WO1994026902A1 - Nucleic acids encoding human astrovirus serotype 2 and uses thereof - Google Patents

Nucleic acids encoding human astrovirus serotype 2 and uses thereof Download PDF

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Publication number
WO1994026902A1
WO1994026902A1 PCT/US1994/005287 US9405287W WO9426902A1 WO 1994026902 A1 WO1994026902 A1 WO 1994026902A1 US 9405287 W US9405287 W US 9405287W WO 9426902 A1 WO9426902 A1 WO 9426902A1
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Prior art keywords
nucleic acid
serotype
leu
human astrovirus
thr
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PCT/US1994/005287
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French (fr)
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Stephan S. Monroe
Roger I. Glass
Marion Koopmans
Baoming Jiang
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United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Priority to AU68321/94A priority Critical patent/AU6832194A/en
Publication of WO1994026902A1 publication Critical patent/WO1994026902A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983Viruses
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011Details
    • C12N2770/12011Astroviridae
    • C12N2770/12022New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to astroviruses.
  • the present invention relates to genomic and subgenomic nucleic acids of Human Astrovirus serotype 2.
  • Astroviruses are 28-nm nonenveloped, viruses that were initially identified from the feces of infants with gastroenteritis by their distinctive ultrastructural features of characteristic five- or six-pointed surface stars (Appleton, J. et al., Lancet, 1:1297 (1975); Madeley, C. R. et al., Lancet, 2:451-452 (1975)). These nonenveloped agents were subsequently determined to be positive-strand RNA viruses (Herring, A. J. et al., J. Gen. Virol., 53:47 (1981); Monroe, S. S. et al., J. Virol, 65:641 (1991); Matsui, S. M. et al., J.
  • Clinical signs associated with astrovirus infection include nausea, vomiting, non-bloody diarrhea, abdominal cramps, headaches, fever, chills and myalgia (LeBaron, C. W. et al., Morbidity and Mortality Weekly Report (Centers for Disease Control), Vol. 39 (April 27, 1990)). Although most transmission is probably person-to-person among children, contaminated water and shellfish have also given rise to outbreaks in England (Kurtz, J. B. et al., (Ciba Foundation Symposium; 128), Chichester, UK: John Wiley & Sons Ltd., pp. 92-107 (1987)). Asymptomatic shedding of astrovirus has been documented (Ashley, C. R. et al., /. Clin.
  • H-Astl human astrovirus serotype 1
  • 1034 nucleotides from the 3' end of genomic RNA Willcocks, M. M. et al., Arch. Virol., 124:279 - 289 (1992), a 289 nucleotide immunoreactive epitope which overlaps the
  • the fastidious nature of the virus coupled with extremely low levels of viral RNA generated by the organism during growth have made conventional sequencing approaches unpredictable and unreliable.
  • the art has yet to provide sequences for human astrovirus type 2.
  • the present invention satisfies this need by utilizing a unique combination of sequencing techniques to identify, diagnose, and treat astrovirus infection by providing nucleotide sequences for the complete genomic and subgenomic RNA of serotype 2 (H-Ast2) and analysis of the entire genomic RNA of H-Ast2.
  • the present invention also provides the surprising discovery of a ribosomal frame shift occurring in one open reading frame which results in encoding of a fusional nonstructural polyprotein.
  • the present invention provides a nucleic acid encoding human Astrovirus serotype 2, or a unique fragment thereof.
  • the sequence for the genomic RNA of human astrovirus was sequenced from virion RNA and cDNA and was found to contain 6797 nucleotides, exclusive of the poly(A) tail, organized into three open reading frames (defined as Open Reading Frames (ORFs) la, lb, and 2)).
  • ORFs Open Reading Frames
  • a ribosomal frameshift site is identified in the overlap region of ORFs la and lb at position 2794. This translation frameshift results in the suppression of in-frame amber termination at the end of ORF la and the synthesis of a nonstructural, fusion polyprotein that contains the putative protease and RNA-dependent RNA polymerase.
  • the present invention also provides the sequence of a nucleic acid encoding a subgenomic RNA of human Astrovirus serotype 2.
  • This 2484-nucleotide RNA contains a single open readmg frame, which encodes a protein with a molecular mass of about 88 kDa.
  • the present invention proides purified antigenic polypeptide fragments encoded by the nucleic acid encoding human Astrovirus serotype 2.
  • the present invention provides a purified antigenic polypeptide fragment encoded by the nucleic acid encoding open reading frame 2, or a unique portion thereof, in a pharmaceutically acceptable carrier.
  • the present invention also provides isolated nucleic acids capable of selectively hybridizing with the nucleic acid of human Astrovirus serotype 2 including, but not limited to, primers and probes for utilization in polymerase chain reaction (PCR) and other nucleic acid amplification techniques.
  • PCR polymerase chain reaction
  • the present invention provides vectors comprising the nucleic acid encoding human Astrovirus serotype 2 or a unique fragment thereof and provides the vector in a host capable of expressing the polypeptide encoded by that nucleic acid.
  • the present invention also provides a purfied monoclonal antibody specifically reactive with human Astrovirus serotype 2 and a method of detection of human Astrovirus serotype 2 utilizing the antibodies of the present invention.
  • Fig. 1 shows the genomic organization of human astrovirus. The locations of three ORFs, the first methionine (Met), and the frameshift site are indicated. The predicted transmembrane helices (MB), protease (Pro), nuclear
  • RECTIFIED SHEET (RULE 91) ISA/EP localization signal (NLS), and RNA-dependent RNA polymerase (Pol) are indicated by stippled boxes.
  • Fig. 2 shows (A) Nucleotide sequence and predicted RNA secondary structure in the overlap region of astrovirus ORFs la and lb.
  • the putative frameshift site ("shifty" heptanucleotide sequence) is underlined and the termination codon for ORF la is boxed.
  • the RNA secondary structure was predicted using the RNAFOLD program (Zuker, M. et al, (1981)).
  • a potential pseudoknot structure was predicted by searching the region downstream of the stem-loop structure for sequences complementary to the loop sequence. Three base pairs may be sufficient for the pseudoknot formation (Pleij, C. W. A. et al., Trends Biochem.
  • Fig. 3 shows the predicted secondary structure at the 3' end of astrovirus RNA sequences.
  • the structures were calculated by the method of Zuker and Stiegler (Zuker, M. et al., Nucleic Acids Res., 9:133 - 148 (1981)).
  • the H-Astl structure contains a total of 154 nucleotides including 134 bases from the reported 3' end sequence (Willcocks, M. M. et al., Arch. Virol., 124:279 - 289 (1992)) plus 20 additional adenine residues.
  • the H-Ast2 structure contains 156 nucleotides corresponding to bases 2349 to 2504 in SEQ ID NO: 3.
  • the region of the poly(A) tract involved in stem I is outlined with a box. The two insertions of the loop between stems I and II are shown with arrowheads. The residues within the
  • RECTIFIED SHEET conserved stem II that vary between the two serotypes are indicated.
  • the present invention provides an isolated nucleic acid encoding human Astrovirus serotype 2 as set forth in the Sequencing Listing as SEQ ID NO: 1, or a unique fragment thereof.
  • the invention also provides a nucleic acid capable of selectively hybridizing the DNA, RNA and cDNA sequences which can be derived from SEQ ID NO: 1. While SEQ ID NO: 1 is an RNA sequence, the invention also provides the corresponding DNA sequence.
  • isolated is meant identifiably separated from other nucleic acids found in the naturally occurring organism.
  • capable of selectively hybridizing is meant a sequence which does not hybridize with other nucleic acids to prevent an adequate positive hybridization with nucleic acids from human Astrovirus serotype 2.
  • unique fragment is meant a fragment that can selectively hybridize with a RNA, DNA or cDNA sequence derived from the novel sequences.
  • nucleic acid is an open reading frame of 2,387 bases comprising nucleotides 4325 through 6712 (designated open reading frame 2 (ORF 2)) as set forth in SEQ ID NO: 1.
  • ORF 2 open reading frame 2
  • This specific nucleic acid can be used to detect human Astrovirus serotype 2 in methods such as polymerase chain reaction, ligase chain reaction and hybridization.
  • the ORF 2 sequence can be utilized to produce an antigentic protein or protein fragment.
  • nucleic acid can be utilized to find sequences homologous with nucleotide sequences present in other human or animal astroviruses. Such an amino acid sequence shared with other astroviruses can be used for example to simultaneously detect related strains or as a basis for a multiprotective vaccine.
  • An isolated nucleic acid capable of selectively hybridizing with or selectively amplifying a nucleic acid encoding the human Astrovirus serotype 2, or unique fragments thereof is also contemplated.
  • the sequences can be selected based on the nucleotide sequence and the utility of the particular sequence.
  • nucleic acids of the invention are also contemplated as long as the essential structure and function of the polypeptide encoded by the nucleic acids is maintained.
  • fragments used as primers or probes can have substitutions so long as enough complementary bases exist for selective hybridization (Kunkel et al. Methods Enzymol. 1987:154:367, (1987)).
  • the present invention provides an isolated nucleic acid encoding open reading frame la of human Astrovirus serotype 2, comprising nucleotides 83 through 2842 contained in the nucleotide sequence as set forth in the Sequencing Listing SEQ ID NO: 1, or a unique fragment thereof.
  • the open reading frame designated "la” is defined as comprising nucleotides 83 through 2842 contained in the nucleotide sequence set forth in the Sequencing Listing SEQ ID NO: 1 and depicted in FIG 1.
  • an isolated nucleic acid capable of selectively hybridizing with the nucleic acid encoding open reading frame la.
  • the present invention provides an isolated nucleic acid encoding open reading frame lb of human Astrovirus serotype 2, comprising nucleotides 2773 through 4329 contained in the nucleotide sequence as set forth in the Sequencing Listing SEQ ID NO: 1, or a unique fragment thereof.
  • the open reading frame designated "lb” is defined as comprising nucleotides 2773 through 4329 contained in the nucleotide sequence set forth in the Sequencing Listing SEQ ID NO: 1 and depicted in FIG 1.
  • an isolated nucleic acid capable of selectively hybridizing with the nucleic acid encoding open reading frame lb.
  • Another embodiment of the present invention provides an isolated nucleic acid encoding open reading frame 2 of human Astrovirus serotype 2, comprising nucleotides 4325 through 6712 contained in the nucleotide sequence as set forth in the Sequencing Listing as SEQ ID NO: 1, or a unique fragment thereof.
  • the open reading frame designated "2" is defined as comprising nucleotides 4325 through 6712 contained in the nucleotide sequence set forth in the Sequencing Listing SEQ ID NO: 1 and depicted in FIG 1.
  • an isolated nucleic acid capable of selectively hybridizing with the nucleic acid encoding open reading frame lb.
  • the present invention also provides an isolated nucleic acid encoding open reading frame la/lb of human Astrovirus serotype 2, as set forth in the nucleotide sequence defined in the Sequencing Listing as SEQ ID NO: 2, or a unique fragment thereof.
  • a minus 1 frame shift occurs at position 2712 of the sequence depicted in SEQ ID NO: 2.
  • the open reading frame la/lb can also be identified in FIG 1 and in SEQ ID NO: 1 wherein it comprises nucleotides 83 through 4329.
  • the minus 1 frameshift occurs at position 2794 of the sequence depicted in SEQ ID NO:l.
  • an isolated nucleic acid capable of selectively hybridizing with the nucleic acid encoding open reading frame la/lb.
  • the present invention provides an isolated nucleic acid encoding a subgenomic RNA of human Astrovirus serotype 2, as set forth in the nucleotide sequence defined in the Sequencing Listing as SEQ ID NO: 3, or a unique fragment thereof.
  • the subgenomic RNA of human Astrovirus serotype 2 can also be identified in FIG 1 and in SEQ ID NO:l wherein it comprises nucleotides 4314 through 6797 exclusive of the poly(A) tail.
  • an isolated nucleic acid capable of selectively hybridizing with the nucleic acid encoding the subgenomic RNA of human Astrovirus serotype
  • nucleic acids can be derived as set forth in the examples, given the sequences, it is also possible to synthesize partial sequences and enzymatically combine the partial sequences to make an entire synthetic gene.
  • antigenic polypeptides encoded by the nucleic acids.
  • the invention also provides these antigenic polypeptides in a pharmaceutically acceptable carrier.
  • the amino acid sequence of these polypeptides can be deduced from the nucleotide sequences set forth in the
  • Purified antigenic polypeptide fragments encoded by the nucleic acids of the present invention are also contemplated.
  • purified means the antigen is sufficiently free of contaminants or cell components with which the antigen normally occurs to distinguish the antigen from the contaminants or components.
  • Purified human Astrovirus serotype 2 antigen and antigenic fragments thereof of the present invention are also referred to herein as “the antigen” or "the H-Ast 2 antigen.” It is contemplated that the antigenic fragments can be encoded from any portion of the nucleic acid encoding human Astrovirus serotype 2 as set forth in SEQ ID NO:l, but especially from fragments encoded by the open reading frames la, lb, la/lb, and 2 as described herein.
  • RECTIFIED SHEET (RULE 91) ISA/EP Specifically, one example includes an approximately 88 kDa antigenic polypeptide encoded by an open reading frame of 2387 bases (ORF 2) consisting essentially of the amino acids encoded by nucleotides 4325 through 6797 contained in the nucleotide sequence set forth in the Sequence Listing as SEQ ID NO: 1 .
  • ORF 2 open reading frame of 2387 bases
  • An antigenic fragment of the antigen can be isolated from the whole antigen by chemical or mechanical disruption. The purified fragments thus obtained can be tested to determine their antigenicity and specificity by the methods taught herein. Antigenic fragments of the antigen can also be synthesized directly.
  • An immunoreactive fragment is generally an amino acid sequence of at least about five consecutive amino acids derived from the antigen amino acid sequence.
  • polypeptide fragments of the present invention can also be recombinant proteins obtained by cloning nucleic acids encoding the polypeptide in an expression system capable of producing the antigenic polypeptide or fragments thereof.
  • amino acid sequence of the antigen it is also possible to synthesize, using standard peptide synthesis techniques, peptide fragments chosen to be homologous to immunoreactive regions of the antigen and to modify these fragments by inclusion, deletion or modification of particular amino acids residues in the derived sequences. Thus, synthesis or purification of an extremely large number of peptides derived from the antigen is possible.
  • the amino acid sequences of the present polypeptides can contain an immunoreactive portion of the H-Ast 2 antigen attached to sequences designed to provide for some additional property, such as solubility.
  • the amino acid sequences of an H-Ast 2 antigen can include sequences in which one or more amino acids have been substituted with another amino acid to provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to
  • RECTIFIED SHEET (RULE 91) ISA/EP increase its bio-longevity, alter enzymatic activity, or alter interactions with gastric acidity.
  • the peptide must possess a bioactive property, such as immunoreactivity, immunogenicity, etc.
  • the purified polypeptide fragments thus obtained can be tested to determine their immunogenicity and specificity. Briefly, various concentrations of a putative immunogenically specific fragment are prepared and administered to an animal and the immunological response (e.g., the production of antibodies or cell mediated immunity) of an animal to each concentration is determined. The amounts of antigen administered depend on the subject, e.g. a human or a guinea pig, the condition of the subject, the size of the subject, etc. Thereafter an animal so inoculated with the antigen can be exposed to the virus to test the potential vaccine effect of the specific immunogenic fragment. The specificity of a putative immunogenic fragment can be ascertained by testing sera, other fluids or lymphocytes from the inoculated animal for cross reactivity with other closely related Astroviruses.
  • a vector comprising the nucleic acids of the present invention is also provided.
  • the vectors of the invention can be in a host capable of expressing the antigenic polypeptide fragments contemplated by the present invention.
  • E. coli expression vectors known to one of ordinary skill in the art useful for the expression of the antigen.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • bacilli such as Bacillus subtilus
  • enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species.
  • prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Tip) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters will typically control expression, optionally with an operator sequence, and have
  • RECTIFIED SHEET (RULE 91) ISA/EP initiating and completing transcription and translation. If necessary an amino terminal methionine can be provided by insertion of a Met codon 5' and in-frame with the antigen. Also, the carboxy-terminal extension of the antigenic fragments can be removed using standard oligonucleotide mutagenesis procedures.
  • yeast expression can be used. There are several advantages to yeast expression systems. First, evidence exists that proteins produced in yeast secretion systems exhibit correct disulfide pairing. Second, post- translational glycosylation is efficiently carried out by yeast secretory systems. The Saccharomyces cerevisiae pre-pro-alpha-factor leader region (encoded by the MFa-1 gene) is routinely used to direct protein secretion from yeast (Brake et al., 1984).
  • the leader region of pre-pro-alpha-factor contains a signal peptide and a pro- segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene: this enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage-signal sequence.
  • the antigen coding sequence can be fused in-frame to the pre-pro-alpha-factor leader region. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter.
  • the antigen coding sequence is followed by a translation termination codon which is followed by transcription termination signals.
  • the antigen coding sequences can be fused to a second protein coding sequence, such as Sj26 or ⁇ -galactosidase, used to facilitate purification of the fusion protein by affinity chromatography.
  • a second protein coding sequence such as Sj26 or ⁇ -galactosidase
  • the insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast.
  • Mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active protein.
  • Vectors useful for the expression of antigens in mammalian cells are characterized by insertion of the antigen coding sequence between a strong viral promoter and a polyadenylation signal.
  • the vectors can contain genes conferring either gentamicin or methotrexate resistance for use as selectable markers.
  • ISA/EP antigen and immunoreactive fragment coding sequence can be introduced into a Chinese hamster ovary cell line using a methotrexate resistance-encoding vector. Presence of the vector RNA in transformed cells can be confirmed by Southern analysis and production of a cDNA or oposite strand RNA corresponding to the antigen coding sequence can be confirmed by Northern analysis.
  • suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc.
  • the vectors containing the nucleic acid segments of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts.
  • vectors for the expression of antigen in mammalian cells those similar to those developed for the expression of human gamma-interferon, tissue plasminogen activator, clotting Factor VIII, hepatitis B virus surface antigen, protease Nexinl, and eosinophil major basic protein, can be employed.
  • the vector can include CMV promoter sequences and a polyadenylation signal available for expression of inserted nucleic acid in mammalian cells (such as COS7).
  • nucleic acid sequences can be expressed in hosts after the sequences have been operably linked to, i.e., positioned to ensure the functioning of, an expression control sequence.
  • These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
  • expression vectors can contain selection markers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired nucleic acid sequences sequences (see, e.g., U.S. Patent 4,704,362).
  • Polynucleotides encoding a variant polypeptide may include sequences that facilitate transcription (expression sequences) and translation of the coding sequences such that the encoded polypeptide product is produced. Construction of such polynucleotides is well known in the art.
  • such polynucleotides can include a promoter, a transcription termination site (polyadenylation site in eukaryotic expression hosts), a ribosome binding site, and, optionally, an enhancer for use in eukaryotic expression hosts, and, optionally, sequences necessary for replication of a vector.
  • a purified monoclonal antibody specifically reactive with human Astrovirus serotype 2 is also provided.
  • the antibodies can be specifically reactive with a unique epitope of the antigen or they can also react with epitopes of other organisms.
  • the term “reactive” means capable of binding or otherwise associating nonrandomly with an antigen.
  • “Specifically reactive” as used herein describes an antibody or other ligand that does not cross react substantially with any antigen other than the one specified, in this case, human Astrovirus serotype 2.
