods.
Two primers, named 35 (CTT GTT GGT TTG AGG CCA TAT, complementary to nt 4944-4924 in the Norwalk virus genome, SEQ ID NO: 15) and 36 (ATA AAA GTT GGC ATG AAC A, nt 4475-4493 in the
Norwalk virus genome, SEQ ID NO: 16), were chosen from the region likely to encode the Norwalk virus RNA polymerase. These primers then were used to prepare a cDNA clone by reverse transcriptase-PCR from the nucleotide sequence of human calicivirus Sapporo strain (HuCV Sapporo), 1982 outbreak (Figure 9, SEQ ID NO:5). The resulting sequence was compared to that of Norwalk virus and of feline and rabbit caliciviruses available from Genbank. The first cDNA clone from Sapporo, named "c-29_4-gel", determined to contain calicivirus sequence was 488 nucleotides long, of which 40 nucleotides were contributed by primers 36 and 35, leaving 448 nucleotides unique to human calicivirus Sapporo. The sequence of clone c-29_4-gel between primers 36 and 35 also is shown in Figure 9, SEQ ID NO:8.
Evidence that the HuCV Sapporo cDNA clone was correct is shown by five facts. First, the sequence exhibits strong homology with Norwalk virus, feline calicivirus, and the rabbit calicivirus at the nucleotide and amino acid levels. (See Figure 10 and Tables 7 and 8). Second, the sequence contains a continuous protein encoding region on the positive strand. In Norwalk, feline, and rabbit caliciviruses continuous protein encoding regions also are found in the region of homology. Third, the sequence contains the amino acid motif YGDD, which is a marker for RNA virus proteins which have RNA-dependent-RNA-polymerase activity. In c-29_4-gel, the YGDD motif is at the predicted distance from the ends of the sequence. Fourth, the same cDNA product was obtained from six different stool specimens. Fifth, no significant homologies were found for other sequences in the Genbank.
The nucleotide sequence of c-29_4-gel was used to synthesize an internal primer. This internal primer was used to prepare a second set of RT-PCR products from human calicivirus Sapporo RNA. A number of new cDNA clones were obtained of which one, named "at23s2m31", contains overlapping sequence which is 5' on the virus genome from that contained in c-29_4-gel. Sequence at23s2m31 is 149 nucleotides long (SEQ ID NO:7) and overlaps c-29_4-gel by 46 nucleotides. See Figure 9 for at23s2m31 sequence and area of overlap with c-29_4-gel. The resulting combined sequence information of c-29_4-gel and at23s2m31 is 551 nucleotides in length, excluding the portion c-29_4-gel contributed by prime 35.
Although the human calicivirus Sapporo sequence was generated from knowledge of the Norwalk virus sequence, the former is distinguishable in the same region (see Table 8 or Figure 9). The known sequence of human calicivirus Sapporo indicates that this virus is more closely related to the animal caliciviruses than to Norwalk virus.
In May, 1987, a child in Houston was infected with a virus which was identified as a calicivirus based on its morphology. Samples containing virus particles from this child failed to react in serologic assays developed for the detection of Norwalk virus and human calicivirus Sapporo. Primers 36 and 35 were used to prepare cDNA from the viral genome of this strain using RT-PCR. The resulting cDNA product, called 4847 complete, is 434 nucleotides long, excluding the primers, and is distinguishable from that of Norwalk virus and human calicivirus Sapporo. (See "Houston" in homology comparison in Figure 10; Table 10 and SEQ ID NO: 10). Evidence that this Houston cDNA is correct is the same as that listed for c-29_4-g above, except that homology with Norwalk virus and human calicivirus Sapporo is not statistically significant.
Use of the sequence from the human calicivirus Sapporo strain to produce an amplification primers for human calicivirus Sapporo and related agents The known sequence of human calicivirus Sapporo overlaps one of the two primers, called primer 36 (see Table 6), used for the initial amplification of cDNA clone c-29_4-gel- Examination of the homology of known calicivirus sequences (Table 8 SEQ ID NOS 57 through 62) in that region indicated that a new 36 primer could be synthesized and used to amplify caliciviruses more closely related to human calicivirus Sapporo than Norwalk virus. A new primer was synthesized and is called primer "new 36" (see Table 6, last line, and SEQ ID NO:37). The new 36 primer was used with primer 35 to generate a cDNA clone from a calicivirus which caused a diarrhea outbreak in November, 1986, in a Houston day care center ("Day care"). The calicivirus strain causing this Day care outbreak was antigenically related to human calicivirus Sapporo but antigenically distinct from Norwalk virus by EIA. The Day care cDNA product obtained from the RT-PCR reaction with primers new 36 and 35 is 445 nucleotides long, excluding the primers (see Figure 9 and SEQ ID NO: 9), and has close homology to human calicivirus Sapporo and a more distant, yet still significant homology with Norwalk virus, as shown in Figure 10. Evidence that this Day care cDNA is correct is the same as that listed for c-29_4-gel above.
Use of primers 35 and 36 derived from the Norwalk virus sequence to derive a cDNA clone from an animal calicivirus A calicivirus was isolated from the mouth of the pygmy chimpanzee, Pan paniscus. This calicivirus is antigenically distinct from the human calicivirus Sapporo strain by EIA. A cDNA was produced from the primate calicivirus (PrCV) RNA using RT-PCR and primers 36 and 35. The complete nucleotide sequence of this cDNA is not yet available. The cDNA, called atprcvw2 (Figure 20; SEQ. ID. NOS 13 and 14), is of the predicted size and has significant nucleotide homology with human calicivirus Sapporo, feline calicivirus(es), and the rabbit calicivirus in the region of known sequence. No significant homology with Norwalk virus has been observed in the region of known sequence. The known amino acid sequence contains the YGDD motif on the positive strand at the predicted distance from primer 35.
Use of multiple primers form the Norwalk virus genomic sequence to detect and characterize KY89. another small round virus associated with an outbreak of gastroenteritis. The known sequence for Norwalk virus is used to obtain the sequence of other viruses such as SRSV/KY/89, an agent from a stool from an outbreak of gastroenteritis in Japan in 1989. Originally, cDNA products and sequence information were obtained using primer sets 36-35. Continued work with another 8 sets of primers (Primers 56 and 23, 42 and 55, 58 and 59, 60 and 61, 72 and 63, 76 and 77, 64 and 75, and 74 and 3 in Table 6 and SEQ ID NOS:21 through 36) allowed the SRSV/KY/89 sequence of 2516 nucleotides to be determined (Figures 11 and 12, SEQ ID NO: 12). This sequence includes the part of the polymerase region and the capsid region of the genome. Figures 14 and 6 (SEQ ID NOS 38 through 50 and 63 through 75) show sequences from other Norwalk- related viruses. Continued use of this approach with other Norwalk- related viruses (such as those shown in Table 7) allows the discovery of the complete sequences of multiple Norwalk-related viruses. Those skilled in the art will realize that the use of such sequence information and expression of fragments and derivatives of Norwalk-related viruses permits development of diagnostic assays to detect antibodies, antigens, viral genetic material or antivirals and to develop vaccines for specific Norwalk-related viruses in the same manner that Norwalk virus fragments and derivatives have been used.
Example 8 Development of diagnostic assays using expressed Norwalk virus proteins to detect immune responses to Norwalk virus
Protein(s) encoded in the Norwalk virus genome or fragments or derivatives thereof is produced in a prokaryotic or eukaryotic expression system and used as antigens in diagnostic assays to detect immune responses following virus infections. Prokaryotic hosts may include Gram negative as well as Gram positive bacteria, such as Escherichia coli, Salmonella tymphimurium, Serratia marcescens, Bacillus subtilis, Staphylococcus aureus and Streptococcus sanguinis. Eukaryotic hosts may include yeast, insect or mammalian cells. Diagnostic assays may include many formats such as enzyme-linked immunosorbent assays, radioimmunoassays, immunoblots or other assays. Figure 15 shows data for a capsid protein encoded from the 3 '-end of the Norwalk virus genome. It is expressed by nucleotides 5337 through 7753 of the DNA sequence shown in Table 2 and Figure 8. This protein has an approximate molecular weight of 58,500 and is hereinafter referred to as the 58,500 mwt protein. It was produced in insect cells infected with baculovirus recombinants (C-6 and C-8). A band (see arrow in Figure 15) representing the 58,500 mwt protein in C-6 and C-8 infected cells is not seen in insect cells infected with wild-type (WT) baculovirus or in mock infected cells. Other proteins encoded by Norwalk virus cDNA or fragments or derivatives are similarly expressed using baculovirus recombinants and other expression systems. Figure 16 shows data using the 58,500 mwt protein produced using the baculovirus expression system to detect immune responses before and after infection of volunteers with Norwalk virus inoculum. Antigen was put on ELISA plates and pre- and post-infection human sera were added. The data show that when an individual has had the infection, the post- serum reacts strongly to the antigen. Other proteins encoded in the Norwalk virus cDNA or fragments or derivatives thereof are similarly used to detect immune responses following Norwalk virus infection.
Some proteins have the intrinsic property of being able to form particles. The 58,500 mwt protein discussed above has that property. Particles formed from proteins are expressed in any expression system and used to produce diagnostic assays based on detection of antibody responses or immune responses. Figure 17 shows an electron micrograph of particles produced using the baculovirus expression system from recombinants containing the 3'-end of the Norwalk genome. These particles are similar in size to the native virus particles. They are antigenic, immunoreactive and immunogenic. They differ from most of the virus particles resulting from natural infection in that many of the expressed particles lack nucleic acids. The rNV particles are highly immunogenic when given parenterally to mice, rabbits and guinea pigs and when given orally to mice.
Figure 18 shows data on the properties of rNV particles following centrifugation in gradients of CsCl. The density of the particles (symbolized by closed boxes) is 1.31 g/cc which is distinct from the 1.38 g/cc density of particles purified from the original infectious Norwalk inoculum given to volunteers. The gradients were fractionated. Each fraction was put on an ELISA plate and human serum was then introduced. The open boxes show that there was no ELISA activity with the pre-infection serum. The closed diamonds show there was reactivity with the post-infection serum. Other particles made from other proteins encoded in the Norwalk virus cDNA or fragments or derivatives thereof are similarly used to detect immune responses following Norwalk virus infection. Figure 19 shows data using purified particles formed by the 58,500 mwt protein to detect immune responses in post-inoculation (but not pre- inoculation) serum samples of 9 volunteers infected with Norwalk virus. One of the volunteers, number 6, exhibited no symptoms of Norwalk virus infection based on monitoring clinical symptoms or measuring an immune response. Purified, expressed particles were put on ELISA plates and one pre- and one post-infection serum samples from each volunteer was added to the particles. The amount of antibody binding to the particles in each pre- and post-infection sample was measured. The data in Figure 19 show that the expressed proteins form particles that are immunoreactive and antigenic. Other proteins encoded in the Norwalk virus cDNA or fragments or derivatives thereof are similarly used to detect immunoreactive and antigenic activity.
Additional developments of diagnostic assays for the detection of Norwalk and Norwalk-related viruses also were pursued. First, new ELISA assays were made based on utilizing the Norwalk virus capsid protein that was engineered to be synthesized from a cDNA fragment that was deduced from the Norwalk virus cDNA sequence and then produced using the baculovirus expression system. This expressed Norwalk virus capsid protein self-assembled into recombinant Norwalk virus particles (rNV). Two new ELISA assays were established using this rNV antigen. One assay detects antiviral antibody and the other detects viral antigen. Both the ELISAs are very sensitive when compared to the previous assays (based on reagents from human volunteers) available to detect such agents. Further characterization of the antibody ELISA has shown this assay detects immune responses following human infections with Norwalk virus and a subset of human infections with viruses in the Norwalk group such as Snow Mountain and Hawaii agents. In contrast, the antigen ELISA is based on use of hyperimmune serum made to the baculovirus expressed recombinant Norwalk virus particles (rNV). This antigen ELISA has been found to be very specific in that is recognizes the prototype Norwalk virus (8FIIa) and a subset of closely related agents, but not all other viruses in the Norwalk group such as the Snow Mountain agent and Hawaii agent (See Tables 1 and 7). While the antigen ELISA does not detect other viruses in the Norwalk group such as the small round structured viruses or caliciviruses, these and other Norwalk-related viruses have been able to be detected using primers selected from the nucleotide sequence of Norwalk virus (See Table 7). To develop more broadly reactive diagnostic assays, ELISAs based on using other fragments of the Norwalk virus genome were developed. The new diagnostic assays are based on detection of antibody responses or of antigens deduced from fragments of the Norwalk virus genome other than the capsid region. An example and data of this approach is the following.
One Norwalk virus nonstructural protein is predicted to be encoded in the first ORF of Norwalk viral genome. This ORF is located at the 5 end of the viral genome and it has a predicated molecular weight of 190,000 (190K). Whether this ORF 1 is useful in diagnostic assays first was evaluated by expressing the protein encoded in the full length viral RNA, and then synthesizing and testing the immunoreactivity of the encoded protein using a cell-free system. This was accomplished by in vitro transcription of a full length cDNA (pGNV-F) of the Norwalk viral genome cDNAs. This full-length cDNA was constructed by ligation of subgenomic derivatives of the original Norwalk virus cDNAs shown in the physical map in Figure 5. The in vitro synthesized NV mRNAs next were examined for their ability to direct the synthesis of a Norwalk virus specific protein by cell-free translation in rabbit reticulocyte lysates in the presence of ^S methionine to produce a radiolabeled protein. The expressed proteins were analyzed by polyacrylamide gel electrophoresis (PAGE). A clear band of approximate molecular weight of 130,000 was observed in the sample containing the viral RNA but not in the negative control (without viral RNA). The immunoreactivity of this protein was examined by reactivity with pre- and post-infection sera from volunteers given Norwalk virus. The 130K protein was precipitated by a convalescent serum of a volunteer infected with Norwalk virus, but not by serum collected before infection, indicating this protein was virus-specific. This showed this 130K protein contains some immunoreactive epitopes. The apparent smaller size of the protein made in this translation system suggested that either the protein migrates aberrantly on gels, or an internal initiation codon was used to begin translation or some type of post translational modification may have occurred after the protein was translated.