  • Antibodies can be made as described in the Examples (see also, Harlow and Lane, Antibodies;
  • antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response.
  • Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells are then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen clone libraries for cells secreting the antigen.
  • the antibody can be bound to a substrate or labeled with a detectable moiety or both bound and labeled.
  • detectable moieties contemplated with the composition of the present invention are those listed below in the description of the diagnostic methods, including fluorescent, enzymatic and radioactive markers.
  • a purified human Astrovirus serotype 2 antigen bound to a substrate and a ligand specifically reactive with the antigen are also contemplated.
  • a purified ligand specifically reactive with the antigen can be an antibody.
  • the antibody can be a monoclonal antibody obtained by standard methods and as described herein.
  • the monoclonal antibody can be secreted by a hybridoma cell line specifically produced for that purpose (Harlow and Lane, 1988).
  • nonhuman polyclonal antibodies specifically reactive with the antigen are within the scope of the present invention.
  • the polyclonal antibody can also be obtained by the standard immunization and purification protocols (Harlow and Lane, 1988).
  • the present invention provides a method of detecting the presence of human Astrovirus serotype 2 in a subject, comprising the steps of contacting an antibody-containing sample from the subject with a detectable amount of the antigenic polypeptide fragment of the present invention and detecting the reaction of the fragment and the antibody, the reaction indicating the presence of the Astrovirus or a previous infection with H-Ast 2.
  • the antigen will be on intact cells containing the antigen, or will be fragments of the antigen.
  • the antibody includes any ligand which binds the antigen, for example, an intact antibody, a fragment of an antibody or another reagent that has reactivity with the antigen.
  • the fluid sample of this method can comprise any body fluid which would contain the antigen or a cell containing the antigen, such as blood, plasma, serum, saliva, feces and urine. Other possible examples of body fluids include sputum, mucus, gastric juice and the like.
  • Enzyme immunoassays such as immunofluorescence assays (IF A), enzyme linked immunosorbent assays (ELISA) and immunoblotting can be readily adapted to accomplish the detection of the antigen.
  • An ELISA method effective for the detection of the antigen can, for example, be as follows: (1) bind the antibody to a substrate; (2) contact the bound antibody with a fluid or tissue sample containing the antigen; (3) contact the above with a secondary antibody bound to a detectable moiety (e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme); (4) contact the above with the substrate for the enzyme; (5) contact the above with a color reagent; (6) observe color change.
  • the above method can be readily modified to detect antibody as well as antigen.
  • MAbs monoclonal antibodies
  • sera or other body fluids from the subject is reacted with the antigen bound to a substrate (e.g. an ELISA 96-well plate). Excess sera is thoroughly washed away.
  • a labeled (enzyme-linked, fluorescent, radioactive, etc.) monoclonal antibody is then reacted with the previously reacted antigen-serum antibody complex. The amount of inhibition of monoclonal antibody binding is measured relative to a control (no patient serum antibody). The degree of monoclonal antibody inhibition is a very specific test for a particular variety or strain since it is based on monoclonal antibody binding specificity.
  • MAbs can also be used for detection directly in cells by lFA.
  • R E CTIFIED SHEET (RULE 91) ISA/EP A micro-agglutination test can also be used to detect the presence of H-Ast 2 in a subject. Briefly, latex beads (or red blood cells) are coated with the antigen and mixed with a sample from the subject, such that antibodies in the tissue or body fluids that are specifically reactive with the antigen crosslink with the antigen, causing agglutination. The agglutinated antigen-antibody complexes form a precipitate, visible with the naked eye or by spectrophotometer. In a modification of the above test, antibodies specifically reactive with the antigen can be bound to the beads and antigen in the tissue or body fluid thereby detected.
  • the antibody can be bound to a substrate and reacted with the antigen. Thereafter, a secondary labeled antibody is bound to epitopes not recognized by the first antibody and the secondary antibody is detected. Since the present invention provides H-Ast 2 antigen for the detection of infectious H-Ast 2 or previous H-Ast 2 infection other serological methods such as flow cytometry and immunoprecipitation can also be used as detection methods.
  • the antigen can be bound to a substrate and contacted by a fluid sample such as serum, urine, saliva, feces or gastric juice.
  • a fluid sample such as serum, urine, saliva, feces or gastric juice.
  • This sample can be taken directly from the patient or in a partially purified form.
  • antibodies specific for the antigen (the primary antibody) will specifically react with the bound antigen.
  • a secondary antibody bound to, or labeled with, a detectable moiety can be added to enhance the detection of the primary antibody.
  • the secondary antibody or other ligand which is reactive either specifically with a different epitope of the antigen or nonspecifically with the ligand or reacted antibody, will be selected for its ability to react with multiple sites on the primary antibody.
  • several molecules of the secondary antibody can react with each primary antibody, making the primary antibody more detectable.
  • detectable moiety will allow visual detection of a precipitate or a color change, visual detection by microscopy, or automated detection by spectrometry, radiometric measurement or the like.
  • detectable moieties include fluorescein and rhodamine (for fluorescence microscopy), horseradish peroxidase (for either light or electron microscopy and biochemical detection), biotin-streptavidin (for light or electron microscopy) and alkaline phosphatase (for biochemical detection by color change).
  • the detection methods and moieties used can be selected, for example, from the list above or other suitable examples by the standard criteria applied to such selections (Harlow and Lane, 1988).
  • the antigen e.g., a purified antigenic polypeptide fragment encoded by open reading frame 2 of this invention can be used in the construction of a vaccine comprising an immunogenic amount of the antigen and a pharmaceutically acceptable carrier.
  • the vaccine can be the entire antigen, the antigen on an intact H-Ast 2 organism, E. coli or other strain, or an epitope specific to the antigen.
  • the vaccine can also be potentially cross-reactive with antibodies to other antigens.
  • the vaccine can then be used in a method of preventing diarrhea or other complications of H-Ast 2 infection.
  • Immunogenic amounts of the antigen can be determined using standard procedures. Briefly, various concentrations of a putative specific immunoreactive epitope are prepared, administered to an animal and the immunological response (e.g., the production of antibodies) of an animal to each concentration is determined.
  • the pharmaceutically acceptable carrier can comprise saline or other suitable carriers (Arnon, R. (Ed.) Synthetic Vaccines, 1:83-92, CRC Press, Inc., Boca
  • An adjuvant can also be a part of the carrier of the vaccine, in which case it can be selected by standard criteria based on the antigen used, the mode of administration and the subject (Arnon, R. (Ed.), 1987).
  • ISA/EP administration can be by oral or sublingual means, or by injection, depending on the particular vaccine used and the subject to whom it is administered.
  • the vaccine can be used as a prophylactic or a therapeutic modality.
  • the invention provides methods of preventing or treating H-Ast 2 infection and the associated diseases by administering the vaccine to a subject.
  • the presence of H-Ast 2 can also be determined by detecting the presence of a nucleic acid specific for H-Ast 2 or the antigens of H-Ast 2 encoded by the nucleic acid.
  • the present invention provides a method of detecting the presence of human Astrovirus serotype 2 in a subject, comprising detecting the presence of the nucleic acid encoding human Astrovirus serotype 2.
  • the specificity of these sequences for H-Ast 2 can be determined by conducting a computerized comparison with known sequences, catalogued in GenBank, a computerized database, using the computer programs Word Search or FASTA of the Genetics Computer Group (Madison, WI), which search the catalogued nucleotide sequences for similarities to the nucleic acid in question.
  • the nucleic acid specific for H-Ast 2 can be detected utilizing a nucleic acid amplification technique, such as polymerase chain reaction or ligase chain reaction.
  • the nucleic acid is detected utilizing direct hybridization or by utilizing a restriction fragment length polymorphism.
  • the present invention provides a method of detecting the presence of H- Ast 2 comprising ascertaining the presence of a nucleotide sequence associated with a restriction endonuclease cleavage site.
  • PCR primers which hybridize only with nucleic acids specific for H-Ast 2 can be utilized. The presence of amplification indicates the presence of the antigen.
  • a restriction fragment of a nucleic acid sample can be sequenced directly using techniques known in the art and described herein and compared to the known unique sequence to detect H-Ast 2.
  • the present invention provides a method of detecting the presence of H-Ast 2 by selective amplification
  • H-Ast 2 can be detected by directly hybridizing the unique sequence with a H-Ast 2 selective nucleic acid probe.
  • the nucleotide sequence could be amplified prior to hybridization by the methods described above.
  • ligase chain reaction involves the use of mismatch probes, i.e., probes which are fully complementary with the target except at the point of the mutation.
  • the target sequence is then allowed to hybridize both with oligonucleotides which are fully complementary and with oligonucleotides containing a mismatch, under conditions which will distinguish between the two.
  • By manipulating the reaction conditions it is possible to obtain hybridization only where there is full complementarity. If a mismatch is present there is significantly reduced hybridization.
  • PCR polymerase chain reaction
  • reverse transcriptase PCR are techniques that amplifies specific nucleic acid sequences with remarkable efficiency.
  • polymerase e.g., a heat stable enzyme Taq polymerase
  • polymerase e.g., a heat stable enzyme Taq polymerase
  • the nucleic acid can be denatured at high temperatures (e.g., 95°C) and then reannealed in the presence of a large molar excess of oligonucleotides.
  • the oligonucleotides oriented with their 3' ends pointing towards each other, hybridize to opposite strands of the target sequence and prime enzymatic extension along the nucleic acid template.
  • the end product is then denatured again for another cycle. After this three-step cycle has been repeated several times, amplification of a nucleic acid segment by more than one million-fold can be achieved.
  • the resulting nucleic acid may then be directly sequenced.
  • PCR may be followed by restriction endonuclease digestion with subsequent analysis of the resultant products. Nucleotide substitutions can result in the gain or loss of specific restriction endonuclease site.
  • the gain or loss of a restriction endonuclease recognition site facilitates the detection of the organism using restriction fragment length polymorphism (RFLP) analysis or by detection of the presence or absence of a polymorphic restriction endonuclease site in a PCR product that spans the sequence of interest.
  • RFLP restriction fragment length polymorphism
  • nucleic acid is obtained, for example from the blood, gastric specimen, saliva, dental plaque, or other bodily fluids of the subject suspected of containing H-Ast 2 is digested with a restriction endonuclease, and subsequently separated on the basis of size by agarose gel electrophoresis.
  • the Southern blot technique can then be used to detect, by hybridization with labeled probes, the products of endonuclease digestion.
  • the patterns obtained from the Southern blot can then be compared.
  • H-Ast 2 nucleic acid is detected by determining the number of bands detected and comparing this number to the nucleic acid from H-Ast 2.
  • SSCA Single strand conformational analysis
  • primers for PCR and LCR are usually about 20 bp in length and the preferable range is from 15-25 bp. Better amplification is obtained when both primers are the same length and with roughly the same nucleotide composition. Denaturation of strands usually takes place at 94°C and extension from the primers is usually at 72°C. The annealing temperature varies according to the sequence under investigation. Examples of reaction times are: 20 mins denaturing; 35 cycles of 2 min, 1 min, 1 min for annealing, extension and denaturation; and finally a 5 min extension step.
  • PASA specific alleles
  • PASA also known as allele specific amplification
  • PASA involves amplification with two oligonucleotide primers such that one is allele-specific.
  • the desired allele is efficiently amplified, while the other allele(s) is poorly amplified because it mismatches with a base at or near the 3* end of the allele-specific primer.
  • PASA or the related method of PAMSA may be used to specifically amplify the mutation sequences of the invention. Where such amplification is done on H-Ast 2 isolates or samples obtained from an individual, it can serve as a method of detecting the presence of H-Ast 2.
  • LCR ligase chain reaction
  • cDNA cloning and RNA blot hybridization Cell-culture-adapted human astrovirus serotype 2 (H-Ast2) was obtained from Dr. John Kurtz (Oxford, England), was plaque purified three times before use, and was propagated in LLCMK2 cells (ATCC CCL7.1) as previously described (Monroe, S. S. et al.). Double-stranded cDNA was synthesized from the polyadenylated fraction of RNA isolated from astrovirus infected cells (cDNA Cloning Kit, Boehringer Mannheim Biochemicals,), and was cloned into the pBluescript II plasmid vector (Stratagene).
  • Recombinant clones were screened for astrovirus specific inserts by hybridization of [ 32 P]-labelled RNA transcribed in vitro from individual cDNA clones to total cytoplasmic RNA isolated from uninfected and astrovirus-infected cells.
  • RNA transcripts from the insert in one cDNA clone hybridized to both the 7.2- and 2.8-kb viral RNAs as evidenced by autoradiography of RNA blot hybridization (not shown). The hybridization reactivity was first detectable at 12
  • RECTIFIED SHEET (RULE 91) ISA/EP hours postinfection, coincident with detection of these RNAs by metabolic labeling (Monroe, S. S. et al.).
  • the hybridization of a cRNA probe to both viral specific RNAs confirmed that the 2.8-kb RNA contains sequences present in the larger species, with the relative intensities indicating that the smaller RNA is present in at least a 10-fold molar excess.
  • Sequence information for the subgenomic RNA was obtained by three approaches: 1) sequencing of supercoiled DNA from two plasmids with cDNA inserts, 2) sequencing of RNA purified from virions, and 3) amplification of genomic RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), followed by sequencing of the double-stranded DNA products. Plasmid DNA and PCR products were sequenced using modified T7 DNA polymerase (Sequenase2 ® , US Biochemicals). Sequence information from the 5' end of the original clone was used to generate oligonucleotide primers for a second round of cDNA cloning from cytoplasmic RNA.
  • RNA sequencing products were purified by gel filtration (Miniprep Spun Column, Pharmacia) before sequencing.
  • RNA was sequenced using reverse transcriptase and dideoxynucleotide terminators (RNA Sequencing Kit, Boehringer Mannheim Biochemicals) with primers derived from the sequence of the cDNA clones.
  • RNA Sequencing Kit reverse transcriptase and dideoxynucleotide terminators
  • ambiguities were resolved through the use of RT-PCR sequencing over the same regions.
  • the information from the three independent sequencing strategies was combined to arrive at a consensus sequence for the entire subgenomic region (Devereux, J. P et al., Nucleic Acids Res., 12:387 - 395 (1984).
  • sequence derived from cDNA clone 16 contains a 29-nucleotide poly(A) tract immediately adjacent to the cloning linker, indicating that this cDNA insert is probably derived from the extreme 3 1 end of viral RNA.
  • the location of the 5' end of the subgenomic RNA was estimated by primer runoff using total cytoplasmic RNA as template.
  • RNA is 2484 nucleotides long and includes the following features: 1) an 11- nucleotide 5'-untranslated region (5'-UTR); 2) a 2388-nucleotide open reading frame (ORF); and 3) an 85-nucleotide 3'-UTR as set forth in SEQ ID NO:3 and deposited with GenBank Data Library as Accession Number L06802.
  • the single ORF in the subgenomic RNA encodes a 796-amino-acid polypeptide with a predicted molecular mass of 88 kDa, consistent with the estimated 90-kDa mass of the capsid protein precursor we observed in infected cells (Monroe, S. S. et al.).
  • the predicted polypeptide has a region of basic amino acids that may play a role as a nucleic acid binding motif.
  • At the carboxy terminus is a region of acidic amino acids.
  • H-Ast 2 subgenomic RNA and deduced protein sequences Comparison of the H-Ast 2 subgenomic RNA and deduced protein sequences to the H-Ast 1 partial sequence.
  • a comparison of the H-Ast 2 RNA and deduced protein sequences to the partial sequences previously reported for H-Ast 1 (MatsuL S. M. et al.; Willcocks, M. M. et al., Program Abstr. Third International Symposium, Clearwater, Florida, abstr, pp. 2 - 47 (1992)) indicated regions of both similarities and differences.
  • nucleotide sequence immediately adjacent to the poly(A) tract including the 3' UTR and the last 8 codons of the predicted ORF is 94% conserved, with only five differences and two single base insertions in the first 109 unique nucleotides. Four of the five differences, including two in the coding region, result in compensating changes that maintain base pairing in predicted stem-loop structures at the 3 1 ends of the RNAs
  • Fig. 3 Predicted secondary structure at the 3' end of astrovirus RNA sequences.
  • the structures were calculated by the method of Zuker and Stiegler (Needleman, S. B. et al., /. Mol BioL, 48:443 - 453 (1970); Zuker, M. et al., Nucleic Acids Res., 9:133 - 148 (1981)).
  • the H-Ast 1 structure contains a total of 154 nucleotides including 134 bases from the reported 3'-end sequence (Willcocks, M. M. et aL) plus 20 additional adenine residues.
  • RECTIFIED SHEET contains 156 nucleotides corresponding to bases 2349 to 2504 in SEQ ID NO:3.
  • the region of the poly(A) tract involved in stem I is outlined with a box.
  • the two insertions in the loop between stems I and II are shown with arrowheads.
  • the residues within the conserved stem II that vary between the two serotypes are indicated.
  • the terminator codons, in the loop of stem II, are marked with asterisks.
  • stem I includes base pairs involving the poly(A) tract.
  • the two insertions in the H-Ast 2 sequence occur in a predicted loop between conserved stems I and II.
  • the terminator UAG codons are located in the loop at the top of stem II, between the conservative changes.
  • the stems marked III although similar in predicted secondary structure, are composed of dissimilar sequences.
  • the conserved primary and secondary structure at the 3' end of the genome may function as a recognition site during RNA replication.
  • H-Ast 2 was propagated in vitro, and virion RNA was extracted and used as template for cDNA synthesis and sequence determination.
  • ISA/EP Human astrovirus was obtained from Dr. John Kurtz (Oxford, England) and propagated in LLCMK2 cells in Earle minimal essential medium (EMEM) supplemented with 5 ⁇ g of trypsin per ml as described (Herring, A. J. et al (1981); Monroe, S. S. et al (1991); Matsui et al. (1993)).
  • Virions were partially purified from infected cell lysates by centrifuging through a 30% (w/v) sucrose cushion, suspended in TNE buffer containing 1% SDS, and extracted with phenol/chloroform.
  • Virion RNA was precipitated with 2 M LiCl and used for both the sequencing and the polymerase chain reaction (PCR) assays.
  • Single-stranded cDNA was synthesized from virion RNA with super reverse transcriptase (Molecular Genetics Resources, Tampa, Florida) using primers derived originally from cDNA sequence and subsequently from sequences determined by directly sequencing virion RNA, using a "primer walking" technique.
  • DNA fragments of varying length were amplified by the PCR assay with Taq polymerase (Perkin-Elmer Co., Norwalk, Connecticut) and virus-specific primers. Sequences were determined from three sources: virion RNA, PCR DNA, and cDNA clones.
  • RNA sequencing kit Boehringer Mannheim, Indianapolis, Indiana. Both the PCR DNA and the cloned cDNA were purified by using miniprep spun columns (Pharmacia, Piscataway, New Jersey) and sequenced by using the Sequenase Version 2 ⁇ 0 DNA Sequencing Kit (USB, Cleveland, Ohio). Sequences on both strands of DNA were determined with each base sequenced an average of at least four times. Sequences were assembled and aligned by using the Genetics Computer Group (GCG) sequence analysis program (Devereux et al., Nucleic Acids Res., 12:387 (1984)) and a consensus sequence was derived.