To further characterize immunoreactive derivatives of the Norwalk virus cDNA useful for diagnostic assays, the 2C region of the Norwalk viral genome (see Figure 8) was expressed using the baculovirus expression system. This region was selected for initial expression because it is located at the 5'-end of the non-structural protein and a high level of conservation was found between the sequence of the predicted Norwalk virus protein, and new sequence published for related caliciviruses and picornavirus. A 5 '-end cDNA fragment of the viral genome was subcloned into the baculovirus transfer vector pVL 1393. After co-transfection of insect Sf9 cells with wild-type baculovirus DNA, recombinants containing the Norwalk viral gene were identified and selected. After three rounds of plaque purification, radiolabeled lysates of recombinant-infected insect cells were prepared, and the radiolabeled proteins were analyzed by PAGE. The results showed that a protein of apparent molecular weight of 57,000 (57K) was made in recombinant-infected but not in uninfected cells. The size of the protein suggested that the internal AUG initiation codon located at nucleotide 953 was used for making this protein. This 57K protein also was precipitated by convalescent serum (but not by pre- infection serum) from a volunteer who was infected with Norwalk virus. This protein mainly remained cell-associated. One skilled in the art will readily see that improvements in the yield and purification of this 2C nonstructural protein are possible and will yield more rapid ELISAs to detect Norwalk and related virus infections. One skilled in the art also will see that by expressing proteins from other regions of the Norwalk viral genome {e.g., 3C-like, 3D-like and the 3d ORF), diagnostic assays are made for Norwalk and related viruses similar to the ELISAs made with the 2C nonstructural and rNV structural protein. These new assays should widen the spectrum in detection of Norwalk-related viruses.
The initial lack of sensitive methods to detect Norwalk and Norwalk- related viruses made the description of the many Norwalk-related viruses difficult to define. However, as shown in Table 7, the methods and data provided here demonstrate how the discovery of the nucleotide sequence of the Norwalk virus genome has led to the ability to develop tests to detect Norwalk virus and other related agents. The data and methods also demonstrate that fragments and derivatives of the Norwalk virus genome can be used to provide evidence of and immunity against Norwalk and related viruses.
Example 9
Development of diagnostic assays using expressed Norwalk virus and Norwalk-related viruses to detect viral antigens Individual proteins, particles or protein aggregates formed from expression of one or more Norwalk virus genes in any prokaryotic or eukaryotic expression system are used as an immunogen or inoculate animals to produce polyclonal and monoclonal antibodies for diagnostic assays to detect viral antigens.
Recombinant Norwalk virus particles (rNV) produced using the baculovirus expression system has been used to produce polyclonal antibodies in mice, guinea pigs and rabbits following parenteral immunization (see Table 9). Mice given rNV orally also have developed serum antibodies. Hybridomas from mice immunized with rNV also have been obtain following fusion with myeloma cells. Use of these antibodies in a capture ELISA has shown NV antigen can be detected. This antigen ELISA based on the antiserum made to the rNV particles is quite specific and it detects only a subset of Norwalk-related viruses (See Table 7). Therefore, additional capsid antigens from other Norwalk-related viruses (such a Snow Mountain, Hawaii etc.) must be expressed to produce a more broadly reactive ELISA for capsid antigen. The ELISA is only one format that can be used to detect virus antigen. Other formats could include immunofluorescence or immunocytochemistry, or immune electron microscopy. The comparison of the capsid sequences of Norwalk virus and Norwalk-related viruses permits the identification of conserved regions of the capsid protein and use of fragments of such sequences to immunize animals and can result in the production of antisera with more broad reactivity to Norwalk-related viruses. Alternatively, sequential immunization of animals with expressed proteins of Norwalk and Norwalk-related viruses will result in antiserum with the desired broad reactivity. Antigen detection assays that are specific to one of a few strains of Norwalk and Norwalk-related viruses and additional assays that are more broadly reactive each will have use. Expression of fragments of proteins encoded in other regions of the genome can be used to produce antiserum to other proteins for use in ELISAs to detect viral antigens. The expression of the first ORF that represents a polyprotein encoded in the 5'-end of the genome and fragment 2C of the polyprotein has shown that each of these nonstructural proteins in immunoreactive and antiserum made to these can be used to develop diagnostic assays to detect these viral proteins. These assays can be broadly reactive and detect many other Norwalk- related viruses because of sequence conservation. Those skilled in the art will recognize that knowledge of the genome organization of Norwalk virus permits similar expression of the same regions of the genomes of other Norwalk-related viruses for use in diagnostic assays to detect viral antigens. Example 10 Development of a vaccine using Norwalk virus expressed antigens Vaccines for Norwalk virus, the Norwalk group of viruses or other small round viruses are made from an expressed Norwalk virus protein. That expressed protein can be a Norwalk virus capsid protein expressed alone or in combination with one or more other Norwalk virus proteins or self-forming particles. For example, the particles shown in Figure 17 were produced using the baculovirus expression system. They are used as a vaccine when expressed alone or in combination with one or more other Norwalk virus proteins. Similarly, the other proteins encoded in the Norwalk virus cDNA or fragments or derivatives thereof are used as a vaccine when expressed alone or in combination with one or more Norwalk virus proteins. Individuals are vaccinated orally, parenterally or by a combination of both methods. For parenteral vaccination, the expressed protein is mixed with an adjuvant and administered in one or more doses in amounts and at intervals that give maximum immune response and protective immunity. Oral vaccination parallels natural infection by Norwalk virus inoculum, i.e. the individual ingests the vaccine with dechlorinated water or buffer. Oral vaccination may follow sodium bicarbonate treatment to neutralize stomach activity. For example, sodium bicarbonate solution is taken by each person 2 minutes before and 5 minutes after vaccine administration.
Example 11
Production of a vaccine for other agents bv using expressed Norwalk virus capsids as a carrier or vehicle for the expression of other antigens or parts of other antigens Identification of the region of the genome that encodes the Norwalk virus capsid protein and that forms particles following expression (i.e., regions 5346 through 6935 and 5337 through 7753) allows genetic engineering of the cDNA that encodes the capsid protein to incorporate one or more heterologous pieces of cDNA that encode antigenic epitopes. Expression of such recombinant genes produces a recombinant capsid that is antigenic, induces antibodies, and protects against Norwalk virus and its antigens, and against the heterologous epitopes or antigens.
Alternatively, the Norwalk virus capsid protein carrier is mixed with or covalently linked to one or more heterologous protein antigens or synthetic peptides containing heterologous epitopes. This mixture is antigenic, induces antibodies, and protects against Norwalk virus and its antigens, and against the heterologous epitopes or antigens.
Individuals are vaccinated using the oral and parenteral methods described above in example 10.
Example 12 Kit Kits for detecting immune responses to Norwalk virus and are prepared by supplying in a container a protein deduced from the Norwalk virus genome shown in Table 2 or fragments or derivatives thereof. Similar proteins are prepared from Norwalk-related viruses to detect immune responses to the Norwalk-related viruses. For example, the protein encoded by Norwalk virus nucleotides 1 through 7753, the protein encoded by Norwalk virus nucleotides 146 through 5359, the protein encoded by Norwalk virus nucleotides 5337 through 7573, the protein encoded by Norwalk virus nucleotides 5346 through 6935, the protein encoded by Norwalk virus nucleotides 6938 through 7573 and any combinations thereof may be used in such kits. The kit can also include controls for false positive and false negatives, reagents and sample collection devices. The kit can be equipped to detect one sample or multiple samples. Example 13
Kit
Kits for detecting Norwalk viruses and Norwalk-related viruses are prepared by supplying in a container at least one antiserum made from a protein expressed from the deduced amino acid sequence of the Norwalk virus genome shown in Tables 3, 4, or 5 or from a fragment or derivative the deduced amino acid sequence. Similar antiserum are made from proteins encoded by Norwalk-related viruse genomes. For example, an antiserum made to the protein encoded by Norwalk virus nucleotides 1 through 7753, the protein encoded by Norwalk virus nucleotides 146 through 5359, the protein encoded by Norwalk virus nucleotides 5337 through 7573, the protein encoded by Norwalk virus nucleotides 5346 through 6935, the protein encoded by Norwalk virus nucleotides 6938 through 7573 and any combination thereof may be used in such kits. The kit can also include controls for false positives and false negatives, reagents and sample collection devices. The kit can be equipped to detect one sample or multiple samples.
In conclusion, it is seen that the present invention and the embodiments disclosed herein are well adapted to carry out the objectives and attain the ends and advantages mentioned as well as other inherent therein. The novel features characteristic of this invention are set forth in the appended claims. While presently preferred embodiments of the invention have been described for the purpose of disclosure, numerous changes in the details of synthesis and use described herein will be apparent to those skilled in the art. It should be understood, however, that there is no intention to limit the invention to the specific form disclosed, but on the contrary, the intention is to cover all modifications, alternative means of synthesis and use and equivalents falling within the spirit and scope of the invention. Table 1. Classification of small round viruses.
Featureless viruses*
Virus Physical features Examples Enterovirus RNA Polio
BD* 1.34 g/cm3 Hepatitis A
Size range 20-30 nm
Parvovirus DNA Feline/mink/canine
BD 1.38-1.46 g/cm3 Bovine
Size range 18-26 nm
Candidate parvovirus DNA? Wollan, Ditchling,
BD 1.38-1.4g/cm3 Parramatta, cockle
Structured viruses
Virus Morphology Physical features Examples Astrovirus 5—6-pointed surface RNA Lamb x star BD 1.36-1.38 g/cm3 Human
Size range 28-30 nm
Calicivirus Surface hollows, RNA Human (Norwalk, UK1-4, and ragged outline BD 1.36-1.39 g/cm3 Sapporo and other Japanese
'Star of David' Size range 30-38 nm strains) configuration Newbury (bovine)
Pig
Small round Amorphous surface, BD 1.36-1.41 g/cm3 Montgomery County, structured virus ragged outline Size range 30-35 nm Hawaii, Taunton, Amulree, (SRSV) Otofuke, Snow Mountain
Smooth outer edge and no discernible surface structure. Surface structure and/or ragged outline BD = buoyant density
Figure imgf000042_0001
Table 2, continued TTTGACCCCT CTGCAGCAGA AGCTAGGGCT GTGGCATACG GGCGGGATCT GCGCTCGTTA AGCCATGGCA TTAGAAGGAC AGCTGGAAAG GGGCCCATAG TGAAGAGGAG GAGAGTGAGG AAAGAACAAA GGCAAGACCA TCGCCGTGGT CTGAGTGATG TGGCAATTAT AGTATACAAG AGAGGTACAG GCAGGTGGTG GGTCTTCTAT AAATCCAAGA AGTGACTGGA TCAGACATCA ATGGGCAGAT GATGACAGAG GACACTATGG AGCCGAGTCA AGTGTTCATC ACAACCACAC CCTATCTAGT ATAGCAATCC AATGCGCCCT GACTTGACAG CTCAGTCCTA ATTAAACGGG TATTGCCTCC ATGAGGATAC AGGGGCCAAT GCAAAGGGGA CGTCCACAAG CGCGGGAATG AGGCAACACC GTGGTCTGCG AGACAAGGGG CATTATGCCG AACTAAAACA AAATTCTGGA AGCATACCTG GGGGGCAAGG ACGTGACCAA CTGAAACCCT GGAGGCTGCG GTTGAGACTG GTGGTCTTAC GCTGATGCCT CCATAAAAGG AAGAATGATG AGCTGCACAC GCCAACAATA AGCCTTAAAA GATGAACTAG ACTATGGGGC GCCGATCTCG TGACGCTATA AAATCACATG AGATGGCCCC CTCATCTATG TACAGCATGG GACTCAACAC GCGCCTTACG GCCTCACCAG TGAGATGGAT GTAGGTGATT ATGTACTTCC CAGGTGAACA GGCCACTGGT TTATCACCTG TGATGAGATT GTGTCAACTG GGAATATGGC CTCAAACCAA AAATGTGGAT GGACTGGTCT AGGCAGGTTA GATAGGGCTT
Figure imgf000043_0001
Table 2, continued TTCAGATCCA TCAGAGACTC TCTAGGGGAA GCTTCACTCC ACATGAAATC AAGACTGGTG CTGGATGCGC TTCCATGACC CGTAAATGAT GATGGCGTCT GTCAGTTGGT ACCGGAGGTT CTTCGACAGC AGTCGCGACT ATTTTGTGCA AGCCCCCCAA TTTTGTTTGA TTTGAGTTTG TGTATAATGG TTGGGTTGGT CTGCGGGGAA GATAATAGTT TAGCACAAGC AACTCTCTTT AGGTGCCTTT GGAAGATGTT CCATGCGCCT TGTGTGCATG CTTTTGTAGT TGCAGGGCGA TAGTCCCTCC TACGGTGGAG GTTCTCTGTC TAACTCACGT ATGTCCAGAG TGTGCAGTTC CCACCCCAGT TTCATTGTCA TCAACCTTAC TGAATTGGAT GGTTTCCAGA CCTCGGTGGT GCCAGACCCA GTATGATGTA TTCAGGCAAA TGGCATTGGC CATCACACCC GTCTGGCTCC TTACGGAGGC AACACATCTA TCTTTTTCAT GTCAAAAATG AAGAGTACAT TTCACATCTT TCCACTATGT TGACCCTGAT GTTTCCTCAC TTGTGTCCCC GGGTCTTTGT CTTTGTTTCA CCAGCTCGGC AAGAGGTAGG AATTGCTGCT TCCACAGCAG GGCCCTCCAA AGCCAAAGGT TGACAGGGAA ATGATTGGGT GGCAACTAGA TATTCACTCC TGTGGCAGGA GCTCCAGTCA CGAGTCCTCT GCTACCACAT CTCTAAGCAA AAACAGGTTC CATTTCTCGA ACCACTAGTT TCCATACCAC GCGGAGGCTC TTCTACACTG TCTTCTGTGC AAATAATAGG CGATGATGTT TAATTAGGTT TAATTTGATG
Figure imgf000044_0001
Table 2, continued
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 7740
AAAAAAAAAA AAA 7753
Table 3. The amino acid sequence deduced from nucleotides 146 through 5359 of the Norwalk virus genome shown in Table 2.
CTCGATAAAG ATAACCAACC AAGAT ATG GCT CTG GGG CTG ATT GGA CAG GTC 172
Met Ala Leu Gly Leu He Gly Gin Val 1 5
CCA GCG CCA AAG GCC ACA TCC GTC GAT GTC CCT AAA CAA CAG AGG GAT 220 Pro Ala Pro Lys Ala Thr Ser Val Asp Val Pro Lys Gin Gin Arg Asp 10 15 20 25
AGA CCA CCA CGG ACT GTT GCC GAA GTT CAA CAA AAT TTG CGT TGG ACT 268 Arg Pro Pro Arg Thr Val Ala Glu Val Gin Gin Asn Leu Arg Trp Thr 30 35 40
GAG AGA CCA CAA GAC CAG AAT GTT AAG ACG TGG GAT GAG CTT GAC CAC 316 Glu Arg Pro Gin Asp Gin Asn Val Lys Thr Trp Asp Glu Leu Asp His 45 50 55
ACA ACA AAA CAA CAG ATA CTT GAT GAA CAC GCT GAG TGG TTT GAT GCC 364 Thr Thr Lys Gin Gin He Leu Asp Glu His Ala Glu Trp Phe Asp Ala 60 65 70
GGT GGC TTA GGT CCA AGT ACA CTA CCC ACT AGT CAT GAA CGG TAC ACA 412 Gly Gly Leu Gly Pro Ser Thr Leu Pro Thr Ser His Glu Arg Tyr Thr 75 80 85
CAT GAG AAT GAT GAA GGC CAC CAG GTA AAG TGG TCG GCT AGG GAA GGT 460 His Glu Asn Asp Glu Gly His Gin Val Lys Trp Ser Ala Arg Glu Gly 90 95 100 105
GTA GAC CTT GGC ATA TCC GGG CTC ACG ACG GTG TCT GGG CCT GAG TGG 508 Val Asp Leu Gly He Ser Gly Leu Thr Thr Val Ser Gly Pro Glu Trp 110 115 120
AAT ATG TGC CCG CTA CCA CCA GTT GAC CAA AGG AGC ACG ACA CCT GCA 556 Asn Met Cys Pro Leu Pro Pro Val Asp Gin Arg Ser Thr Thr Pro Ala 125 130 135
ACT GAG CCC ACA ATT GGT GAC ATG ATC GAA TTC TAT GAA GGG CAC ATC 604 Thr Glu Pro Thr He Gly Asp Met He Glu Phe Tyr Glu Gly His He 140 145 150
TAT CAT TAT GCT ATA TAC ATA GGT CAA GGC AAG ACG GTG GGT GTA CAC 652 Tyr His Tyr Ala He Tyr He Gly Gin Gly Lys Thr Val Gly Val His 155 160 165
TCC CCT CAA GCA GCC TTC TCA ATA ACG AGG ATC ACC ATA CAG CCC ATA 700 Ser Pro Gin Ala Ala Phe Ser He Thr Arg He Thr He Gin Pro He 170 175 180 185
TCA GCT TGG TGG CGA GTC TGT TAT GTC CCA CAA CCA AAA CAG AGG CTC 748 Ser Ala Trp Trp Arg Val Cys Tyr Val Pro Gin Pro Lys Gin Arg Leu 190 195 200
ACA TAC GAC CAA CTC AAA GAA TTA GAA AAT GAA CCA TGG CCG TAT GCC 796 Thr Tyr Asp Gin Leu Lys Glu Leu Glu Asn Glu Pro Trp Pro Tyr Ala 205 210 215
GCA GTC ACG AAC AAC TGC TTC GAA TTT TGT TGC CAG GTC ATG TGC TTG 844 Ala Val Thr Asn Asn Cys Phe Glu Phe Cys Cys Gin Val Met Cys Leu 220 225 230 Table 3, continued
GAA GAT ACT TGG TTG CAA AGG AAG CTC ATC TCC TCT GGC CGG TTT TAC 892 Glu Asp Thr Trp Leu Gin Arg Lys Leu He Ser Ser Gly Arg Phe Tyr 235 240 245
CAC CCG ACC CAA GAT TGG TCC CGA GAC ACT CCA GAA TTC CAA CAA GAC 940 His Pro Thr Gin Asp Trp Ser Arg Asp Thr Pro Glu Phe Gin Gin Asp 250 255 260 265
AGC AAG TTA GAG ATG GTT AGG GAT GCA GTG CTA GCC GCT ATA AAT GGG 988 Ser Lys Leu Glu Met Val Arg Asp Ala Val Leu Ala Ala He Asn Gly 270 275 280
TTG GTG TCG CGG CCA TTT AAA GAT CTT CTG GGT AAG CTC AAA CCC TTG 1036 Leu Val Ser Arg Pro Phe Lys Asp Leu Leu Gly Lys Leu Lys Pro Leu 285 290 295
AAC GTG CTT AAC TTA CTT TCA AAC TGT GAT TGG ACG TTC ATG GGG GTC 1084 Asn Val Leu Asn Leu Leu Ser Asn Cys Asp Trp Thr Phe Met Gly Val 300 305 310
GTG GAG ATG GTG GTC CTC CTT TTA GAA CTC TTT GGA ATC TTT TGG AAC 1132 Val Glu Met Val Val Leu Leu Leu Glu Leu Phe Gly He Phe Trp Asn 315 320 325