  • GCG Genetics Computer Group
  • Sequences of the 5' and 3' ends of the genomic RNA were determined by following the procedure of Lambden et al., /. Virol, 66:1817 (1992). Briefly, a synthetic primer 1 was ligated to the 3' ends of virion RNA or cDNA corresponding to the 5' end of virion RNA with T4 RNA ligase (GIBCO BRL, Gaithersburg, Maryland). cDNA fragments (400- to 600-bp) spanning either the 5* or the 3' ends were produced by the PCR amplification using a primer 2 complementary to the primer 1 and virus-specific primers, and sequenced by using internal primers.
  • ORF la is preceded by 82 untranslated nucleotides and encodes a polypeptide of 920 amino acids.
  • the 5' untranslated region of the genomic RNA was analyzed using the RNAFOLD program (Zuker, M. et al., Nucleic Acids Res., 9:133 (1981)). This region was predicted to contain extensive secondary structure, as demonstrated by the characteristic stem-loop structures preceding the initiation AUG codon.
  • ORF lb which overlaps ORF la by 70 nucleotides, is in reading frame +1 and its first AUG codon, which is predicted to be weak, is located 380 nucleotides downstream of the ORF la termination codon.
  • ORF 2 present also in the subgenomic RNA, overlaps ORF lb by 5 nucleotides, begins with a start codon at nucleotide 4325, and ends with a stop codon 82 bases from the 3' end.
  • ORF 2 codes for a capsid protein precursor of 796 amino acids with a predicted molecular mass of 88 kDa.
  • a ribosomal frameshift signal was identified, consisting of the "shifty" heptanucleotide (AAAAAAC) from position 2791 to 2797, followed by a stem-loop structure that may form a pseudoknot with a downstream sequence.
  • the putative frameshift signal of the astrovirus showed a striking resemblance to those at the gag-pro junction of some retroviruses, such as mouse mammary tumor virus (MMTV) (Fig. 2B), and fit perfectly the simultaneous tRNA slippage model of -1 frameshifting described for the synthesis of the g ⁇ g-related polyproteins (Jacks, et
  • Ribosomal frameshifting recently has been shown to be a normal expression mechanism in several groups of positive-strand RNA viruses, namely animal coronaviruses and arteriviruses, and plant luteoviruses and dianthoviruses (Brierly, I. et al., Cell, 57:537 (1989); den Boon, J. et al., /. Virol. , (1991); Prufer, D. et al., EMBO J., 11:1111 (1992)).
  • the putative frameshifting signal of astrovirus was much less similar to the frameshift regions of these viruses than to those of some retroviruses (not shown).
  • the ribosomal frameshifting during translation of astrovirus RNA directs the synthesis of an ORF la/lb fusion nonstructural polyprotein of 1416 amino acids with a predicted molecular mass of 161 kDa as set forth in SEQ ID NO: 4.
  • the predicted transmembrane ⁇ -helices occur at residues 156 - 172, 308 - 333, 343 - 362, and 369 - 387, the predicted cleavage site at the N-terminus of the putative VPg-protease occurs at residues 419 - 420, the putative nuclear localization signal occurs at residues 666 - 682, and the fusion dipeptide (KK) occurs at residues 904 - 905.
  • nucleotide sequence of the astrovirus genomic RNA and the deduced amino acid sequences of the nonstructural polyprotein and the capsid protein were compared with the current sequence databases (Altschul et al.,/. Mol Biol, 215:403 (1990); Henikoff, S. et al., Proc. Natl. Acad. ScL USA, 89:(1992)).
  • H-Ast 1 sequences Monroe, S. S. et al., (1991); MatsuL S. M. et al., (1993); Willcocks, M. M. et al., (1992); Jiang, B.
  • H-Ast 2 An important feature of the putative protease of H-Ast 2 is the substitution of serine for the catalytic cysteine found in the majority of positive-strand RNA virus proteases of superfamily I. Previously, an analogous substitution has been found in the putative proteases of sobemoviruses, luteoviruses, and arteriviruses (Gorbalenya, A. E. et al (1989 and 1988); Bazan, J. F. et al, (1989 and 1990); den Boon, J. A. et al. (1991)). However, the putative protease of H-Ast 2 showed lower similarity to these viral proteases than to the cysteine proteases of caliciviruses.
  • RECTIFIED SHEET (RULE 91) than 6000 nucleotides (Gorbalenya, A. E. et al., Nucleic Acids Res., 17:8413 (1989)).
  • the absence of the methyltransferase domain suggested that the astrovirus encodes VPg, a protein covalently linked to the 5' end of the viral genome (Wimmer, E. et al., Cell, 28:199 (1982); Vartapetian, A.B. et al., Prog. Nucl. Acids Res. Molec.
  • transmembrane ⁇ -helices were located in the region upstream of the protease and they may be involved in membrane anchoring of the viral RNA replication complex, as described for the 3A or 3AB proteins of poliovirus (Giachetti, C et al.,/. Virol, 65:2647 (1991); Giachetti, C. et al.,/. Virol, 66:6046 (1992)).
  • VPg is linked to the 5' end of the viral RNA via a tyrosine or a serine residue (Wimmer, E. (1982); Vartapetian, A. B. et al. (1987)). Inspection of the respective region of the H-Ast 2 polyprotein revealed no appropriately located tyrosines and only one serine (Ser 420). It is plausible to speculate that this serine may be the RNA-linking amino acid of VPg.
  • the active serine is located at the very N-terminus of the astrovirus VPg, similar to the VPg of comoviruses (Hellen, C. U. T. et al., Biochemistry, 28:9881 (1990); Palmenberg, A. et al., A. Rev. MicrobioL, 44:603 (1990); Eggen, R. et al., in RNA Genetics, P. Ahlquist, J. et al., eds., Vol. 1, p.
  • the nuclear localization signal (NLS), spanning amino acids 666 to 682, is identical to that of H-Ast 1 (Willcocks, M. M. et al., (1992)). This signal may be involved in transport of astrovirus proteins to the nucleus, as substantiated by the observation that astrovirus products were detected by immunofluorescence in the
  • RECTIFIED SHEET (RULE 91) nucleus of bovine astrovirus-infected cells ((Aroonprasert, D. et al., Vet. MicrobioL, 19:113 (1989)).
  • the astrovirus NLS perfectly fits the consensus for the bipartite signal motif comprising two clusters of basic amino acid residues separated by a ten-residue spacer region (Dingwall, C. et al., Trends Biochem. ScL, 16:478 (1991)).
  • both the protease and the RdRp of potyviruses contain similar NLS and are accumulated in the nuclei of infected plant cells (Carrington, J. C. et al., Plant Cell, 3:953 (1991); Li, X. H. et al., Virology, 193:951 (1993)).
  • astroviruses have no close relatives among other viruses, as demonstrated by comparative sequence analysis, and that their genomic organization is novel among animal viruses. It is remarkable, however, that astroviruses combine features typical of several very different groups of positive-strand RNA viruses and even retroviruses (the frameshift signal). Of special interest is the similarity of the genomic organization and expression strategy of astrovirus and plant luteoviruses (Martin, R. R. et al., Annu. Rev. Phytopathol,
  • MOLECULE TYPE RNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • AAUCUUCCCC ACCACUGGAA AAACUUAUUU CCAACGAGUU GUUGUGAUUA CCGGUGGGCU 300
  • MOLECULE TYPE RNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ACUUAUUUCC AACGAGUUGU UGUGAUUACC GGUGGGCUUG AGGAUGGAAC AUAUGGCUCA 240

Abstract

The present invention provides a nucleic acid endocing human Astrovirus serotype 2, or a unique fragment thereof. The sequence, a genomic RNA of human astovirus serotype 2 contains 6,797 nucleotides, and is organized into three open reading frames. Also provided are purified antigenic polypeptide fragments encoded by the nucleic acid encoding human Astrovirus serotype 2, or unique portions thereof. The present invention also provides a monoclonal antibody specific for human astrovirus serotype 2 and isolated nucleic acids capable of selectively hybridizing with the nucleic acid of serotype 2, including methods for detecting the presence of serotype 2 utilizing these products.

Description

NUCLEIC ACE S ENCODING HUMAN ASTROVIRUS SEROTYPE 2 AND USES THEREOF
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The present invention relates to astroviruses. In particular, the present invention relates to genomic and subgenomic nucleic acids of Human Astrovirus serotype 2.
BACKGROUND ART
Astroviruses are 28-nm nonenveloped, viruses that were initially identified from the feces of infants with gastroenteritis by their distinctive ultrastructural features of characteristic five- or six-pointed surface stars (Appleton, J. et al., Lancet, 1:1297 (1975); Madeley, C. R. et al., Lancet, 2:451-452 (1975)). These nonenveloped agents were subsequently determined to be positive-strand RNA viruses (Herring, A. J. et al., J. Gen. Virol., 53:47 (1981); Monroe, S. S. et al., J. Virol, 65:641 (1991); Matsui, S. M. et al., J. Virol., 67:1712 (1993)). Immune electron microscopy and immunofluorescence techniques have now identified five serotypes of human astroviruses, currently designated H-Astl to H-Ast5 (Kurtz, J. B. et al., Lancet, 2:1405 (1984).
Astroviruses cause acute gastroenteritis in children and adults worldwide (Cruz, J. R. et al., J. Clin. MicrobioL, 30:1140 (1992); Greenberg, H. B. et al., Infect. Agents Dis. 1:71 (1992); Moe et al.,/. Clin. MicrobioL, 29:2390 (1991), but the disease burden has been difficult to determine because of the lack of sensitive diagnostic assays. Recent studies have demonstrated that astroviruses were more frequently found in children with diarrhea than was previously thought (Herrmann et al., /. Infect. Dis. 161:226 (1990); Herrmann et al., N. Engl. J. Med., 324:1757 (1991); Lew, J. F. et al., /. Infect. Dis., 164:673 (1991)). Outbreaks have been reported in kindergartens, (Konno, T. et al., /. Med. Virol. , 9:11-17 (1982)) pediatric wards (Kurtz, J. B. et al., /. Clin. PathoL, 30:948-952 (1977)) and also in
RECTIFIED SHEET (RULE 91) ISA EP nursing homes (Gary, J. J. et al.,/. Med. Virol., 23:377-381 (1987); Oshiro, L. S. et al., /. Infect. Dis., 143:791-795 (1981)).
Clinical signs associated with astrovirus infection include nausea, vomiting, non-bloody diarrhea, abdominal cramps, headaches, fever, chills and myalgia (LeBaron, C. W. et al., Morbidity and Mortality Weekly Report (Centers for Disease Control), Vol. 39 (April 27, 1990)). Although most transmission is probably person-to-person among children, contaminated water and shellfish have also given rise to outbreaks in Britain (Kurtz, J. B. et al., (Ciba Foundation Symposium; 128), Chichester, UK: John Wiley & Sons Ltd., pp. 92-107 (1987)). Asymptomatic shedding of astrovirus has been documented (Ashley, C. R. et al., /. Clin. PathoL, 31:939-943 (1978) and infectivity can last as long as two days after clinical symptoms (White, K. E. et al., Am. J. Epidemiol., 124:120-126 (1986)). Immuno-compromised individuals, e.g., AIDS patients, especially risk infection from astroviruses.
Previous studies of the biochemical properties of purified astrovirus particles have provided divergent results concerning the number and size of the proteins present in astroviruses; from two to as many as six polypeptides have been reported, ranging in size from 5.5 kDa to 42 kDa (Willcocks, M. M. et al., Rev. Med. Virol., 2:97 - 106 (1992)). Likewise, there have been conflicting reports of the presence of subgenomic RNA present in astroviruses (Monroe, S. S. et al., /. of Virol., 65(2):641 - 648 (1991); Willcocks et al., Arch. Virol., 124:279 - 289 (1992). Moreover, characterization of the genome has been hindered because of the fastidious growth of astroviruses in vitro.
Investigators have reported partial sequence information from internal regions and at the 3' end of human astrovirus serotype 1 (H-Astl) including: 1034 nucleotides from the 3' end of genomic RNA (Willcocks, M. M. et al., Arch. Virol., 124:279 - 289 (1992), a 289 nucleotide immunoreactive epitope which overlaps the
3' end sequence (Matsui, S. M. et al., /. of Virol., 67:1712 - 1715 (1993)), and two
RECTIFIED SHEET (RULE 91) ISA/EP overlapping regions which hybridize only to genomic RNA (Matsui, S. M. et al. (1993)).
The fastidious nature of the virus coupled with extremely low levels of viral RNA generated by the organism during growth have made conventional sequencing approaches unpredictable and unreliable. Thus, despite a great need, the art has yet to provide sequences for human astrovirus type 2. The present invention satisfies this need by utilizing a unique combination of sequencing techniques to identify, diagnose, and treat astrovirus infection by providing nucleotide sequences for the complete genomic and subgenomic RNA of serotype 2 (H-Ast2) and analysis of the entire genomic RNA of H-Ast2. The present invention also provides the surprising discovery of a ribosomal frame shift occurring in one open reading frame which results in encoding of a fusional nonstructural polyprotein.
SUMMARY OF THE INVENTION
The present invention provides a nucleic acid encoding human Astrovirus serotype 2, or a unique fragment thereof. The sequence for the genomic RNA of human astrovirus was sequenced from virion RNA and cDNA and was found to contain 6797 nucleotides, exclusive of the poly(A) tail, organized into three open reading frames (defined as Open Reading Frames (ORFs) la, lb, and 2)). A ribosomal frameshift site is identified in the overlap region of ORFs la and lb at position 2794. This translation frameshift results in the suppression of in-frame amber termination at the end of ORF la and the synthesis of a nonstructural, fusion polyprotein that contains the putative protease and RNA-dependent RNA polymerase.
RECTIFIED SHEET (RULE 91) ISA/EP The present invention also provides the sequence of a nucleic acid encoding a subgenomic RNA of human Astrovirus serotype 2. This 2484-nucleotide RNA contains a single open readmg frame, which encodes a protein with a molecular mass of about 88 kDa.
The present invention proides purified antigenic polypeptide fragments encoded by the nucleic acid encoding human Astrovirus serotype 2. In particular, the present invention provides a purified antigenic polypeptide fragment encoded by the nucleic acid encoding open reading frame 2, or a unique portion thereof, in a pharmaceutically acceptable carrier.
The present invention also provides isolated nucleic acids capable of selectively hybridizing with the nucleic acid of human Astrovirus serotype 2 including, but not limited to, primers and probes for utilization in polymerase chain reaction (PCR) and other nucleic acid amplification techniques.
Further, the present invention provides vectors comprising the nucleic acid encoding human Astrovirus serotype 2 or a unique fragment thereof and provides the vector in a host capable of expressing the polypeptide encoded by that nucleic acid.
Finally, the present invention also provides a purfied monoclonal antibody specifically reactive with human Astrovirus serotype 2 and a method of detection of human Astrovirus serotype 2 utilizing the antibodies of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the genomic organization of human astrovirus. The locations of three ORFs, the first methionine (Met), and the frameshift site are indicated. The predicted transmembrane helices (MB), protease (Pro), nuclear
RECTIFIED SHEET (RULE 91) ISA/EP localization signal (NLS), and RNA-dependent RNA polymerase (Pol) are indicated by stippled boxes.
Fig. 2 shows (A) Nucleotide sequence and predicted RNA secondary structure in the overlap region of astrovirus ORFs la and lb. The putative frameshift site ("shifty" heptanucleotide sequence) is underlined and the termination codon for ORF la is boxed. The RNA secondary structure was predicted using the RNAFOLD program (Zuker, M. et al, (1981)). A potential pseudoknot structure was predicted by searching the region downstream of the stem-loop structure for sequences complementary to the loop sequence. Three base pairs may be sufficient for the pseudoknot formation (Pleij, C. W. A. et al., Trends Biochem. ScL, 16:143 (1990)) but the formation of a larger "secondary" stem with a non-canonical G-A pair (shown by a dotted line) and two additional canonical base pairs is also possible. The deduced amino acid sequences of ORFs la, lb, and la-lb surrounding the frameshift site are shown.
(B) Nucleotide sequence and predicted RNA secondary structure in the gag-pro overlap region of MMTV (Jacks, T. et al., Cell, 55:447 (1988)); Hatfield, D. L. et al., Trends Biochem. ScL, 15:186 (1990); Chamorro, M. et al., Proc. Natl. Acad. ScL USA, 89:713 - 717 (1992) are shown for comparison. The frameshift site, the termination codon, and the RNA pseudoknot are indicated or described as in (A).
Fig. 3 shows the predicted secondary structure at the 3' end of astrovirus RNA sequences. The structures were calculated by the method of Zuker and Stiegler (Zuker, M. et al., Nucleic Acids Res., 9:133 - 148 (1981)). The H-Astl structure contains a total of 154 nucleotides including 134 bases from the reported 3' end sequence (Willcocks, M. M. et al., Arch. Virol., 124:279 - 289 (1992)) plus 20 additional adenine residues. The H-Ast2 structure contains 156 nucleotides corresponding to bases 2349 to 2504 in SEQ ID NO: 3. The region of the poly(A) tract involved in stem I is outlined with a box. The two insertions of the loop between stems I and II are shown with arrowheads. The residues within the
RECTIFIED SHEET (RULE 91) conserved stem II that vary between the two serotypes are indicated. The terminator codons, in the loop of stem II, are marked with asterisks.
DETAILED DESCRIPΗON OF THE INVENTION
The present invention may be understood more readily by reference to the following detailed description of specific embodiments and the Examples included therein.
As used in the claims, "a" can mean one or more.
The present invention provides an isolated nucleic acid encoding human Astrovirus serotype 2 as set forth in the Sequencing Listing as SEQ ID NO: 1, or a unique fragment thereof. The invention also provides a nucleic acid capable of selectively hybridizing the DNA, RNA and cDNA sequences which can be derived from SEQ ID NO: 1. While SEQ ID NO: 1 is an RNA sequence, the invention also provides the corresponding DNA sequence.
By "isolated" is meant identifiably separated from other nucleic acids found in the naturally occurring organism. By "capable of selectively hybridizing" is meant a sequence which does not hybridize with other nucleic acids to prevent an adequate positive hybridization with nucleic acids from human Astrovirus serotype 2. By "unique fragment" is meant a fragment that can selectively hybridize with a RNA, DNA or cDNA sequence derived from the novel sequences.
An example of such a nucleic acid is an open reading frame of 2,387 bases comprising nucleotides 4325 through 6712 (designated open reading frame 2 (ORF 2)) as set forth in SEQ ID NO: 1. This specific nucleic acid can be used to detect human Astrovirus serotype 2 in methods such as polymerase chain reaction, ligase chain reaction and hybridization. Alternatively, the ORF 2 sequence can be utilized to produce an antigentic protein or protein fragment.
RECTIFIED SHEET (RULE 91) ISA/EP In addition, the nucleic acid can be utilized to find sequences homologous with nucleotide sequences present in other human or animal astroviruses. Such an amino acid sequence shared with other astroviruses can be used for example to simultaneously detect related strains or as a basis for a multiprotective vaccine.
An isolated nucleic acid capable of selectively hybridizing with or selectively amplifying a nucleic acid encoding the human Astrovirus serotype 2, or unique fragments thereof is also contemplated. The sequences can be selected based on the nucleotide sequence and the utility of the particular sequence.
Modifications to the nucleic acids of the invention are also contemplated as long as the essential structure and function of the polypeptide encoded by the nucleic acids is maintained. Likewise, fragments used as primers or probes can have substitutions so long as enough complementary bases exist for selective hybridization (Kunkel et al. Methods Enzymol. 1987:154:367, (1987)).