CCA CCT GAT GTT TCC AAC TTT ATA GCT TCA CTC CTG CCA GAT TTC CAT 1180 Pro Pro Asp Val Ser Asn Phe He Ala Ser Leu Leu Pro Asp Phe His 330 335 340 345
CTA CAG GGC CCC GAG GAC CTT GCC AGG GAT CTC GTG CCA ATA GTA TTG 1228 Leu Gin Gly Pro Glu Asp Leu Ala Arg Asp Leu Val Pro He Val Leu 350 355 360
GGG GGG ATC GGC TTA GCC ATA GGA TTC ACC AGA GAC AAG GTA AGT AAG 1276 Gly Gly He Gly Leu Ala He Gly Phe Thr Arg Asp Lys Val Ser Lys 365 370 375
ATG ATG AAG AAT GCT GTT GAT GGA CTT CGT GCG GCA ACC CAG CTC GGT 1324 Met Met Lys Asn Ala Val Asp Gly Leu Arg Ala Ala Thr Gin Leu Gly 380 385 390
CAA TAT GGC CTA GAA ATA TTC TCA TTA CTA AAG AAG TAC TTC TTC GGT 1372 Gin Tyr Gly Leu Glu He Phe Ser Leu Leu Lys Lys Tyr Phe Phe Gly 395 400 405
GGT GAT CAA ACA GAG AAA ACC CTA AAA GAT ATT GAG TCA GCA GTT ATA 1420 Gly Asp Gin Thr Glu Lys Thr Leu Lys Asp He Glu Ser Ala Val He 410 415 420 425
GAT ATG GAA GTA CTA TCA TCT ACA TCA GTG ACT CAG CTC GTG AGG GAC 1468 Asp Met Glu Val Leu Ser Ser Thr Ser Val Thr Gin Leu Val Arg Asp
430 435 440
AAA CAG TCT GCA CGG GCT TAT ATG GCC ATC TTA GAT AAT GAA GAA GAA 1516 Lys Gin Ser Ala Arg Ala Tyr Met Ala He Leu Asp Asn Glu Glu Glu 445 450 455
AAG GCA AGG AAA TTA TCT GTC AGG AAT GCC GAC CCA CAC GTA GTA TCC 1564 Lys Ala Arg Lys Leu Ser Val Arg Asn Ala Asp Pro His Val Val Ser 460 465 470
TCT ACC AAT GCT CTC ATA TCC CGG ATC TCA ATG GCT AGG GCT GCA TTG 1612 Ser Thr Asn Ala Leu He Ser Arg He Ser Met Ala Arg Ala Ala Leu 475 480 485 Table 3, continued
GCC AAG GCT CAA GCT GAA ATG ACC AGC AGG ATG CGT CCT GTG GTC ATT 1660 Ala Lys Ala Gin Ala Glu Met Thr Ser Arg Met Arg Pro Val Val He 490 495 500 505
ATG ATG TGT GGG CCC CCT GGT ATA GGT AAA ACC AAG GCA GCA GAA CAT 1708 Met Met Cys Gly Pro Pro Gly He Gly Lys Thr Lys Ala Ala Glu His 510 515 520
CTG GCT AAA CGC CTA GCC AAT GAG ATA CGG CCT GGT GGT AAG GTT GGG 1756 Leu Ala Lys Arg Leu Ala Asn Glu He Arg Pro Gly Gly Lys Val Gly 525 530 535
CTG GTC CCA CGG GAG GCA GTG GAT CAT TGG GAT GGA TAT CAC GGA GAG 1804 Leu Val Pro Arg Glu Ala Val Asp His Trp Asp Gly Tyr His Gly Glu 540 545 550
GAA GTG ATG CTG TGG GAC GAC TAT GGA ATG ACA AAG ATA CAG GAA GAC 1852 Glu Val Met Leu Trp Asp Asp Tyr Gly Met Thr Lys He Gin Glu Asp 555 560 565
TGT AAT AAA CTG CAA GCC ATA GCC GAC TCA GCC CCC CTA ACA CTC AAT 1900 Cys Asn Lys Leu Gin Ala He Ala Asp Ser Ala Pro Leu Thr Leu Asn 570 575 580 585
TGT GAC CGA ATA GAA AAC AAG GGA ATG CAA TTT GTG TCT GAT GCT ATA 1948 Cys Asp Arg He Glu Asn Lys Gly Met Gin Phe Val Ser Asp Ala He 590 595 600
GTC ATC ACC ACC AAT GCT CCT GGC CCA GCC CCA GTG GAC TTT GTC AAC 1996 Val He Thr Thr Asn Ala Pro Gly Pro Ala Pro Val Asp Phe Val Asn 605 610 615
CTC GGG CCT GTT TGC CGA AGG GTG GAC TTC CTT GTG TAT TGC ACG GCA 2044 Leu Gly Pro Val Cys Arg Arg Val Asp Phe Leu Val Tyr Cys Thr Ala 620 625 630
CCT GAA GTT GAA CAC ACG AGG AAA GTC AGT CCT GGG GAC ACA ACT GCA 2092 Pro Glu Val Glu His Thr Arg Lys Val Ser Pro Gly Asp Thr Thr Ala 635 640 645
CTG AAA GAC TGC TTC AAG CCC GAT TTC TCA CAT CTA AAA ATG GAG TTG 2140 Leu Lys Asp Cys Phe Lys Pro Asp Phe Ser His Leu Lys Met Glu Leu 650 655 660 665
GCT CCC CAA GGG GGC TTT GAT AAC CAA GGG AAT ACC CCG TTT GGT AAG 2188 Ala Pro Gin Gly Gly Phe Asp Asn Gin Gly Asn Thr Pro Phe Gly Lys 670 675 680
GGT GTG ATG AAG CCC ACC ACC ATA AAC AGG CTG TTA ATC CAG GCT GTA 2236 Gly Val Met Lys Pro Thr Thr He Asn Arg Leu Leu He Gin Ala Val 685 690 695
GCC TTG ACG ATG GAG AGA CAG GAT GAG TTC CAA CTC CAG GGG CCT ACG 2284 Ala Leu Thr Met Glu Arg Gin Asp Glu Phe Gin Leu Gin Gly Pro Thr 700 705 710
TAT GAC TTT GAT ACT GAC AGA GTA GCT GCG TTC ACG AGG ATG GCC CGA 2332 Tyr Asp Phe Asp Thr Asp Arg Val Ala Ala Phe Thr Arg Met Ala Arg 715 720 725
GCC AAC GGG TTG GGT CTC ATA TCC ATG GCC TCC CTA GGC AAA AAG CTA 2380 Ala Asn Gly Leu Gly Leu He Ser Met Ala Ser Leu Gly Lys Lys Leu 730 735 7.40 745 Table 3, continued
CGC AGT GTC ACC ACT ATT GAA GGA TTA AAG AAT GCT CTA TCA GGC TAT 2428 Arg Ser Val Thr Thr He Glu Gly Leu Lys Asn Ala Leu Ser Gly Tyr 750 755 760
AAA ATA TCA AAA TGC AGT ATA CAA TGG CAG TCA AGG GTG TAC ATT ATA 2476 Lys He Ser Lys Cys Ser He Gin Trp Gin Ser Arg Val Tyr He He 765 770 775
GAA TCA GAT GGT GCC AGT GTA CAA ATC AAA GAA GAC AAG CAA GCT TTG 2524 Glu Ser Asp Gly Ala Ser Val Gin He Lys Glu Asp Lys Gin Ala Leu 780 785 790
ACC CCT CTG CAG CAG ACA ATT AAC ACG GCC TCA CTT GCC ATC ACT CGA 2572 Thr Pro Leu Gin Gin Thr He Asn Thr Ala Ser Leu Ala He Thr Arg 795 800 805
CTC AAA GCA GCT AGG GCT GTG GCA TAC GCT TCA TGT TTC CAG TCC GCC 2620 Leu Lys Ala Ala Arg Ala Val Ala Tyr Ala Ser Cys Phe Gin Ser Ala 810 815 820 825
ATA ACT ACC ATA CTA CAA ATG GCG GGA TCT GCG CTC GTT ATT AAT CGA 2668 He Thr Thr He Leu Gin Met Ala Gly Ser Ala Leu Val He Asn Arg 830 835 840
GCG GTC AAG CGT ATG TTT GGT ACC CGT ACA GCA GCC ATG GCA TTA GAA 2716 Ala Val Lys Arg Met Phe Gly Thr Arg Thr Ala Ala Met Ala Leu Glu 845 850 855
GGA CCT GGG AAA GAA CAT AAT TGC AGG GTC CAT AAG GCT AAG GAA GCT 2764 Gly Pro Gly Lys Glu His Asn Cys Arg Val His Lys Ala Lys Glu Ala 860 865 870
GGA AAG GGG CCC ATA GGT CAT GAT GAC ATG GTA GAA AGG TTT GGC CTA 2812 Gly Lys Gly Pro He Gly His Asp Asp Met Val Glu Arg Phe Gly Leu 875 880 885
TGT GAA ACT GAA GAG GAG GAG AGT GAG GAC CAA ATT CAA ATG GTA CCA 2860 Cys Glu Thr Glu Glu Glu Glu Ser Glu Asp Gin He Gin Met Val Pro 890 895 900 905
AGT GAT GCC GTC CCA GAA GGA AAG AAC AAA GGC AAG ACC AAA AAG GGA 2908 Ser Asp Ala Val Pro Glu Gly Lys Asn Lys Gly Lys Thr Lys Lys Gly 910 915 920
CGT GGT CGC AAA AAT AAC TAT AAT GCA TTC TCT CGC CGT GGT CTG AGT 2956 Arg Gly Arg Lys Asn Asn Tyr Asn Ala Phe Ser Arg Arg Gly Leu Ser 925 930 935
GAT GAA GAA TAT GAA GAG TAC AAA AAG ATC AGA GAA GAA AAG AAT GGC 3004 Asp Glu Glu Tyr Glu Glu Tyr Lys Lys He Arg Glu Glu Lys Asn Gly 940 945 950
AAT TAT AGT ATA CAA GAA TAC TTG GAG GAC CGC CAA CGA TAT GAG GAA 3052 Asn Tyr Ser He Gin Glu Tyr Leu Glu Asp Arg Gin Arg Tyr Glu Glu 955 960 965
GAA TTA GCA GAG GTA CAG GCA GGT GGT GAT GGT GGC ATA GGA GAA ACT 3100 Glu Leu Ala Glu Val Gin Ala Gly Gly Asp Gly Gly He Gly Glu Thr 970 975 980 985
GAA ATG GAA ATC CGT CAC AGG GTC TTC TAT AAA TCC AAG AGT AAG AAA 3148 Glu Met Glu He Arg His Arg Val Phe Tyr Lys Ser Lys Ser Lys Lys 990 995 1000 Table 3, continued
CAC CAA CAA GAG CAA CGG CGA CAA CTT GGT CTA GTG ACT GGA TCA GAC 3196 His Gin Gin Glu Gin Arg Arg Gin Leu Gly Leu Val Thr Gly Ser Asp 1005 1010 1015
ATC AGA AAA CGT AAG CCC ATT GAC TGG ACC CCG CCA AAG AAT GAA TGG 3244 He Arg Lys Arg Lys Pro He Asp Trp Thr Pro Pro Lys Asn Glu Trp 1020 1025 1030
GCA GAT GAT GAC AGA GAG GTG GAT TAT AAT GAA AAG ATC AAT TTT GAA 3292 Ala Asp Asp Asp Arg Glu Val Asp Tyr Asn Glu Lys He Asn Phe Glu 1035 1040 1045
GCT CCC CCG ACA CTA TGG AGC CGA GTC ACA AAG TTT GGA TCA GGA TGG 3340 Ala Pro Pro Thr Leu Trp Ser Arg Val Thr Lys Phe Gly Ser Gly Trp 1050 1055 1060 1065
GGC TTT TGG GTC AGC CCG ACA GTG TTC ATC ACA ACC ACA CAT GTA GTG 3388 Gly Phe Trp Val Ser Pro Thr Val Phe He Thr Thr Thr His Val Val 1070 1075 1080
CCA ACT GGT GTG AAA GAA TTC TTT GGT GAG CCC CTA TCT AGT ATA GCA 3436 Pro Thr Gly Val Lys Glu Phe Phe Gly Glu Pro Leu Ser Ser He Ala 1085 1090 1095
ATC CAC CAA GCA GGT GAG TTC ACA CAA TTC AGG TTC TCA AAG AAA ATG 3484 He His Gin Ala Gly Glu Phe Thr Gin Phe Arg Phe Ser Lys Lys Met 1100 1105 1110
CGC CCT GAC TTG ACA GGT ATG GTC CTT GAA GAA GGT TGC CCT GAA GGG 3532 Arg Pro Asp Leu Thr Gly Met Val Leu Glu Glu Gly Cys Pro Glu Gly 1115 1120 1125
ACA GTC TGC TCA GTC CTA ATT AAA CGG GAT TCG GGT GAA CTA CTT CCG 3580 Thr Val Cys Ser Val Leu He Lys Arg Asp Ser Gly Glu Leu Leu Pro 1130 1135 1140 1145
CTA GCC GTC CGT ATG GGG GCT ATT GCC TCC ATG AGG ATA CAG GGT CGG 3628 Leu Ala Val Arg Met Gly Ala He Ala Ser Met Arg He Gin Gly Arg 1150 1155 1160
CTT GTC CAT GGC CAA TCA GGG ATG TTA CTG ACA GGG GCC AAT GCA AAG 3676 Leu Val His Gly Gin Ser Gly Met Leu Leu Thr Gly Ala Asn Ala Lys 1165 1170 1175
GGG ATG GAT CTT GGC ACT ATA CCA GGA GAC TGC GGG GCA CCA TAC GTC 3724 Gly Met Asp Leu Gly Thr He Pro Gly Asp Cys Gly Ala Pro Tyr Val 1180 1185 1190
CAC AAG CGC GGG AAT GAC TGG GTT GTG TGT GGA GTC CAC GCT GCA GCC 3772 His Lys Arg Gly Asn Asp Trp Val Val Cys Gly Val His Ala Ala Ala 1195 1200 1205
ACA AAG TCA GGC AAC ACC GTG GTC TGC GCT GTA CAG GCT GGA GAG GGC 3820 Thr Lys Ser Gly Asn Thr Val Val Cys Ala Val Gin Ala Gly Glu Gly 1210 1215 1220 1225
GAA ACC GCA CTA GAA GGT GGA GAC AAG GGG CAT TAT GCC GGC CAC GAG 3868 Glu Thr Ala Leu Glu Gly Gly Asp Lys Gly His Tyr Ala Gly His Glu 1230 1235 1240
ATT GTG AGG TAT GGA AGT GGC CCA GCA CTG TCA ACT AAA ACA AAA TTC 3916 He Val Arg Tyr Gly Ser Gly Pro Ala Leu Ser Thr Lys Thr Lys Phe 1245 1250 1255 Table 3, continued
TGG AGG TCC TCC CCA GAA CCA CTG CCC CCC GGA GTA TAT GAG CCA GCA 3964
Trp Arg Ser Ser Pro Glu Pro Leu Pro Pro Gly Val Tyr Glu Pro Ala 1260 1265 1270
TAC CTG GGG GGC AAG GAC CCC CGT GTA CAG AAT GGC CCA TCC CTA CAA 4012 Tyr Leu Gly Gly Lys Asp Pro Arg Val Gin Asn Gly Pro Ser Leu Gin 1275 1280 1285
CAG GTA CTA CGT GAC CAA CTG AAA CCC TTT GCG GAC CCC CGC GGC CGC 4060 Gin Val Leu Arg Asp Gin Leu Lys Pro Phe Ala Asp Pro Arg Gly Arg 1290 1295 1300 1305
ATG CCT GAG CCT GGC CTA CTG GAG GCT GCG GTT GAG ACT GTA ACA TCC 4108 Met Pro Glu Pro Gly Leu Leu Glu Ala Ala Val Glu Thr Val Thr Ser 1310 1315 1320
ATG TTA GAA CAG ACA ATG GAT ACC CCA AGC CCG TGG TCT TAC GCT GAT 4156 Met Leu Glu Gin Thr Met Asp Thr Pro Ser Pro Trp Ser Tyr Ala Asp 1325 1330 1335
GCC TGC CAA TCT CTT GAC AAA ACT ACT AGT TCG GGG TAC CCT CAC CAT 4204 Ala Cys Gin Ser Leu Asp Lys Thr Thr Ser Ser Gly Tyr Pro His His 1340 1345 1350
AAA AGG AAG AAT GAT GAT TGG AAT GGC ACC ACC TTC GTT GGA GAG CTC 4252 Lys Arg Lys Asn Asp Asp Trp Asn Gly Thr Thr Phe Val Gly Glu Leu 1355 1360 1365
GGT GAG CAA GCT GCA CAC GCC AAC AAT ATG TAT GAG AAT GCT AAA CAT 4300 Gly Glu Gin Ala Ala His Ala Asn Asn Met Tyr Glu Asn Ala Lys His 1370 1375 1380 1385
ATG AAA CCC ATT TAC ACT GCA GCC TTA AAA GAT GAA CTA GTC AAG CCA 4348 Met Lys Pro He Tyr Thr Ala Ala Leu Lys Asp Glu Leu Val Lys Pro 1390 1395 1400
GAA AAG ATT TAT CAA AAA GTC AAG AAG CGT CTA CTA TGG GGC GCC GAT 4396 Glu Lys He Tyr Gin Lys Val Lys Lys Arg Leu Leu Trp Gly Ala Asp 1405 1410 1415
CTC GGA ACA GTG GTC AGG GCC GCC CGG GCT TTT GGC CCA TTT TGT GAC 4444 Leu Gly Thr Val Val Arg Ala Ala Arg Ala Phe Gly Pro Phe Cys Asp 1420 1425 1430
GCT ATA AAA TCA CAT GTC ATC AAA TTG CCA ATA AAA GTT GGC ATG AAC 4492 Ala He Lys Ser His Val He Lys Leu Pro He Lys Val Gly Met Asn 1435 1440 1445
ACA ATA GAA GAT GGC CCC CTC ATC TAT GCT GAG CAT GCT AAA TAT AAG 4540 Thr He Glu Asp Gly Pro Leu He Tyr Ala Glu His Ala Lys Tyr Lys 1450 1455 1460 1465
AAT CAT TTT GAT GCA GAT TAT ACA GCA TGG GAC TCA ACA CAA AAT AGA 4588 Asn His Phe Asp Ala Asp Tyr Thr Ala Trp Asp Ser Thr Gin Asn Arg 1470 1475 1480
CAA ATT ATG ACA GAA TCC TTC TCC ATT ATG TCG CGC CTT ACG GCC TCA 4636 Gin He Met Thr Glu Ser Phe Ser He Met Ser Arg Leu Thr Ala Ser 1485 1490 1495
CCA GAA TTG GCC GAG GTT GTG GCC CAA GAT TTG CTA GCA CCA TCT GAG 4684 Pro Glu Leu Ala Glu Val Val Ala Gin Asp Leu Leu Ala Pro Ser Glu 1500 1505 1510 Table 3, continued
ATG GAT GTA GGT GAT TAT GTC ATC AGG GTC AAA GAG GGG CTG CCA TCT 4732 Met Asp Val Gly Asp Tyr Val He Arg Val Lys Glu Gly Leu Pro Ser 1515 1520 1525
GGA TTC CCA TGT ACT TCC CAG GTG AAC AGC ATA AAT CAC TGG ATA ATT 4780 Gly Phe Pro Cys Thr Ser Gin Val Asn Ser He Asn His Trp He He 1530 1535 1540 1545
ACT CTC TGT GCA CTG TCT GAG GCC ACT GGT TTA TCA CCT GAT GTG GTG 4828 Thr Leu Cys Ala Leu Ser Glu Ala Thr Gly Leu Ser Pro Asp Val Val 1550 1555 1560
CAA TCC ATG TCA TAT TTC TCA TTT TAT GGT GAT GAT GAG ATT GTG TCA 4876 Gin Ser Met Ser Tyr Phe Ser Phe Tyr Gly Asp Asp Glu He Val Ser 1565 1570 1575
ACT GAC ATA GAT TTT GAC CCA GCC CGC CTC ACT CAA ATT CTC AAG GAA 4924 Thr Asp He Asp Phe Asp Pro Ala Arg Leu Thr Gin He Leu Lys Glu 1580 1585 1590
TAT GGC CTC AAA CCA ACA AGG CCT GAC AAA ACA GAA GGA CCA ATA CAA 4972 Tyr Gly Leu Lys Pro Thr Arg Pro Asp Lys Thr Glu Gly Pro He Gin 1595 1600 1605
GTG AGG AAA AAT GTG GAT GGA CTG GTC TTC TTG CGG CGC ACC ATT TCC 5020 Val Arg Lys Asn Val Asp Gly Leu Val Phe Leu Arg Arg Thr He Ser 1610 1615 1620 1625
CGT GAT GCG GCA GGG TTC CAA GGC AGG TTA GAT AGG GCT TCG ATT GAA 5068 Arg Asp Ala Ala Gly Phe Gin Gly Arg Leu Asp Arg Ala Ser He Glu 1630 1635 1640
CGC CAA ATC TTC TGG ACC CGC GGG CCC AAT CAT TCA GAT CCA TCA GAG 5116 Arg Gin He Phe Trp Thr Arg Gly Pro Asn His Ser Asp Pro Ser Glu 1645 1650 1655
ACT CTA GTG CCA CAC ACT CAA AGA AAA ATA CAG TTG ATT TCA CTT CTA 5164 Thr Leu Val Pro His Thr Gin Arg Lys He Gin Leu He Ser Leu Leu 1660 1665 1670
GGG GAA GCT TCA CTC CAT GGT GAG AAA TTT TAC AGA AAG ATT TCC AGC 5212 Gly Glu Ala Ser Leu His Gly Glu Lys Phe Tyr Arg Lys He Ser Ser 1675 1680 1685
AAG GTC ATA CAT GAA ATC AAG ACT GGT GGA TTG GAA ATG TAT GTC CCA 5260 Lys Val He His Glu He Lys Thr Gly Gly Leu Glu Met Tyr Val Pro 1690 1695 1700 1705
GGA TGG CAG GCC ATG TTC CGC TGG ATG CGC TTC CAT GAC CTC GGA TTG 5308 Gly Trp Gin Ala Met Phe Arg Trp Met Arg Phe His Asp Leu Gly Leu 1710 1715 1720
TGG ACA GGA GAT CGC GAT CTT CTG CCC GAA TTC GTA AAT GAT GAT GGC 5356 Trp Thr Gly Asp Arg Asp Leu Leu Pro Glu Phe Val Asn Asp Asp Gly 1725 1730 1735
GTC TAAGGACGCT ACATCAAGCG TGGATGGCGC TAGTGGCGCT GGTCAGTTGG 5409
Val Table 4. The amino acid sequence deduced from nucleotides 5346 through 6935 of the Norwalk virus genome shown in Table 2.
CGTAA ATG ATG ATG GCG TCT AAG GAC GCT ACA TCA AGC GTG GAT GGC 5387 Met Met Met Ala Ser Lys Asp Ala Thr Ser Ser Val Asp Gly
1 5 10
GCT AGT GGC GCT GGT CAG TTG GTA CCG GAG GTT AAT GCT TCT GAC CCT 5435 Ala Ser Gly Ala Gly Gin Leu Val Pro Glu Val Asn Ala Ser Asp Pro 15 20 25 30
CTT GCA ATG GAT CCT GTA GCA GGT TCT TCG ACA GCA GTC GCG ACT GCT 5483 Leu Ala Met Asp Pro Val Ala Gly Ser Ser Thr Ala Val Ala Thr Ala
35 40 45
GGA CAA GTT AAT CCT ATT GAT CCC TGG ATA ATT AAT AAT TTT GTG CAA 5531 Gly Gin Val Asn Pro He Asp Pro Trp He He Asn Asn Phe Val Gin 50 55 60
GCC CCC CAA GGT GAA TTT ACT ATT TCC CCA AAT AAT ACC CCC GGT GAT 5579 Ala Pro Gin Gly Glu Phe Thr He Ser Pro Asn Asn Thr Pro Gly Asp 65 70 75
GTT TTG TTT GAT TTG AGT TTG GGT CCC CAT CTT AAT CCT TTC TTG CTC 5627 Val Leu Phe Asp Leu Ser Leu Gly Pro His Leu Asn Pro Phe Leu Leu 80 85 90
CAT CTA TCA CAA ATG TAT AAT GGT TGG GTT GGT AAC ATG AGA GTC AGG 5675 His Leu Ser Gin Met Tyr Asn Gly Trp Val Gly Asn Met Arg Val Arg 95 100 105 110
ATT ATG CTA GCT GGT AAT GCC TTT ACT GCG GGG AAG ATA ATA GTT TCC 5723 He Met Leu Ala Gly Asn Ala Phe Thr Ala Gly Lys He He Val Ser
115 120 125
TGC ATA CCC CCT GGT TTT GGT TCA CAT AAT CTT ACT ATA GCA CAA GCA 5771 Cys He Pro Pro Gly Phe Gly Ser His Asn Leu Thr He Ala Gin Ala 130 135 140
ACT CTC TTT CCA CAT GTG ATT GCT GAT GTT AGG ACT CTA GAC CCC ATT 5819 Thr Leu Phe Pro His Val He Ala Asp Val Arg Thr Leu Asp Pro He 145 150 155
GAG GTG CCT TTG GAA GAT GTT AGG AAT GTT CTC TTT CAT AAT AAT GAT 5867 Glu Val Pro Leu Glu Asp Val Arg Asn Val Leu Phe His Asn Asn Asp 160 165 170
AGA AAT CAA CAA ACC ATG CGC CTT GTG TGC ATG CTG TAC ACC CCC CTC 5915 Arg Asn Gin Gin Thr Met Arg Leu Val Cys Met Leu Tyr Thr Pro Leu 175 180 185 190
CGC ACT GGT GGT GGT ACT GGT GAT TCT TTT GTA GTT GCA GGG CGA GTT 5963 Arg Thr Gly Gly Gly Thr Gly Asp Ser Phe Val Val Ala Gly Arg Val 195 200 205
ATG ACT TGC CCC AGT CCT GAT TTT AAT TTC TTG TTT TTA GTC CCT CCT 6011 Met Thr Cys Pro Ser Pro Asp Phe Asn Phe Leu Phe Leu Val Pro Pro 210 215 220
ACG GTG GAG CAG AAA ACC AGG CCC TTC ACA CTC CCA AAT CTG CCA TTG 6059 Thr Val Glu Gin Lys Thr Arg Pro Phe Thr Leu Pro Asn Leu Pro Leu 225 230 235 Table 4, continued
AGT TCT CTG TCT AAC TCA CGT GCC CCT CTC CCA ATC AGT AGT ATG GGC 6107 Ser Ser Leu Ser Asn Ser Arg Ala Pro Leu Pro He Ser Ser Met Gly 240 245 250
ATT TCC CCA GAC AAT GTC CAG AGT GTG CAG TTC CAA AAT GGT CGG TGT 6155 He Ser Pro Asp Asn Val Gin Ser Val Gin Phe Gin Asn Gly Arg Cys 255 260 265 270
ACT CTG GAT GGC CGC CTG GTT GGC ACC ACC CCA GTT TCA TTG TCA CAT 6203 Thr Leu Asp Gly Arg Leu Val Gly Thr Thr Pro Val Ser Leu Ser His 275 280 285
GTT GCC AAG ATA AGA GGG ACC TCC AAT GGC ACT GTA ATC AAC CTT ACT 6251 Val Ala Lys He Arg Gly Thr Ser Asn Gly Thr Val He Asn Leu Thr 290 295 300
GAA TTG GAT GGC ACA CCC TTT CAC CCT TTT GAG GGC CCT GCC CCC ATT 6299 Glu Leu Asp Gly Thr Pro Phe His Pro Phe Glu Gly Pro Ala Pro He 305 310 315
GGG TTT CCA GAC CTC GGT GGT TGT GAT TGG CAT ATC AAT ATG ACA CAG 6347 Gly Phe Pro Asp Leu Gly Gly Cys Asp Trp His He Asn Met Thr Gin 320 325 330
TTT GGC CAT TCT AGC CAG ACC CAG TAT GAT GTA GAC ACC ACC CCT GAC 6395 Phe Gly His Ser Ser Gin Thr Gin Tyr Asp Val Asp Thr Thr Pro Asp 335 340 345 350
ACT TTT GTC CCC CAT CTT GGT TCA ATT CAG GCA AAT GGC ATT GGC AGT 6443 Thr Phe Val Pro His Leu Gly Ser He Gin Ala Asn Gly He Gly Ser 355 360 365
GGT AAT TAT GTT GGT GTT CTT AGC TGG ATT TCC CCC CCA TCA CAC CCG 6491 Gly Asn Tyr Val Gly Val Leu Ser Trp He Ser Pro Pro Ser His Pro 370 375 380
TCT GGC TCC CAA GTT GAC CTT TGG AAG ATC CCC AAT TAT GGG TCA AGT 6539 Ser Gly Ser Gin Val Asp Leu Trp Lys He Pro Asn Tyr Gly Ser Ser 385 390 395
ATT ACG GAG GCA ACA CAT CTA GCC CCT TCT GTA TAC CCC CCT GGT TTC 6587 He Thr Glu Ala Thr His Leu Ala Pro Ser Val Tyr Pro Pro Gly Phe 400 405 410
GGA GAG GTA TTG GTC TTT TTC ATG TCA AAA ATG CCA GGT CCT GGT GCT 6635 Gly Glu Val Leu Val Phe Phe Met Ser Lys Met Pro Gly Pro Gly Ala 415 420 425 430
TAT AAT TTG CCC TGT CTA TTA CCA CAA GAG TAC ATT TCA CAT CTT GCT 6683 Tyr Asn Leu Pro Cys Leu Leu Pro Gin Glu Tyr He Ser His Leu Ala 435 440 445
AGT GAA CAA GCC CCT ACT GTA GGT GAG GCT GCC CTG CTC CAC TAT GTT 6731 Ser Glu Gin Ala Pro Thr Val Gly Glu Ala Ala Leu Leu His Tyr Val 450 455 460
GAC CCT GAT ACC GGT CGG AAT CTT GGG GAA TTC AAA GCA TAC CCT GAT 6779 Asp Pro Asp Thr Gly Arg Asn Leu Gly Glu Phe Lys Ala Tyr Pro Asp 465 470 475
GGT TTC CTC ACT TGT GTC CCC AAT GGG GCT AGC TCG GGT CCA CAA CAG 6827 Gly Phe Leu Thr Cys Val Pro Asn Gly Ala Ser Ser Gly Pro Gin Gin 480 485 490 Table 4, continued
CTG CCG ATC AAT GGG GTC TTT GTC TTT GTT TCA TGG GTG TCC AGA TTT 6875 Leu Pro He Asn Gly Val Phe Val Phe Val Ser Trp Val Ser Arg Phe 495 500 505 510
TAT CAA TTA AAG CCT GTG GGA ACT GCC AGC TCG GCA AGA GGT AGG CTT 6923 Tyr Gin Leu Lys Pro Val Gly Thr Ala Ser Ser Ala Arg Gly Arg Leu 515 520 525
GGT CTG CGC CGA TAATGGCCCA AGCCATAATT GGTGCAATTG CTGCTTCCAC 6975
Gly Leu Arg Arg 530
Table 5. The amino acid sequence deduced from nucleotides 6938 through 7573 of the Norwalk virus genome shown in Table 2.
CCAGCTCGGC AAGAGGTAGG CTTGGTCTGC GCCGATA ATG GCC CAA GCC ATA ATT 6955
Met Ala Gin Ala He He 1 5
GGT GCA ATT GCT GCT TCC ACA GCA GGT AGT GCT CTG GGA GCG GGC ATA 7003 Gly Ala He Ala Ala Ser Thr Ala Gly Ser Ala Leu Gly Ala Gly He 10 15 20
CAG GTT GGT GGC GAA GCG GCC CTC CAA AGC CAA AGG TAT CAA CAA AAT 7051 Gin Val Gly Gly Glu Ala Ala Leu Gin Ser Gin Arg Tyr Gin Gin Asn 25 30 35
TTG CAA CTG CAA GAA AAT TCT TTT AAA CAT GAC AGG GAA ATG ATT GGG 7099 Leu Gin Leu Gin Glu Asn Ser Phe Lys His Asp Arg Glu Met He Gly 40 45 50
TAT CAG GTT GAA GCT TCA AAT CAA TTA TTG GCT AAA AAT TTG GCA ACT 7147
Tyr Gin Val Glu Ala Ser Asn Gin Leu Leu Ala Lys Asn Leu Ala Thr
55 60 65 70
AGA TAT TCA CTC CTC CGT GCT GGG GGT TTG ACC AGT GCT GAT GCA GCA 7195 Arg Tyr Ser Leu Leu Arg Ala Gly Gly Leu Thr Ser Ala Asp Ala Ala 75 80 85
AGA TCT GTG GCA GGA GCT CCA GTC ACC CGC ATT GTA GAT TGG AAT GGC 7243 Arg Ser Val Ala Gly Ala Pro Val Thr Arg He Val Asp Trp Asn Gly 90 95 100
GTG AGA GTG TCT GCT CCC GAG TCC TCT GCT ACC ACA TTG AGA TCC GGT 7291 Val Arg Val Ser Ala Pro Glu Ser Ser Ala Thr Thr Leu Arg Ser Gly 105 110 115
GGC TTC ATG TCA GTT CCC ATA CCA TTT GCC TCT AAG CAA AAA CAG GTT 7339 Gly Phe Met Ser Val Pro He Pro Phe Ala Ser Lys Gin Lys Gin Val 120 125 130
CAA TCA TCT GGT ATT AGT AAT CCA AAT TAT TCC CCT TCA TCC ATT TCT 7387 Gin Ser Ser Gly He Ser Asn Pro Asn Tyr Ser Pro Ser Ser He Ser 135 140 145 150
CGA ACC ACT AGT TGG GTC GAG TCA CAA AAC TCA TCG AGA TTT GGA AAT 7435 Arg Thr Thr Ser Trp Val Glu Ser Gin Asn Ser Ser Arg Phe Gly Asn 155 160 165
CTT TCT CCA TAC CAC GCG GAG GCT CTC AAT ACA GTG TGG TTG ACT CCA 7483 Leu Ser Pro Tyr His Ala Glu Ala Leu Asn Thr Val Trp Leu Thr Pro 170 175 180
CCC GGT TCA ACA GCC TCT TCT ACA CTG TCT TCT GTG CCA CGT GGT TAT 7531 Pro Gly Ser Thr Ala Ser Ser Thr Leu Ser Ser Val Pro Arg Gly Tyr 185 190 195
TTC AAT ACA GAC AGG TTG CCA TTA TTC GCA AAT AAT AGG CGA 7573 Phe Asn Thr Asp Arg Leu Pro Leu Phe Ala Asn Asn Arg Arg 200 205 210 imers used for detection of Norwalk-related us by PCR
CTT GTT GGT TTG AGG CCA TAT 4924 ATA AAA GTT GGC ATG AAC A 4493 GTT GAC ACA ATC TCA TCA TC 4859 GGC CTG CCA TCT GGA TTG CC 4740 GGG CCC CCT GGT ATA GGT AA 1689
TGG TGA TGA CTA TAG CAT CAG ACA CAA A 1958 ACT CAC CCA AAT CCT CCA 4920 GTT CTG ACC ACC TAA CCT 5247 AGT TTG GGT CCC CAT CTT AAT CCT TT 5620 TGA ACC AAA ACC AGG GGG 5747 AGC AAA GTC ATA CAT GAA AT 5229 CCA TTA TAC ATT TGT AG 5650 ATT ATA GTT TCT TGC ATA 5729 CAC ACT CTG GAC ATT GTC TG 6134 CAT TGG GTT TCC AGA CCT A 6313 ATA ATT GGG GAT CTT CCA AA 6530 TAG TGG CAT GGG TAT TTC 6114 TAT GCC AAT CAC AGC CAC 6333 GTC TGG CTC CCA AGT TGA CC 6510 CGG TAT CAG GGT CAA CAT 6744 TGA GGC TGC CCT GCT CCA 6724 CCA CCG CTG TCC GGG AGG 7027 GTT GCT GTT GGC ATT AAC A 4493
Figure imgf000057_0001
♦Based on KY89 sequence # Based HuVc Sapporo sequence Table 7. Detection of Norwalk and Norwalk-Related Viruses
Figure imgf000058_0001
ND Not done. N/A = Not available Internal primers were used to amplify this agent.
# The primate CV 35-36 PCR product sequence is not yet complete. Similarity information is based upon the partial sequence.
Size of 69-39 PCR cDNA product. Primers 69-39 are located inside 36-35 on the Norwalk virus genome sequence. All others are the sizes of the cDNA products made using primers 36-35.
Table 8. Nucleotide Homologies of Different Caliciviruses in the Primer 36 Region.
Virus Strain Nucleotide*
Norwalk Virus A T A A A A G T T G G C A T G A A C A C A A A T
HuCV Sapporo G - T G C T - - - - - - - - T - - - - T G G - C
Feline CV F4 - T T C C T - - - - - - - - A - - - - T G G - C
Feline CV CFI - - - C C G - - - - - - - - T - - - - T G G - C
Feline CV F9 - - - C - G - - - - - T - - C - - - - T G G - C
Rabbit CV - - T G C - - - - - - - G - T G - - - T G - C A
"-" means nucleotide is identical at that site for the strain. A new letter at a site indicates the nucleotide differs at that site. Primer 36 extends across the first 19 nucleotides in the Norwalk sequence above. Primer "new 36" is the first 19 - nucleotides of the HuCV Sapporo sequence,
Table 9. Characterization of Serum from Animals Immunized Parenterally with Recombinant Norwalk Virus Particles3
Figure imgf000060_0001
ELISA Titer of Indicated Serum with Norwalk Virus Particles
Species Immunized Pre-immune Post-immune0
6 mice <100b >10c
4 guinea pigs <100 >106
2 rabbits <100 >10
Two to six animals of each species were immunized with purified recombinant Norwalk virus protein produced using the baculovirus expression system. Serum was collected before or after three immunizations with antigen (80mg for mice, 200mg for guinea pigs and 300mg for rabbits) and tested for reactivity with antigen coated on ELISA plates.
Lowest dilution tested was 1:100.
These sera also were used as capture and detector antibodies to establish an ELISA to detect Norwalk virus antigen.
Table 10. Nucleotide and amino acid sequence of human calicivirus Houston cDNA:
44
GG CCA TGT TAT AGT GGT GTT CAC ATG AAA GAT GGC GAC AAG ATG HuCV Houston
P C Y S G V H M K D G D K M HuCV Houston amino acid
89 TTG ATA GAT GCC AAT CTT CCT TAC AAC CAG AAA TTA ACT ACT ATG HuCV Houston L I D A N L P Y N Q K L T T M HuCV Houston amino acid
134 ATT CAT GAG ACT AGG CAT AGG ATA GGA CAG TAT ATA GAT AAT ACT HuCV Houston I H E T R H R I G Q Y I D N T HuCV Houston amino acid
179 TTT GGA AAG ACA TTT AGA CAT GGA TTG ACA AAA CCT GCT GAC AAG HuCV Houston F G K T F R H G L T K P A D K HuCV Houston amino acid
224 ACT GTA GAT TTG ATC TAT AAG ACA TTG AAT TAT GAT GAT TTT CTG HuCV Houston
T V D L I Y K T L N Y D D F L HuCV Houston amino acid '
269 GCA ATA ATG CTA ATC ATA TAT GGG CAA AAG TCG GCC ACT AAT ACG HuCV Houston A I M L I I Y G Q K S A T N T HuCV Houston amino acid
314 GAG TTG CAA TTC TTG ATG GAG AAA CTT AGA GGT TAT GAA TCT ACA HuCV Houston E L Q F L M E K L R G Y E S T HuCV Houston amino acid
359 ATG GAT GAC ATA GGG AAA GTC TAT GGA GAT GAT AAA ATG AGA GAT HuCV Houston M D D I G K V Y G D D K M R D HuCV Houston amino acid
404 ATA ATC AAG AAT ATT TCT GAT GAT GAC ATA AAG AGT CTT TTA GGG HuCV Houston I I K N I S D D D I K S L L G HuCV Houston amino acid
434
GAG ATA AAT AGT GAT TAT TCT GGT AAG NAT HuCV Houston
E I N S D Y S G K X Houston amino acid
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Matson, David O Esteε, Mary K Jiang, Xi Graham, David Y
(ii) TITLE OF INVENTION: Methods and Reagents to Detect and Characterize Norwalk and Related Viruses
(iii) NUMBER OF SEQUENCES: 75
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Fulbright & Jaworski Patent Dept
(B) STREET: 1301 McKinney, Suite 5100
(C) CITY: Houston
(D) STATE: Texas
(E) COUNTRY: USA
(F) ZIP: 77010-3095
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Launer, Charlene A
(B) REGISTRATION NUMBER: 33,035
(C) REFERENCE/DOCKET NUMBER: D-5526
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 713-651-3634
(B) TELEFAX: 713-651-5246
(C) TELEX: Western Union 762829
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7753 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Norwalk virus
(B) STRAIN: 8FIIa
(C) INDIVIDUAL ISOLATE: 8FIIa
(vii) IMMEDIATE SOURCE:
(B) CLONE: pUCNV-953 and its derivatives
(ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 146..5359
(D) OTHER INFORMATION: /note= "The protein encoded by nucleotides 146 through 5359 is eventually cleaved to make at least a picornavirus 2c-like protein, a 3C-like protease and an RNA-dependent RNA plymerase.
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 5346..6935
(D) OTHER INFORMATION: /note= "Nucleotides 5346 through 5359 are used for coding two different amino acid sequences: the first is the amino acid is coded by nucleotide 146 through 5359, the second by nucleotides 5346 through 6935.
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 6938. .7573
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GGCGTCAAAA GACGTCGTTC CTACTGCTGC TAGCAGTGAA TATTAAGTCT CGTCTATTGG CGAGACTCAA GGGTTCAGGT CTCGATAAAG ATAACCAACC AAGATATGGC TCTGGGGCTG AAAGGCCACA TCCGTCGATG TCCCTAAACA ACAGAGGGAT CGAAGTTCAA CAAAATTTGC GTTGGACTGA GAGACCACAA GGATGAGCTT GACCACACAA CAAAACAACA GATACTTGAT TGCCGGTGGC TTAGGTCCAA GTACACTACC CACTAGTCAT TGATGAAGGC CACCAGGTAA AGTGGTCGGC TAGGGAAGGT GCTCACGACG GTGTCTGGGC CTGAGTGGAA TATGTGCCCG GAGCACGACA CCTGCAACTG AGCCCACAAT TGGTGACATG CATCTATCAT TATGCTATAT ACATAGGTCA AGGCAAGACG AGCASCCTTC TCAAISHSGA- GGATCACCAT^ACAGCCCATA TTATGTCCCA CAACCAAAAC AGAGGCTCAC ATACGACCAA ACCATGGCCG TATGCCGCAG TCACGAACAA CTGCTTCGAA CTTGGAAGAT ACTTGGTTGC AAAGGAAGCT CATCTCCTCT CCAAGATTGG TCCCGAGACA CTCCAGAATT CCAACAAGAC GGATGCAGTG CTAGCCGCTA TAAATGGGTT GGTGTCGCGG TAAGCTCAAA CCCTTGAACG TGCTTAACTT ACTTTCAAAC GGTCGTGGAG ATGGTGGTCC TCCTTTTAGA ACTCTTTGGA TGTTTCCAAC TTTATAGCTT CACTCCTGCC AGATTTCCAT TGCCAGGGAT CTCGTGCCAA TAGTATTGGG GGGGATCGGC AGACAAGGTA AGTAAGATGA TGAAGAATGC TGTTGATGGA CGGTCAATAT GGCCTAGAAA TATTCTCATT ACTAAAGAAG AACAGAGAAA ACCCTAAAAG ATATTGAGTC AGCAGTTATA TACATCAGTG ACTCAGCTCG TGAGGGACAA ACAGTCTGCA AGATAATGAA GAAGAAAAGG CAAGGAAATT ATCTGTCAGG ATCCTCTACC AATGCTCTCA TATCCCGGAT CTCAATGGCT TCAAGCTGAA ATGACCAGCA GGATGCGTCC TGTGGTCATT TATAGGTAAA ACCAAGGCAG CAGAACATCT GGCTAAACGC TGGTGGTAAG GTTGGGCTGG TCCCACGGGA GGCAGTGGAT AGAGGAAGTG ATGCTGTGGG ACGACTATGG AATGACAAAG ACTGCAAGCC ATAGCCGACT CAGCCCCCCT AACACTCAAT GGGAATGCAA TTTGTGTCTG ATGCTATAGT CATCACCACC AGTGGACTTT GTCAACCTCG GGCCTGTTTG CCGAAGGGTG GGCACCTGAA GTTGAACACA CGAGGAAAGT CAGTCCTGGG CTGCTTCAAG CCCGATTTCT CACATCTAAA AATGGAGTTG TAACOKAGGG AATACCCCGT~TTGGTAAGGG~TGTGATGAAG GTTAATCCAG GCTGTAGCCT TGACGATGGA GAGACAGGAT TACGTATGAC TTTGATACTG ACAGAGTAGC TGCGTTCACG GTTGGGTCTC ATATCCATGG CCTCCCTAGG CAAAAAGCTA AGGATTAAAG AATGCTCTAT CAGGCTATAA AATATCAAAA AAGGGTGTAC ATTATAGAAT CAGATGGTGC CAGTGTACAA TTTGACCCCT CTGCAGCAGA CAATTAACAC GGCCTCACTT
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1738 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ala Leu Gly Leu He Gly Gin Val Pro Ala Pro Lys Ala Thr Ser 1 5 10 15
Val Asp Val Pro Lys Gin Gin Arg Asp Arg Pro Pro Arg Thr Val Ala 20 25 30
Glu Val Gin Gin Asn Leu Arg Trp Thr Glu Arg Pro Gin Asp Gin Asn 35 40 45
Val Lys Thr Trp Asp Glu Leu Asp His Thr Thr Lys Gin Gin He Leu 50 55 60
Asp Glu His Ala Glu Trp Phe Asp Ala Gly Gly Leu Gly Pro Ser Thr 65 70 75 80
Leu Pro Thr Ser His Glu Arg Tyr Thr His Glu Asn Asp Glu Gly His 85 90 95
Gin Val Lys Trp Ser Ala Arg Glu Gly Val Asp Leu Gly He Ser Gly 100 105 110
Leu Thr Thr Val Ser Gly Pro Glu Trp Asn Met Cys Pro Leu Pro Pro 115 120 125
Val Asp Gin Arg Ser Thr Thr Pro Ala Thr Glu Pro Thr He Gly Asp 130 135 140
Met He Glu Phe Tyr Glu Gly His He Tyr His Tyr Ala He Tyr He 145 150 155 160 Gly Gin Gly Lys Thr Val Gly Val His Ser Pro Gin Ala Ala Phe Ser 165 170 175
He Thr Arg He Thr He Gin Pro He Ser Ala Trp Trp Arg Val Cys 180 185 190
Tyr Val Pro Gin Pro Lys Gin Arg Leu Thr Tyr Asp Gin Leu Lys Glu 195 200 205
Leu Glu Asn Glu Pro Trp Pro Tyr Ala Ala Val Thr Asn Asn Cys Phe 210 215 220
Glu Phe Cys Cys Gin Val Met Cys Leu Glu Asp Thr Trp Leu Gin Arg 225 230 235 240
Lys Leu He Ser Ser Gly Arg Phe Tyr His Pro Thr Gin Asp Trp Ser 245 250 255
Arg Asp Thr Pro Glu Phe Gin Gin Asp Ser Lys Leu Glu Met Val Arg 260 265 270
Asp Ala Val Leu Ala Ala He Asn Gly Leu Val Ser Arg Pro Phe Lys 275 280 285
Asp Leu Leu Gly Lys Leu Lys Pro Leu Asn Val Leu Asn Leu Leu Ser 290 295 300
Asn Cys Asp Trp Thr Phe Met Gly Val Val Glu Met Val Val Leu Leu 305 310 315 320
Leu Glu Leu Phe Gly He Phe Trp Asn Pro Pro Asp Val Ser Asn Phe 325 330 335
He Ala Ser Leu Leu Pro Asp Phe His Leu Gin Gly Pro Glu Asp Leu 340 345 350
Ala Arg Asp Leu Val Pro He Val Leu Gly Gly He Gly Leu Ala He 355 360 365
Gly Phe Thr Arg Asp Lys Val Ser Lys Met Met Lys Asn Ala Val Asp 370 375 380
Gly Leu Arg Ala Ala Thr Gin Leu Gly Gin Tyr Gly Leu Glu He Phe 385 390 395 400
Ser Leu Leu Lys Lys Tyr Phe Phe Gly Gly Asp Gin Thr Glu Lys Thr
405 410 415
Leu Lys Asp He Glu Ser Ala Val He Asp Met Glu Val Leu Ser Ser 420 425 430
Thr Ser Val Thr Gin Leu Val Arg Asp Lys Gin Ser Ala Arg Ala Tyr
435 440 445
Met Ala He Leu Asp Asn Glu Glu Glu Lys Ala Arg Lys Leu Ser Val 450 455 460
Arg Asn Ala Asp Pro His Val Val Ser Ser Thr Asn Ala Leu He Ser 465 470 475 480
Arg He Ser Met Ala Arg Ala Ala Leu Ala Lys Ala Gin Ala Glu Met 485 490 495
Thr Ser Arg Met Arg Pro Val Val He Met Met Cys Gly Pro Pro Gly 500 505 510 He Gly Lys Thr Lys Ala Ala Glu His Leu Ala Lys Arg Leu Ala Asn 515 520 525
Glu He Arg Pro Gly Gly Lys Val Gly Leu Val Pro Arg Glu Ala Val 530 535 540
Asp His Trp Asp Gly Tyr His Gly Glu Glu Val Met Leu Trp Asp Asp 545 550 555 560
Tyr Gly Met Thr Lys He Gin Glu Asp Cys Asn Lys Leu Gin Ala He 565 570 575
Ala Asp Ser Ala Pro Leu Thr Leu Asn Cys Asp Arg He Glu Asn Lys 580 585 590
Gly Met Gin Phe Val Ser Asp Ala He Val He Thr Thr Asn Ala Pro 595 600 605
Gly Pro Ala Pro Val Asp Phe Val Asn Leu Gly Pro Val Cys Arg Arg 610 615 620
Val Asp Phe Leu Val Tyr Cys Thr Ala Pro Glu Val Glu His Thr Arg 625 630 635 640
Lys Val Ser Pro Gly Asp Thr Thr Ala Leu Lys Asp Cys Phe Lys Pro 645 650 655
Asp Phe Ser His Leu Lys Met Glu Leu Ala Pro Gin Gly Gly Phe Asp 660 665 670
Asn Gin Gly Asn Thr Pro Phe Gly Lys Gly Val Met Lys Pro Thr Thr 675 680 685
He Asn Arg Leu Leu He Gin Ala Val Ala Leu Thr Met Glu Arg Gin 690 695 700
Asp Glu Phe Gin Leu Gin Gly Pro Thr Tyr Asp Phe Asp Thr Asp Arg 705 710 715 720
Val Ala Ala Phe Thr Arg Met Ala Arg Ala Asn Gly Leu Gly Leu He 725 730 735
Ser Met Ala Ser Leu Gly Lys Lys Leu Arg Ser Val Thr Thr He Glu 740 745 750
Gly Leu Lys Asn Ala Leu Ser Gly Tyr Lys He Ser Lys Cys Ser He 755 760 765
Gin Trp Gin Ser Arg Val Tyr He He Glu Ser Asp Gly Ala Ser Val 770 775 780
Gin He Lys Glu Asp Lys Gin Ala Leu Thr Pro Leu Gin Gin Thr He 785 790 795 800
Asn Thr Ala Ser Leu Ala He Thr Arg Leu Lys Ala Ala Arg Ala Val 805 810 815
Ala Tyr Ala Ser Cys Phe Gin Ser Ala He Thr Thr He Leu Gin Met 820 825 830
Ala Gly Ser Ala Leu Val He Asn Arg Ala Val Lys Arg Met Phe Gly 835 840 845
Thr Arg Thr Ala Ala Met Ala Leu Glu Gly Pro Gly Lys Glu His Asn 850 855 860 Cys Arg Val His Lys Ala Lys Glu Ala Gly Lys Gly Pro He Gly His 865 870 875 880
Asp Asp Met Val Glu Arg Phe Gly Leu Cys Glu Thr Glu Glu Glu Glu 885 890 895
Ser Glu Asp Gin He Gin Met Val Pro Ser Asp Ala Val Pro Glu Gly 900 905 910
Lys Asn Lys Gly Lys Thr Lys Lys Gly Arg Gly Arg Lys Asn Asn Tyr 915 920 925
Asn Ala Phe Ser Arg Arg Gly Leu Ser Asp Glu Glu Tyr Glu Glu Tyr 930 935 940
Lys Lys He Arg Glu Glu Lys Asn Gly Asn Tyr Ser He Gin Glu Tyr 945 950 955 960
Leu Glu Asp Arg Gin Arg Tyr Glu Glu Glu Leu Ala Glu Val Gin Ala 965 970 975
Gly Gly Asp Gly Gly He Gly Glu Thr Glu Met Glu He Arg His Arg 980 985 990
Val Phe Tyr Lys Ser Lys Ser Lys Lys His Gin Gin Glu Gin Arg Arg 995 1000 1005
Gin Leu Gly Leu Val Thr Gly Ser Asp He Arg Lys Arg Lys Pro He 1010 1015 1020
Asp Trp Thr Pro Pro Lys Asn Glu Trp Ala Asp Asp Asp Arg Glu Val 1025 1030 1035 1040
Asp Tyr Asn Glu Lys He Asn Phe Glu Ala Pro Pro Thr Leu Trp Ser 1045 1050 1055
Arg Val Thr Lys Phe Gly Ser Gly Trp Gly Phe Trp Val Ser Pro Thr 1060 1065 1070
Val Phe He Thr Thr Thr His Val Val Pro Thr Gly Val Lys Glu Phe 1075 1080 1085