In one embodiment the present invention provides an isolated nucleic acid encoding open reading frame la of human Astrovirus serotype 2, comprising nucleotides 83 through 2842 contained in the nucleotide sequence as set forth in the Sequencing Listing SEQ ID NO: 1, or a unique fragment thereof. The open reading frame designated "la" is defined as comprising nucleotides 83 through 2842 contained in the nucleotide sequence set forth in the Sequencing Listing SEQ ID NO: 1 and depicted in FIG 1. Also contemplated by the present invention is an isolated nucleic acid capable of selectively hybridizing with the nucleic acid encoding open reading frame la.
RECTIFIED SHEET (RULE 91) ISA/EP In another embodiment, the present invention provides an isolated nucleic acid encoding open reading frame lb of human Astrovirus serotype 2, comprising nucleotides 2773 through 4329 contained in the nucleotide sequence as set forth in the Sequencing Listing SEQ ID NO: 1, or a unique fragment thereof. The open reading frame designated "lb" is defined as comprising nucleotides 2773 through 4329 contained in the nucleotide sequence set forth in the Sequencing Listing SEQ ID NO: 1 and depicted in FIG 1. Also contemplated by the present invention is an isolated nucleic acid capable of selectively hybridizing with the nucleic acid encoding open reading frame lb.
Another embodiment of the present invention provides an isolated nucleic acid encoding open reading frame 2 of human Astrovirus serotype 2, comprising nucleotides 4325 through 6712 contained in the nucleotide sequence as set forth in the Sequencing Listing as SEQ ID NO: 1, or a unique fragment thereof. The open reading frame designated "2" is defined as comprising nucleotides 4325 through 6712 contained in the nucleotide sequence set forth in the Sequencing Listing SEQ ID NO: 1 and depicted in FIG 1. Also contemplated by the present invention is an isolated nucleic acid capable of selectively hybridizing with the nucleic acid encoding open reading frame lb.
The present invention also provides an isolated nucleic acid encoding open reading frame la/lb of human Astrovirus serotype 2, as set forth in the nucleotide sequence defined in the Sequencing Listing as SEQ ID NO: 2, or a unique fragment thereof. A minus 1 frame shift occurs at position 2712 of the sequence depicted in SEQ ID NO: 2. The open reading frame la/lb can also be identified in FIG 1 and in SEQ ID NO: 1 wherein it comprises nucleotides 83 through 4329. The minus 1 frameshift occurs at position 2794 of the sequence depicted in SEQ ID NO:l. Also contemplated by the present invention is an isolated nucleic acid capable of selectively hybridizing with the nucleic acid encoding open reading frame la/lb.
RECTIFIED SHEET (RULE 91) ISA/EP In another embodiment, the present invention provides an isolated nucleic acid encoding a subgenomic RNA of human Astrovirus serotype 2, as set forth in the nucleotide sequence defined in the Sequencing Listing as SEQ ID NO: 3, or a unique fragment thereof. The subgenomic RNA of human Astrovirus serotype 2 can also be identified in FIG 1 and in SEQ ID NO:l wherein it comprises nucleotides 4314 through 6797 exclusive of the poly(A) tail. Also contemplated by the present invention is an isolated nucleic acid capable of selectively hybridizing with the nucleic acid encoding the subgenomic RNA of human Astrovirus serotype
2. While the nucleic acids can be derived as set forth in the examples, given the sequences, it is also possible to synthesize partial sequences and enzymatically combine the partial sequences to make an entire synthetic gene.
Also provided are purified antigenic polypeptides encoded by the nucleic acids. The invention also provides these antigenic polypeptides in a pharmaceutically acceptable carrier. The amino acid sequence of these polypeptides can be deduced from the nucleotide sequences set forth in the
Sequence Listing. One example is set forth in SEQ ID NO: 4.
Purified antigenic polypeptide fragments encoded by the nucleic acids of the present invention are also contemplated. As used herein, "purified" means the antigen is sufficiently free of contaminants or cell components with which the antigen normally occurs to distinguish the antigen from the contaminants or components. Purified human Astrovirus serotype 2 antigen and antigenic fragments thereof of the present invention are also referred to herein as "the antigen" or "the H-Ast 2 antigen." It is contemplated that the antigenic fragments can be encoded from any portion of the nucleic acid encoding human Astrovirus serotype 2 as set forth in SEQ ID NO:l, but especially from fragments encoded by the open reading frames la, lb, la/lb, and 2 as described herein.
RECTIFIED SHEET (RULE 91) ISA/EP Specifically, one example includes an approximately 88 kDa antigenic polypeptide encoded by an open reading frame of 2387 bases (ORF 2) consisting essentially of the amino acids encoded by nucleotides 4325 through 6797 contained in the nucleotide sequence set forth in the Sequence Listing as SEQ ID NO: 1 .
An antigenic fragment of the antigen can be isolated from the whole antigen by chemical or mechanical disruption. The purified fragments thus obtained can be tested to determine their antigenicity and specificity by the methods taught herein. Antigenic fragments of the antigen can also be synthesized directly. An immunoreactive fragment is generally an amino acid sequence of at least about five consecutive amino acids derived from the antigen amino acid sequence.
The polypeptide fragments of the present invention can also be recombinant proteins obtained by cloning nucleic acids encoding the polypeptide in an expression system capable of producing the antigenic polypeptide or fragments thereof.
Once the amino acid sequence of the antigen is provided, it is also possible to synthesize, using standard peptide synthesis techniques, peptide fragments chosen to be homologous to immunoreactive regions of the antigen and to modify these fragments by inclusion, deletion or modification of particular amino acids residues in the derived sequences. Thus, synthesis or purification of an extremely large number of peptides derived from the antigen is possible.
The amino acid sequences of the present polypeptides can contain an immunoreactive portion of the H-Ast 2 antigen attached to sequences designed to provide for some additional property, such as solubility. The amino acid sequences of an H-Ast 2 antigen can include sequences in which one or more amino acids have been substituted with another amino acid to provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to
RECTIFIED SHEET (RULE 91) ISA/EP increase its bio-longevity, alter enzymatic activity, or alter interactions with gastric acidity. In any case, the peptide must possess a bioactive property, such as immunoreactivity, immunogenicity, etc.
The purified polypeptide fragments thus obtained can be tested to determine their immunogenicity and specificity. Briefly, various concentrations of a putative immunogenically specific fragment are prepared and administered to an animal and the immunological response (e.g., the production of antibodies or cell mediated immunity) of an animal to each concentration is determined. The amounts of antigen administered depend on the subject, e.g. a human or a guinea pig, the condition of the subject, the size of the subject, etc. Thereafter an animal so inoculated with the antigen can be exposed to the virus to test the potential vaccine effect of the specific immunogenic fragment. The specificity of a putative immunogenic fragment can be ascertained by testing sera, other fluids or lymphocytes from the inoculated animal for cross reactivity with other closely related Astroviruses.
A vector comprising the nucleic acids of the present invention is also provided. The vectors of the invention can be in a host capable of expressing the antigenic polypeptide fragments contemplated by the present invention.
There are numerous E. coli expression vectors known to one of ordinary skill in the art useful for the expression of the antigen. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Tip) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences for example, for
RECTIFIED SHEET (RULE 91) ISA/EP initiating and completing transcription and translation. If necessary an amino terminal methionine can be provided by insertion of a Met codon 5' and in-frame with the antigen. Also, the carboxy-terminal extension of the antigenic fragments can be removed using standard oligonucleotide mutagenesis procedures.
Additionally, yeast expression can be used. There are several advantages to yeast expression systems. First, evidence exists that proteins produced in yeast secretion systems exhibit correct disulfide pairing. Second, post- translational glycosylation is efficiently carried out by yeast secretory systems. The Saccharomyces cerevisiae pre-pro-alpha-factor leader region (encoded by the MFa-1 gene) is routinely used to direct protein secretion from yeast (Brake et al., 1984). The leader region of pre-pro-alpha-factor contains a signal peptide and a pro- segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene: this enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage-signal sequence. The antigen coding sequence can be fused in-frame to the pre-pro-alpha-factor leader region. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter. The antigen coding sequence is followed by a translation termination codon which is followed by transcription termination signals. Alternatively, the antigen coding sequences can be fused to a second protein coding sequence, such as Sj26 or β-galactosidase, used to facilitate purification of the fusion protein by affinity chromatography. The insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast.
Mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active protein. Vectors useful for the expression of antigens in mammalian cells are characterized by insertion of the antigen coding sequence between a strong viral promoter and a polyadenylation signal. The vectors can contain genes conferring either gentamicin or methotrexate resistance for use as selectable markers. The
RECTIFIED SHEET (RULE 91) ISA/EP antigen and immunoreactive fragment coding sequence can be introduced into a Chinese hamster ovary cell line using a methotrexate resistance-encoding vector. Presence of the vector RNA in transformed cells can be confirmed by Southern analysis and production of a cDNA or oposite strand RNA corresponding to the antigen coding sequence can be confirmed by Northern analysis. A number of other suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc. The vectors containing the nucleic acid segments of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts.
Alternative vectors for the expression of antigen in mammalian cells, those similar to those developed for the expression of human gamma-interferon, tissue plasminogen activator, clotting Factor VIII, hepatitis B virus surface antigen, protease Nexinl, and eosinophil major basic protein, can be employed. Further, the vector can include CMV promoter sequences and a polyadenylation signal available for expression of inserted nucleic acid in mammalian cells (such as COS7).
RECTIFIED SHEET (RULE 91) ISA/EP The nucleic acid sequences can be expressed in hosts after the sequences have been operably linked to, i.e., positioned to ensure the functioning of, an expression control sequence. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors can contain selection markers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired nucleic acid sequences sequences (see, e.g., U.S. Patent 4,704,362).
Polynucleotides encoding a variant polypeptide may include sequences that facilitate transcription (expression sequences) and translation of the coding sequences such that the encoded polypeptide product is produced. Construction of such polynucleotides is well known in the art. For example, such polynucleotides can include a promoter, a transcription termination site (polyadenylation site in eukaryotic expression hosts), a ribosome binding site, and, optionally, an enhancer for use in eukaryotic expression hosts, and, optionally, sequences necessary for replication of a vector.
A purified monoclonal antibody specifically reactive with human Astrovirus serotype 2 is also provided. The antibodies can be specifically reactive with a unique epitope of the antigen or they can also react with epitopes of other organisms. The term "reactive" means capable of binding or otherwise associating nonrandomly with an antigen. "Specifically reactive" as used herein describes an antibody or other ligand that does not cross react substantially with any antigen other than the one specified, in this case, human Astrovirus serotype 2. Antibodies can be made as described in the Examples (see also, Harlow and Lane, Antibodies;
A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New
York, 1988). Briefly purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells are then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen clone libraries for cells secreting the antigen.
RECTIFIED SHEET (RULE 91) ISA/EP Those positive clones can then be sequenced (see, for example, Kelly et al., BiolTechnology 10:163-167, 1992 and Bebbington et al., BiolTechnology 10:169-175, 1992).
The antibody can be bound to a substrate or labeled with a detectable moiety or both bound and labeled. The detectable moieties contemplated with the composition of the present invention are those listed below in the description of the diagnostic methods, including fluorescent, enzymatic and radioactive markers.
A purified human Astrovirus serotype 2 antigen bound to a substrate and a ligand specifically reactive with the antigen are also contemplated. Such a purified ligand specifically reactive with the antigen can be an antibody. The antibody can be a monoclonal antibody obtained by standard methods and as described herein. The monoclonal antibody can be secreted by a hybridoma cell line specifically produced for that purpose (Harlow and Lane, 1988). Likewise, nonhuman polyclonal antibodies specifically reactive with the antigen are within the scope of the present invention. The polyclonal antibody can also be obtained by the standard immunization and purification protocols (Harlow and Lane, 1988).
The present invention provides a method of detecting the presence of human Astrovirus serotype 2 in a subject, comprising the steps of contacting an antibody-containing sample from the subject with a detectable amount of the antigenic polypeptide fragment of the present invention and detecting the reaction of the fragment and the antibody, the reaction indicating the presence of the Astrovirus or a previous infection with H-Ast 2.
One example of the method of detecting human Astrovirus serotype
2 is performed by contacting a fluid or tissue sample from the subject with an amount of a purified antibody specifically reactive with the antigen as defined herein, and detecting the reaction of the ligand with the antigen. It is contemplated
FCTIFIED SHEET (RULE 91) ISA EP that the antigen will be on intact cells containing the antigen, or will be fragments of the antigen. As contemplated herein, the antibody includes any ligand which binds the antigen, for example, an intact antibody, a fragment of an antibody or another reagent that has reactivity with the antigen. The fluid sample of this method can comprise any body fluid which would contain the antigen or a cell containing the antigen, such as blood, plasma, serum, saliva, feces and urine. Other possible examples of body fluids include sputum, mucus, gastric juice and the like.
Enzyme immunoassays such as immunofluorescence assays (IF A), enzyme linked immunosorbent assays (ELISA) and immunoblotting can be readily adapted to accomplish the detection of the antigen. An ELISA method effective for the detection of the antigen can, for example, be as follows: (1) bind the antibody to a substrate; (2) contact the bound antibody with a fluid or tissue sample containing the antigen; (3) contact the above with a secondary antibody bound to a detectable moiety (e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme); (4) contact the above with the substrate for the enzyme; (5) contact the above with a color reagent; (6) observe color change. The above method can be readily modified to detect antibody as well as antigen.
Another immunologic technique that can be useful in the detection of H-Ast 2 or previous H-Ast 2 infection utilizes monoclonal antibodies (MAbs) for detection of antibodies specifically reactive with H-Ast 2 antigen. Briefly, sera or other body fluids from the subject is reacted with the antigen bound to a substrate (e.g. an ELISA 96-well plate). Excess sera is thoroughly washed away. A labeled (enzyme-linked, fluorescent, radioactive, etc.) monoclonal antibody is then reacted with the previously reacted antigen-serum antibody complex. The amount of inhibition of monoclonal antibody binding is measured relative to a control (no patient serum antibody). The degree of monoclonal antibody inhibition is a very specific test for a particular variety or strain since it is based on monoclonal antibody binding specificity. MAbs can also be used for detection directly in cells by lFA.
RECTIFIED SHEET (RULE 91) ISA/EP A micro-agglutination test can also be used to detect the presence of H-Ast 2 in a subject. Briefly, latex beads (or red blood cells) are coated with the antigen and mixed with a sample from the subject, such that antibodies in the tissue or body fluids that are specifically reactive with the antigen crosslink with the antigen, causing agglutination. The agglutinated antigen-antibody complexes form a precipitate, visible with the naked eye or by spectrophotometer. In a modification of the above test, antibodies specifically reactive with the antigen can be bound to the beads and antigen in the tissue or body fluid thereby detected.
In addition, as in a typical sandwich assay, the antibody can be bound to a substrate and reacted with the antigen. Thereafter, a secondary labeled antibody is bound to epitopes not recognized by the first antibody and the secondary antibody is detected. Since the present invention provides H-Ast 2 antigen for the detection of infectious H-Ast 2 or previous H-Ast 2 infection other serological methods such as flow cytometry and immunoprecipitation can also be used as detection methods.
In the diagnostic methods taught herein, the antigen can be bound to a substrate and contacted by a fluid sample such as serum, urine, saliva, feces or gastric juice. This sample can be taken directly from the patient or in a partially purified form. In this manner, antibodies specific for the antigen (the primary antibody) will specifically react with the bound antigen. Thereafter, a secondary antibody bound to, or labeled with, a detectable moiety can be added to enhance the detection of the primary antibody. Generally, the secondary antibody or other ligand which is reactive, either specifically with a different epitope of the antigen or nonspecifically with the ligand or reacted antibody, will be selected for its ability to react with multiple sites on the primary antibody. Thus, for example, several molecules of the secondary antibody can react with each primary antibody, making the primary antibody more detectable.
RECTIFIED SHEET (RULE 91) ISA/EP The detectable moiety will allow visual detection of a precipitate or a color change, visual detection by microscopy, or automated detection by spectrometry, radiometric measurement or the like. Examples of detectable moieties include fluorescein and rhodamine (for fluorescence microscopy), horseradish peroxidase (for either light or electron microscopy and biochemical detection), biotin-streptavidin (for light or electron microscopy) and alkaline phosphatase (for biochemical detection by color change). The detection methods and moieties used can be selected, for example, from the list above or other suitable examples by the standard criteria applied to such selections (Harlow and Lane, 1988).
The antigen, e.g., a purified antigenic polypeptide fragment encoded by open reading frame 2 of this invention can be used in the construction of a vaccine comprising an immunogenic amount of the antigen and a pharmaceutically acceptable carrier. The vaccine can be the entire antigen, the antigen on an intact H-Ast 2 organism, E. coli or other strain, or an epitope specific to the antigen. The vaccine can also be potentially cross-reactive with antibodies to other antigens. The vaccine can then be used in a method of preventing diarrhea or other complications of H-Ast 2 infection.
Immunogenic amounts of the antigen can be determined using standard procedures. Briefly, various concentrations of a putative specific immunoreactive epitope are prepared, administered to an animal and the immunological response (e.g., the production of antibodies) of an animal to each concentration is determined.
The pharmaceutically acceptable carrier can comprise saline or other suitable carriers (Arnon, R. (Ed.) Synthetic Vaccines, 1:83-92, CRC Press, Inc., Boca
Raton, Florida, 1987). An adjuvant can also be a part of the carrier of the vaccine, in which case it can be selected by standard criteria based on the antigen used, the mode of administration and the subject (Arnon, R. (Ed.), 1987). Methods of
RECTIFIED SHEET (RULE 91) ISA/EP administration can be by oral or sublingual means, or by injection, depending on the particular vaccine used and the subject to whom it is administered.
It can be appreciated from the above that the vaccine can be used as a prophylactic or a therapeutic modality. Thus, the invention provides methods of preventing or treating H-Ast 2 infection and the associated diseases by administering the vaccine to a subject.
The presence of H-Ast 2 can also be determined by detecting the presence of a nucleic acid specific for H-Ast 2 or the antigens of H-Ast 2 encoded by the nucleic acid. The present invention provides a method of detecting the presence of human Astrovirus serotype 2 in a subject, comprising detecting the presence of the nucleic acid encoding human Astrovirus serotype 2. The specificity of these sequences for H-Ast 2 can be determined by conducting a computerized comparison with known sequences, catalogued in GenBank, a computerized database, using the computer programs Word Search or FASTA of the Genetics Computer Group (Madison, WI), which search the catalogued nucleotide sequences for similarities to the nucleic acid in question.
The nucleic acid specific for H-Ast 2 can be detected utilizing a nucleic acid amplification technique, such as polymerase chain reaction or ligase chain reaction. Alternatively, the nucleic acid is detected utilizing direct hybridization or by utilizing a restriction fragment length polymorphism. For example, the present invention provides a method of detecting the presence of H- Ast 2 comprising ascertaining the presence of a nucleotide sequence associated with a restriction endonuclease cleavage site. In addition, PCR primers which hybridize only with nucleic acids specific for H-Ast 2 can be utilized. The presence of amplification indicates the presence of the antigen. In another embodiment a restriction fragment of a nucleic acid sample can be sequenced directly using techniques known in the art and described herein and compared to the known unique sequence to detect H-Ast 2. In a further embodiment, the present invention provides a method of detecting the presence of H-Ast 2 by selective amplification
RECTLFIED SHEET (RULE 91) ISA/EP by the methods described herein. In yet another embodiment H-Ast 2 can be detected by directly hybridizing the unique sequence with a H-Ast 2 selective nucleic acid probe. Furthermore, the nucleotide sequence could be amplified prior to hybridization by the methods described above.