Phe Gly Glu Pro Leu Ser Ser He Ala He His Gin Ala Gly Glu Phe 1090 1095 1100
Thr Gin Phe Arg Phe Ser Lys Lys Met Arg Pro Asp Leu Thr Gly Met 1105 1110 1115 1120
Val Leu Glu Glu Gly Cys Pro Glu Gly Thr Val Cys Ser Val Leu He
1125 1130 1135
Lys Arg Asp Ser Gly Glu Leu Leu Pro Leu Ala Val Arg Met Gly Ala 1140 1145 1150
He Ala Ser Met Arg He Gin Gly Arg Leu Val His Gly Gin Ser Gly 1155 1160 1165
Met Leu Leu Thr Gly Ala Asn Ala Lys Gly Met Asp Leu Gly Thr He 1170 1175 1180
Pro Gly Asp Cys Gly Ala Pro Tyr Val His Lys Arg Gly Asn Asp Trp 1185 1190 1195 1200
Val Val Cys Gly Val His Ala Ala Ala Thr Lys Ser Gly Asn Thr Val 1205 1210 1215 Val Cys Ala Val Gin Ala Gly Glu Gly Glu Thr Ala Leu Glu Gly Gly 1220 1225 1230
Asp Lys Gly His Tyr Ala Gly His Glu He Val Arg Tyr Gly Ser Gly 1235 1240 1245
Pro Ala Leu Ser Thr Lys Thr Lys Phe Trp Arg Ser Ser Pro Glu Pro 1250 1255 1260
Leu Pro Pro Gly Val Tyr Glu Pro Ala Tyr Leu Gly Gly Lys Asp Pro 1265 1270 1275 1280
Arg Val Gin Asn Gly Pro Ser Leu Gin Gin Val Leu Arg Asp Gin Leu 1285 1290 1295
Lys Pro Phe Ala Asp Pro Arg Gly Arg Met Pro Glu Pro Gly Leu Leu 1300 1305 1310
Glu Ala Ala Val Glu Thr Val Thr Ser Met Leu Glu Gin Thr Met Asp 1315 1320 1325
Thr Pro Ser Pro Trp Ser Tyr Ala Asp Ala Cys Gin Ser Leu Asp Lys 1330 1335 1340
Thr Thr Ser Ser Gly Tyr Pro His His Lys Arg Lys Asn Asp Asp Trp 1345 1350 1355 1360
Asn Gly Thr Thr Phe Val Gly Glu Leu Gly Glu Gin Ala Ala His Ala 1365 1370 1375
Asn Asn Met Tyr Glu Asn Ala Lys His Met Lys Pro He Tyr Thr Ala 1380 1385 1390
Ala Leu Lys Asp Glu Leu Val Lys Pro Glu Lys He Tyr Gin Lys Val 1395 1400 1405
Lys Lys Arg Leu Leu Trp Gly Ala Asp Leu Gly Thr Val Val Arg Ala 1410 1415 1420
Ala Arg Ala Phe Gly Pro Phe Cys Asp Ala He Lys Ser His Val He 1425 1430 1435 1440
Lys Leu Pro He Lys Val Gly Met Asn Thr He Glu Asp Gly Pro Leu 1445 1450 1455
He Tyr Ala Glu His Ala Lys Tyr Lys Asn His Phe Asp Ala Asp Tyr 1460 1465 1470
Thr Ala Trp Asp Ser Thr Gin Asn Arg Gin He Met Thr Glu Ser Phe 1475 1480 1485
Ser He Met Ser Arg Leu Thr Ala Ser Pro Glu Leu Ala Glu Val Val 1490 1495 1500
Ala Gin Asp Leu Leu Ala Pro Ser Glu Met Asp Val Gly Asp Tyr Val 1505 1510 1515 1520
He Arg Val Lys Glu Gly Leu Pro Ser Gly Phe Pro Cys Thr Ser Gin 1525 1530 1535
Val Asn Ser He Asn His Trp He He Thr Leu Cys Ala Leu Ser Glu 1540 1545 1550
Ala Thr Gly Leu Ser Pro Asp Val Val Gin Ser Met Ser Tyr Phe Ser 1555 1560 1565 Phe Tyr Gly Asp Asp Glu He Val Ser Thr Asp He Asp Phe Asp Pro 1570 1575 1580
Ala Arg Leu Thr Gin He Leu Lys Glu Tyr Gly Leu Lys Pro Thr Arg 1585 1590 1595 1600
Pro Asp Lys Thr Glu Gly Pro He Gin Val Arg Lys Asn Val Asp Gly 1605 1610 1615
Leu Val Phe Leu Arg Arg Thr He Ser Arg Asp Ala Ala Gly Phe Gin 1620 1625 1630
Gly Arg Leu Asp Arg Ala Ser He Glu Arg Gin He Phe Trp Thr Arg 1635 1640 1645
Gly Pro Asn His Ser Asp Pro Ser Glu Thr Leu Val Pro His Thr Gin 1650 1655 1660
Arg Lys He Gin Leu He Ser Leu Leu Gly Glu Ala Ser Leu His Gly 1665 1670 1675 1680
Glu Lys Phe Tyr Arg Lys He Ser Ser Lys Val He His Glu He Lys 1685 1690 1695
Thr Gly Gly Leu Glu Met Tyr Val Pro Gly Trp Gin Ala Met Phe Arg 1700 1705 1710
Trp Met Arg Phe His Asp Leu Gly Leu Trp Thr Gly Asp Arg Asp Leu 1715 1720 1725
Leu Pro Glu Phe Val Asn Asp Asp Gly Val 1730 1735
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 530 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Met Met Ala Ser Lys Asp Ala Thr Ser Ser Val Asp Gly
1 5 10
Ala Ser Gly Ala Gly Gin Leu Val Pro Glu Val Asn Ala Ser Asp Pro 15 20 25 30
Leu Ala Met Asp Pro Val Ala Gly Ser Ser Thr Ala Val Ala Thr Ala
35 40 45
Gly Gin Val Asn Pro He Asp Pro Trp He He Asn Asn Phe Val Gin 50 55 60
Ala Pro Gin Gly Glu Phe Thr He Ser Pro Asn Asn Thr Pro Gly Asp 65 70 75
Val Leu Phe Asp Leu Ser Leu Gly Pro His Leu Asn Pro Phe Leu Leu 80 85 90
His Leu Ser Gin Met Tyr Asn Gly Trp Val Gly Asn Met Arg Val Arg 95 100 105 110 He Met Leu Ala Gly Asn Ala Phe Thr Ala Gly Lys He He Val Ser
115 120 125
Cys He Pro Pro Gly Phe Gly Ser His Asn Leu Thr He Ala Gin Ala
130 135 140
Thr Leu Phe Pro His Val He Ala Asp Val Arg Thr Leu Asp Pro He 145 150 155
Glu Val Pro Leu Glu Asp Val Arg Asn Val Leu Phe His Asn Asn Asp 160 165 170
Arg Asn Gin Gin Thr Met Arg Lou Val Cys Met Leu Tyr Thr Pro Leu 175 180 185 190
Arg Thr Gly Gly Gly Thr Gly Asp Ser Phe Val Val Ala Gly Arg Val 195 200 205
Met Thr Cys Pro Ser Pro Asp Phe Asn Phe Leu Phe Leu Val Pro Pro 210 215 220
Thr Val Glu Gin Lys Thr Arg Pro Phe Thr Leu Pro Asn Leu Pro Leu 225 230 235
Ser Ser Leu Ser Asn Ser Arg Ala Pro Leu Pro He Ser Ser Met Gly 240 245 250
He Ser Pro Asp Asn Val Gin Ser Val Gin Phe Gin Asn Gly Arg Cys 255 260 265 270
Thr Leu Asp Gly Arg Leu Val Gly Thr Thr Pro Val Ser Leu Ser His 275 280 285
Val Ala Lys He Arg Gly Thr Ser Asn Gly Thr Val He Asn Leu Thr 290 295 300
Glu Leu Asp Gly Thr Pro Phe His Pro Phe Glu Gly Pro Ala Pro He 305 310 315
Gly Phe Pro Asp Leu Gly Gly Cys Asp Trp His He Asn Met Thr Gin 320 325 330
Phe Gly His Ser Ser Gin Thr Gin Tyr Asp Val Asp Thr Thr Pro Asp 335 340 345 350
Thr Phe Val Pro His Leu Gly Ser He Gin Ala Asn Gly He Gly Ser 355 360 365
Gly Asn Tyr Val Gly Val Leu Ser Trp He Ser Pro Pro Ser His Pro 370 375 380
Ser Gly Ser Gin Val Asp Leu Trp Lys He Pro Asn Tyr Gly Ser Ser 385 390 395
He Thr Glu Ala Thr His Leu Ala Pro Ser Val Tyr Pro Pro Gly Phe 400 405 410
Gly Glu Val Leu Val Phe Phe Met Ser Lys Met Pro Gly Pro Gly Ala 415 420 425 430
Tyr Asn Leu Pro Cys Leu Leu Pro Gin Glu Tyr He Ser His Leu Ala 435 440 445
Ser Glu Gin Ala Pro Thr Val Gly Glu Ala Ala Leu Leu His Tyr Val 450 455 460 Asp Pro Asp Thr Gly Arg Asn Leu Gly Glu Phe Lys Ala Tyr Pro Asp 465 470 475
Gly Phe Leu Thr Cys Val Pro Asn Gly Ala Ser Ser Gly Pro Gin Gin 480 485 490
Leu Pro He Asn Gly Val Phe Val Phe Val Ser Trp Val Ser Arg Phe 495 500 505 510
Tyr Gin Leu Lys Pro Val Gly Thr Ala Ser Ser Ala Arg Gly Arg Leu 515 520 525
Gly Leu Arg Arg 530
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 212 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Ala Gin Ala He He 1 5
Gly Ala He Ala Ala Ser Thr Ala Gly Ser Ala Leu Gly Ala Gly He 10 15 20
Gin Val Gly Gly Glu Ala Ala Leu Gin Ser Gin Arg Tyr Gin Gin Asn 25 30 35
Leu Gin Leu Gin Glu Asn Ser Phe Lys His Asp Arg Glu Met He Gly 40 45 50
Tyr Gin Val Glu Ala Ser Asn Gin Leu Leu Ala Lys Asn Leu Ala Thr 55 60 65 70
Arg Tyr Ser Leu Leu Arg Ala Gly Gly Leu Thr Ser Ala Asp Ala Ala 75 80 85
Arg Ser Val Ala Gly Ala Pro Val Thr Arg He Val Asp Trp Asn Gly 90 95 100
Val Arg Val Ser Ala Pro Glu Ser Ser Ala Thr Thr Leu Arg Ser Gly 105 110 115
Gly Phe Met Ser Val Pro He Pro Phe Ala Ser Lys Gin Lys Gin Val 120 125 130
Gin Ser Ser Gly He Ser Asn Pro Asn Tyr Ser Pro Ser Ser He Ser 135 140 145 150
Arg Thr Thr Ser Trp Val Glu Ser Gin Asn Ser Ser Arg Phe Gly Asn 155 160 165
Leu Ser Pro Tyr His Ala Glu Ala Leu Asn Thr Val Trp Leu Thr Pro 170 175 180
Pro Gly Ser Thr Ala Ser Ser Thr Leu Ser Ser Val Pro Arg Gly Tyr 185 190 195 Phe Asn Thr Asp Arg Leu Pro Leu Phe Ala Asn Asn Arg Arg 200 205 210
(2) INFORMATION FOR SEQ ID HO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 551 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: human calicivirus Sapporo
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..549
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
TGTGATGCTG CCACCACGCT TATAGCCACC GCGGCTTTTA AGGCCGTGGC TACNAGGCTA 60
CAGGTGGTGA CACCAATGAC ACCAGTTGCT GTTGGCATTA ACATGGACTC TGTTCAGATG 120
CAAGTGATGA ATGACTCTTT AAAGGGGGGT GTTCTTTACT GTTTGGATTA TTCCAAATGG 180
GATTCCACAC AAAACCCTGC AGTGACAGCA GCCTCCCTGG CAATATTGGA GAGATTTGCT 240
GAGCCCCATC CAATTGTGTC TTGTGCCATT GAGGCTCTTT CCTCCCCTGC AGAGGGCTAT 300
GTCAATGATA TCAAATTTGT GACACGCGGC GGCCTACCAT CTGGGATGCC ATTTACATCT 360
GTCGTCAATT CTATCAACCA TATGATATAC GTGGCGGCAG CCATCCTGCA GGCATACGAA 420
AGCCACAATG TCCCATATAC TGGAAACGTC TTCCAAGTGG AGACCGTTCA CACGTATGGT 480
GATGATTGCA TGTACAGCGT GTGCCCTGCC ACTGCATCAA TTTTCCACAC TGTGCTTGCA 540
AACCTAACGT C 551
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 183 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Cys Asp Ala Ala Thr Thr Leu He Ala Thr Ala Ala Phe Lys Ala Ala 1 5 10 15
Val Xaa Arg Leu Gin Val Val Thr Pro Met Thr Pro Val Ala Val Gly 20 25 30
He Asn Met Asp Ser Val Gin Met Gin Val Met Asn Asp Ser Leu Lys 35 40 45 Gly Gly Val Leu Tyr Cys Leu Asp Tyr Ser Lys Trp Asp Ser Thr Gin 50 55 60
Asn Pro Ala Val Thr Ala Ala Ser Leu Ala He Leu Glu Arg Phe Ala 65 70 75 80
Glu Pro His Pro He Val Ser Cys Ala He Glu Ala Leu Ser Ser Pro 85 90 95
Ala Glu Gly Tyr Val Asn Asp He Lys Phe Val Thr Arg Gly Gly Leu 100 105 110
Pro Ser Gly Met Pro Phe Thr Ser Val Val Asn Ser He Asn His Met 115 120 125
He Tyr Val Ala Ala Ala He Leu Gly Ala Tyr Glu Ser His Asn Val 130 135 140
Pro Tyr Thr Gly Asn Val Phe Gin Val Glu Thr Val His Thr Tyr Gly 145 150 155 160
Asp Asp Cys Met Tyr Ser Val Cys Pro Ala Thr Ala Ser He Phe His 165 170 175
Thr Val Leu Ala Asn Leu Thr 180
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 148 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TGTGATGCTG CCACCACGCT TATAGCCACC GCGGCTTTTA AGGCCGTGGC TACAGGCTAC 60 AGGTGGTGAC ACCAATGACA CCAGTTGCTG TTGGCATTAA CATGGACTCT GTTCAGATGC 120 AAGTGATGAA TGACTCTTTA AAGGGGGG 148
(2) INFORMATION FOR SEQ ID NO:8
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 449 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
ATGGACTCTG TTCAGATGCA AGTGATGAAT GACTCTTTAA AGGGGGGTGT TCTTTACTGT 60
TTGGATTATT CCAAATGGGA TTCCACACAA AACCCTGCAG TGACAGCAGC CTCCCTGGCA 120
ATATTGGAGA GATTTGCTGA GCCCCATCCA ATTGTGTCTT GTGCCATTGA GGCTCTTTCC 180
TCCCCTGCAG AGGGCTATGT CAATGATATC AAATTTGTGA CACGCGGCGG CCTACCATCT 240 GGGATGCCAT TTACATCTGT CGTCAATTCT ATCAACCATA TGATATACGT GGCGGCAGCC 300
ATCCTGCAGG CATACGAAAG CCACAATGTC CCATATACTG GAAACGTCTT CCAAGTGGAG 360
ACCGTTCACA CGTATGGTGA TGATTGCATG TACAGCGTGT GCCCTGCCAC TGCATCAATT 420
TTCCACACTG TGCTTGCAAA CCTAACGTC 449
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 446 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: human calicivirus Saporro (Day care)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ATGGACTCTG TTCAGATGCA AGTGATGAAT GACTCTTTAA AGGGAGGTGT TCTCTACTGC 60
CTGGATTACT CCAAATGGGA CTCCACACAA AATGCTGCAG TGACAGCAGC ATCCCTNNCA 120
ATATTGGAGA GATTTGCTGA ACCCCACCCA ATTGTGTCTT GTGCCATTGA GGCCCTGNNC 180
TCNNCTGCAG AGGGTTACGT TAATGATATC AAGTTTGTGA CACGTGGCGG CCTACCATGT 240
GGGATGCCAT TCACATCTGT TGTCAATTCC ATCAACCACA TNATATACGT GGCAGCCGCC 300
ATCCTGCAGG CATACGAAAG CCACAATGTT CCATACACTG GAAATGTCTT CCAAGTGGAG 360
ACTGTTCACA CGTATGGTGA CGATTGCATG TACAGCGTGT GCCCTGCCAC CGCATCAATT ' 420
TTCCACACTG TACTTGCAAA CCTAAC 446
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 434 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: human calicivirus Houston
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..434
( i) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GGCCATGTTA TAGTGGTGTT CACATGAAAG ATGGCGACAA GATGTTGATA GATGCCAATC 60
TTCCTTACAA CCAGAAATTA ACTACTATGA TTCATGAGAC TAGGCATAGG ATAGGACAGT 120
ATATAGATAA TACTTTTGGA AAGACATTTA GACATGGATT GACAAAACCT GCTGACAAGA 180 CTGTAGATTT GATCTATAAG ACATTGAATT ATGATGATTT TCTGGCAATA ATGCTAATCA 240
TATATGGGCA AAAGTCGGCC ACTAATACGG AGTTGCAATT CTTGATGGAG AAACTTAGAG 300
GTTATGAATC TACAATGGAT GACATAGGGA AAGTCTATGG AGATGATAAA ATGAGAGATA 360
TAATCAAGAA TATTTCTGAT GATGACATAA AGAGTCTTTT AGGGGAGATA AATAGTGATT 420
ATTCTGGTAA GNAT 434
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 144 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
Pro Cys Tyr Ser Gly Val His Met Lys Asp Gly Asp Lys Met Leu He 1 5 10 15
Asp Ala Asn Leu Pro Tyr Asn Gin Lys Leu Thr Thr Met He His Glu 20 25 30
Thr Arg His Arg He Gly Gin Tyr He Asp Asn Thr Phe Gly Lys Thr 35 40 45
Phe Arg His Gly Leu Thr Lys Pro Ala Asp Lys Thr Val Asp Leu He 50 55 60
Tyr Lys Thr Leu Asn Tyr Asp Asp Phe Leu Ala He Met Leu He He 65 70 75 80
Tyr Gly Gin Lys Ser Ala Thr Asn Thr Glu Leu Gin Phe Leu Met Glu 85 90 95
Lys Leu Arg Gly Tyr Glu Ser Thr Met Asp Asp He Gly Lys Val Tyr 100 105 110
Gly Asp Asp Lys Met Arg Asp He He Lys Asn He Ser Asp Asp Asp 115 120 125
He Lys Ser Leu Leu Gly Glu He Asn Ser Asp Tyr Ser Gly Lys Xaa 130 135 140
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2516 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: SRSV/KY/89
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CAATAGAGGA TGGCCCTTTA ATTTATGCTG AACATGCCAA GTACAAAAAT CATTTTGATG 60
CAGATTACAC AGCATGGGAC TCTACACAAA ATAGACAAAT TATGACAGAA TCCTTCTCCA 120
TCATGTCACG CCTTACGGCC TCTCCAGAAC TAGCTGAGGT TGTAGCCCAG GACTTACTAG 180
CACCATCCGA GATGGATGTG GGCGACTATG TTATAAGGGT CAAAGAAGGC CTACCATCAG 240
GATTTCCCTG CACTTCTCAA GTGAATAGCA TAAATCACTG GATAATCACC CTTTGTGCAT 300
TGTCTGAGGC TACTGGCTTA TCACCTGATG TGGTACAGTC CATGTCATAC TTCTCATTCT 360
ACGGTGATGA TGAGATCGTA TCAACTGACA TAGACTTTGA CCCAACTCGC CTCACCCAAA 420
TTCTCAAGGA ATACGGCCTC AAGCCAACAA GGCCAGACAA AACAGAAGGA CCAATACAGG 480
TGAGGAAGAA TGTGGATGGG CTAGTTTTTC TGCGGCGCAC CATCTCCCGG GACGCAGCAG 540
GGTTCCAAGG TAGACTGGAT AGAGCCTCAA TTGAACGTCA AATTTTCTGG ACCCGCGGGC 600
CCAACCATTC AGACCCATCA GAGACTCTGG TACCACACAC CCAAAGGAAA GTCCAGCTGA 660
TCTCACTTCT AGGAGAAGCC TCACTCCACG GGGAAAAATT TTACAGGAAA ATATCTAGCA 720
AAGTCATACA TGAAATTAAG ACTGGTGGGC TGGAGATGTA TGTCCCAGGG TGGCAGGCCA 780
TGTTCCGCTG GATGCGCTTC CATGACCTCG GATTGTGGAC AGGAGATCGC AATCTCCTGC 840
CCGAATTCGT AAATGATGAT GGCGTCTAAG GACGCTACGT CAAGCGTGGA TGGCGCCAGT 900
GCGTCGGTTC AGTTGGTACC GGAGGTTAAT GCTTCTGACC CTCTTGCAAT GGATCCTGTG 960
GCGGGTTCTT CAACAGCAGT TGCAACCGCT GGACAAGTTA ACCCTATTGA CCCTTGGATA 1020
ATCAATAACT TTGTGCAGGC TCCCCAAGGT GAATTTACTA TTTCTCCAAA TAATACCCCC 1080
GGTGATGTTT TGTTTGATTT GAGTCTAGGC CCTCATCTTA ATCCCTTCTT GTTACATTTG 1140
TCACAAATGT ATAATGGCTG GGTTGGCAAC ATGAGAGTTA GGATTATGCT GGCTGGTAAT 1200
GCATTTACTG CAGGCAAAAT TATAGTTTCT TGCATACCTC CTGGCTTTGG CTCCCAACAA 1260
CTTACTATAG CACAAGCAAC TCTCTTCCCG CATGTGATTG CTGATGTTAG GACTTTAGAC 1320
CCAATTGAAG TACCCTTGGA AGATGTAAGG AATGTTCTCT TTCATAATAA TGATAGAAAT 1380
CAACAAACTA TGCGCCTTGT GTGCATGCTT TATACCCCCC TCAGCACTGG TGGCGGTACA 1440
GGTGATTCTT TTGTGGTTGC AGGGCGAGTC ATGACTTGTC CTAGCCCCGA CTTTAATTTC 1500
TTGTTCTTGG TTCCTCCCAC AGTGGAACAG AAGACTAGGC CTTTCACCCT CCCAAATTTA 1560
CCGCTGAGTT CTTTGTCTAA TTCACGTGCT CCTCTTCCAA TTAGTGGCAT GGGTATTTCT 1620
CCAGATAATG TTCAGAGTGT GCAGTTCCAA AATGGCCGAT GTACCTTAGA TGGACGTCTT 1680
GTTGGCACCA CCCCAGTTTC CCTCTCCCAT GTTGCTAAGA TAAGGGGTAC TTCTAATGGT 1740
ACAGTAATCA ATCTCACCGA ATTGGATGGC ACCCCCTTCC ACCCTTTTGA AGGCCCTGCC 1800
CCTATTGGTT TTCCAGATCT TGGTGGCTGT GATTGGCATA TTAATATGAC ACAATTTGGA 1860
CATTCCAGTC AGACTCAGTA TGATGTAGAC ACCACCCCCG ACACCTCCGT CCCTCACTTA 1920
GGTTCAATCC AGGCGAATGG CATTGGTAGT GGCAACTATA TTGGTGTTCT TAGCTGGGTC 1980 TCCCCCCCAT CACATCCATC TGGCTCTCAA GTTGATCTCT GGAAGATCCC CAACTATGGG 2040
TCTAGTATCA CAGAGGCAAC CCATCTAGCT CCCTCTGTCT ATTCTCCTGG CTTTGGAGAG 2100
GTGCTAGTCT TTTTCATGTC AAAGATACCA GGTCCTGGTG GTGATAGTCT GCCCTGTTTA 2160
CTGCCACAAG GATATATCTC ACACCTTGCA AGTGAACAAG CCCCAACTGT TGGTGAGGGT 2220
CCCCTGCTCC ACTATGTTGA CCCTGACACG GACCGGAATC TTGGGGAGTT TAAGGCTTAC 2280
CCTGATGGTT TCCTAACCTG TGTCCCTAAT GGGGCCAGCT CGGGCCCACA ACAACTACCA 2340
ATCAATGGAG TCTTTGTCTT TGTTTCATGG GTGTCCAGAT TTTATCAGTT AAAGCCTGTG 2400
GGAACTGCCA GTACGGCAAG AGGTAGGCTT GGTTTGCGCC GATAATGGCT CAGGCTATAA 2460
TTGGTGCAAT TGCCGCCTCT ACAGCAGGTA GTGCTTTAGG GGCAGGTATA CAGGTT 2516
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 124 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: primate calcicvirus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
TGGACGGACC TGCTGTTGAA GATCTCTTCA AAGGCTCGAA CGACCAAAGC ACGATCGGTA 60
TTGTGTTGAC TACGCAAAGT GGGACTCAAC CCACCACCAA AAGTAACATC CAATCAATGA 120
CATC 124
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 110 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: primate calcicvirus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GTGAATGACA TCTTCGACTC GATGGACCTA TTCACATATG GTGATGACGG TGTCTACATC 60
GTCCCACCAC TATATCATCT GTCATGCCCA AGTCTTCACC AACCTGAAAC 110
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: CTTGTTGGTT TGAGGCCATA T 21
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: ATAAAAGTTG GCATGAACA 19
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GTTGACACAA TCTCATCATC 20
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GGCCTGCCAT CTGGATTGCC 20
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GGGCCCCCTG GTATAGGTAA 20
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: TGGTGATGAC TATAGCATCA GACACAAA 28
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: ACTCACCCAA ATCCTCCA 18
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GTTCTGACCA CCTAACCT 18
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: AGTTTGGGTC CCCATCTTAA TCCTTT 26 (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: TGAACCAAAA CCAGGGGG 18
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: AGCAAAGTCA TACATGAAAT 20
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CCATTATACA TTTGTAG 17
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: ATTATAGTTT CTTGCATA 18
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: CACACTCTGG ACATTGTCTG 20
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: CATTGGGTTT CCAGACCTA 19
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: ATAATTGGGG ATCTTCCAAA 20
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: TAGTGGCATG GGTATTTC 18
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: TATGCCAATC ACAGCCAC 18
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: GTCTGGCTCC CAAGTTGACC 20
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: CGGTATCAGG GTCAACAT 18
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: TGAGGCTGCC CTGCTCCA 18
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: CCACCGCTGT CCGGGAGG 18 (2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: GTTGCTGTTG GCATTAACA 19
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 126 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:38:
His Phe Asp Ala Asp Tyr Thr Ala Trp Asp Ser Thr Gin Asn Arg Gin 1 5 10 15
He Met Thr Glu Ser Phe Ser He Met Ser Arg Leu Thr Ala Ser Pro 20 25 30
Glu Leu Ala Glu Val Val Ala Gin Asp Leu Leu Ala Pro Ser Glu Met 35 40 45
Asp Val Gly Asp Tyr Val He Arg Val Lys Glu Gly Pro Ser Gly Phe 50 55 60
Pro Cys Thr Ser Gin Val Asn Ser He Asn His Trp He He Thr Leu 65 70 75 80
Cys Ala Leu Ser Glu Ala Thr Gly Leu Ser Pro Asp Val Val Gin Ser 85 90 95
Met Ser Tyr Phe Ser Phe Tyr Gly Asp Asp Glu He Val Ser Thr Asp
100 105 110
He Asp Phe Asp Pro Ala Arg Leu Thr Gin He Leu Lys Glu 115 120 125
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 121 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: hepatitis E virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
Val Phe Glu Asn Asp Phe Ser Glu Phe Asp Ser Thr Gin Asn Asn Phe 1 5 10 15
Ser Leu Gly Leu Glu Cys Ala He Met Glu Glu Cys Gly Met Pro Gin 20 25 30
Trp Leu He Arg Leu Tyr His Leu He Arg Ser Ala Trp He Leu Gin 35 40 45
Ala Pro Lys Glu Ser Leu Arg Gly Phe Trp Lys Lys His Ser Lys His 50 55 60
Ser Gly Glu Pro Gly Thr Leu Leu Trp Asn Thr Val Trp Asn Met Ala 65 70 75 80
Val He Thr His Cys Tyr Asp Phe Arg Asp Phe Gin Val Ala Ala Phe 85 90 95
Lys Gly Asp Asp Ser He Val Leu Cys Ser Glu Tyr Arg Gin Ser Pro 100 105 110
Gly Ala Ala Val Leu He Ala Gly Cys 115 120
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 127 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(Vi) ORIGINAL SOURCE:
(A) ORGANISM: hepatitis C virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
Gly Phe Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Ser 1 5 10 15
Asp He Arg Thr Glu Glu Ala He Tyr Gin Cys Cys Asp Leu Asp Pro 20 25 30
Gin Ala Arg Val Ala He Lys Ser Leu Thr Glu Arg Leu Tyr Val Gly 35 40 45
Gly Pro Leu Thr Asn Ser Arg Gly Glu Asn Cys Gly Tyr Arg Arg Cys 50 55 60
Arg Ala Ser Arg Ala Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr 65 70 75 80
Leu Thr Cys Tyr He Lys Ala Arg Ala Ala Cys Arg Ala Ala Gly Leu 85 90 95
Gin Asp Cys Thr Met Leu Val Cys Gly Asp Asp Leu Val Val He Cys 100 105 110
Glu Ser Ala Gly Val Gin Glu Asp Ala Ala Ser Leu Arg Ala Phe 115 120 125 (2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 132 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: hepatitis A virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
Gly Leu Asp Leu Asp Phe Ser Ala Phe Asp Ala Ser Leu Ser Pro Phe 1 5 10 15
Met He Arg Glu Ala Gly Arg He Met Ser Glu Leu Ser Gly Thr Pro 20 25 30
Ser His Phe Gly Thr Ala Leu He Asn Thr He He Tyr Ser Lys His 35 40 45
Leu Leu Tyr Asn Cys Cys Tyr His Val Cys Gly Ser Met Pro Ser Gly 50 55 60
Ser Pro Cys Thr Ala Leu Leu Asn Ser He He Asn Asn Val Asn Leu 65 70 75 80
Tyr Tyr Val Phe Ser Lys He Phe Gly Lys Ser Pro Val Phe Phe Cys 85 90 95
Gin Ala Leu Lys He Leu Cys Tyr Gly Asp Asp Val Leu He Val Phe 100 105 110
Ser Arg Asp Val Gin He Asp Asn Leu Asp Leu He Gly Gin Lys He 115 120 125
Val Asp Glu Phe 130
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 158 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Japanese encephalitis virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg He Thr Arg Thr
1 5 10 15
Asp Leu Glu Asn Glu Ala Lys Val Leu Glu Leu Leu Asp Gly Glu His 20 25 30
Arg Met Leu Ala Arg Ala He He Glu Leu Thr Tyr Arg His Lys Val 35 40 45
Figure imgf000087_0001
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 117 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Poliovirus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
Phe Ala Phe Asp Tyr Thr Gly Tyr Asp Ala Ser Leu Ser Pro Ala Trp
1 5 10 15
Phe Glu Ala Leu Lys Met Val Leu Glu Lys He Gly Phe Gly Asp Arg 20 25 30
Val Asp Tyr He Asp Tyr Leu Asn His Ser His His Leu Tyr Lys Asn 35 40 45
Lys Thr Tyr Cys Val Lys Gly Gly Met Pro Ser Gly Cys Ser Gly Thr 50 55 60
Ser He Phe Asn Ser Met He Asn Asn Leu He He Arg Thr Leu Leu 65 70 75 80
Leu Lys Thr Tyr Lys Gly He Asp Leu Asp His Leu Lys Met He Ala 85 90 95
Tyr Gly Asp Asp Val He Ala Ser Tyr Pro His Glu Val Asp Ala Ser 100 105 110
Leu Leu Ala Gin Ser 115
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 121 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Foot-and-mouth disease virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
Val Trp Asp Val Asp Tyr Ser Ala Phe Asp Ala Asn His Cys Ser Asp 1 5 10 15
Ala Met Asn He Met Phe Glu Glu Val Phe Arg Thr Asp Phe Gly Phe 20 25 30
His Pro Asn Ala Glu Trp He Leu Lys Thr Leu Val Asn Thr Glu His 35 40 45
Ala Tyr Glu Asn Lys Arg He Thr Val Glu Gly Gly Met Pro Ser Gly 50 55 60
Cys Ser Ala Thr Ser He He Asn Thr He Leu Asn Asn He Tyr Val 65 70 75 80
Leu Tyr Ala Leu Arg Arg His Tyr Glu Gly Val Glu Leu Asp Thr Tyr 85 90 95
Thr Met He Ser Tyr Gly Asp Asp He Val Val Ala Ser Asp Tyr Asp 100 105 110
Leu Asp Phe Glu Ala Leu Lys Pro His 115 120
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 126 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: encephalomyocarditis virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
Val Tyr Asp Val Asp Tyr Ser Asn Phe Asp Ser Thr His Ser Val Ala
1 5 10 15
Met Phe Arg Leu Leu Ala Glu Glu Phe Phe Thr Pro Glu Asn Gly Phe 20 25 30
Asp Pro Leu Thr Arg Glu Tyr Leu Glu Ser Leu Ala He Ser Thr His 35 40 45
Ala Phe Glu Glu Lys Arg Phe Leu He Thr Gly Gly Leu Pro Ser Gly 50 55 60
Cys Ala Ala Thr Ser Met Leu Asn Thr He Met Asn Asn He He He 65 70 75 80 Arg Ala Gly Leu Tyr Leu Thr Tyr Lys Asn Phe Glu Phe Asp Asp Val 85 90 95
Lys Val Leu Ser Tyr Gly Asp Asp Leu Leu Val Ala Thr Asn Tyr Gin 100 105 110
Leu Asp Phe Asp Lys Val Arg Ala Ser Leu Ala Lys Thr Gly 115 120 125
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 122 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Sindbis virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
Val Leu Glu Thr Asp He Ala Ser Phe Asp Lys Ser Gin Asp Asp Ala
1 5 10 15
Met Ala Leu Thr Gly Leu Met He Leu Glu Asp Leu Gly Val Asp Gin 20 25 30
Pro Leu Leu Asp Leu He Glu Cys Ala Phe Gly Glu He Ser Ser Thr 35 40 45
His Leu Pro Thr Gly Thr Arg Phe Lys Phe Gly Ala Met Met Lys Ser 50 55 60
Gly Met Phe Leu Thr Leu Phe Val Asn Thr Val Leu Asn Val Val He 65 70 75 80
Ala Ser Arg Val Leu Glu Glu Arg Leu Lys Thr Ser Arg Cys Ala Ala 85 90 95
Phe He Gly Asp Asp Asn He He His Gly Val Val Ser Asp Lys Glu 100 105 110
Met Ala Glu Arg Cys Ala Thr Trp Leu Asn 115 120
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 124 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: tobacco mosaic virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
Val Leu Glu Leu Asp He Ser Lys Tyr Asp Lys Ser Gin Asn Glu Phe
1 5 10 15 His Cys Ala Val Glu Tyr Glu He Trp Arg Arg Leu Gly Phe Glu Asp 20 25 30
Phe Leu Gly Glu Val Trp Lys Gin Gly His Arg Lys Thr Thr Leu Lys 35 40 45
Asp He Thr Ala Gly Tyr Lys Thr Cys He Trp Tyr Gin Arg Lys Ser
50 55 60
Gly Asp Val Thr Thr Phe He Gly Asn Thr Val He He Ala Ala Cys 65 70 75 80
Leu Ala Ser Met Leu Pro Met Glu Lys He He Lys Gly Ala Phe Cys 85 90 95
Gly Asp Asp Ser Leu Leu Tyr Phe Pro Lys Gly Cys Glu Phe Pro Asp 100 105 110
Val Gin His Ser Ala Asn Leu Met Trp Asn Phe Glu 115 120
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 125 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: alfalfa mosaic virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
Phe Lys Glu He Asp Phe Ser Lys Phe Asp Lys Ser Gin Asn Glu Leu
1 5 10 15
His His Leu He Gin Glu Arg Phe Leu Lys Tyr Leu Gly He Pro Asn 20 25 30
Glu Phe Leu Thr Leu Trp Phe Asn Ala His Arg Lys Ser Arg He Ser 35 40 45
Asp Ser Lys Asn Gly Val Phe Phe Asn Val Asp Phe Gin Arg Arg Thr 50 55 60
Gly Asp Ala Leu Thr Tyr Leu Gly Asn Thr He Val Thr Leu Ala Cys 65 70 75 80
Leu Cys His Val Tyr Asp Leu Met Asp Pro Asn Val Lys Phe Val Val 85 90 95
Ala Ser Gly Asp Asp Ser Leu He Gly Thr Val Glu Glu Leu Pro Arg 100 105 110
Asp Gin Glu Phe Leu Phe Thr Thr Leu Phe Asn Leu Glu
115 120 125
(2) INFORMATION FOR SEQ ID NO:49: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 122 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: 1inear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: brome mosaic virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
Phe Leu Glu Ala Asp Leu Ser Lys Phe Asp Lys Ser Gin Gly Glu Leu 1 5 10 15
His Leu Glu Phe Gin Arg Glu He Leu Leu Ala Leu Gly Phe Pro Ala 20 25 30
Pro Leu Thr Asn Trp Trp Ser Asp Phe His Arg Asp Ser Tyr Leu Ser 35 40 45
Asp Pro His Ala Lys Val Gly Met Ser Val Ser Phe Gin Arg Arg Thr 50 55 60
Gly Asp Ala Phe Thr Tyr Phe Gly Asn Thr Leu Val Thr Met Ala Met 65 70 75 80
He Ala Tyr Ala Ser Asp Leu Ser Asp Cys Asp Cys Ala He Phe Ser 85 90 95
Gly Asp Asp Ser Leu He He Ser Lys Val Lys Pro Val Leu Asp Thr 100 105 110
Asp Met Phe Thr Ser Leu Phe Asn Met Glu
115 120
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 142 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: cowpea mosaic virus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
Val Leu Cys Cys Asp Tyr Ser Ser Phe Asp Gly Leu Leu Ser Lys Gin
1 5 10 15
Val Met Asp Val He Ala Ser Met He Asn Glu Leu Cys Gly Gly Glu 20 25 30
Asp Gin Leu Lys Asn Ala Arg Arg Asn Leu Leu Met Ala Cys Cys Ser 35 40 45
Arg Leu Ala He Cys Lys Asn Thr Val Trp Arg Val Glu Cys Gly He 50 55 60 Pro Ser Gly Phe Pro Met Thr Val He Val Asn Ser He Phe Asn Glu 65 70 75 80
He Leu He Arg Tyr His Tyr Lys Lys Leu Met Arg Glu Gin Gin Ala 85 90 95
Pro Glu Leu Met Val Gin Ser Phe Asp Lys Leu He Gly Leu Val Thr 100 105 110
Tyr Gly Asp Asp Asn Leu He Ser Val Asn Ala Val Val Thr Pro Tyr 115 120 125
Phe Asp Gly Lys Lys Leu Lys Gin Ser Leu Ala Gin Gly Gly 130 135 140
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: CACGCGGAGG CTCTCAAT 18
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE- CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: GGTGGCGAAG CGGCCCTC 18
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: TCAGCAGTTA TAGATATG 18
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: ATGCTATATA CATAGGTC 18
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: CAACAGGTAC TACGTGAC 18
(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: TGTGGCCCAA GATTTGCT 18
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOG : unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: ATAAAAGTTG GCATGAACAC AAAT 24
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: GTTGCTGTTG GCATTAACAT GGAC 24
(2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: GTTCCTGTTG GCATAAACAT GGAC 24
(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: GTTCCGGTTG GCATTAACAT GGAC 24
(2) INFORMATION FOR SEQ ID NO:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: GTTCCGGTTG GTATCAACAT GGAC 24
(2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: GTTGCGGTTG GTGTTGACAT GACA 24 (2) INFORMATION FOR SEQ ID NO:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: SRSV/CDC 6/91
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
ATGCACTTCA CAGGTGAATA GCATCAACCA CTGGATCCTA ACTCTATGTG CATTGTCAGA 60
AGTCACTGGC TTGTCCCCTG ATGTGATACA ATCACAATCT TATTTCTCAT TTTATGGT 118
(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: SRSV/UT/88
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
ATGTACCTCA CAAGTGAACA GCATCAATCA CTGGATTTTG ACCTTGTGGG GCCTATCAGA 60
AGTTACTGGT CTGGCTCCTG ATGTAATACA GTCACAATCT TACTTTTCAT TCTATGGT 118
(2) INFORMATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 117 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Snow Mountain Agent/78
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
CTGCACATCA CAGTGGAATT CCATGCCCAC TGGCTCCTCA CACTCTGTGC ACTATCTGAA 60
GTCACAAACC TGGCTCCTGA CATCATACAA GCTAACTCCT TGTTCTCTTT CTATGGT 117
(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: SRSV/CAMBRIDGE, UK 92
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:
CTGCACCTCA CAGTGGAACT CCATTGCCCA CTGGTTGCTT ACTCTGTGTG CCCTTTCTGA 60
AGTGACAGGA CTAGGCCCCG ACATCATACA AGCTAATTCC ATGTACTCTT TCTATGGT 118
(2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: SRSV/CDC 32
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
TTGCACCTCA CAGTGGAACT CCATTGCCCT CTGGTTGCTT ACTCTGTGTG CCCTTTCTGA 60
AGTGACAGGA CTAGGCCCCG ACATCATACA AGCTAATTCC ATGTACTCTT TCTATGGT 118
(2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Norwalk virus/8FIIa/68
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
ATGTACTTCC CAGGTGAACA GCATAAATCA CTGGATAATT ACTCTCTGTG CACTGTCTGA 60
GGCCACTGGT TTATCACCTG ATGTGGTGCA ATCCATGTCA TATTTCTCAT TTTATGGT 118
(2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:
(A) ORGANISM: SRSV-3/88
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:
CTGCACTTCT CAAGTAAATA GCATAAATCA CTGGATAATC ACCCTTTGTG CACTGTCTGA 60
GGCTACTGGC TTATCACCTG ATGTGGTGCA GTCCATGTCA TACTTCTCAT TTTACGGT 118
(2) INFORMATION FOR SEQ ID NO:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: SRSV/KY89/89
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
CTGCACTTCT CAAGTGAATS GCATAAATCA CTGGATAATC ACCCTTTGTG CATTGTCTGA 60
GGCTACTGGC TTATCACCTG ATGTGGTACA GTCCATGTCA TACTTCTCAT TCTACGGT 118
(2) INFORMATION FOR SEQ ID NO:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 279 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Norwalk Virus/8FIIa/68
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
CAATAGAAGA TGGCCCCCTC ATCTATGCTG AGCATGCTAA ATATAAGAAT CATTTTGATG 60
CAGATTATAC AGCATGGGAC TCAACACAAA ATAGACAAAT TATGACAGAA TCCTTCTCCA 120
TTATGTCGCG CCTTACGGCC TCACCAGAAT TGGCCGAGGT TGTGGCCCAA GATTTGCTAG 180
CACCATCTGA GATGGATGTA GGTGATTATG TCATCAGGGT CAAAGAGGGG CTGCCATCTG 240
GATTCCCATG TACTTCCCAG GTGAACAGCA TAAATCACT 279
(2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE" CHARACTERISTICS:
(A) LENGTH: 279 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:
(A) ORGANISM: SRSV-3/88
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
CAATAGAGGA TGGCCCTTTA ATTTATGCTG AGCATGCCAA GTACAAAAAT CATTTTGATG 60
CAGATTACAC AGCATGGGAC TCTACACAAA ATAGACAAAT AATGACAGAA TCCTTTTCCA 120
TCATGTCACG CCTCACGGCC TCTCCAGAAC TAGCTGAGGT TGTAGCCCAG GACTTGCTAG 180
CACCATCCGA GATGGATGTG GGTGACTATG TTATAAGGGT CAAAGAAGGC CTACCATCAG 240
GATTTCCCTG CACTTCTCAA GTAAATAGCA TAAATCACT 279
(2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 279 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: SRSV/KY89
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
CAATAGAGGA TGGCCCTTTA ATTTATGCTG AACATGCCAA GTACAAAAAT CATTTTGATG 60
CAGATTACAC AGCATGGGAC TCTACACAAA ATAGACAAAT TATGACAGAA TCCTTCTCCA 120
TCATGTCACG CCTTACGGCC TCTCCAGAAC TAGCTGAGGT TGTAGCCCAG GACTTACTAG 180
CACCATCCGA GATGGATGTG GGCGACTATG TTATAAGGGT CAAAGAAGGC CTACCATCAG 240
GATTTCCCTG CACTTCTCAA GTGAATAGCA TAAATCACT 279
(2) INFORMATION FOR SEQ ID NO:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 279 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: SRSV/Cambridge, UK/92
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
TGTATGAAGA TGGTACCATA ATATTTGAGA AACATTCCAG ATACAGATAC CACTATGATG 60
CAGATTATCC CGCTGGGTAC TCCACGCAGC AACGGGCAGT GTTGGCAGCA GCACTTGAAA 120
TCATGGTGAG GTTCTCTGCT GAACCACAGC TAGCGCAAAT AGTAGCTGAA GATCTGCTAG 180
CACCAAGTGT AGTTGATGTG GGTGACTTCA AGATCACCAT TAATGAAGGC CTACCTTCTG 240 GTGTGCCCTG CACCTCACAG TGGAACTCCA TTGCCCACT 279
(2) INFORMATION FOR SEQ ID NO:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 277 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Snow Mountain Agent/78
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:
GAATGAGGAT GGACCCATAA TTTTTGAAAA GCACTCCAGG TTCTCATACC ACTATGATGC 60
AGATTACTCA CGCTGGGACT CAACCCAACA GAGGGCAGTG CTAGCTGCAG CCTTGGAAAT 120
CATGGTAAAA TTCTCACCAG AACCACATTT GGCCCAAATT GTTGCAGAGG ATCTCCTAGC 180
CCCCAGTGTG ATGGATGTAG GTGATTTCAA AATAACAATT AATGAGGGAC TGCCCTCGGG 240
AGTACCCTGC ACATCACAGT GGAATTCCAT GCCCACT 277