Alternative probing techniques, such as ligase chain reaction (LCR), involve the use of mismatch probes, i.e., probes which are fully complementary with the target except at the point of the mutation. The target sequence is then allowed to hybridize both with oligonucleotides which are fully complementary and with oligonucleotides containing a mismatch, under conditions which will distinguish between the two. By manipulating the reaction conditions, it is possible to obtain hybridization only where there is full complementarity. If a mismatch is present there is significantly reduced hybridization.
The polymerase chain reaction (PCR) and reverse transcriptase PCR are techniques that amplifies specific nucleic acid sequences with remarkable efficiency. Repeated cycles of denaturation, primer annealing and extension carried out with polymerase, e.g., a heat stable enzyme Taq polymerase, leads to exponential increases in the concentration of desired nucleic acid sequences. Given a knowledge of the nucleotide sequence of H-Ast 2, synthetic oligonucleotides can be prepared which are complementary to sequences which flank the nucleic acid of interest. Each oligonucleotide is complementary to one of the two strands. The nucleic acid can be denatured at high temperatures (e.g., 95°C) and then reannealed in the presence of a large molar excess of oligonucleotides. The oligonucleotides, oriented with their 3' ends pointing towards each other, hybridize to opposite strands of the target sequence and prime enzymatic extension along the nucleic acid template. The end product is then denatured again for another cycle. After this three-step cycle has been repeated several times, amplification of a nucleic acid segment by more than one million-fold can be achieved. The resulting nucleic acid may then be directly sequenced.
RECTIFIED SHEET (RULE 91) ISA/EP In yet another method, PCR may be followed by restriction endonuclease digestion with subsequent analysis of the resultant products. Nucleotide substitutions can result in the gain or loss of specific restriction endonuclease site. The gain or loss of a restriction endonuclease recognition site facilitates the detection of the organism using restriction fragment length polymorphism (RFLP) analysis or by detection of the presence or absence of a polymorphic restriction endonuclease site in a PCR product that spans the sequence of interest.
For RFLP analysis, nucleic acid is obtained, for example from the blood, gastric specimen, saliva, dental plaque, or other bodily fluids of the subject suspected of containing H-Ast 2 is digested with a restriction endonuclease, and subsequently separated on the basis of size by agarose gel electrophoresis. The Southern blot technique can then be used to detect, by hybridization with labeled probes, the products of endonuclease digestion. The patterns obtained from the Southern blot can then be compared. Using such an approach, H-Ast 2 nucleic acid is detected by determining the number of bands detected and comparing this number to the nucleic acid from H-Ast 2.
Similar creation of additional restriction sites by nucleotide substitutions at the disclosed mutation sites can be readily calculated by reference to the genetic code and a list of nucleotide sequences recognized by restriction endonucleases.
Single strand conformational analysis (SSCA) offers a relatively quick method of detecting sequence changes which may be appropriate in at least some instances.
RECTIFIED SHEET (RULE 91) ISA/EP In general, primers for PCR and LCR are usually about 20 bp in length and the preferable range is from 15-25 bp. Better amplification is obtained when both primers are the same length and with roughly the same nucleotide composition. Denaturation of strands usually takes place at 94°C and extension from the primers is usually at 72°C. The annealing temperature varies according to the sequence under investigation. Examples of reaction times are: 20 mins denaturing; 35 cycles of 2 min, 1 min, 1 min for annealing, extension and denaturation; and finally a 5 min extension step.
PCR amplification of specific alleles (PASA) is a rapid method of detecting single-base mutations or polymorphisms. PASA (also known as allele specific amplification) involves amplification with two oligonucleotide primers such that one is allele-specific. The desired allele is efficiently amplified, while the other allele(s) is poorly amplified because it mismatches with a base at or near the 3* end of the allele-specific primer. Thus, PASA or the related method of PAMSA may be used to specifically amplify the mutation sequences of the invention. Where such amplification is done on H-Ast 2 isolates or samples obtained from an individual, it can serve as a method of detecting the presence of H-Ast 2.
As mentioned above, a method known as ligase chain reaction (LCR) can be used to successfully detect a single-base substitution. LCR probes may be combined or multiplexed for simultaneously screening for multiple different mutations. Thus, LCR can be particularly useful where, as here, multiple mutations are predictive of the same disease.
The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.
RECTIFIED SHEET (RULE 91) ISA/EP EXAMPLES
Example 1: Subgenomic RNA
To first examine the mechanism of replication of astroviruses, we analyzed the synthesis of proteins and RNA during a single-cycle infection of cultured cells (Monroe, S. S. et al. /. Virol. , 65:641 - 648 (1991)). We detected a previously unreported 90-kDa protein that, by virtue of its reactivity with hyperimmune rabbit serum, is presumed to be a capsid protein precursor. This 90- kDa precursor could be cleaved by trypsin in vitro, with the appearance of three smaller proteins (31 kDa, 29 kDa, and 20 kDa). A second observation of our in vitro studies was a previously unreported 2.8-kb RNA that is polyadenylated and that we presumed to be a subgenomic mRNA encoding the 90-kDa precursor polypeptide.
cDNA cloning and RNA blot hybridization. Cell-culture-adapted human astrovirus serotype 2 (H-Ast2) was obtained from Dr. John Kurtz (Oxford, England), was plaque purified three times before use, and was propagated in LLCMK2 cells (ATCC CCL7.1) as previously described (Monroe, S. S. et al.). Double-stranded cDNA was synthesized from the polyadenylated fraction of RNA isolated from astrovirus infected cells (cDNA Cloning Kit, Boehringer Mannheim Biochemicals,), and was cloned into the pBluescript II plasmid vector (Stratagene). Recombinant clones were screened for astrovirus specific inserts by hybridization of [32P]-labelled RNA transcribed in vitro from individual cDNA clones to total cytoplasmic RNA isolated from uninfected and astrovirus-infected cells.
Total cytoplasmic RNA isolated from astrovirus infected cells at the indicated times post infection and unlabeled RNA transcribed in vitro from cDNA clone 16, were resolved in a 1.2% agarose gel, transferred to a nylon membrane, and probed with [32P]-labelled RNA transcribed from cDNA clone 16. The RNA transcripts from the insert in one cDNA clone (number 16) hybridized to both the 7.2- and 2.8-kb viral RNAs as evidenced by autoradiography of RNA blot hybridization (not shown). The hybridization reactivity was first detectable at 12
RECTIFIED SHEET (RULE 91) ISA/EP hours postinfection, coincident with detection of these RNAs by metabolic labeling (Monroe, S. S. et al.). The hybridization of a cRNA probe to both viral specific RNAs confirmed that the 2.8-kb RNA contains sequences present in the larger species, with the relative intensities indicating that the smaller RNA is present in at least a 10-fold molar excess. These observations support our earlier conclusion that the 2.8-kb RNA is a subgenomic mRNA (Monroe, S. S. et al.).
Nucleotide sequence analysis. Sequence information for the subgenomic RNA was obtained by three approaches: 1) sequencing of supercoiled DNA from two plasmids with cDNA inserts, 2) sequencing of RNA purified from virions, and 3) amplification of genomic RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), followed by sequencing of the double-stranded DNA products. Plasmid DNA and PCR products were sequenced using modified T7 DNA polymerase (Sequenase2®, US Biochemicals). Sequence information from the 5' end of the original clone was used to generate oligonucleotide primers for a second round of cDNA cloning from cytoplasmic RNA. PCR products were purified by gel filtration (Miniprep Spun Column, Pharmacia) before sequencing. RNA was sequenced using reverse transcriptase and dideoxynucleotide terminators (RNA Sequencing Kit, Boehringer Mannheim Biochemicals) with primers derived from the sequence of the cDNA clones. Although direct RNA sequencing often resulted in regions of the gels that were difficult to interpret, ambiguities were resolved through the use of RT-PCR sequencing over the same regions. The information from the three independent sequencing strategies was combined to arrive at a consensus sequence for the entire subgenomic region (Devereux, J. P et al., Nucleic Acids Res., 12:387 - 395 (1984). The sequence derived from cDNA clone 16 contains a 29-nucleotide poly(A) tract immediately adjacent to the cloning linker, indicating that this cDNA insert is probably derived from the extreme 31 end of viral RNA. The location of the 5' end of the subgenomic RNA was estimated by primer runoff using total cytoplasmic RNA as template.
RECTIFIED SHEET (RULE 91) The consensus sequence for the unique region of the subgenomic
RNA is 2484 nucleotides long and includes the following features: 1) an 11- nucleotide 5'-untranslated region (5'-UTR); 2) a 2388-nucleotide open reading frame (ORF); and 3) an 85-nucleotide 3'-UTR as set forth in SEQ ID NO:3 and deposited with GenBank Data Library as Accession Number L06802.
Analysis of the predicted capsid precursor polypeptide. The single ORF in the subgenomic RNA encodes a 796-amino-acid polypeptide with a predicted molecular mass of 88 kDa, consistent with the estimated 90-kDa mass of the capsid protein precursor we observed in infected cells (Monroe, S. S. et al.). At the amino terminus, the predicted polypeptide has a region of basic amino acids that may play a role as a nucleic acid binding motif. At the carboxy terminus is a region of acidic amino acids.
Comparison of the H-Ast 2 subgenomic RNA and deduced protein sequences to the H-Ast 1 partial sequence. A comparison of the H-Ast 2 RNA and deduced protein sequences to the partial sequences previously reported for H-Ast 1 (MatsuL S. M. et al.; Willcocks, M. M. et al., Program Abstr. Third International Symposium, Clearwater, Florida, abstr, pp. 2 - 47 (1992)) indicated regions of both similarities and differences. The nucleotide sequence immediately adjacent to the poly(A) tract, including the 3' UTR and the last 8 codons of the predicted ORF is 94% conserved, with only five differences and two single base insertions in the first 109 unique nucleotides. Four of the five differences, including two in the coding region, result in compensating changes that maintain base pairing in predicted stem-loop structures at the 31 ends of the RNAs
Referring to Fig. 3. Predicted secondary structure at the 3' end of astrovirus RNA sequences. The structures were calculated by the method of Zuker and Stiegler (Needleman, S. B. et al., /. Mol BioL, 48:443 - 453 (1970); Zuker, M. et al., Nucleic Acids Res., 9:133 - 148 (1981)). The H-Ast 1 structure contains a total of 154 nucleotides including 134 bases from the reported 3'-end sequence (Willcocks, M. M. et aL) plus 20 additional adenine residues. The H-Ast 2 structure
RECTIFIED SHEET (RULE 91) contains 156 nucleotides corresponding to bases 2349 to 2504 in SEQ ID NO:3. The region of the poly(A) tract involved in stem I is outlined with a box. The two insertions in the loop between stems I and II are shown with arrowheads. The residues within the conserved stem II that vary between the two serotypes are indicated. The terminator codons, in the loop of stem II, are marked with asterisks.
Note that stem I includes base pairs involving the poly(A) tract. The two insertions in the H-Ast 2 sequence occur in a predicted loop between conserved stems I and II. The terminator UAG codons are located in the loop at the top of stem II, between the conservative changes. The stems marked III, although similar in predicted secondary structure, are composed of dissimilar sequences. The conserved primary and secondary structure at the 3' end of the genome may function as a recognition site during RNA replication. As a further indication that the primary sequence information in this 3' region is conserved among astroviruses, we have used oligonucleotide primers derived from this region to amplify RNA from all five reference serotypes of human astrovirus.
In contrast to the high degree of primary sequence conservation at the 31 end of the genome, there is only 59% nμcleotide sequence identity in the consensus coding region sequence from H-Ast 1 (Matsui, S. M. et al. (1993); Willcocks, M. M. et a\., Arch. Virol. , 124:279 - 289 (1992)) and the corresponding region of H-Ast 2. Alignment of the 392 amino acid partial H-Ast 1 sequence with the corresponding region of the H-Ast 2 amino acid sequence indicates an overall similarity of 67%, with 52% identical residues. The proteins are more conserved at their carboxy termini, which both include the highly acidic region, with 80% similarity and 62% identity over the terminal 114 residues.
Example 2: Genomic RNA
H-Ast 2 was propagated in vitro, and virion RNA was extracted and used as template for cDNA synthesis and sequence determination.
RECTIFIED SHEET (RULE 91) ISA/EP Human astrovirus was obtained from Dr. John Kurtz (Oxford, England) and propagated in LLCMK2 cells in Earle minimal essential medium (EMEM) supplemented with 5 μg of trypsin per ml as described (Herring, A. J. et al (1981); Monroe, S. S. et al (1991); Matsui et al. (1993)). Virions were partially purified from infected cell lysates by centrifuging through a 30% (w/v) sucrose cushion, suspended in TNE buffer containing 1% SDS, and extracted with phenol/chloroform. Virion RNA was precipitated with 2 M LiCl and used for both the sequencing and the polymerase chain reaction (PCR) assays. Single-stranded cDNA was synthesized from virion RNA with super reverse transcriptase (Molecular Genetics Resources, Tampa, Florida) using primers derived originally from cDNA sequence and subsequently from sequences determined by directly sequencing virion RNA, using a "primer walking" technique. DNA fragments of varying length were amplified by the PCR assay with Taq polymerase (Perkin-Elmer Co., Norwalk, Connecticut) and virus-specific primers. Sequences were determined from three sources: virion RNA, PCR DNA, and cDNA clones. Virion RNA was directly sequenced by using an RNA sequencing kit (Boehringer Mannheim, Indianapolis, Indiana). Both the PCR DNA and the cloned cDNA were purified by using miniprep spun columns (Pharmacia, Piscataway, New Jersey) and sequenced by using the Sequenase Version 2^0 DNA Sequencing Kit (USB, Cleveland, Ohio). Sequences on both strands of DNA were determined with each base sequenced an average of at least four times. Sequences were assembled and aligned by using the Genetics Computer Group (GCG) sequence analysis program (Devereux et al., Nucleic Acids Res., 12:387 (1984)) and a consensus sequence was derived. Sequences of the 5' and 3' ends of the genomic RNA were determined by following the procedure of Lambden et al., /. Virol, 66:1817 (1992). Briefly, a synthetic primer 1 was ligated to the 3' ends of virion RNA or cDNA corresponding to the 5' end of virion RNA with T4 RNA ligase (GIBCO BRL, Gaithersburg, Maryland). cDNA fragments (400- to 600-bp) spanning either the 5* or the 3' ends were produced by the PCR amplification using a primer 2 complementary to the primer 1 and virus-specific primers, and sequenced by using internal primers.
RECTIFIED SHEET (RULE 91) ISA/EP The genomic RNA of H-Ast 2 is 6797 nucleotides in length, excluding
31 adenines (poly(A) tail) at the 3' end as set forth in SEQ ID NO:l and deposited with GenBank Data Library as Accession Number L13745. The genome possesses three overlapping open reading frames (ORFs) designated la, lb, and 2 and depicted in Fig 2.
Referring to Fig. 1, the sequences sunounding the first AUG codons of ORFs la and 2 are predicted to be optimal for the initiation of translation (Kozak, M. et al., /. Biol. Chem., 266:19867 (1991)). ORF la is preceded by 82 untranslated nucleotides and encodes a polypeptide of 920 amino acids. The 5' untranslated region of the genomic RNA was analyzed using the RNAFOLD program (Zuker, M. et al., Nucleic Acids Res., 9:133 (1981)). This region was predicted to contain extensive secondary structure, as demonstrated by the characteristic stem-loop structures preceding the initiation AUG codon.
ORF lb, which overlaps ORF la by 70 nucleotides, is in reading frame +1 and its first AUG codon, which is predicted to be weak, is located 380 nucleotides downstream of the ORF la termination codon. ORF 2, present also in the subgenomic RNA, overlaps ORF lb by 5 nucleotides, begins with a start codon at nucleotide 4325, and ends with a stop codon 82 bases from the 3' end. As we recently reported, ORF 2 codes for a capsid protein precursor of 796 amino acids with a predicted molecular mass of 88 kDa.
The existence of two separate ORFs (la and lb) located in two different reading frames prompted us to examine the 70-nucleotide overlap region in greater detail. A ribosomal frameshift signal was identified, consisting of the "shifty" heptanucleotide (AAAAAAC) from position 2791 to 2797, followed by a stem-loop structure that may form a pseudoknot with a downstream sequence. The putative frameshift signal of the astrovirus showed a striking resemblance to those at the gag-pro junction of some retroviruses, such as mouse mammary tumor virus (MMTV) (Fig. 2B), and fit perfectly the simultaneous tRNA slippage model of -1 frameshifting described for the synthesis of the gαg-related polyproteins (Jacks, et
RECTIFIED SHEET (RULE 91) ISA/EP al., Cell, 55:447 (1988)). Ribosomal frameshifting recently has been shown to be a normal expression mechanism in several groups of positive-strand RNA viruses, namely animal coronaviruses and arteriviruses, and plant luteoviruses and dianthoviruses (Brierly, I. et al., Cell, 57:537 (1989); den Boon, J. et al., /. Virol. , (1991); Prufer, D. et al., EMBO J., 11:1111 (1992)). However, the putative frameshifting signal of astrovirus was much less similar to the frameshift regions of these viruses than to those of some retroviruses (not shown). The ribosomal frameshifting during translation of astrovirus RNA directs the synthesis of an ORF la/lb fusion nonstructural polyprotein of 1416 amino acids with a predicted molecular mass of 161 kDa as set forth in SEQ ID NO: 4. The predicted transmembrane α-helices occur at residues 156 - 172, 308 - 333, 343 - 362, and 369 - 387, the predicted cleavage site at the N-terminus of the putative VPg-protease occurs at residues 419 - 420, the putative nuclear localization signal occurs at residues 666 - 682, and the fusion dipeptide (KK) occurs at residues 904 - 905.
The nucleotide sequence of the astrovirus genomic RNA and the deduced amino acid sequences of the nonstructural polyprotein and the capsid protein were compared with the current sequence databases (Altschul et al.,/. Mol Biol, 215:403 (1990); Henikoff, S. et al., Proc. Natl. Acad. ScL USA, 89:(1992)). Apart from the obvious similarity to several partial H-Ast 1 sequences (Monroe, S. S. et al., (1991); MatsuL S. M. et al., (1993); Willcocks, M. M. et al., (1992); Jiang, B. et al., unpublished), statistically significant sequence similarity was observed between a region in the C-terminal portion of the nonstructural polyprotein and the putative RNA-dependent RNA polymerases (RdRps) of plant bymoviruses and potyviruses (score of 75 corresponding to the Poisson probability of random matching (P) of 0.015 was observed with the putative RdRp of barley yellow mosaic bymovirus, and score of 73 (P= 0.095) was found with Ornithogalum mosaic potyvirus RdRp). Further analysis using the previously published multiple alignment revealed in the putative astrovirus polymerase the eight conserved motifs typical of the positive-strand RNA virus RdRps and showed that it belongs to the so-called supergroup I, which includes the polymerases of picornaviruses, caliciviruses, potyviruses, and several other groups of plant viruses
RECTIFIED SHEET (RULE 91) (Koonin, E. V. et al., /. Gen. Virol. , 72:2197 (1991); Dolja, V. V. et al., Semin. Virol, 3:315 (1992); Koonin, E. V. et al., Crit. Rev. Biochem. Mol. Biol, in press).
A more sensitive analysis performed by comparing the astrovirus protein sequences with a database of positive-strand RNA virus sequences showed a region of the similarity between the polyproteins of H-Ast 2 and rabbit hemorrhagic disease virus (RHDV). This region included the putative catalytic cysteine of the RHDV protease. Using the previously published alignments of chymotrypsin-related proteases of positive-strand RNA viruses, we identified, in the putative protease domain of astrovirus, the conserved segments surrounding the three catalytic amino acid residues and a fourth distal segment implicated in substrate binding (Gorbalenya, A. E. et al., FEBS Lett., 243:103 (1989)). A triple alignment of moderate statistical significance could be generated for the putative proteases of H-Ast 2 and two caliciviruses (Gorbalenya, A. E. et al., (1989)).
An important feature of the putative protease of H-Ast 2 is the substitution of serine for the catalytic cysteine found in the majority of positive-strand RNA virus proteases of superfamily I. Previously, an analogous substitution has been found in the putative proteases of sobemoviruses, luteoviruses, and arteriviruses (Gorbalenya, A. E. et al (1989 and 1988); Bazan, J. F. et al, (1989 and 1990); den Boon, J. A. et al. (1991)). However, the putative protease of H-Ast 2 showed lower similarity to these viral proteases than to the cysteine proteases of caliciviruses.
An extensive search of the astrovirus nonstructural polyprotein sequence for the motifs defining other conserved domains of positive-strand RNA viruses, namely RNA helicase, methyltransferase, and papain-like protease (Gorbalenya, A. E. et al., Nucleic Acids Res., 17:4713 (1989); Gorbalenya, A. E. et al., FEBS Lett., 252:145 (1990); Gorbalenya, A. E. et al., FEBS Lett., 288:201 (1991); Rozanov, M. N. et al., /. Gen. Virol, 73:2129 (1992)), failed to identify any candidate regions. The absence of the helicase domain is remarkable as so far this domain has been identified in all positive-strand RNA viruses with genomes larger
RECTIFIED SHEET (RULE 91) than 6000 nucleotides (Gorbalenya, A. E. et al., Nucleic Acids Res., 17:8413 (1989)). The absence of the methyltransferase domain suggested that the astrovirus encodes VPg, a protein covalently linked to the 5' end of the viral genome (Wimmer, E. et al., Cell, 28:199 (1982); Vartapetian, A.B. et al., Prog. Nucl. Acids Res. Molec. Biol, 34:209 (1987)), compatible with the affinity of the putative H-Ast 2 polymerase with supergroup I RdRps, which mostly belong to VPg-containing viruses (Koonin, E. V. et al., (1991); Dolja, V. V. et al., (1992); Koonin, E. V. et al., in press)).
Additional features detected by analysis of the nonstructural polyprotein of H-Ast 2 included four transmembrane α-helices and a nuclear localization signal (Fig. 1). The transmembrane helices were located in the region upstream of the protease and they may be involved in membrane anchoring of the viral RNA replication complex, as described for the 3A or 3AB proteins of poliovirus (Giachetti, C et al.,/. Virol, 65:2647 (1991); Giachetti, C. et al.,/. Virol, 66:6046 (1992)). In all positive-strand RNA viruses for which the location of the VPg domain in the polyprotein is known, the domain is found within a short region between a (putative) transmembrane segment and the protease (Koonin, E.V., unpublished observations). VPg is linked to the 5' end of the viral RNA via a tyrosine or a serine residue (Wimmer, E. (1982); Vartapetian, A. B. et al. (1987)). Inspection of the respective region of the H-Ast 2 polyprotein revealed no appropriately located tyrosines and only one serine (Ser 420). It is tempting to speculate that this serine may be the RNA-linking amino acid of VPg. Moreover, as it is preceded by a glutamine residue, thus forming a canonical cleavage site for the viral protease, it is possible that the active serine is located at the very N-terminus of the astrovirus VPg, similar to the VPg of comoviruses (Hellen, C. U. T. et al., Biochemistry, 28:9881 (1990); Palmenberg, A. et al., A. Rev. MicrobioL, 44:603 (1990); Eggen, R. et al., in RNA Genetics, P. Ahlquist, J. et al., eds., Vol. 1, p. 49, CRC Press, Boca Raton, Florida (1988); Chen, X. et al., Virology, 191:607 (1992)) . The nuclear localization signal (NLS), spanning amino acids 666 to 682, is identical to that of H-Ast 1 (Willcocks, M. M. et al., (1992)). This signal may be involved in transport of astrovirus proteins to the nucleus, as substantiated by the observation that astrovirus products were detected by immunofluorescence in the
RECTIFIED SHEET (RULE 91) nucleus of bovine astrovirus-infected cells ((Aroonprasert, D. et al., Vet. MicrobioL, 19:113 (1989)). The astrovirus NLS perfectly fits the consensus for the bipartite signal motif comprising two clusters of basic amino acid residues separated by a ten-residue spacer region (Dingwall, C. et al., Trends Biochem. ScL, 16:478 (1991)). In a curious analogy, both the protease and the RdRp of potyviruses contain similar NLS and are accumulated in the nuclei of infected plant cells (Carrington, J. C. et al., Plant Cell, 3:953 (1991); Li, X. H. et al., Virology, 193:951 (1993)).
Screening failed to detect other sequences significantly similar to the capsid protein of H-Ast 2, direct comparison of this capsid sequence with the sequences of other positive-strand RNA virus capsid proteins identified a conserved domain with hepatitis E virus (HEV), an agent phylogenetically remote from astrovirus and other supergroup I viruses in terms of the comparison of RdRps and the other principal nonstructural domains (Koonin, E. V. et al., Proc. Natl. Acad. ScL USA, 89:8259 (1992)). Since both astrovirus and HEV replicate in the human gut, this conserved domain might have resulted from a recombinational event during coinfection. Of interest, astrovirus has previously been reported in association with fatal hepatitis in ducklings, suggesting a possible hepatic tropism for this virus (Gough, R. C. et al., Vet. Rec, 114:279 (1984)).
To gain further insight into the evolutionary relationship of astroviruses, we generated a tentative phylogenetic tree (Felsenstein, J. et al., Cladistics, 5:164 (1989)) for the supergroup I RdRps, including the H-Ast 2 sequence. The result showed that astroviruses constitute a distinct evolutionary lineage not closely associated with any other group of viruses.
RECTIFIED SHEET (RULE 91) ISA/EP Our data show that astroviruses have no close relatives among other viruses, as demonstrated by comparative sequence analysis, and that their genomic organization is novel among animal viruses. It is remarkable, however, that astroviruses combine features typical of several very different groups of positive-strand RNA viruses and even retroviruses (the frameshift signal). Of special interest is the similarity of the genomic organization and expression strategy of astrovirus and plant luteoviruses (Martin, R. R. et al., Annu. Rev. Phytopathol,
28:341 (1990)). Both groups of viruses lack the helicase domain, while the protease and the polymerase domains are apparently fused via ribosome frameshifting. Moreover, this analogy correlates with the substitution of serine for the catalytic cysteine in the viral proteases.
The present findings strongly support the classification of astroviruses in a new family, Astroviridae. The availability of sequence information will be useful in the development of sensitive new diagnostic assays to further our understanding of the importance of this group of viruses as a cause of disease in humans and animals.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Although the present process has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims.
RECTIFIED SHEET (RULE 91) ISA/EP SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Monroe, Stephan S. Glass, Roger I. Koopmans, Marion Jiang, Baoming
(ii) TITLE OF INVENTION: NUCLEIC ACIDS ENCODING HUMAN ASTROVIRUS SEROTYPE II AND USES THEREOF
(iii) NUMBER OF SEQUENCES: 4
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version#1.25
(v) CURRENT APPLICATION DATA:
Application Number PCT/US94/05287
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: USSN 08/061,465
(B) FILING DATE: May 12, 1993
(C) CLASSIFICATION:
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6828 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Human Astrovirus
(B) STRAIN: Serotype 2
(viii) POSITION IN GENOME:
(C) UNITS: 100%
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CCAAGAGGGG GGUGGUGAUU GGCCUUUGGC UUAUCAGUGU GUAUAUAAGA UUUCUACACU 60 CUUUUAUCAA GUACUCUACA GGAUGGCACA CGGUGAGCCA UACUACAGUU CUAAACCUGA 120
RECTIFIED SHEET (RULE 91) CAAAGAUUUC AAUUUUGGAA GCACAAUGGC ACGUAGGCAA AUGACACCUA CCAUGGUUAC 180
AAAGCUUCCC AAGUUUGUUA GGAAUUCUCC ACAAGCCUAU GAUUGGAUCG UAAGAGGUCU 240
AAUCUUCCCC ACCACUGGAA AAACUUAUUU CCAACGAGUU GUUGUGAUUA CCGGUGGGCU 300
UGAGGAUGGA ACAUAUGGCU CAUUCGCAUU UGAUGGUAGA GAAUGGGUAG AGAUCUACCC 360
AAUAGAGCAU CUAAAUCUCA UGUCAUCUUU GAAACUAAUA CACAAAGCCA AUGCUCUUCA 420
GGAGAGAUUA CGUCUCUCCC AAGAAGAGAA AGCCACCCUU GCUCUUGAUG UGCAAUUCCU 480
UCAGCAUGAA AACGUGCGAC UGAAGGAAUU GAUUCCAAAA CCAGAGCCAC GGAAGAUACA 540
GAUGAAGUGG AUAAUUGUAG GAGCAGUGCU UACAUUUUUA UCUCUAAUAC CUGGGGGCUA 600
UGCGCAAAGU CAGACCAACA ACACUAUAUU UACAGAUGUG AUAGCUGCCU GCAAAUAUUC 660
AACUGAGACA UUAACAGAAA ACCUUGACCU UAGAAUCAAG CUCGCACUAG CAAACAUAAC 720
CAUUAGUGAC AAGUUAGACG CUGUGAGGCA AAUUCUUAAC UUUGCCUUUG UACCUAGAGC 780
UCAUUGGUUG AGAACUGUUU UCUACUACAU CCAUUAUUAU GAAAUGUGGA AUAUUUUUAU 840
GUUUGUUCUU GCAAUUGGCA CUGUCAUGAG GAGCGCCCGC CCCGGUACAG ACUUAAUCAC 900
ACUUGCAACG UCCCACUUGU CUGGUUUUAG GCUGGCUGUU UUACCCACAA UUCCAUUCCA 960
UACCACUAUG ACUUUGUGGG UCAUGAACAC UCUUAUGGUU UGUUACUAUU UUGAUAAUUU 1020
GCUAGCAAUA ACAAUGGCAA UCUUAGCACC AAUCCUUGGC AUCAUCUUCU UGUGCUUCAU 1080
GGAAGACUCC AAUUAUGUGA GCCAGAUACG UGGUCUUAUU GCCACAGCAA UAUUAAUUGC 1140
UGGUGGGCAU GCCUGUUUGA CACUCACAGG CACAACCACG UCAUUAUUUG CUGUCAUACU 1200
AACUUGUAGG UUCAUACGUA UGGCGACGGU UUUUAUUGGC ACCAGAUUCG AGAUCCGUGA 1260
UGCUAAUGGG AAGGUCGUGG CUACUGUACC AACUAGGAUC AAAAAUGUUG CAUUUGACUU 1320
CUUCCAGAAG CUAAAACAGU CAGGGGUGAG AGUUGGAGUC AACGAAUUCG UUGUUAUAAA 1380
ACCAGGUGCA UUAUGUGUCA UAGACACCCC UGAAGGGAAA GGAACAGGUU UCUUUUCUGG 1440
CAAUGACAUA GUAACAGCAG CACAUGUUGU UGGCAAUAAU ACUUUUGUGA AUGUGUGCUA 1500
CGAGGGCUUG AUGUACGAAG CGAAAGUUCG UUACAUGCCU GAAAAGGACA UAGCAUUCAU 1560
AACUUGUCCU GGUGACUUGC AUCCAACAGC AAGAUUAAAA UUAUCAAAGA ACCCAGAUUA 1620
UAGUUAUGUC ACAGUCAUGG CUUACGUGAA UGAAGAUCUU GUGGUUUCAA CCGCAGCUGC 1680
CAUGGUGCAU GGUAACACUC UCUCAUAUGC AGUUCGCACC CAAGACGGGA UGUCGGGUGC 1740
ACCAGUUUGU GACAAGUAUG GUCGGGUGUU GGCAGUCCAU CAAACCAAUA CUGGGUACAC 1800
RECTIFIED SHEET (RULE 91)
ISA/EP UGGAGGUGCU GUCAUAAUAG ACCCAGCAGA CUUUCAUCCA GUGAAGGCCC CAUCUCAGGU 1860
GGAAUUGCUC AAAGAGGAAA UAGAGCGACU AAAAGCCCAA UUGAAUUCCG CCGCUGAGAA 1920
CCCAGCGACU GUUGCUACAC AACAACCUGC CAUUACAUUA GAACAGAAAA GUGUUAGCGA 1980
CAGUGAUGUU GUUGACCUUG UCAGAACUGC AAUGGAACGU GAGAUGAAGG UACUGCGUGA 2040
UGAAAUCAAU GGGAUACUUG CACCAUUUCU ACAAAAAAAG AAAGGUAAGA CCAAGCAUGG 2100
UAGGGGUAGA GUCAGACGUA ACCUUAGAAA AGGCGUGAAA CUCCUUACUG AGGAAGAGUA 2160
UCGAGAACUC UUAGAGAAAG GUCUAGAUCG UGAGACAUUC CUUGACCUUA UAGACCGCAU 2220
UAUUGGAGAG AGGUCUGGCU ACCCUGACUA UGAUGAUGAG GAUUAUUAUG AUGAAGAUGA 2280
UGAUGGAUGG GGAAUGGUUG GUGAUGAUGU AGAAUUUGAU UAUACUGAAG UAAUUAAUUU 2340
UGACCAAGCA AAACCAACUC CUGCCCCAAG AACAACCAAG CCAAAACCUU GCCCCGAGCC 2400
AGAAACUGAA ACACAACCAC UUGAUUUGUC UCAGAAGAAA GAGAAACAAC CAGAACAUGA 2460
ACAACAAGUG GUGAAGUCUA CCAAGCCUCA GAAGAAUGAA CCUCAGCCAU AUUCACAAAC 2520
UUAUGGCAAG GCACCAAUCU GGGAAUCUUA UGAUUUUGAC UGGGACGAGG AUGAUGCCAA 2580
GUUCAUCCUG CCAGCACCAC ACCGGUUAAC UAAGGCAGAU GAAAUAGUUC UUGGGUCAAA 2640
AAUUGUCAAG CUUAGGACGA UUAUUGAAAC AGCCAUUAAG ACCCAGAACU AUAGUGCACU 2700
ACCUGAAGCU GUGUUUGAGC UCGACAAAGC AGCUUAUGAA GCAGGUCUAG AAGGUUUCCU 2760
CCAAAGAGUU AAAUCGAAAA ACAAGGCCCC AAAAAACUAC AAAGGGCCCC AGAAGACCAA 2820
GGGGCCCAAA AUUAUCACUC AUUAGAUGCA UGGAAAUCAU UGCUAGAACC UCCACGUGAG 2880
CGGAGGUGCG UACCUGCUAA UUUUCCAUUG UUAGGUCAUU UACCAAUUAA UAGACCCAUC 2940
UUUGAUGAUA AGAAACCCAG GGAUGAUCUC CUUGGAUUAC UUCCAGAACC AACCUGGCAU 3000
GCUUUUGAGG AAUAUGGACC AACUACAUGG GGCCCACAAG CUUUCAUUAA GUCUUUUGAU 3060
AAAUUCUUUU AUGCAGAACC AAUUGAUUUU UUUUCAGAAU AUCCACAGUU GUGUGCUUUC 3120
GCUGAUUGGG CAACUUAUCG CGAGUUUCGG UAUCUAGAGG ACACUAGAGU GAUACACAUA 3180
ACUGCAACUG AGAAGAAUAC UGAUUCAACA CCUGCAUAUC CUAAAAUGAA UUAUUUUGAU 3240
ACUGAAGAAA GUUAUUUGGA AGCACAUGGG UGGGCUCCAU AUAUUAGAGA AUUCACUAGG 3300
GUCUUCAAAG GAGACAAACC UGAAGUACUG UGGUACCUAU UUCUUAAGAA AGAGAUCAUU 3360
AAGGAGGAAA AAGUUAAAAA UUCUGAUAUC CGGCAGAUAG UAUGUGCCGA UCCCAUUUAC 3420
ACCAGGAUAG GGGCGUGCUU AGAGGCACAU CAGAAUGCUU UGAUGAAACA GCAUACCGAU 3480
RECTIFIED SHEET (RULE 91) ACUUCAGUUG GUCAGUGUGG GUGGUCACCA AUGGAAGGCG GCUUUAAAAA AACAAUGCAA 3540
CGCCUAGUAA AUAAAGGGAA UAAGUACUUU AUUGAAUUUG ACUGGACCCG CUAUGAUGGA 3600
ACUAUACCAC CAGCACUUUU CAAACACAUC AAAGAAAUUA GGUGGAAUUU CAUCAAUAAA 3660
GACCAACGUG AAAAGUACAG ACAUGUGCAU GACUGGUAUG UUGACAACCU CCUUAACCGC 3720
CAUGUACUUC UACCAUCUGG UGAAGUUACC UUGCAGACAC GAGGCAAUCC AUCUGGGCAG 3780
UUUUCAACAA CAAUGGAUAA UAACAUGGUC AAUUUUUGGC UACAAGCUUU UGAGUUCGCU 3840
UAUUUCAAUG GCCCAGACAA AGACCUUUGG AAGACCUAUG ACACUGUGGU UUAUGGAGAU 3900
GACAGGCUCU CUACAACACC UUCGGUACCU GAUGAUUAUG AGGAGAGAGU GAUCACUAUG 3960
UAUAGAGACA UCUUUGGCAU GUGGGUUAAG CCCGGGAAGG UCAUCUGUAG AAACAGCAUA 4020
GUUGGAUUAU CCUUUUGUGG CUUUACUGUU AAUGAAAAUC UUGAACCUGU GCCAACCUCU 4080
CCGGAAAAGU UGAUGGCAUC ACUGCUAAAG CCUUAUAAAG UUUUACCUGA UCUUGAAUCA 4140
CUCCAUGGGA AGCUCCUAUG CUAUCAGUUG CUUGCUGCGU UCAUGGCAGA AGAUCACCCU 4200
UUUAAGGUGU AUAUAGAACA CUGCCUAUCA CGGACUGCAA AGCAGCUUCG UGACUCUGGC 4260
CUACCGGCCA GGCUCACAGA AGAGCAACUC CAUCGCAUUU GGAGGGGAGG ACCAAAGAAG 4320
UGUGAUGGCU AGCAAGUCUG ACAAGCAAGU CACUGUUGAG GUCAAUAACA AUGGCCGAAA 4380
CAGGAGCAAA UCCAGAGCUC GAUCACAAUC UAGAGGUCGA GGUAGAUCAG UCAAAAUCAC 4440
AGUCAAUUCU CACAACAAAG GCAGAAGACA AAACGGACGC AACAAAUAUC AAUCUAAUCA 4500
GCGUGUCCGU AAAAUUGUCA AUAAACAACU CAGGAAACAG GGUGUCACAG GACCAAAACC 4560
UGCAAUAUGC CAGAGAGCCA CAGCAACACU UGGGACAAUU GGAUCAAACA CAACAGGAGC 4620
AACAGAGAUC GAGGCGUGCA UACUCCUUAA UCCCGUCCUG GUUAAGGACG CUACUGGAAG 4680
UACUCAGUUU GGGCCAGUGC AGGCGCUAGG UGCUCAGUAU UCAAUGUGGA AACUAAAGUA 4740
UUUGAAUGUU AAACUGACUU CCAUGGUGGG CGCCUCAGCU GUUAACGGGA CUGUACUCCG 4800
CAUCUCGCUC AACCCUACAU CCACUCCAUC AUCAACUAGC UGGUCUGGAC UUGGUGCUCG 4860
UAAGCACAUG GAUGUUACAG UGGGCAGGAA UGCAGUCUUU AAACUUAGAC CAUCAGACCU 4920
UGGAGGGCCA AGGGAUGGCU GGUGGCUCAC UAAUACCAAU GACAAUGCAU CUGAUACAUU 4980
AGGCCCAUCU AUUGAAAUUC ACACCCUUGG UAAAACCAUG UCUUCAUAUA AAAAUGAGCA 5040
AUUUACAGGU GGACUAUUUC UUGUUGAGCU UGCUUCAGAA UGGUGUUUUA CUGGCUAUGC 5100-
AGCUAAUCCA AAUUUAGUUA AUUUGGUUAA AUCCACUGAU CAUGAGGUGA AUGUCACUUU 5160
RECTIFIED SHEET (RULE 91) UGAGGGCUCA AAAGGUACGC CCCUAAUAAU GAAUGUCGCA GAGCACAGCC ACUUUGCAAG 5220
AAUGGCUGAA CAACAUUCCU CCAUCUCAAC AACAUUUUCA AGAGCUGGAG GCGAUGCAAC 5280
AUCUGACACU GUUUGGCAGG UGCUGAACAC AGCAGUCUCA GCAGCAGAGC UUGUAGCCCC 5340
ACCACCGUUC AAUUGGCUUA UAAAGGGUGG CUGGUGGUUU GUAAAGUUGA UUGCAGGUAG 5400
AACUAGAACU GGUACCAAGC AAUUUUAUGU UUAUCCUAGU UAUCAGGAUG CUUUAUCAAA 5460
UAAACCAGCU CUUUGCACUG GUGGAGUUAC AGGUGGCGUU CUACGUACCA CACCGGUAAC 5520
AACUCUACAG UUCACUCAAA UGAACCAGCC AAGCCUUGGG CAUGGUGAGC ACACUGCCAC 5580
CAUUGGCAGU AUUGUGCAAG AUCCAAGUGG GGAACUGCGU GUGCUGCUAA CAGUUGGCUC 5640
AAUCAUGAGC CCGAAUUCAG CUGAUAGGCA AGUUUGGCUG AACAAAACUC UGACAGCGCC 5700
AGGAACAAAU UCAAAUGACA AUCUUGUAAA GAUAGCCCAC GACUUGGGUC ACUAUUUGAU 5760
CAUGCAAGGG UUUAUGCAUA UAAAGACAGU AGAGUGGUAU ACUCCUGAUU UUCAACCUUC 5820
GCGUGACCCA ACCCCUAUUG CUGGCAUGUC AGUGAUGGUU AACAUAACAA AGAAGGCUGA 5880
UGUCUACUUC AUGAAGCAAU UCAAAAAUUC UUACACCAAC AACCGCCAUC AAAUAACAAG 5940
CAUCUUUUUA AUUAAACCAU UGGCAGAUUU UAAGGUGCAA UGUUAUAUGA GCUACUUUAA 6000
AAGAGAGUCA CAUGACAAUG AUGGGGUUGC CAAUCUUACA GUGAGAAGUA UGACCAGCCC 6060
GGAGACUAUC AGGUUUCAAG UUGGAGAAUG GUAUUUGCUA ACAAGUACCA CACUUAAGGA 6120
GAACAACCUA CCAGAGGGCU GGGUUUGGGA UAGGGUGGAG CUUAAGAGUG ACACACCAUA 6180
CUAUGCUGAU CAAGCAUUGA CAUAUUUCAU AACACCACCC CCAGUGGACU CCCAAAUUUU 6240
AUUUGAAGGU AACACCACAU UGCCCAGAAU UUCCUCUCCG CCUGACAAUC CCAGCGGGCG 6300
AUAUAUGGAA AGCCACCAGC AAGACUGUGA CUCUUCUGAU GAUGAGGAUG AUUGUGAAAA 6360
UGUUUCAGAG GAGACAGAAA CUGAGGAUGA GGAAGAUGAG GACGAAGACG AUGAAGCGGA 6420
CAGGUUUGAU CUCCACAGCC CCUAUAGUUC UGAACCUGAG GACUCUGAUG AGAACAACCG 6480
UGUAACCCUC CUCUCUACAC UCAUAAACCA AGGAAUGACA GUGGAGCGCG CAACAAGAAU 6540
AACUAAACGC GCUUUCCCAA CCUGCGCUGA GAAACUGAAG CGCAGCGUGU ACAUGGACCU 6600
GCUUGCCUCC GGUGCAUCGC CGAGCAGUGC AUGGUCAAAC GCGUGUGAUG AAGCACGCAA 6660
UGUGGGCAGC AAUCAGCUGG CCAAACUUUC UGGAGACCGC GGCCACGCCG AGUAGGAUCG 6720
AGGGUACAGU CUCCAUUACU UUUCUGUCUC UGUUUAGAUU AUUUUAAUCA CCAUUUAAAA 6780
UUGAUUUAAU CAGAAGCAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA 6828
RECTIFIED SHEET (RULE 91) (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4247 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Human Astrovirus
(B) STRAIN: Serotype 2
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
AUGGCACACG GUGAGCCAUA CUACAGUUCU AAACCUGACA AAGAUUUCAA UUUUGGAAGC 60
ACAAUGGCAC GUAGGCAAAU GACACCUACC AUGGUUACAA AGCUUCCCAA GUUUGUUAGG 120
AAUUCUCCAC AAGCCUAUGA UUGGAUCGUA AGAGGUCUAA UCUUCCCCAC CACUGGAAAA 180
ACUUAUUUCC AACGAGUUGU UGUGAUUACC GGUGGGCUUG AGGAUGGAAC AUAUGGCUCA 240
UUCGCAUUUG AUGGUAGAGA AUGGGUAGAG AUCUACCCAA UAGAGCAUCU AAAUCUCAUG 300
UCAUCUUUGA AACUAAUACA CAAAGCCAAU GCUCUUCAGG AGAGAUUACG UCUCUCCCAA 360
GAAGAGAAAG CCACCCUUGC UCUUGAUGUG CAAUUCCUUC AGCAUGAAAA CGUGCGACUG 420
AAGGAAUUGA UUCCAAAACC AGAGCCACGG AAGAUACAGA UGAAGUGGAU AAUUGUAGGA 480
GCAGUGCUUA CAUUUUUAUC UCUAAUACCU GGGGGCUAUG CGCAAAGUCA GACCAACAAC 540
ACUAUAUUUA CAGAUGUGAU AGCUGCCUGC AAAUAUUCAA CUGAGACAUU AACAGAAAAC 600
CUUGACCUUA GAAUCAAGCU CGCACUAGCA AACAUAACCA UUAGUGACAA GUUAGACGCU 660
GUGAGGCAAA UUCUUAACUU UGCCUUUGUA CCUAGAGCUC AUUGGUUGAG AACUGUUUUC 720
UACUACAUCC AUUAUUAUGA AAUGUGGAAU AUUUUUAUGU UUGUUCUUGC AAUUGGCACU 780
GUCAUGAGGA GCGCCCGCCC CGGUACAGAC UUAAUCACAC UUGCAACGUC CCACUUGUCU 840
GGUUUUAGGC UGGCUGUUUU ACCCACAAUU CCAUUCCAUA CCACUAUGAC UUUGUGGGUC 900
AUGAACACU UUAUGGUUUG UUACUAUUUU GAUAAUUUGC UAGCAAUAAC AAUGGCAAUC 960
UUAGCACCAA UCCUUGGCAU CAUCUUCUUG UGCUUCAUGG AAGACUCCAA UUAUGUGAGC 1020
CAGAUACGUG GUCUUAUUGC CACAGCAAUA UUAAUUGCUG GUGGGCAUGC CUGUUUGACA 1080
RECTIFIED SHEET (RULE 91) CUCACAGGCA CAACCACGUC AUUAUUUGCU GUCAUACUAA CUUGUAGGUU CAUACGUAUG 1140
GCGACGGUUU UUAUUGGCAC CAGAUUCGAG AUCCGUGAUG CUAAUGGGAA GGUCGUGGCU 1200
ACUGUACCAA CUAGGAUCAA AAAUGUUGCA UUUGACUUCU UCCAGAAGCU AAAACAGUCA 1260
GGGGUGAGAG UUGGAGUCAA CGAAUUCGUU GUUAUAAAAC CAGGUGCAUU AUGUGUCAUA 1320
GACACCCCUG AAGGGAAAGG AACAGGUUUC UUUUCUGGCA AUGACAUAGU AACAGCAGCA 1380
CAUGUUGUUG GCAAUAAUAC UUUUGUGAAU GUGUGCUACG AGGGCUUGAU GUACGAAGCG 1440
AAAGUUCGUU ACAUGCCUGA AAAGGACAUA GCAUUCAUAA CUUGUCCUGG UGACUUGCAU 1500
CCAACAGCAA GAUUAAAAUU AUCAAAGAAC CCAGAUUAUA GUUAUGUCAC AGUCAUGGCU 1560
UACGUGAAUG AAGAUCUUGU GGUUUCAACC GCAGCUGCCA UGGUGCAUGG UAACACUCUC 1620
UCAUAUGCAG UUCGCACCCA AGACGGGAUG UCGGGUGCAC CAGUUUGUGA CAAGUAUGGU 1680
CGGGUGUUGG CAGUCCAUCA AACCAAUACU GGGUACACUG GAGGUGCUGU CAUAAUAGAC 1740
CCAGCAGACU UUCAUCCAGU GAAGGCCCCA UCUCAGGUGG AAUUGCUCAA AGAGGAAAUA 1800
GAGCGACUAA AAGCCCAAUU GAAUUCCGCC GCUGAGAACC CAGCGACUGU UGCUACACAA 1860
CAACCUGCCA UUACAUUAGA ACAGAAAAGU GUUAGCGACA GUGAUGUUGU UGACCUUGUC 1920
AGAACUGCAA UGGAACGUGA GAUGAAGGUA CUGCGUGAUG AAAUCAAUGG GAUACUUGCA 1980
CCAUUUCUAC AAAAAAAGAA AGGUAAGACC AAGCAUGGUA GGGGUAGAGU CAGACGUAAC 2040
CUUAGAAAAG GCGUGAAACU CCUUACUGAG GAAGAGUAUC GAGAACUCUU AGAGAAAGGU 2100
CUAGAUCGUG AGACAUUCCU UGACCUUAUA GACCGCAUUA UUGGAGAGAG GUCUGGCUAC 2160
CCUGACUAUG AUGAUGAGGA UUAUUAUGAU GAAGAUGAUG AUGGAUGGGG AAUGGUUGGU 2220
GAUGAUGUAG AAUUUGAUUA UACUGAAGUA AUUAAUUUUG ACCAAGCAAA ACCAACUCCU 2280
GCCCCAAGAA CAACCAAGCC AAAACCUUGC CCCGAGCCAG AAACUGAAAC ACAACCACUU 2340
GAUUUGUCUC AGAAGAAAGA GAAACAACCA GAACAUGAAC AACAAGUGGU GAAGUCUACC 2400
AAGCCUCAGA AGAAUGAACC UCAGCCAUAU UCACAAACUU AUGGCAAGGC ACCAAUCUGG 2460
GAAUCUUAUG AUUUUGACUG GGACGAGGAU GAUGCCAAGU UCAUCCUGCC AGCACCACAC 2520
CGGUUAACUA AGGCAGAUGA AAUAGUUCUU GGGUCAAAAA UUGUCAAGCU UAGGACGAUU 2580
AUUGAAACAG CCAUUAAGAC CCAGAACUAU AGUGCACUAC CUGAAGCUGU GUUUGAGCUC 2640
GACAAAGCAG CUUAUGAAGC AGGUCUAGAA GGUUUCCUCC AAAGAGUUAA AUCGAAAAAC 2700
AAGGCCCCAA AAAACUACAA AGGGCCCCAG AAGACCAAGG GGCCCAAAAU UAUCACUCAU 2760
RECTIFIED SHEET (RULE 91)
ISA/EP UAGAUGCAUG GAAAUCAUUG CUAGAACCUC CACGUGAGCG GAGGUGCGUA CCUGCUAAUU 2820
UUCCAUUGUU AGGUCAUUUA CCAAUUAAUA GACCCAUCUU UGAUGAUAAG AAACCCAGGG 2880
AUGAUCUCCU UGGAUUACUU CCAGAACCAA CCUGGCAUGC UUUUGAGGAA UAUGGACCAA 2940
CUACAUGGGG CCCACAAGCU UUCAUUAAGU CUUUUGAUAA AUUCUUUUAU GCAGAACCAA 3000
UUGAUUUUUU UUCAGAAUAU CCACAGUUGU GUGCUUUCGC UGAUUGGGCA ACUUAUCGCG 3060
AGUUUCGGUA UCUAGAGGAC ACUAGAGUGA UACACAUAAC UGCAACUGAG AAGAAUACUG 3120
AUUCAACACC UGCAUAUCCU AAAAUGAAUU AUUUUGAUAC UGAAGAAAGU UAUUUGGAAG 3180
CACAUGGGUG GGCUCCAUAU AUUAGAGAAU UCACUAGGGU CUUCAAAGGA GACAAACCUG 3240
AAGUACUGUG GUACCUAUUU CUUAAGAAAG AGAUCAUUAA GGAGGAAAAA GUUAAAAAUU 3300
CUGAUAUCCG GCAGAUAGUA UGUGCCGAUC CCAUUUACAC CAGGAUAGGG GCGUGCUUAG 3360
AGGCACAUCA GAAUGCUUUG AUGAAACAGC AUACCGAUAC UUCAGUUGGU CAGUGUGGGU 3420
GGUCACCAAU GGAAGGCGGC UUUAAAAAAA CAAUGCAACG CCUAGUAAAU AAAGGGAAUA 3480
AGUACUUUAU UGAAUUUGAC UGGACCCGCU AUGAUGGAAC UAUACCACCA GCACUUUUCA 3540
AACACAUCAA AGAAAUUAGG UGGAAUUUCA UCAAUAAAGA CCAACGUGAA AAGUACAGAC 3600
AUGUGCAUGA CUGGUAUGUU GACAACCUCC UUAACCGCCA UGUACUUCUA CCAUCUGGUG 3660
AAGUUACCUU GCAGACACGA GGCAAUCCAU CUGGGCAGUU UUCAACAACA AUGGAUAAUA 3720
ACAUGGUCAA UUUUUGGCUA CAAGCUUUUG AGUUCGCUUA UUUCAAUGGC CCAGACAAAG 3780
ACCUUUGGAA GACCUAUGAC ACUGUGGUUU AUGGAGAUGA CAGGCUCUCU ACAACACCUU 3840
CGGUACCUGA UGAUUAUGAG GAGAGAGUGA UCACUAUGUA UAGAGACAUC UUUGGCAUGU 3900
GGGUUAAGCC CGGGAAGGUC AUCUGUAGAA ACAGCAUAGU UGGAUUAUCC UUUUGUGGCU 3960
UUACUGUUAA UGAAAAUCUU GAACCUGUGC CAACCUCUCC GGAAAAGUUG AUGGCAUCAC 4020
UGCUAAAGCC UUAUAAAGUU UUACCUGAUC UUGAAUCACU CCAUGGGAAG CUCCUAUGCU 4080
AUCAGUUGCU UGCUGCGUUC AUGGCAGAAG AUCACCCUUU UAAGGUGUAU AUAGAACACU 4140
GCCUAUCACG GACUGCAAAG CAGCUUCGUG ACUCUGGCCU ACCGGCCAGG CUCACAGAAG 4200
AGCAACUCCA UCGCAUUUGG AGGGGAGGAC CAAAGAAGUG UGAUGGC 4247 (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2515 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
RECTIFIED SHEET (RULE 91) (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Human Astrovirus
(B) STRAIN: Serotype 2
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
AAAGAAGUGU GAUGGCUAGC AAGUCUGACA AGCAAGUCAC UGUUGAGGUC AAUAACAAUG 60
GCCGAAACAG GAGCAAAUCC AGAGCUCGAU CACAAUCUAG AGGUCGAGGU AGAUCAGUCA 120
AAAUCACAGU CAAUUCUCAC AACAAAGGCA GAAGACAAAA CGGACGCAAC AAAUAUCAAU 180
CUAAUCAGCG UGUCCGUAAA AUUGUCAAUA AACAACUCAG GAAACAGGGU GUCACAGGAC 240
CAAAACCUGC AAUAUGCCAG AGAGCCACAG CAACACUUGG GACAAUUGGA UCAAACACAA 300
CAGGAGCAAC AGAGAUCGAG GCGUGCAUAC UCCUUAAUCC CGUCCUGGUU AAGGACGCUA 360
CUGGAAGUAC UCAGUUUGGG CCAGUGCAGG CGCUAGGUGC UCAGUAUUCA AUGUGGAAAC 420
UAAAGUAUUU GAAUGUUAAA CUGACUUCCA UGGUGGGCGC CUCAGCUGUU AACGGGACUG 480
UACUCCGCAU CUCGCUCAAC CCUACAUCCA CUCCAUCAUC AACUAGCUGG UCUGGACUUG 540
GUGCUCGUAA GCACAUGGAU GUUACAGUGG GCAGGAAUGC AGUCUUUAAA CUUAGACCAU 600
CAGACCUUGG AGGGCCAAGG. GAUGGCUGGU GGCUCACUAA UACCAAUGAC AAUGCAUCUG 660
AUACAUUAGG CCCAUCUAUU GAAAUUCACA CCCUUGGUAA AACCAUGUCU UCAUAUAAAA 720
AUGAGCAAUU UACAGGUGGA CUAUUUCUUG UUGAGCUUGC UUCAGAAUGG UGUUUUACUG 780
GCUAUGCAGC UAAUCCAAAU UUAGUUAAUU UGGUUAAAUC CACUGAUCAU GAGGUGAAUG 840
UCACUUUUGA GGGCUCAAAA GGUACGCCCC UAAUAAUGAA UGUCGCAGAG CACAGCCACU 900
UUGCAAGAAU GGCUGAACAA CAUUCCUCCA UCUCAACAAC AUUUUCAAGA GCUGGAGGCG 960
AUGCAACAUC UGACACUGUU UGGCAGGUGC UGAACACAGC AGUCUCAGCA GCAGAGCUUG 1020
UAGCCCCACC ACCGUUCAAU UGGCUUAUAA AGGGUGGCUG GUGGUUUGUA AAGUUGAUUG 1080
CAGGUAGAAC UAGAACUGGU ACCAAGCAAU UUUAUGUUUA UCCUAGUUAU CAGGAUGCUU 1140
UAUCAAAUAA ACCAGCUCUU UGCACUGGUG GAGUUACAGG UGGCGUUCUA CGUACCACAC 1200
RECTIFIED SHEET (RULE 91) CGGUAACAAC UCUACAGUUC ACUCAAAUGA ACCAGCCAAG CCUUGGGCAU GGUGAGCACA 1260
CUGCCACCAU UGGCAGUAUU GUGCAAGAUC CAAGUGGGGA ACUGCGUGUG CUGCUAACAG 1320
UUGGCUCAAU CAUGAGCCCG AAUUCAGCUG AUAGGCAAGU UUGGCUGAAC AAAACUCUGA 1380
CAGCGCCAGG AACAAAUUCA AAUGACAAUC UUGUAAAGAU AGCCCACGAC UUGGGUCACU 1440
AUUUGAUCAU GCAAGGGUUU AUGCAUAUAA AGACAGUAGA GUGGUAUACU CCUGAUUUUC 1500
AACCUUCGCG UGACCCAACC CCUAUUGCUG GCAUGUCAGU GAUGGUUAAC AUAACAAAGA 1560
AGGCUGAUGU CUACUUCAUG AAGCAAUUCA AAAAUUCUUA CACCAACAAC CGCCAUCAAA 1620
UAACAAGCAU CUUUUUAAUU AAACCAUUGG CAGAUUUUAA GGUGCAAUGU UAUAUGAGCU 1680
ACUUUAAAAG AGAGUCACAU GACAAUGAUG GGGUUGCCAA UCUUACAGUG AGAAGUAUGA 1740
CCAGCCCGGA GACUAUCAGG UUUCAAGUUG GAGAAUGGUA UUUGCUAACA AGUACCACAC 1800
UUAAGGAGAA CAACCUACCA GAGGGCUGGG UUUGGGAUAG GGUGGAGCUU AAGAGUGACA 1860
CACCAUACUA UGCUGAUCAA GCAUUGACAU AUUUCAUAAC ACCACCCCCA GUGGACUCCC 1920
AAAUUUUAUU UGAAGGUAAC ACCACAUUGC CCAGAAUUUC CUCUCCGCCU GACAAUCCCA 1980
GCGGGCGAUA UAUGGAAAGC CACCAGCAAG ACUGUGACUC UUCUGAUGAU GAGGAUGAUU 2040
GUGAAAAUGU UUCAGAGGAG ACAGAAACUG AGGAUGAGGA AGAUGAGGAC GAAGACGAUG 2100
AAGCGGACAG GUUUGAUCUC CACAGCCCCU AUAGUUCUGA ACCUGAGGAC UCUGAUGAGA 2160
ACAACCGUGU AACCCUCCUC UCUACACUCA UAAACCAAGG AAUGACAGUG GAGCGCGCAA 2220
CAAGAAUAAC UAAACGCGCU UUCCCAACCU GCGCUGAGAA ACUGAAGCGC AGCGUGUACA 2280
UGGACCUGCU UGCCUCCGGU GCAUCGCCGA GCAGUGCAUG GUCAAACGCG UGUGAUGAAG 2340
CACGCAAUGU GGGCAGCAAU CAGCUGGCCA AACUUUCUGG AGACCGCGGC CACGCCGAGU 2400
AGGAUCGAGG GUACAGUCUC CAUUACUUUU CUGUCUCUGU UUAGAUUAUU UUAAUCACCA 2460
UUUAAAAUUG AUUUAAUCAG AAGCAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAA 2515 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1416 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
RECTIFIED SHEET (RULE 91) ISA/EP (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Human Astrovirus
(B) STRAIN: Serotype 2
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Ala His Gly Glu Pro Tyr Tyr Ser Ser Lys Pro Asp Lys Asp Phe 1 5 10 15
Asn Phe Gly Ser Thr Met Ala Arg Arg Gin Met Thr Pro Thr Met Val 20 25 30
Thr Lys Leu Pro Lys Phe Val Arg Asn Ser Pro Gin Ala Tyr Asp Trp 35 40 45
He Val Arg Gly Leu He Phe Pro Thr Thr Gly Lys Thr Tyr Phe Gin 50 - 55 60
Arg Val Val Val He Thr Gly Gly Leu Glu Asp Gly Thr Tyr Gly Ser 65 70 75 80
Phe Ala Phe Asp Gly Arg Glu Trp Val Glu He Tyr Pro He Glu His 85 90 95
Leu Asn Leu Met Ser Ser Leu Lys Leu He His Lys Ala Asn Ala Leu 100 105 110
Gin Glu Arg Leu Arg Leu Ser Gin Glu Glu Lys Ala Thr Leu Ala Leu 115 120 125
Asp Val Gin Phe Leu Gin His Glu Asn Val Arg Leu Lys Glu Leu He 130 135 140
Pro Lys Pro Glu Pro Arg Lys He Gin Met Lys Trp He He Val Gly 145 150 155 160
Ala Val Leu Thr Phe Leu Ser Leu He Pro Gly Gly Tyr Ala Gin Ser 165 170 175
Gin Thr Asn Asn Thr He Phe Thr Asp Val He Ala Ala Cys Lys Tyr 180 185 190
Ser Thr Glu Thr Leu Thr Glu Asn Leu Asp Leu Arg He Lys Leu Ala 195 200 205
Leu Ala Asn He Thr He Ser Asp Lys Leu Asp Ala Val Arg Gin He 210 215 220
Leu Asn Phe Ala Phe Val Pro Arg Ala His Trp Leu Arg Thr Val Phe 225 230 235 240
Tyr Tyr He His Tyr Tyr Glu Met Trp Asn He Phe Met Phe Val Leu
RECTIFIED SHEET (RULE 91) 245 250 255
Ala He Gly Thr Val Met Arg Ser Ala Arg Pro Gly Thr Asp Leu He 260 265 270
Thr Leu Ala Thr Ser His Leu Ser Gly Phe Arg Leu Ala Val Leu Pro 275 280 285
Thr He Pro Phe His Thr Thr Met Thr Leu Trp Val Met Asn Thr Leu 290 295 300
Met Val Cys Tyr Tyr Phe Asp Asn Leu Leu Ala He Thr Met Ala He 305 310 315 320
Leu Ala Pro He Leu Gly He He Phe Leu Cys Phe Met Glu Asp Ser 325 330 335
Asn Tyr Val Ser Gin He Arg Gly Leu He Ala Thr Ala He Leu He 340 345 350
Ala Gly Gly His Ala Cys Leu Thr Leu Thr Gly Thr Thr Thr Ser Leu 355 360 365
Phe Ala Val He Leu Thr Cys Arg Phe He Arg Met Ala Thr Val Phe 370 375 380
He Gly Thr Arg Phe Glu He Arg Asp Ala Asn Gly Lys Val Val Ala 385 390 395 400
Thr Val Pro Thr Arg He Lys Asn Val Ala Phe Asp Phe Phe Gin Lys 405 410 415
Leu Lys Gin Ser Gly Val Arg Val Gly Val Asn Glu Phe Val Val He 420 425 430
Lys Pro Gly Ala Leu Cys Val He Asp Thr Pro Glu Gly Lys Gly Thr 435 440 445
Gly Phe Phe Ser Gly Asn Asp He Val Thr Ala Ala His Val Val Gly 450 455 460
Asn Asn Thr Phe Val Asn Val Cys Tyr Glu Gly Leu Met Tyr Glu Ala 465 470 475 480
Lys Val Arg Tyr Met Pro Glu Lys Asp He Ala Phe He Thr Cys Pro 485 490 495
Gly Asp Leu His Pro Thr Ala Arg Leu Lys Leu Ser Lys Asn Pro Asp 500 505 510
Tyr Ser Tyr Val Thr Val Met Ala Tyr Val Asn Glu Asp Leu Val Val 515 520 525
Ser Thr Ala Ala Ala Met Val His Gly Asn Thr Leu Ser Tyr Ala Val 530 535 540
RECTIFIED SHEET (RULE 91) Arg Thr Gin Asp Gly Met Ser Gly Ala Pro Val Cys Asp Lys Tyr Gly 545 550 555 560
Arg Val Leu Ala Val His Gin Thr Asn Thr Gly Tyr Thr Gly Gly Ala 565 570 575
Val He He Asp Pro Ala Asp Phe His Pro Val Lys Ala Pro Ser Gin 580 585 590
Val Glu Leu Leu Lys Glu Glu He Glu Arg Leu Lys Ala Gin Leu Asn 595 600 605
Ser Ala Ala Glu Asn Pro Ala Thr Val Ala Thr Gin Gin Pro Ala He 610 615 620
Thr Leu Glu Gin Lys Ser Val Ser Asp Ser Asp Val Val Asp Leu Val 625 630 635 640
Arg Thr Ala Met Glu Arg Glu Met Lys Val Leu Arg Asp Glu He Asn 645 650 655
Gly He Leu Ala Pro Phe Leu Gin Lys Lys Lys Gly Lys Thr Lys His 660 665 670
Gly Arg Gly Arg Val Arg Arg Asn Leu Arg Lys Gly Val Lys Leu Leu 675 680 685
Thr Glu Glu Glu Tyr Arg Glu Leu Leu Glu Lys Gly Leu Asp Arg Glu 690 695 700
Thr Phe Leu Asp Leu He Asp Arg He He Gly Glu Arg Ser Gly Tyr 705 710 715 720
Pro Asp Tyr Asp Asp Glu Asp Tyr Tyr Asp Glu Asp Asp Asp Gly Trp 725 730 735
Gly Met Val Gly Asp Asp Val Glu Phe Asp Tyr Thr Glu Val He Asn 740 745 750
Phe Asp Gin Ala Lys Pro Thr Pro Ala Pro Arg Thr Thr Lys Pro Lys 755 760 765
Pro Cys Pro Glu Pro Glu Thr Glu Thr Gin Pro Leu Asp Leu Ser Gin 770 775 780
Lys Lys Glu Lys Gin Pro Glu His Glu Gin Gin Val Val Lys Ser Thr 785 790 795 800
Lys Pro Gin Lys Asn Glu Pro Gin Pro Tyr Ser Gin Thr Tyr Gly Lys 805 810 815
Ala Pro He Trp Glu Ser Tyr Asp Phe Asp Trp Asp Glu Asp Asp Ala 820 825 830
Lys Phe He Leu Pro Ala Pro His Arg Leu Thr Lys Ala Asp Glu He 835 840 845
RECTiF1ED SHEET (RULE 91) Val Leu Gly Ser Lys He Val Lys Leu Arg Thr He He Glu Thr Ala 850 855 860
He Lys Thr Gin Asn Tyr Ser Ala Leu Pro Glu Ala Val Phe Glu Leu 865 870 875 880
Asp Lys Ala Ala Tyr Glu Ala Gly Leu Glu Gly Phe Leu Gin Arg Val 885 890 895
Lys Ser Lys Asn Lys Ala Pro Lys Lys Leu Gin Arg Ala Pro Glu Asp 900 905 910
Gin Gly Ala Gin Asn Tyr His Ser Leu Asp Ala Trp Lys Ser Leu Leu 915 920 925
Glu Pro Pro Arg Glu Arg Arg Cys Val Pro Ala Asn Phe Pro Leu Leu 930 935 940
Gly His Leu Pro He Asn Arg Pro He Phe Asp Asp Lys Lys Pro Arg 945 950 955 960
Asp Asp Leu Leu Gly Leu Leu Pro Glu Pro Thr Trp His Ala Phe Glu 965 970 975
Glu Tyr Gly Pro Thr Thr Trp Gly Pro Gin Ala Phe He Lys Ser Phe 980 985 990
Asp Lys Phe Phe Tyr Ala Glu Pro He Asp Phe Phe Ser Glu Tyr Pro 995 1000 1005
Gin Leu Cys Ala Phe Ala Asp Trp Ala Thr Tyr Arg Glu Phe Arg Tyr 1010 1015 1020
Leu Glu Asp Thr Arg Val He His He Thr Ala Thr Glu Lys Asn Thr 1025 1030 1035 1040
Asp Ser Thr Pro Ala Tyr Pro Lys Met Asn Tyr Phe Asp Thr Glu Glu 1045 1050 1055
Ser Tyr Leu Glu Ala His Gly Trp Ala Pro Tyr He Arg Glu Phe Thr 1060 1065 1070
Arg Val Phe Lys Gly Asp Lys Pro Glu Val Leu Trp Tyr Leu Phe Leu 1075 1080 1085
Lys Lys Glu He He Lys Glu Glu Lys Val Lys Asn Ser Asp He Arg 1090 1095 1100
Gin He Val Cys Ala Asp Pro He Tyr Thr Arg He Gly Ala Cys Leu 1105 1110 1115 1120
Glu Ala His Gin Asn Ala Leu Met Lys Gin His Thr Asp Thr Ser Val 1125 1130 1135
Gly Gin Cys Gly Trp Ser Pro Met Glu Gly Gly Phe Lys Lys Thr Met 1140 1145 1150
RECTIFIED SHEET (RULE 91) Gin Arg Leu Val Asn Lys Gly Asn Lys Tyr Phe He Glu Phe Asp Trp 1155 1160 1165
Thr Arg Tyr Asp Gly Thr .He Pro Pro Ala Leu Phe Lys His He Lys 1170 1175 1180
Glu He Arg Trp Asn Phe He Asn Lys Asp Gin Arg Glu Lys Tyr Arg 1185 1190 1195 1200
His Val His Asp Trp Tyr Val Asp Asn Leu Leu Asn Arg His Val Leu 1205 1210 1215
Leu Pro Ser Gly Glu Val Thr Leu Gin Thr Arg Gly Asn Pro Ser Gly 1220 1225 1230
Gin Phe Ser Thr Thr Met Asp Asn Asn Met Val Asn Phe Trp Leu Gin 1235 1240 1245
Ala Phe Glu Phe Ala Tyr Phe Asn Gly Pro Asp Lys Asp Leu Trp Lys 1250 1255 1260
Thr Tyr Asp Thr Val Val Tyr Gly Asp Asp Arg Leu Ser Thr Thr Pro 1265 1270 1275 1280
Ser Val Pro Asp Asp Tyr Glu Glu Arg Val He Thr Met Tyr Arg Asp 1285 1290 1295
He Phe Gly Met Trp Val Lys Pro Gly Lys Val He Cys Arg Asn Ser 1300 1305 1310
He Val Gly Leu Ser Phe Cys Gly Phe Thr Val Asn Glu Asn Leu Glu 1315 1320 1325
Pro Val Pro Thr Ser Pro Glu Lys Leu Met Ala Ser Leu Leu Lys Pro 1330 1335 1340
Tyr Lys Val Leu Pro Asp Leu Glu Ser Leu His Gly Lys Leu Leu Cys 1345 1350 1355 1360
Tyr Gin Leu Leu Ala Ala Phe Met Ala Glu Asp His Pro Phe Lys Val 1365 1370 1375
Tyr He Glu His Cys Leu Ser Arg Thr Ala Lys Gin Leu Arg Asp Ser 1380 1385 1390
Gly Leu Pro Ala Arg Leu Thr Glu Glu Gin Leu His Arg He Trp Arg 1395 1400 1405
Gly Gly Pro Lys Lys Cys Asp Gly 1410 1415
RECTIFIED SHEET (RULE 91) ISA/EP

Claims

What is claimed is:
1. A nucleic acid encoding human Astrovirus serotype 2 as set forth in the Sequencing Listing as SEQ ID NO: 1, or a unique fragment thereof.
2. A purified antigenic polypeptide fragment encoded by the nucleic acid of claim 1.
3. An isolated nucleic acid capable of selectively hybridizing with the nucleic acid of claim 1.
4. An isolated nucleic acid encoding open reading frame la of human Astrovirus serotype 2, comprising nucleotides 83 through 2842 contained in the nucleotide sequence as set forth in the Sequencing Listing SEQ ID NO: 1, or a unique fragment thereof.
5. A purified antigenic polypeptide encoded by the nucleic acid of claim 4.
6. An isolated nucleic acid capable of selectively hybridizing with the nucleic acid of claim 4.
7. An isolated nucleic acid encoding open reading frame lb of human Astrovirus serotype 2, comprising nucleotides 2773 through 4329 contained in the nucleotide sequence as set forth in the Sequencing Listing SEQ ID NO: 1, or a unique fragment thereof.
8. A purified antigenic polypeptide fragment encoded by the nucleic acid of claim 7.
9. An isolated nucleic acid capable of selectively hybridizing with the nucleic acid of claim 7.
RECTIFIED SHEET (RULE 91) ISA/EP
10. An isolated nucleic acid encoding open reading frame 2 of human Astrovirus serotype 2, comprising nucleotides 4325 through 6712 contained in the nucleotide sequence defined in the Sequencing Listing as SEQ ID NO: 1, or a unique fragment thereof.
11. A purified antigenic polypeptide fragment encoded by the nucleic acid of claim 10.
12. An isolated nucleic acid capable of selectively hybridizing with the nucleic acid of claim 10.
13. The polypeptide fragment of claim 11 in a pharmaceutically acceptable carrier.
14. An isolated nucleic acid encoding open reading frame la/lb of human Astrovirus serotype 2, as set forth in the nucleotide sequence defined in the Sequencing Listing as SEQ ID NO: 2, or a unique fragment thereof.
15. A purified antigenic polypeptide fragment encoded by the nucleic acid of claim 14.
16. An isolated nucleic acid capable of selectively hybridizing with the nucleic acid of claim 14.
17. An isolated nucleic acid encoding subgenomic RNA of human Astrovirus serotype 2, as set forth in the nucleotide sequence defined in the Sequencing Listing as SEQ ID NO: 3, or a unique fragment thereof.
18. A purified antigenic polypeptide fragment encoded by the nucleic acid of claim 17.
RECTIFIED SHEET (RULE 91) ISA EP
19. The polypeptide fragment of claim 18 in a pharmaceutically acceptable carrier.
20. An isolated nucleic acid capable of selectively hybridizing with the nucleic acid of claim 17.
21. A vector comprising the nucleic acid of claim 1.
22. The vector of claim 21 in a host capable of expressing the polypeptide encoded by the nucleic acid.
23. A purified monoclonal antibody specifically reactive with human Astrovirus serotype 2.
24. A method of detecting the presence of human Astrovirus serotype 2 in a subject, comprising the steps of: a) contacting an antibody-containing sample from the subject with a detectable amount of the fragment of claim 2; b) detecting the reaction of the fragment and the antibody, the reaction indicating the presence of the Astrovirus.
25. A method of detecting the presence of human Astrovirus serotype 2 in a subject comprising the steps of: a) contacting a sample from the subject suspected of containing human Astrovirus serotype 2 with a detectable amount of the monoclonal antibody of claim 23; and b) detecting the reaction of the human Astrovirus serotype 2 antigen and the antibody, the reaction indicating the presence of human Astrovirus serotype 2.
26. A method of detecting the presence of human Astrovirus serotype 2 in a subject, comprising detecting the presence of the nucleic acid of claim 1.
RECTIFIED SHEET (RULE 91)
27. The method of claim 26, wherein the nucleic acid is detected utilizing a nucleic acid amplification technique.
28. The method of claim 26, wherein the nucleic acid is detected utilizing a restriction fragment length polymorphism.
RECTIFIED SHEET (RULE 91) P
PCT/US1994/005287 1993-05-12 1994-05-12 Nucleic acids encoding human astrovirus serotype 2 and uses thereof WO1994026902A1 (en)

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