Claims

1. A cDNA sequence of the formula shown in Table 2 and fragments and derivatives thereof having sufficient size to bind a Norwalk or Norwalk-related virus genome.
2. A protein encoded by nucleotides including nucleotides 1 through 7753 of the Norwalk virus genome shown in Table 2 or fragments or derivatives thereof.
3. The protein of claim -2, wherein said protein is produced in a prokaryotic expression system or a eukaryotic expression system.
4. The protein of claim 2, wherein said protein is produced by chemical methods.
5. A protein encoded by nucleotides 146 through 5359 of the Norwalk virus genome shown in Table 2 or fragments or derivatives thereof.
6. The protein of claim 5, wherein said protein is produced in a prokaryotic expression system or eukaryotic expression system.
7. The protein of claim 5, wherein said protein is produced by chemical methods.
8. A RNA-dependent RNA polymerase encoded by nucleotides 4543 to 4924 of the Norwalk virus genome shown in Table 2 or fragments.
9. The RNA polymerase of claim 8, wherein said RNA polymerase is produced in a prokaryotic expression system or a eukaryotic expression system.
10. The RNA polymerase of claim 8, wherein said RNA polymerase is produced by chemical methods.
11. A protein encoded by nucleotides 5337 through 7573 of the Norwalk virus genome shown in Table 2 or fragments or derivatives thereof.
12. The protein of claim 11, wherein said protein is produced in a prokaryotic expression system or eukaryotic expression system.
13. The protein of claim 11, wherein said protein is produced by chemical methods.
14. A protein encoded by nucleotides 5346 through 6935 of the
Norwalk virus genome shown in Table 2 or fragments or derivatives thereof.
15. The protein of claim 14, wherein said protein is produced in a prokaryotic expression system or eukaryotic expression system.
16. The protein of claim 14. wherein said protein is produced by chemical methods.
17. A protein encoded by nucleotides 6938 through 7573 of the Norwalk virus genome shown in Table 2 or fragments or derivatives thereof.
18. The protein of claim 17, wherein said protein is produced in a prokaryotic expression system or eukaryotic expression system.
19. The protein of claim 17, wherein said protein is produced by chemical methods.
20. A method of making a RNA probe to detect Norwalk or Norwalk-related viruses, comprising the steps of: subcloning a Norwalk virus cDNA clone into a transcription vector; growing said cDNA containing transcription vector; adding RNA polymerase to generate single stranded RNA by in vitro transcription; and isolating said single stranded RNA.
21. A method of identifying Norwalk or Norwalk-related viruses in a sample suspected of containing Norwalk or Norwalk-related viruses, comprising the steps of: adding a cDNA or a RNA probe specific to Norwalk virus or a Norwalk-related virus to said sample to be tested under conditions in which the cDNA or RNA probe will bind to the Norwalk or Norwalk-related virus genome; and measuring the amount of binding of said cDNA or RNA probe.
22. The method of claim 21 , wherein said sample is selected from the group consisting of food, water and stool.
23. The method of claim 21, wherein said cDNA is selected from a group consisting of pUCNV-953, pUCNV-4145, pUCNV-4095, pUCNV- 5030 and pUCNV-5101 or fragments or derivatives thereof.
24. A method of identifying Norwalk or Norwalk-related viruses in a sample suspected of containing Norwalk or Norwalk-related viruses comprising the steps of: adding at least two oligonucleotides each of about 10 nucleotides or greater to said sample under conditions in which said oligonucleotides bind to the Norwalk or Norwalk-related virus genome; amplifying a nucleotide sequence between said bound oligonucleotides; and measuring the amount of amplified sequence.
25. A method of identifying Norwalk or Norwalk-related viruses in a sample suspected of containing Norwalk or Norwalk-related viruses comprising the steps of: isolating said nucleic acids using CTAB procedure; amplifying nucleic acid; and measuring the amplified product.
26. The method of claim 25, wherein the CTAB procedure includes: extracting said sample with genetron; removing the supernatant of said genetron extracted sampled; precipitating viruses in said supernatant with polyethylene glycol; treating said precipitate with proteinase K in the presence of SDS at about 30° minutes; sequentially extracting said treated precipitate with phenol- chloroform and then chloroform; forming a mixture by adding a solution of about 5% CTAB and about 0.4M NaCl to said supernatant of said sequentially extracted sample at a ratio of about 5:2 sample:CTAB; incubating said mixture; centrifuging said mixture to collect nucleic acids; suspending said nucleic acids in IM NaCL and thereafter extracting with chloroform.
27. A method of claim 25 further comprising: performing reverse transcription on said nucleic acids; amplifying nucleic acids using primers; and detecting the amplified nucleic acids using agarose gel electrophoresis.
28. A method of cloning Norwalk or pathogens from food, biological and environmental samples, comprising: isolating said nucleic acids using CTAB procedure; amplifying nucleic acids; and incorporating said amplified nucleic acids into vectors.
29. A primer sequence of the formula CTT GTT GGT TTG AGG CCA TAT.
30. A primer sequence of the formula ATA AAA GTT GGC ATG
AAC A.
31. A primer sequence of the formula GTT GAC ACA ATC TCA TCA TC.
32. A primer sequence of the formula GGC CTG CCA TCT GGA TTG CC.
33. A primer sequence of the formula GGG CCC CCT GGT ATA GGT AA.
34. A primer sequence of the formula TGG TGA TGA CTA TAG CAT CAG ACA CAA A.
35. A primer sequence of the formula ACT CAC CCA AAT CCT
CCA.
36. A primer sequence of the formula GTT CTG ACC ACC TAA CCT.
37. A primer sequence of the formula AGT TTG GGT CCC CAT CTT AAT CCT TT.
38. A primer sequence of the formula TGA ACC AAA ACC AGG GGG.
39. A primer sequence of the formula AGC AAA GTC ATA CAT
GAA AT.
40. A primer sequence of the formula CCA TTA TAC ATT TGT AG.
41. A primer sequence of the formula ATT ATA GTT TCT TGC ATA.
42. A primer sequence of the formula CAC ACT CTG GAC ATT GTC TG.
43. A primer sequence of the formula CAT TGG GTT TCC AGA CCT A.
44. A primer sequence of the formula ATA ATT GGG GAT CTT
CCA AA.
45. A primer sequence of the formula TAG TGG CAT GGG TAT TTC.
46. A primer sequence of the formula TAT GCC AAT CAC AGC CAC.
47. A primer sequence of the formula GTC TGG CTC CCA AGT TGA CC.
48. A primer sequence of the formula CGG TAT CAG GGT CAA CAT.
49. A primer sequence of the formula TGA GGC TGC CCT GCT CCA.
50. A primer sequence of the formula CCA CCG CTG TCC GGG
AGG.
51. A primer sequence of the formula GTT GCT GTT GGC ATT AAC A.
52. A method of making a probe to detect Norwalk or Norwalk- related viruses, comprising the steps of: synthesizing one or more short or long nucleotides from the Norwalk virus genome shown in Table 2 or fragments or derivatives thereof.
53. The probe produced by the method of claim 52.
54. A method of making a probe to detect Norwalk or Norwalk- related viruses, comprising the step of: synthesizing one or more short or long nucleotides from a subgenomic region of the Norwalk virus genome shown in Table 2 or fragments or derivatives thereof.
55. The probe produced by the method of claim 54.
56. The probe of claim 55, wherein said subgenomic region includes a sequence of the formula CTT GTT GGT TTG AGG CCA TAT.
57. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula ATA AAA GTT GGC ATG AAC A.
58. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula GTT GAC ACA ATC TCA
TCA TC.
59. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula GGC CTG CCA TCT GGA TTG CC.
60. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula GGG CCC CCT GGT ATA GGT AA.
61. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula TGG TGA TGA CTA TAG CAT CAG ACA CAA A.
62. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula GTT CTG ACC ACC TAA CCT.
63. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula AGT TTG GGT CCC CAT
CTT AAT CCT TT.
64. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula TGA ACC AAA ACC AGG GGG.
65. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula AGC AAA GTC ATA CAT GAA AT.
66. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula CCA TTA TAC ATT TGT
AG.
67. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula CAC ACT CTG GAC ATT GTC TG.
68. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula CAT TGG GTT TCC AGA CCT A.
69. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula ATA ATT GGG GAT CTT CCA AA.
70. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula TAT GCC AAT CAC AGC CAC.
71. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula GTC TGG CTC CCA AGT
TGA CC.
72. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula CGG TAT CAG GGT CAA CAT.
73. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula TGA GGC TGC CCT GCT CCA.
74. The probe of claim 55, wherein said subgenomic region includes a nucleotide sequence of the formula CCA CCG CTG TCC GGG
AGG.
75. The method of claim 54, wherein said subgenomic region includes said Norwalk genome's first open reading frame.
76. The probe produced by the method of claim 75.
77. The method of claim 54, wherein said subgenomic region includes nucleotides 146 through 5359.
78. The probe produced by the method of claim 77.
79. The method of claim 54, wherein said nucleotides code for a picornavirus 2C-like protein, a 3C-like protease, an RNA-dependent RNA polymerase or any combination thereof.
80. The probe produced by the method of claim 79.
81. The method of claim 54, wherein said nucleotide codes for a capsid protein.
82. The probe produced by the method of claim 81.
83. The method of claim 54, wherein said subgenomic region includes nucleotides 5337 through 7573.
84. The probe produced by the method of claim 83.
85. The method of claim 54, wherein said subgenomic region includes nucleotides 5346 through 6935.
86. The probe produced by the method of claim 85.
87. The method of claim 54, wherein said subgenomic region includes nucleotides 6938 through 7573.
88. The probe produced by the method of claim 87.
89. A method of making a probe to detect Norwalk-related viruses, comprising the steps of: selecting one or more nucleotide sequences from the group consisting of GTTGCTGTTGGCATTAACA,
TAGTGGCATGGGTATTTC, ATTATAGTTTCTTGCATA, AGCAAAGTCATACATGAAAT, and ACTCACCCAAATCCTCCA; producing said nucleotide sequence by chemical methods or in an expression system.
90. The probe produced by the method of claim 89.
91. A kit for detecting an immune response to Norwalk virus, comprising: a container including a protein encoded by the Norwalk virus genome shown in Table 2 or fragments or derivatives thereof.
92. The kit of claim 91, wherein said protein is selected from the group consisting of the protein encoded by nucleotides 1 through 7753, the protein encoded by nucleotides 146 through 5359, the protein encoded by nucleotides 5337 through 7573, the protein encoded by nucleotides 5346 through 6935, the protein encoded by nucleotides 6938 through 7573 and any combination thereof.
93. A kit for detecting an immune response to a Norwalk-related virus, comprising: a container including a protein encoded by the genome for said Norwalk-related virus.
94. A method of detecting an immune response to Norwalk virus, comprising the steps of: collecting a serum sample from an individual suspected of having been exposed to Norwalk virus; selecting a protein encoded by the Norwalk virus genome shown in Table 2 or fragments or derivatives thereof; adding said selected protein to said serum in a diagnostic assay under conditions allowing said selected protein and the serum to react; and measuring the amount of reaction of said serum and said selected protein.
95. The method of claim 94, wherein said diagnostic assay is selected from the group consisting of enzyme-linked immunosorbent assays, radioimmunoassays and immunoblots.
96. The method of claim 94, wherein said selected protein is a capsid protein.
97. The method of claim 94, wherein said selected protein has the intrinsic property of being able to form particle(s).
98. The method of claim 94, wherein said selected protein is selected from the group consisting of the protein encoded by nucleotides 1 through 7753, the protein encoded by nucleotides 146 through 5359, the protein encoded by nucleotides 5337 through 7573, the protein encoded by nucleotides 5346 through 6935, the protein encoded by nucleotides 6938 through 7573 and any combination thereof.
99. A diagnostic assay to detect an immune response to Norwalk virus, comprising: selecting a protein encoded in Norwalk virus genome shown in Table 2 or fragments or derivatives thereof; using said protein as an antigen; adding post-infection serum from a Norwalk infected individual under conditions allowing said serum to react with said antigen; and measuring the amount of reaction of said serum and said antigen.
100. The method of claim 99, wherein said protein is a capsid protein.
101. The method of claim 99, wherein said protein has the intrinsic property of being able to form particle(s).
102. The method of claim 99, selected from the group consisting of the protein encoded by nucleotides 1 through 7753, the protein encoded by nucleotides 146 through 5359, the protein encoded by nucleotides 5337 through 7573, the protein encoded by nucleotides 5346 through 6935, the protein encoded by nucleotides 6938 through 7573 and any combination thereof.
103. A kit for detecting Norwalk viruses and Norwalk-related viruses, comprising: a container including at least one antiserum made from a protein encoded by the Norwalk virus genome shown in Table 2 or from a fragment or derivative of said genome.
104. The kit of claim 102, wherein said protein is selected from the group consisting of the protein encoded by nucleotides 1 through 7753, the protein encoded by nucleotides 146 through 5359, the protein encoded by nucleotides 5337 through 7573, the protein encoded by nucleotides 5346 through 6935, the protein encoded by nucleotides 6938 through 7573 and any combination thereof.
105. A method of producing antibodies to Norwalk and Norwalk- related viruses, comprising: immunizing animals with a protein encoded by the Norwalk virus genome shown in Table 2 or fragments or derivatives thereof.
106. The method of claim 105, wherein said protein is selected from the group consisting of the protein encoded by nucleotides 1 through 7753, the protein encoded by nucleotides 146 through 5359, the protein encoded by nucleotides 5337 through 7573, the protein encoded by nucleotides 5346 through 6935, the protein encoded by nucleotides 6938 through 7573 and any combination thereof.
107. A vaccine for Norwalk virus, comprising: a Norwalk virus antigen encoded by the cDNA sequence of Norwalk virus shown in Table 2 or fragments or derivatives thereof.
108. The vaccine of claim 107, wherein said antigen is produced using nucleotides 146 through 5359 of the Norwalk virus genome shown in Table 2 or a derivative thereof.
109. The vaccine of claim 107, wherein said antigen is produced using nucleotides 5337 through 7573 of the Norwalk virus genome shown in Table 2 or a derivative thereof.
110. The vaccine of claim 107, wherein said antigen is produced using nucleotides 5346 through 6935 of the Norwalk virus genome shown in Table 2 or a derivative thereof.
111. The vaccine of claim 107, wherein said antigen is produced using nucleotides 6938 through 7573 of the Norwalk virus genome shown in Table 2 or a derivative thereof.
112. The vaccine of claim 107, wherein said antigen has the intrinsic property of being able to form particle (s).
113. A method of immunizing an individual against Norwalk virus, comprising the step of: orally or parenterally administering an immunologically effective dose(s) of the vaccine of claim 107.
114. A method of immunizing an individual against Norwalk virus, comprising the steps of: orally and parenterally administering an immunologically effective dose of the vaccine of claim 107.
115. A cDNA sequence of the human calicivirus Sopporo genome shown in Figure 9 and fragments and derivatives thereof, said fragments and derivatives having sufficient size and nucleotide homology to bind a Norwalk or Norwalk-related virus genome.
116. A protein encoded by nucleotides including nucleotides 1 through 551 of the human calicivirus Sopporo genome shown in Figure 9 or fragments or derivatives thereof.
117. A cDNA subclone of the human calicivirus Sopporo genome comprising nucleotides 1 through 149 and fragments and derivatives thereof, said fragments and derivatives having sufficient size and nucleotide homology to bind a Norwalk or Norwalk-related virus genome.
118. A cDNA subclone of the human calcicivirus Sopporo genome comprising nucleotides 113 through 551 and fragments and derivatives thereof, said fragments and derivatives having sufficient size and nucleotide homology to bind a Norwalk or Norwalk-related virus genome.
119. A cDNA sequence of the Day care calicivirus genome shown in Figure 9 and fragments and derivatives thereof, said fragments and derivatives having sufficient size and nucleotide homology to bind a Norwalk or Norwalk-related virus genome.
120. A cDNA sequence of the SRSV/KY/89 genome shown in Figure 12 and fragments and derivatives thereof, said fragments and derivatives having sufficient size and nucleotide homology to bind a Norwalk or Norwalk-related virus genome.
121. A cDNA sequence of the human calicivirus Houston shown in Table 10 and fragments and derivatives thereof, said fragments and derivatives having sufficient size and nucleotide homology to bind a Norwalk or Norwalk-related virus genome.
122. A cDNA subclone of a primate calicivirus comprising the sequence TGGACGGACC TGCTGTTGAA GATCTCTTCA
AANGGCTCGA ACGACCAAAG CACGATCGGT ATTGTGTTGA CTACGCAAAG TGGGACTCAA CCCANCCACCA AAAGTAACAT CCAATCAATN GACATC and fragments and derivatives thereof, said fragments and derivatives having sufficient size and nucleotide homology to bind a Norwalk or Norwalk-related virus genome.
123. A cDNA subclone of a primate calicivirus comprising the sequence GTGANATGNN ACATCTTCGA CTCGATGGAC CTATTCACAT ATGGTGATGA CGGTGTCTAC ATCGTCCCAC CACTATATCA TCTGTCATGC CCAAGTCTTC ACCAACCTGA AAC and fragments and derivatives thereof, said fragments and derivatives having sufficient size and nucleotide homology to bind a Norwalk or Norwalk-related virus genome.
124. A method of detecting an immune response to Norwalk or a Norwalk related virus, comprising the steps of: collecting a serum sample from an individual suspected of having been exposed to Norwalk or a Norwalk related virus; selecting a protein encoded by the genomic sequence of a
Norwalk-related virus or fragments or derivatives thereof, said fragments and derivatives having sufficient size and nucleotide homology to bind a Norwalk or Norwalk-related virus genome; adding said selected protein to said serum in a diagnostic assay under conditions allowing the selected protein and the serum to react; and measuring the amount of reaction of said serum and said selected protein.
125. The method of claim 124, wherein said diagnostic assay is selected from the group consisting of enzyme-linked immunosorbent assays, radioimmunoassays and immunoblots.
126. The method of claim 124, wherein said genomic sequence is the cDNA sequence of claim 117.
127. The method of claim 124, wherein said genomic sequence is the cDNA sequence of claim 119.
128. The method of claim 124, wherein said genomic sequence is the cDNA sequence of claim 120.
129. The method of claim 124, wherein said genomic sequence is the cDNA sequence of claim 121.
130. The method of claim 124, wherein said genomic sequence is the cDNA sequence of claim 122.
131. The method of claim 124, wherein said genomic sequence is the cDNA sequence of claim 123.
132. A kit for detecting Norwalk viruses and Norwalk-related viruses, comprising: a container including at least one antiserum made from a protein encoded by genomic sequence of a Norwalk-related virus genome or from a fragment or derivative said genomic sequence, said fragments and derivatives having sufficient size and nucleotide homology to bind a Norwalk or Norwalk-related virus genome.
133. The kit of claim 132, wherein said genomic sequence is the cDNA sequence of claim 117.
134. The kit of claim 132, wherein said genomic sequence is the cDNA sequence of claim 119.
135. The kit of claim 132, wherein said genomic sequence is the cDNA sequence of claim 120.
136. The kit of claim 132, wherein said genomic sequence is the cDNA sequence of claim 121.
137. The kit of claim 132, wherein said genomic sequence is the cDNA sequence of claim 122.
138. The kit of claim 132, wherein said genomic sequence is the cDNA sequence of claim 123.
139. A chimeric protein, comprising: a protein encoded by a Norwalk virus genome combined with a protein encoded by a genome of a Norwalk-related virus.
140. A method of detecting an immune response to Norwalk virus, comprising the steps of: collecting a serum sample from an individual suspected of having been exposed to Norwalk virus; adding said the chimeric protein of claim 139 to said serum in a diagnostic assay under conditions allowing chimeric protein and the serum to react; and measuring the amount of reaction of said serum and said chimeric protein.
141. A vaccine for Norwalk or Norwalk related viruses, comprising the chimeric protein of claim 139 used as an antigen.
142. A kit for detecting Norwalk or Norwalk-related related viruses, comprising: a container including at least one antiserum made from the chimeric protein of claim 139.
PCT/US1993/008447 1992-09-07 1993-09-07 Methods and reagents to detect and characterize norwalk and related viruses WO1994005700A2 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU48514/93A AU4851493A (en) 1992-09-07 1993-09-07 Methods and reagents to detect and characterize norwalk and related viruses
JP6507522A JPH08500250A (en) 1992-09-07 1993-09-07 Methods and reagents for detecting and characterizing Norwalk and related viruses

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US94136592A 1992-09-07 1992-09-07
US07/941,365 1992-09-08

Publications (2)

Publication Number Publication Date
WO1994005700A2 true WO1994005700A2 (en) 1994-03-17
WO1994005700A3 WO1994005700A3 (en) 1994-08-04

Family

ID=25476348

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/008447 WO1994005700A2 (en) 1992-09-07 1993-09-07 Methods and reagents to detect and characterize norwalk and related viruses

Country Status (3)

Country Link
JP (1) JPH08500250A (en)
AU (1) AU4851493A (en)
WO (1) WO1994005700A2 (en)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1186890A1 (en) * 1999-06-22 2002-03-13 Japan as represented by Director General of National Institute of Infectious Diseases Srsv detection kit
WO2002029119A1 (en) 2000-09-29 2002-04-11 Bml, Inc. Method of detecting norwalk-like virus (gi)
WO2002040509A1 (en) * 2000-11-15 2002-05-23 Bml, Inc. Antibody against norwalk virus and method of detecting virus by using the antibody
US6503717B2 (en) 1999-12-06 2003-01-07 Sangamo Biosciences, Inc. Methods of using randomized libraries of zinc finger proteins for the identification of gene function
US6630300B2 (en) 2000-11-21 2003-10-07 Tosoh Corporation Oligonucleotides and method for characterizing and detecting Genogroup II type small round structured virus
US6942865B2 (en) * 1989-11-08 2005-09-13 Baylor College Of Medicine Methods and reagents to detect and characterize norwalk and related viruses
US7527801B2 (en) 2005-11-22 2009-05-05 Novartis Vaccines And Diagnostics, Inc. Norovirus and Sapovirus antigens
US7534775B2 (en) 2004-04-08 2009-05-19 Sangamo Biosciences, Inc. Methods and compositions for modulating cardiac contractility
WO2012006293A1 (en) 2010-07-06 2012-01-12 Novartis Ag Norovirus derived immunogenic compositions and methods
US20130052216A1 (en) * 2010-01-21 2013-02-28 Ligocyte Pharmaceuticals, Inc. Targeted heterologous antigen presentation on calicivirus virus-like particles
EP3299030A1 (en) 2011-07-11 2018-03-28 Takeda Vaccines, Inc. Parenteral norovirus vaccine formulations
EP3382011A1 (en) 2008-08-08 2018-10-03 Takeda Vaccines, Inc. Virus-like particles comprising composite capsid amino acid sequences for enhanced cross reactivity
CN112522276A (en) * 2020-12-15 2021-03-19 武汉纽福斯生物科技有限公司 EMC1 nucleotide sequence and application thereof
WO2023060086A1 (en) 2021-10-04 2023-04-13 Takeda Vaccines, Inc. Methods for determining norovirus-reactive antibodies
US11926817B2 (en) 2019-08-09 2024-03-12 Nutcracker Therapeutics, Inc. Microfluidic apparatus and methods of use thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3887340B2 (en) * 2003-03-31 2007-02-28 デンカ生研株式会社 Norovirus or Sapovirus specimen dilution and virus detection reagent

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991007502A1 (en) * 1989-11-08 1991-05-30 Baylor College Of Medicine Methods and reagents to detect and characterize norwalk and related viruses
WO1992016543A1 (en) * 1991-03-25 1992-10-01 Genelabs Incorporated NORWALK VIRUS HUMAN GASTROENTERITIS AGENT AND MOLECULAR CLONING OF CORRESPONDING cDNAs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991007502A1 (en) * 1989-11-08 1991-05-30 Baylor College Of Medicine Methods and reagents to detect and characterize norwalk and related viruses
WO1992016543A1 (en) * 1991-03-25 1992-10-01 Genelabs Incorporated NORWALK VIRUS HUMAN GASTROENTERITIS AGENT AND MOLECULAR CLONING OF CORRESPONDING cDNAs

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DE LEON R. ET AL: 'DETECTION OF NORWALK VIRUS IN STOOL SPECIMENS BY REVERSE TRANSCRIPTASE- POLYMERASE CHAIN REACTION AND NONRADIOACTIVE OLIGOPROBES.' J CLIN MICROBIOL vol. 30, no. 12, 1992, *
MATSUI S.M. ET AL: 'THE ISOLATION AND CHARACTERIZATION OF A NORWALK VIRUS-SPECIFIC COMPLEMENTARY DNA.' J CLIN INVEST vol. 87, no. 4, 1991, pages 1456 - 1461 *

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6942865B2 (en) * 1989-11-08 2005-09-13 Baylor College Of Medicine Methods and reagents to detect and characterize norwalk and related viruses
US8357792B2 (en) 1999-06-22 2013-01-22 Japan As Represented By Director-General National Institute Of Infectious Diseases SRSV detection kit
US8067560B2 (en) 1999-06-22 2011-11-29 Japan As Represented By Director-General National Institute Of Infectious Diseases SRSV detection kit
EP1186890A1 (en) * 1999-06-22 2002-03-13 Japan as represented by Director General of National Institute of Infectious Diseases Srsv detection kit
US7575753B2 (en) 1999-06-22 2009-08-18 Japan As Represented By Director-General National Institute Of Infectious Diseases SRSV detection kit
US7067638B1 (en) * 1999-06-22 2006-06-27 Japan As Represented By Director-General National Institute Of Infectious Diseases Small round structured virus (SRSV) detection kit
EP1186890A4 (en) * 1999-06-22 2005-05-04 Jp Nat Inst Infectious Disease Srsv detection kit
US7943553B2 (en) 1999-12-06 2011-05-17 Sangamo Biosciences, Inc. Randomized libraries of zinc finger proteins
US7491531B2 (en) 1999-12-06 2009-02-17 Sangamo Biosciences, Inc. Randomized libraries of zinc finger proteins
US6503717B2 (en) 1999-12-06 2003-01-07 Sangamo Biosciences, Inc. Methods of using randomized libraries of zinc finger proteins for the identification of gene function
US7202032B2 (en) 2000-09-29 2007-04-10 Bml, Inc. Method of detecting Norwalk-like virus (GI)
WO2002029119A1 (en) 2000-09-29 2002-04-11 Bml, Inc. Method of detecting norwalk-like virus (gi)
EP1329522A1 (en) * 2000-09-29 2003-07-23 BML, Inc. Method of detecting norwalk-like virus (gi)
EP1329522A4 (en) * 2000-09-29 2005-07-13 Bml Inc Method of detecting norwalk-like virus (gi)
WO2002040509A1 (en) * 2000-11-15 2002-05-23 Bml, Inc. Antibody against norwalk virus and method of detecting virus by using the antibody
US6630300B2 (en) 2000-11-21 2003-10-07 Tosoh Corporation Oligonucleotides and method for characterizing and detecting Genogroup II type small round structured virus
US7339041B2 (en) 2000-11-21 2008-03-04 Tosoh Corporation Oligonucleotides and method for characterizing and detecting Genogroup II type small round structured virus
US7534775B2 (en) 2004-04-08 2009-05-19 Sangamo Biosciences, Inc. Methods and compositions for modulating cardiac contractility
US8119145B2 (en) 2005-11-22 2012-02-21 Novartis Vaccines & Diagnostics, Inc. Methods for producing norovirus VLPS
EP2360175A2 (en) 2005-11-22 2011-08-24 Novartis Vaccines and Diagnostics, Inc. Norovirus and Sapovirus virus-like particles (VLPs)
US8124104B2 (en) 2005-11-22 2012-02-28 Novartis Vaccines & Diagnostics, Inc Norovirus and sapovirus antigens
US8142793B2 (en) 2005-11-22 2012-03-27 Novartis Vaccines & Diagnostics, Inc. Norovirus and Sapovirus antigens
US7527801B2 (en) 2005-11-22 2009-05-05 Novartis Vaccines And Diagnostics, Inc. Norovirus and Sapovirus antigens
US9428739B2 (en) 2005-11-22 2016-08-30 Novartis Vaccines And Diagnostics, Inc. Norovirus and Sapovirus antigens
EP3382011A1 (en) 2008-08-08 2018-10-03 Takeda Vaccines, Inc. Virus-like particles comprising composite capsid amino acid sequences for enhanced cross reactivity
US20130052216A1 (en) * 2010-01-21 2013-02-28 Ligocyte Pharmaceuticals, Inc. Targeted heterologous antigen presentation on calicivirus virus-like particles
US8980275B2 (en) * 2010-01-21 2015-03-17 Takeda Vaccines, Inc. Targeted heterologous antigen presentation on calicivirus virus-like particles
EP3153578A1 (en) 2010-07-06 2017-04-12 Novartis Ag Norovirus derived immunogenic compositions and methods
WO2012006293A1 (en) 2010-07-06 2012-01-12 Novartis Ag Norovirus derived immunogenic compositions and methods
EP3299030A1 (en) 2011-07-11 2018-03-28 Takeda Vaccines, Inc. Parenteral norovirus vaccine formulations
EP4112074A1 (en) 2011-07-11 2023-01-04 Takeda Vaccines, Inc. Parenteral norovirus vaccine formulations
US11926817B2 (en) 2019-08-09 2024-03-12 Nutcracker Therapeutics, Inc. Microfluidic apparatus and methods of use thereof
CN112522276A (en) * 2020-12-15 2021-03-19 武汉纽福斯生物科技有限公司 EMC1 nucleotide sequence and application thereof
CN112522276B (en) * 2020-12-15 2022-07-15 武汉纽福斯生物科技有限公司 EMC1 nucleotide sequence and application thereof
WO2023060086A1 (en) 2021-10-04 2023-04-13 Takeda Vaccines, Inc. Methods for determining norovirus-reactive antibodies

Also Published As

Publication number Publication date
WO1994005700A3 (en) 1994-08-04
AU4851493A (en) 1994-03-29
JPH08500250A (en) 1996-01-16

Similar Documents

Publication Publication Date Title
US6942865B2 (en) Methods and reagents to detect and characterize norwalk and related viruses
CA2065287C (en) New hcv isolates
US9945856B2 (en) Coronavirus, nucleic acid, protein, and methods for the generation of vaccine, medicaments and diagnostics
Blake et al. Phylogenetic relationships of aquatic birnaviruses based on deduced amino acid sequences of genome segment A cDNA
JP2693908B2 (en) NANBV diagnostics
WO1994005700A2 (en) Methods and reagents to detect and characterize norwalk and related viruses
US5559014A (en) Methods and reagents to detect and characterize Norwalk and related viruses
US6156883A (en) Polyclonal and monoclonal antibodies to Norwalk virus and methods for making them
IE83377B1 (en) New HCV isolate J-1
KR20070028547A (en) Identifying virally infected and vaccinated organisms
Collins et al. Bovine viral diarrhea virus quasispecies during persistent infection
Weston et al. Nucleotide sequence variation in salmonid alphaviruses from outbreaks of salmon pancreas disease and sleeping disease
KR0178399B1 (en) Enterically transmitted non-a/non-b hepatitis viral agent and characteristic epitopes thereof
US7320857B2 (en) Characterization of the earliest stages of the severe acute respiratory syndrome (SARS) virus and uses thereof
WO1995021922A2 (en) Non-a, non-b, non-c, non-d, non-e hepatitis reagents and methods for their use
Schaffer Caliciviruses
Wang et al. Hepatitis E virus
JP2007513604A (en) New enteroviruses, vaccines, medicines and diagnostic kits
US6696562B1 (en) Avian astrovirus
US6451578B1 (en) Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6720166B2 (en) Non-a, non-b, non-c, non-c, non-d, non-e hepatitis reagents and methods for their use
Ohyama et al. Detection and nucleotide sequence analysis of human caliciviruses (HuCVs) from samples in non-bacterial gastroenteritis outbreaks in Hokkaido, Japan
Jiang IV, 4. Development of serological and molecular tests for the diagnosis of calicivirus infections
US6586568B1 (en) Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
Banda Characterization of field strains of Infectious bursal disease virus (IBDV) using molecular techniques

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA