CN1768074A - Diagnostic test for the human virus causing severe acute respiratory syndrome (SARS) - Google Patents

Abstract

The present invention relates to a diagnostic test for the virus that causes severe acute respiratory syndrome (SARS) in humans ("hSARS virus"). In particular, the present invention relates to a real-time quantitative PCR test using reverse transcription and polymerase chain reaction to detect hSARS virus. Specifically, the quantitative test is a TaqMan ^ test. The present invention further relates to a diagnostic kit comprising a nucleic acid molecule for detecting hSARS virus.

Description

Diagnostic test for the human virus causing severe acute respiratory syndrome (SARS)

This application claims priority to U.S. Provisional Application 60/457,031, filed March 24, 2003, U.S. Provisional Application 60/457,730, filed March 26, 2003, U.S. Provisional Application, filed Apr. 2, 2003 60/459,931, U.S. Provisional Application 60/460,357 filed April 3, 2003, U.S. Provisional Application 60/461,265, filed April 8, 2003, U.S. Provisional Application 60/462,805, filed April 14, 2003, 2003 U.S. Provisional Application 60/464,886, filed April 23, 2003, U.S. Provisional Application 60/468,139, filed May 5, 2003, and U.S. Provisional Application 60/471,200, filed May 16, 2003, each of which is incorporated by reference in its entirety to this article.

This application contains a long sequence listing, which is hereby incorporated by reference in its entirety, on CD-R in triplicate instead of printed paper. Said CD-R was burnt on March 22, 2004, respectively labeled "CRF", "Copy1" and "Copy2", each containing only one identical 1.58MB file (V9661078.APP).

1. Field of invention

The present invention relates to a diagnostic test for the virus that causes severe acute respiratory syndrome (SARS) in humans ("hSARS virus"). In particular, the present invention relates to a quantitative assay method for the determination of hSARS virus, its natural or artificial variants, analogs or derivatives using reverse transcription and polymerase chain reaction (RT-PCR). Specifically, the quantitative assay is a ^TaqMan® assay. The invention also relates to a diagnostic kit comprising nucleic acid molecules for the detection of hSARS virus.

2. Background

Recently, there was an outbreak of atypical pneumonia in Guangdong Province in mainland China. Between November 2002 and March 2003, 792 cases were reported, including 31 deaths (WHO. Severe Acute Respiratory Syndrome (SARS) Weekly Epidemiol Rec. 2003; 78:86). In response to the crisis, the Hospital Authority of Hong Kong has stepped up surveillance of patients with severe atypical pneumonia. During the course of this investigation, multiple health care workers were identified as having the disease. In addition, multiple cases of pneumonia have been reported in persons who have been in close contact with individuals infected with the disease. Despite typical antibiotic therapy against bacterial pathogens known to be commonly associated with atypical pneumonia, the severity and progression of the disease remained extraordinary. The inventor of the present invention was one of the groups involved in investigating these patients. In these patients, all identification tests for common viruses and bacteria were negative. The disease is named after the acronym for Severe Acute Respiratory Syndrome ("SARS"). After the inventor of the present invention isolated the hSARS virus from a SARS patient, the pathogenic factor of the disease was understood. The present invention provides the rapid and specific real-time quantitative PCR method disclosed herein. The present invention can be used for clinical and scientific research purposes.

3. Outline of the invention

The present invention relates to the use of the sequence information of the isolated hSARS virus in a diagnostic method. In a preferred embodiment, the isolated hSARS virus is deposited at Genbank, NCBI, with accession number AY278491 (SEQ ID NO: 15), which is incorporated herein by reference. The isolated hSARS virus was deposited at the China Center for Type Cultures (CCTCC) on April 2, 2003, given accession number CCTCC-V200303, as described in Section 7 below, which is incorporated herein by reference.

In a specific embodiment, the present invention provides a diagnostic test method for hSARS virus, its natural or artificial variants, analogs or derivatives. In particular, the present invention relates to a quantitative assay for the determination of hSARS virus nucleic acid molecules using reverse transcription and polymerase chain reaction (RT-PCR). Specifically, the quantitative assay is a ^TaqMan® assay. The present invention also provides nucleic acid molecules suitable for hybridization with hSARS nucleic acid, for example including but not limited to PCR primers, reverse transcriptase primers, probes for Southern analysis or other nucleic acid hybridization analysis to detect hSARS nucleic acid. The hSARS nucleic acid comprises or consists of a nucleic acid sequence, the nucleic acid sequence being SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590, 1965, 2471, 2472, 2473, 2474, 2475 or 2476 nucleic acid sequence or its complement, analogue, derivative, fragment or part. In a preferred embodiment, the primer comprises the nucleic acid sequence of SEQ ID NO: 2471 and/or 2472. In a preferred embodiment, the primer comprises the nucleic acid sequence of SEQ ID NO: 2474 and/or 2475. In a most preferred embodiment, described nucleic acid molecule comprises SEQ IDNO:2473 nucleotide sequence or its part, in RT-PCR test, use comprises the nucleic acid molecule of SEQ IDNO:2471 and/or 2472 nucleotide sequence and can be used for detecting hSARS virus. In another most preferred embodiment, said nucleic acid molecule comprises SEQ ID NO: 2476 nucleic acid sequence or its part, in RT-PCR experiment, use comprises SEQ ID NO: 2474 and/or 2475 nucleic acid molecule of nucleic acid sequence can be used as primer for the detection of hSARS virus. In yet another most preferred embodiment, said assay is a ^TaqMan® quantitative assay.

In one embodiment, the present invention provides methods for detecting the presence or expression of hSARS virus or its natural or artificial variants, analogs, derivatives in biological material such as cells, blood, serum, plasma, Saliva, urine, feces, sputum, nasopharyngeal aspirates, etc. An increase or decrease in hSARS virus activity or expression in a sample relative to a control sample can be determined by contacting the biological material with an agent that can directly or indirectly detect the presence or expression of hSARS virus. In a specific embodiment, the detection reagent is a nucleic acid molecule of the invention. In another specific embodiment, the detection nucleic acid molecules are immobilized on a DNA microarray chip.

In a specific embodiment, the present invention provides a diagnostic kit comprising nucleic acid molecules suitable for detecting hSARS virus or its natural or artificial variants, analogs, derivatives. In a specific embodiment, said nucleic acid molecule has SEQ ID NO: 2471 and/or 2472 nucleic acid sequence. In specific embodiments, described nucleic acid molecule has SEQ ID NO:2473 nucleic acid sequence. In another specific embodiment, said nucleic acid molecule has SEQ ID NO: 2474 and/or 2475 nucleic acid sequence. In specific embodiments, described nucleic acid molecule has SEQ ID NO:2476 nucleotide sequence.

In one aspect, the invention relates to the use of an isolated hSARS virus in a diagnostic method. In a specific embodiment, the present invention provides a method for detecting hSARS virus mRNA or genomic RNA of the present invention in biological materials, such as cells, blood, serum, plasma, saliva, urine, feces, sputum, nasopharyngeal Aspirates, etc. An increase or decrease in hSARS virus mRNA or genomic RNA in a sample relative to a control sample can be determined by contacting the biological material with a reagent that can directly or indirectly detect hSARS virus mRNA or genomic RNA. In a specific embodiment, the detection reagent is a nucleic acid molecule of the invention. In another specific embodiment, the detection nucleic acid molecules are immobilized on a DNA microarray chip.

In another aspect, the invention relates to the use of the isolated hSARS virus in a diagnostic method, such as the detection of antibodies that immunospecifically bind hSARS virus in a biological sample. In a specific embodiment, the detection reagent is hSARS virus, such as the virus with the deposit number CCTCC-V200303, or a virus with SEQ ID NO: 15 genome nucleic acid sequence, or SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590, 1965, 2471, 2472, 2473, 2474, 2475 or 2476 nucleic acid sequence encoded polypeptide.

In yet another aspect, the invention provides antibodies or antigen-binding fragments thereof that immunospecifically bind to SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590, 1965, 2471, 2472, 2473, The polypeptide of the present invention encoded by 2474, 2475 or 2476 nucleotide sequence, comprising under stringent conditions and SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590, 1965, 2471, 2472, 2473 , 2474, 2475 or 2476 nucleotide sequence hybrid nucleic acid encoding polypeptide of the present invention, and/or any hSARS epitope having one or more biological activities of the polypeptide of the present invention. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, Fab fragments, F(ab') ₂ Fragments, disulfide-linked Fvs, intrabodies, and fragments containing VL or VH domains or even complementarity determining regions (CDRs) that specifically bind a polypeptide of the invention.

The present invention also relates to a method for identifying subjects infected with hSARS virus or its natural or artificial variants, analogs, and derivatives. In a specific embodiment, the method comprises obtaining total RNA from a biological sample from the subject; reverse transcribing the total RNA to obtain cDNA; and subjecting the cDNA to a PCR assay with a set of primers derived from the nucleotide sequence of hSARS virus.

The present invention further relates to a diagnostic kit comprising primers and nucleic acid probes to detect mRNA or genomic RNA of hSARS virus.

3.1 Definition

The term "variant" as used herein refers to natural genetic variants of hSARS virus or recombinantly prepared hSARS virus variants, each of which contains one or more mutations in its genome compared to the hSARS virus of CCTCC-V200303. The term "variant" may also refer to natural variants of a particular peptide or recombinantly produced variants of a particular peptide or protein in which one or more amino acid residues have been modified by amino acid substitution, insertion or deletion.

As used herein, the term "analog" with respect to non-protein analogs refers to a second organic or inorganic molecule that has similar or the same function as the first organic or inorganic molecule and that is structurally similar to the first organic or inorganic molecule.

As used herein, the term "derivative" refers to a non-proteinaceous derivative of a second organic or inorganic molecule based on the structure of a first organic or inorganic molecule. Derivatives of organic molecules include, but are not limited to, molecules modified, for example, by adding or removing hydroxyl, methyl, ethyl, carboxyl, or amine groups. Organic molecules can also be esterified, alkylated and/or phosphorylated.

The term "mutant" as used herein refers to an organism that has a mutation in its nucleotide sequence compared to a wild-type organism.

The term "antibody" as used herein refers to a monoclonal antibody, a bispecific antibody, a multispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, a camelised antibody, a single domain antibody, a single chain Fv ( scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against antibodies of the invention), and any of the above Epitope-binding fragments of antibodies. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules (ie, molecules that contain an antigen combining site). Immunoglobulin molecules can be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass.

The term "antibody fragment" as used herein refers to an antibody fragment that immunospecifically binds hSARS virus or any epitope of hSARS virus. Antibody fragments can be produced by any technique known to those of skill in the art. For example, Fab and F(ab') ₂ fragments can be produced by enzymatic digestion of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') ₂ fragments). The F(ab') ₂ fragment contains the complete variable, CH1 and hinge regions of the light and heavy chains. Antibody fragments can also be produced by recombinant DNA techniques. An antibody fragment can be one or more complementarity determining regions (CDRs) of an antibody.

As used herein, the term "antibody or antibody fragment that immunospecifically binds a polypeptide of the present invention" refers to an antibody or fragment thereof that immunospecifically binds to SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590 , 1965, 2471, 2472, 2473, 2474, 2475 or 2476 nucleotide sequence or a polypeptide encoded by its complement, analog, derivative, fragment or part thereof, or immunospecifically bound to a polypeptide having SEQ ID NO: 2, 12, 14 , 17-239, 241-736, 738-1107, 1109-1589, 1591-1964 or 1966-2470 amino acid sequence or a variant, analog, derivative or fragment thereof, and does not non-specifically bind to other polypeptides. Antibodies or fragments thereof that immunospecifically bind a polypeptide of the invention may cross-react with other antigens. Preferably, an antibody or fragment thereof that immunospecifically binds a polypeptide of the invention does not cross-react with other antigens. Antibodies or fragments thereof that immunospecifically bind a polypeptide of the invention can be identified, for example, by immunoassays or other techniques known to those skilled in the art.

The term "epitope" as used herein refers to a fragment of hSARS virus, polypeptide or protein that is antigenic or immunogenic in animals, preferably mammals, most preferably humans. An immunogenic epitope is a fragment of a polypeptide that elicits an antibody response in an animal. An antigenic epitope is a fragment of a polypeptide or protein to which an antibody immunospecifically binds, as determined by any method known in the art, eg, by an immunoassay described herein. An antigenic epitope need not be immunogenic.

The term "antigenicity" as used herein refers to the ability of a substance (such as a foreign body, microorganism, drug, antigen, protein, peptide, polypeptide, nucleic acid, DNA, RNA, etc.) to elicit an immune response in a specific organism, tissue and/or cell. Sometimes the term "antigenic" is synonymous with the term "immunogenic".

The term "immunogenicity" as used herein refers to the property of a substance (such as a foreign substance, microorganism, drug, antigen, protein, peptide, polypeptide, nucleic acid, DNA, RNA, etc.) to induce an immune response in an organism. Immunogenicity depends partly on the size of the substance in question and partly on how dissimilar the substance is to host molecules. Highly conserved proteins tend to be less immunogenic.

An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules that exist in its natural source. Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, and can be substantially free of chemical precursors or other chemicals when synthesized by chemical methods. In a preferred embodiment of the invention, the nucleic acid molecule encoding the polypeptide/protein of the invention is isolated or purified. The term "isolated" nucleic acid molecule does not include nucleic acid that is a member of a library and has not been separated from other library clones containing other nucleic acid molecules.

The term "hybridizes under stringent conditions" as used herein describes at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, Nucleotide sequences that are 85%, 90% or 95% identical typically remain hybridized and washed under conditions that hybridize to each other. Such hybridization conditions are described, for example, but not limited to, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6; Basic Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., N.Y. (1986), Pages 75-78 and 84-87; and Molecular Cloning, Cold Spring Harbor Laboratory, N.Y. (1982), pages 387-389, are well known to those skilled in the art. A preferred, non-limiting example of stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC), 0.5% SDS at about 68°C, followed by one or more washes in 2X SSC, 0.5% SDS at room temperature. Another preferred non-limiting example of stringent hybridization conditions is hybridization in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at about 50-65°C.

"Isolated" or "purified" peptides or proteins are substantially free of cellular material or other contaminating proteins from the cellular or tissue origin of the protein, or when they are chemically synthesized, chemical precursors or other Chemical material. The phrase "substantially free of cellular material" includes polypeptide/protein preparations in which the polypeptide/protein is separated from the cellular components of the cells from which it was isolated or recombinantly produced. Thus, a polypeptide/protein that is substantially free of cellular material includes polypeptide/protein preparations that contain less than about 30%, 20%, 10%, 5%, 2.5%, or 1% (dry weight) of contaminating protein. When the polypeptide/protein is produced recombinantly, it is also preferably substantially free of medium, ie, the medium comprises less than about 20%, 10%, or 5% by volume of the protein preparation. When the polypeptide/protein is produced by chemical synthesis, it is preferably substantially free of, ie, separated from, chemical precursors or other chemicals used to carry out protein synthesis. Accordingly, such polypeptide/protein preparations contain less than about 30%, 20%, 10%, 5% (dry weight) of chemical precursors or compounds that are not fragments of the polypeptide/protein of interest. In preferred embodiments of the invention, the polypeptide/protein is isolated or purified.

As used herein, the term "isolated" virus is a virus separated from other organisms present in its natural source, for example, biological material such as cells, blood, serum, plasma, saliva, urine, feces, sputum, nasopharyngeal Suction etc. The isolated virus can be used to infect a subject.

As used herein, the term "having the biological activity of a polypeptide of the present invention" refers to the characteristics of a polypeptide or protein having a common biological activity, having similar or identical structural domains and/or having sufficient amino acid identity compared to the following polypeptide: SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590, 1965, 2471, 2472, 2473, 2474, 2475 or 2476 nucleotide sequences or their complements, analogs, derivatives, fragments or partial codes or a polypeptide having an amino acid sequence of SEQ ID NO: 2, 12, 14, 17-239, 241-736, 738-1107, 1109-1589, 1591-1964 or 1966-2470 or variants, analogs, derivatives thereof or fragments of polypeptides. Such common biological activities of the polypeptides of the invention include antigenicity and immunogenicity.

As used herein, the term "portion" or "fragment" refers to a fragment comprising at least about 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 26,000, 28,000, 29,000 or more continuous nucleic acids, and there are related nucleic acids. A fragment of a nucleic acid molecule that has at least one functional characteristic of the molecule (or a protein encoded by it that has a functional characteristic of the protein encoded by the related nucleic acid molecule); or refers to a nucleic acid molecule that contains at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 400, 500, 600, 800, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 9,500 or more amino acid residues and have at least one function of a related protein or polypeptide Characteristic protein or polypeptide fragments.

As used herein, the term "analogue" refers to proteinaceous substances (such as proteins, polypeptides, peptides, and antibodies) that have similar or identical functions to a second proteinaceous substance, but do not necessarily contain similar or identical amino acid sequence to a second proteinaceous substance, or have a similar or identical structure to a second proteinaceous substance. In a specific embodiment, the antibody analog immunospecifically binds to the same epitope as the antibody from which the analog is derived. In another embodiment, the antibody analog binds immunospecifically to a different epitope than the antibody from which the analog is derived. A proteinaceous substance having a similar amino acid sequence refers to a second proteinaceous substance that satisfies at least one of the following conditions: (a) the amino acid sequence of the proteinaceous substance is at least 30%, at least 35%, At least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% % identical; (b) its encoding nucleotide sequence of the proteinaceous substance hybridizes under stringent conditions to the nucleotide sequence encoding at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues of the second proteinaceous substance consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues Amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, or at least 150 contiguous amino acid residues (c) a proteinaceous substance whose encoding nucleotide sequence is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% of the nucleotide sequence encoding a second proteinaceous substance , at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% the same. A proteinaceous substance having a similar structure to the second proteinaceous substance refers to a proteinaceous substance having a similar secondary, tertiary or quaternary structure to the second proteinaceous substance. The structure of a proteinaceous material can be determined by methods known to those skilled in the art including, but not limited to, peptide sequencing, X-ray crystallography, nuclear magnetic resonance, circular dichroism, and crystallographic electron microscopy.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, aligning the sequences for optimal comparison purposes (e.g., introducing gaps in a first amino acid sequence or nucleic acid sequence to achieve optimal alignment with a second amino acid or nucleic acid sequence) good alignment). The amino acid residues or nucleotides are then compared at corresponding amino acid positions or nucleotide positions. When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions that the two sequences share (ie % identity = number of identical overlapping positions/total number of positions x 100%). In one embodiment, both sequences are of the same length.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Modified in .Acad.Sci.U.S.A.90:58735877. This algorithm is incorporated into the NBLAST and BLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program with parameters set, eg, score=100, wordlength=12, to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program with parameters set, eg, score=50, wordlength=3, to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison, Gapped BLAST as described in Altschul et al., 1997, Nucleic Acids Res. 25:33893402 can be used. Alternatively, PSI BLAST can be used to perform an iterative search to detect distant relationships (Id.) between molecules. When using the BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used (see, eg, the NCBI website). Another preferred, non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:1117. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without gaps allowed. When calculating percent identity, generally only exact matches are counted.

The term "derivative" as used herein refers to proteinaceous substances (eg proteins, polypeptides, peptides and antibodies) which comprise amino acid sequences altered by introducing substitutions, deletions and/or insertions of amino acid residues. The term "derivative" as used herein also refers to proteinaceous substances that are modified, ie by covalently linking molecules of any type to proteinaceous substances. For example and without limitation in any way, antibodies may be modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolysis, attachment of cellular ligands or other proteins etc. Derivatives of proteinaceous substances can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, derivatives of proteinaceous substances may contain one or more atypical amino acids. Derivatives of proteinaceous substances have similar or identical functions to the proteinaceous substances from which they are derived.

As used herein, the terms "subject" and "patient" are used interchangeably. The term "subject" as used herein refers to an animal, preferably including non-primates (e.g. cattle, pigs, horses, goats, sheep, cats, dogs, birds and rodents) and primates (e.g. monkeys such as cynomolgous monkeys and Human), more preferably human.

4. Brief description of the drawings

Figure 1 shows the partial DNA sequence (SEQ ID NO: 1) and its deduced amino acid sequence (SEQ ID NO: 2) obtained from the SARS virus, which shares the RNA-dependent RNA polymerase protein of known coronaviruses 57% homology.

Figure 2 shows an electron micrograph of the novel hSARS virus with coronavirus-like morphological features.

Figure 3 shows immunofluorescence staining of IgG antibodies bound to FrHK-4 cells infected with novel coronavirus family (Coronaviridae) human respiratory virus.

Figure 4 shows an electron micrograph of an ultracentrifuge sediment of hSARS virus grown in cell culture, negatively stained with 3% potassium phosphotungstate at pH 7.0.

Figure 5A shows a thin-section electron micrograph of a lung biopsy from a SARS patient; Figure 5B shows a thin-section electron micrograph of hSARS virus-infected cells.

Figure 6 shows the phylogenetic analysis results of the partial protein sequence (215 amino acids; SEQ ID NO: 2) of hSARS virus (GenBank accession number AY268070). The phylogenetic tree is constructed by the neighbor joining method. The horizontal line distance indicates the number of positions at which the two compared sequences differ. Bootstrap values were extrapolated from 500 replicates.

Figure 7A shows the amplification graph of fluorescence intensity versus PCR cycles in a real-time quantitative PCR assay capable of quantitatively detecting hSARS virus in a sample. The copy number of input plasmid DNA in the reaction is indicated. The X-axis represents the cycle number of the quantitative PCR assay, and the Y-axis represents the fluorescence intensity (FI) above background. Figure 7B shows the results of melting curve analysis of PCR products of clinical samples. Signals from positive (+ve) samples, negative (-ve) samples and water control (water) are indicated. The X-axis represents temperature (° C.) and the Y-axis represents fluorescence intensity (FI) above background.

Figure 8 shows another partial DNA sequence (SEQ ID NO: 11) and its deduced amino acid sequence (SEQ ID NO: 12) obtained from hSARS virus.

Figure 9 shows another partial DNA sequence (SEQ ID NO: 13) and its deduced amino acid sequence (SEQ ID NO: 14) obtained from hSARS virus.

Figure 10 shows the complete genomic DNA sequence (SEQ ID NO: 15) of hSARS virus.

Figure 11 shows the deduced amino acid sequence obtained from SEQ ID NO: 15 in three reading frames (see SEQ ID NO: 16, 240 and 737). An asterisk (*) indicates a stop codon marking the end of the peptide. Amino acid sequence of the first reading frame: SEQ ID NO: 17-239; amino acid sequence of the second reading frame: SEQ ID NO: 241-736; amino acid sequence of the third reading frame: SEQ ID NO: 738-1107.

Figure 12 shows the deduced amino acid sequence obtained from the complement of SEQ ID NO: 15 in three reading frames (see SEQ ID NO: 1108, 1590 and 1965). An asterisk (*) indicates a stop codon marking the end of the peptide. Amino acid sequence of the first reading frame: SEQ ID NO: 1109-1589; amino acid sequence of the second reading frame: SEQ ID NO: 1591-1964; amino acid sequence of the third reading frame: SEQ ID NO: 1966-2470.

Figure 13 shows the nucleotide sequence of forward primer (SEQ ID NO:2471 and 2474), reverse primer (SEQ IDNO:2472 and 2475) and hybridization probe (SEQ ID NO:2473 and 2476), for quantitative TaqMan ^- assay To detect hSARS virus.

Figure 14 shows the standard curve used for real-time quantitative RT-PCR assay SAS-CoV. The threshold cycle number (Ct) is the number of PCR cycles required for the reaction fluorescence intensity to reach a predetermined threshold. Ct is inversely proportional to the logarithm of the starting concentration of plasmid DNA. Correlation coefficients are given. Cts were calculated based on calculated thresholds for different starting copy numbers by the maximum curvature method in standard amplification plots. The X-axis represents the logarithm of the copy number of the standard, and the Y-axis represents the Ct.

Figure 15 shows a typical amplification plot of fluorescence intensity versus PCR cycle number for NPA samples isolated from SARS patients using the modified RT-PCR detection method of the present invention. With the modified RNA extraction method, 40 of the 50 NPA samples were positive in the real-time assay. For the samples that were negative in the first-generation RT-PCR test, it was found that they all contained a small amount of viral RNA through the detection method of the present invention. The X-axis represents the number of PCR cycles and the Y-axis represents the fluorescence intensity over background (ΔRn).

Figure 16 shows a graph of viral load of SARS-CoV in clinical samples versus date of onset. The results showed that viral load increased with disease progression. Certain samples that were positive in the first generation assay were found to contain significant amounts of viral RNA. The X-axis represents the start date and the Y-axis represents the copy number of each reaction in the sample.

5. Detailed Description of the Invention

The present invention relates to the use of the sequence information of the isolated hSARS virus in a diagnostic method. Specifically, the present invention provides methods for detecting the presence or absence of nucleic acid molecules of hSARS virus or natural or artificial variants, analogs or derivatives thereof in biological samples. The method comprises obtaining a biological sample from various sources, contacting the sample with a compound or reagent capable of detecting nucleic acids (such as mRNA, genomic RNA) of the hSARS virus or its natural or artificial variants, analogs, derivatives, thereby detecting the sample The presence of hSARS virus or its natural or artificial variants, analogs and derivatives. Preferably, the reagent for detecting hSARS mRNA or genomic RNA is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic RNA. In a preferred embodiment, the nucleic acid probe is a nucleic acid molecule comprising or consisting of a SEQ ID NO: 2473 or 2476 nucleic acid sequence or a part thereof, which is sufficient to specifically hybridize with hSARS mRNA or genomic RNA under stringent conditions. In a preferred specific embodiment, the hSARS virus or its natural or artificial variants, similar substances or derivatives. In a non-limiting specific embodiment, the primers used in the preferred RT-PCR method are:

5'-CAGAACGCTGTAGCTTCAAAAATCT-3' (SEQ ID NO: 2471) and 5'-TCAGAACCCTGTGATGAATCAACAG-3' (SEQ ID NO: 2472), in the presence of MgCl ₂ thermal cycling such as but not limited to 50°C for 2 minutes, 95°C 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute (see also Sections 6.7, 6.8, 6.9 below). In a preferred embodiment, the primers comprise the nucleic acid sequences of SEQ ID NO: 2471 and 2472. In another non-limiting specific embodiment, the primers used in the preferred RT-PCR method are: 5'-ACCAGAATGGAGGACGCAATG-3' (SEQ ID NO: 2474) and 5'-GCTGTGAACCAAGACGCAGTATTAT-3' (SEQ ID NO: 2475), In the presence of MgCl, thermal cycles are, for example but not limited to, ₄₅ cycles of 50°C for 2 minutes, 95°C for 10 minutes, then 95°C for 15 seconds, and 60°C for 1 minute (see also Sections 6.7, 6.8, 6.9 below). In a preferred embodiment, the primers comprise the nucleic acid sequences of SEQ ID NO: 2474 and 2475.

The methods of the invention may include real-time quantitative PCR assays. In a preferred embodiment, the quantitative PCR used in the present invention is a ^TaqMan® assay (Holland et al., Proc Natl AcadSci USA 88(16):7276 (1991 )). The assay can be performed on an instrument used to perform such assays, such as that available from Applied Biosystems (Foster City, CA). In a more preferred embodiment, the present invention provides a real-time quantitative PCR test, by reverse-transcribing the extracted total RNA from the sample, performing a PCR reaction on the obtained cDNA with specific primers, and using the probe to detect the presence of the amplified product, Thereby detecting the presence of hSARS virus or its natural or artificial variants, analogs or derivatives in biological samples. In a preferred embodiment, the probe is a ^TaqMan® probe consisting of an oligonucleotide with a 5'-reporter dye and a 3'-quencher dye. In a preferred embodiment, the probe has the nucleotide sequence of 5'-TCTGCGTAGGCAATCC-3' (SEQ ID NO: 2473). In another preferred embodiment, the probe has the nucleotide sequence of 5'-ACCCCAAGGTTTA-CCC-3' (SEQ D NO: 2476). A fluorescent reporter dye, such as a ^FAM® dye, is covalently attached to the 5' end of the oligonucleotide probe. Other dyes such as TET ^- dye or VIC can be used as reporter dyes. Each reporter dye is quenched by a 3' ^TAMRA® dye or a non-fluorescent quencher. In a preferred embodiment, the 3' end is labeled with NFQ-MGB. As PCR products are generated after a certain number of thermal cycles, the fluorescent signal of these reactions is captured at the end of the elongation step, allowing quantification of the viral load in the sample based on the amplification profile.

Other techniques for detecting RNA can also be used. For example, in vitro techniques for detecting mRNA include Northern hybridization, in situ hybridization, RT-PCR, and RNase protection. In vitro techniques for the detection of genomic RNA include Northern blot, RT-PCT, and RNase protection.

As noted above, in a preferred embodiment, polynucleotides from the hSARS virus are amplified prior to their detection. The term "amplification" refers to the process of making multiple copies of a nucleic acid from a single polynucleotide molecule. Amplification of polynucleotides can be performed in vitro by biochemical methods known to those skilled in the art. Amplification reagents can be any compound or system that can perform the function of synthesizing primer extension products, including enzymes. Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Taq polymerase, the Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, polymerase muteins, reverse transcriptase , ligases, and other enzymes including thermo-stable enzymes (ie, enzymes that perform primer extension after being raised to a temperature sufficient to cause denaturation). Appropriate enzymes facilitate the incorporation of nucleotides in an appropriate manner to form primer extension products complementary to each mutant nucleotide strand. In a preferred embodiment, the enzyme is ^{AmpliTaqGold®} DNA polymerase from Applied Biosystems. Typically, the synthesis proceeds from the 3'-end of each primer and proceeds in the 5'-direction along the template strand until the end of the synthesis, resulting in molecules of different lengths. However, some amplification reagents use the same process as above, starting synthesis at the 5'-end and proceeding in the other direction. In any event, the methods of the invention are not limited to the specific embodiments of amplification described herein.

One method of in vitro amplification that can be used in the present invention is the polymerase chain reaction (PCR) described in US Pat. Nos. 4,683,202 and 4,683,195. The term "polymerase chain reaction" refers to a method of amplifying a DNA base sequence using a thermostable DNA polymerase and two oligonucleotide primers, one of which is complementary to the (+)-strand at one end of the sequence to be amplified , and the other is complementary to the (-)-strand at the other end. Since the newly synthesized DNA strands can then serve as additional templates for the same primer sequences, successive cycles of primer annealing, strand elongation, and dissociation result in rapid and highly specific amplification of the desired sequence. Polymerase chain reaction is used to detect the presence of polynucleotides encoding cytokines in a sample. Many polymerase chain methods are known to those skilled in the art and can be used in the methods of the present invention. For example, DNA can be subjected to 30-35 cycles of the following amplification in a thermal cycler: 95°C for 30 seconds, 52-60°C for 1 minute, and 72°C for 1 minute, with a final extension step of 72°C for 5 minutes. As another example, DNA can be subjected to 35 polymerase chain reaction cycles in a thermal cycler with a denaturation temperature of 95°C for 30 seconds, followed by a variable annealing temperature of 54-58°C for 1 minute, an extension step of 70°C for 1 minute, and a final extension step at 70°C for 5 minutes.

Primers for amplifying hSARS virus mRNA or genomic RNA can be prepared using any suitable method, such as traditional phosphotriester and phosphodiester methods or automated embodiments thereof, as long as the primers hybridize to the target polynucleotide. One method of synthesizing oligonucleotides on modified solid supports is described in US Patent 4,458,066. The exact length of a primer depends on many factors, including temperature, buffer, and nucleotide composition. Primers must prime the synthesis of elongated products in the presence of amplification-inducing reagents.

The primers used in the method of the present invention are complementary to each strand of the nucleotide sequence to be amplified. The term "complementary" indicates that the primers must hybridize to their respective strands under conditions that allow the reagents to polymerize. In other words, a primer complementary to the flanking sequence hybridizes to the flanking sequence, allowing the amplification of the nucleotide sequence. The 3' end of the continuation primer preferably has full base pairing complementarity to the side strand. Primers and probes for hSARS viral polynucleotides can be developed using known methods in conjunction with the present disclosure. In a preferred embodiment, primers are designed according to the ^TaqMan® Primer Protocol (Applied Biosystems). The primers were designed using Primer Express software as described in Primer Express UserBulletin (Applied Biosystems). Simply put, when designing primers, they should be chosen after the probe. Primers are preferably as close as possible to the probe without overlapping the probe. The GC content of the primers should be in the range of 20%-80%. Multiple identical nucleotides are preferably avoided. Especially for guanine, 4 or more consecutive G's are preferably avoided. The melting temperature of each primer is preferably 58°C to 60°C. Preferably, there are no more than 2 G and/or C bases among the 5 nucleotides at the 3' end of each primer.

Probes can be designed using the Primer Express software described in the Primer Express User Bulletin (P/N 4317594) (Applied Biosystems). Briefly, it is preferred to keep the G-C content in the range of 20%-80%. Multiple identical nucleotides are preferably avoided. Especially for guanine, 4 or more consecutive G's are preferably avoided. It is preferred not to place the G base at the 5' end. It is preferred to choose a chain such that C exceeds G in the probe. Preferably both probes are on the same strand. For single probe assays, the melting temperature is preferably between 68°C and 70°C.

Those of ordinary skill in the art are aware of various amplification methods that can be used to increase the copy number of a nucleic acid of interest. The polynucleotides detected in the method of the present invention can be further evaluated, detected, cloned, sequenced, etc. in solution or after binding to a solid support, which can be done by any method commonly used to detect specific nucleotide sequences, such as another polymeric Enzyme chain reaction, oligo restriction enzyme digestion (Saiki et al., Bio/Technology 3:1008-1012 (1985)), allele-specific oligonucleotide (ASO) probe analysis (Conner et al., Proc.Natl .Acad Sci.USA 80: 278 (1983), oligonucleotide ligation assay (OLA) (Landegren et al., Science 241: 1077 (1988)), RNase protection test, etc. A review of molecular techniques for existing DNA analysis ( Landegren et al., Science 242:229-237 (1988). After DNA amplification, the reaction product is detected by DNA hybridization analysis without using radioactive probes. In this method, for example, polynuclear cells containing A small amount of DNA sample of nucleotide is amplified and analyzed by Southern blotting. The highly amplified signal promotes the use of non-radioactive probes or labels. In one embodiment of the invention, a nucleoside triphosphate is radiolabeled, thereby allowing The amplification products are directly displayed by autoradiography. In another embodiment, the amplification primers are fluorescently labeled and electrophoresis passes through the electrophoresis system. The amplification products are displayed by computer-aided graphic display instead of radioactive signals after laser detection.

Primers for amplifying portions of hSARS viral mRNA or genomic RNA are of a size of at least 10, 15, 20, 25 or 30 nucleotides in length. Preferably the GC ratio should be higher than 30%, 35%, 40%, 45%, 50%, 55% or 60% to prevent hairpin structures on the primers. In addition, amplicons should be of sufficient length to be detected by standard molecular biology methods. Preferred amplicon lengths are at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200, 250, 300, 350, 400, 450, 500 , 550, 600, 700, 800, or 1000 base pairs.

In a specific embodiment, the methods of the present invention further involve obtaining a control sample from a control subject, contacting the control sample with a compound or reagent capable of detecting the presence of mRNA or genomic RNA in the sample, and comparing the presence of mRNA or genomic RNA in the control sample with that to be tested. The presence of mRNA or genomic DNA in the test sample is compared.

The invention also includes a kit for detecting the presence of hSARS virus nucleic acid in the sample to be tested. The kit may include, for example, labeled compounds or reagents capable of detecting nucleic acid molecules in the sample to be tested, and in some embodiments includes means for detecting the amount of mRNA in the sample (oligonucleotide probes that bind to DNA or mRNA).

For oligonucleotide-based kits, the kit may, for example, comprise: (1) an oligonucleotide, such as a detectably labeled oligonucleotide, which hybridizes to the nucleotide sequence of the hSARS virus; and/or ( 2) A pair of primers for amplifying nucleic acid molecules containing hSARS virus sequences. The kit may also contain, for example, buffering agents, preservatives, or protein stabilizing agents. The kit may also contain components necessary to detect a detectable substance (eg, an enzyme or a substrate). The kit may also contain a control sample or series of control samples, which can be tested and compared to the samples to be tested. The various components of the kit are usually enclosed in individual containers, all of the various containers and instructions for use being enclosed together in a single package.

5.1 Nucleic acid L sequence of hSARS virus

The present invention relates to the use of sequence information of isolated viruses in diagnostic and therapeutic methods. The complete genome sequence of hSARS virus CCTCC-V200303 is disclosed in US Patent Application, Attorney Docket No. V9661.0069, filed contemporaneously with this document on March 24, 2004, which is hereby incorporated by reference in its entirety. In a specific embodiment, the present invention provides the complete nucleotide sequence SEQ ID NO of hSARS virus CCTCC-V200303: 15 or its complement, analog, derivative, fragment or part. In addition, the present invention relates to a nucleic acid molecule that hybridizes to any part of SEQ ID NO: 15 of the hSARS virus CCTCC-V200303 genome under stringent conditions. In a specific embodiment, the present invention provides nucleic acid molecules suitable for use as primers, which nucleic acid molecules comprise or are derived from the nucleotide sequence of SEQ ID NO: 1, 3, 4, 11 or 13 or their complements, analogs, substances, fragments or parts. In a preferred specific embodiment, said primer comprises SEQ ID NO: 2471, 2472, 2474 or 2475 nucleic acid sequence. In another specific embodiment, the present invention provides nucleic acid molecules suitable for use as hybridization probes for the detection of compounds comprising or composed of SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590, 1965 , 2471, 2472, 2473, 2474, 2475 or 2476 nucleic acid sequence or its complement, analogue, derivative, fragment or part of the nucleic acid encoding the polypeptide of the present invention. In another embodiment, the present invention relates to a kit comprising primers whose nucleic acid sequence is SEQ ID NO: 2471 and/or 2472 to detect hSARS virus or its natural or artificial variant, analog or derivative. In a preferred embodiment, the test kit also comprises a probe with SEQ ID NO: 2473 nucleotide sequence. In another embodiment, the present invention relates to a kit comprising primers whose nucleic acid sequence is SEQ ID NO: 2474 and/or 2475 to detect hSARS virus or its natural or artificial variant, analog or derivative. In a preferred embodiment, the test kit also comprises a probe with SEQ ID NO: 2476 nucleotide sequence. In another preferred embodiment, the kit further comprises reagents for detecting genes that are absent in the hSARS virus used as a negative control. The present invention further includes chimeric or recombinant viruses or viral proteins encoded by said nucleotide sequences.

The present invention also relates to an isolated nucleic acid molecule of hSARS virus comprising or consisting of SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590, 1965, 2471, 2472, 2473, 2474 , 2475 or 2476 nucleic acid sequence or its complement, analog, derivative, fragment or part. In another specific embodiment, the present invention provides a combination having SEQ ID NO: 1, 11, 15, 13, 16, 240, 737, 1108, 1590, 1965, 2471, 2472, 2473, under stringent conditions as defined herein. A nucleic acid molecule of the 2474, 2475 or 2476 nucleic acid sequence, or a specific gene of a known member of the Coronaviridae family, or an isolated nucleic acid molecule hybridized to a complement, analog, derivative, fragment or part thereof. In another specific embodiment, the present invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule comprising at least about 5, 10 of the nucleic acid sequence of SEQ ID NO: 1 or its complement, analog, derivative or fragment. , 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600 or more contiguous nucleotides. In another specific embodiment, the present invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule comprising at least about 5, 10, 10, or 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, A nucleotide sequence of 1050, 1100, 1150, 1200 or more contiguous nucleotides. In yet another specific embodiment, the present invention provides the isolated polypeptide or protein of nucleic acid molecule encoding, and described nucleic acid molecule comprises SEQ ID NO:13 nucleic acid sequence or its complement, analogue, derivative or fragment at least about 5,10 , 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more consecutive nucleotides sequence. In yet another specific embodiment, the present invention provides nucleic acid molecules encoded isolated polypeptides or proteins, said nucleic acid molecules comprising SEQ ID NO: 15 nucleic acid sequence or its complement, analog, derivative or fragment at least about 5,10 ,15,20,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000 , 1050, 1100, 1150, 1200, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000 A nucleotide sequence of 23000, 24000, 25000, 26000, 27000, 28000, 29000 or more contiguous nucleotides. Polypeptides include those shown in Figures 11 (SEQ ID NOs: 17-239, 241-736, and 738-1107) and 12 (SEQ ID NOs: 1109-1589, 1591-1964, and 1966-2470). The polypeptide or protein of the present invention preferably has one or more biological activities of the following proteins: represented by SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590, 1965, 2471, 2472, 2473, 2474 , 2475 or 2476 nucleic acid sequence encoding; or contain by SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590, 1965, 2471, 2472, 2473, 2474, 2475 or 2476 nucleic acid sequence The encoded amino acid sequence of the native viral protein.

The present invention further provides the present invention capable of specifically binding to the nucleic acid sequence encoded by SEQ ID NO: 1, 11, 13, 16, 240, 737, 1108, 1590, 1965, 2471, 2472, 2473, 2474, 2475 or 2476 or a fragment thereof. Invention polypeptide or any hSARS epitope antibody. The present invention also provides an antibody that specifically binds to the polypeptide of the present invention or any hSARS epitope encoded by the nucleic acid sequence of SEQ ID NO: 15 or a fragment thereof. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, Fab fragments, F(ab') ₂ Fragments, disulfide-linked Fvs, intrabodies, and fragments containing VL or VH domains or even complementarity determining regions (CDRs) that specifically bind to the polypeptides of the invention.

In another embodiment, the present invention provides a vaccine preparation comprising hSARS virus or natural or artificial variants, analogs or derivatives thereof. In yet another embodiment, the invention provides vaccine preparations comprising recombinant and chimeric forms of hSARS virus or subunits of the virus. In a specific embodiment, the vaccine preparation of the invention comprises live but attenuated hSARS virus, with or without pharmaceutically acceptable excipients, including adjuvants. In another specific embodiment, the vaccine preparation of the invention comprises inactivated or killed hSARS virus, with or without pharmaceutically acceptable carriers, including adjuvants. Vaccine preparations according to the invention may also contain adjuvants. Accordingly, the present invention further provides methods for preparing recombinant or chimeric forms of hSARS virus. In another specific embodiment, the vaccine preparation of the present invention comprises one or more vaccines comprising or consisting of SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590, 1965, 2471, 2472, 2473 , 2474, 2475 or 2476 nucleic acid sequence or a nucleic acid molecule composed of a fragment thereof. In another embodiment, the invention provides a vaccine preparation comprising one or more polypeptides of the invention comprising or consisting of SEQ ID NO: 1, 11, 13, 16, 240, 737, 1108, 1590, 1965, 2471, 2472, 2473, 2474, 2475 or 2476 nucleic acid sequence or a nucleotide sequence code composed of a fragment thereof. In another embodiment, the present invention provides a vaccine preparation comprising one or more polypeptides of the present invention encoded by a nucleotide sequence comprising or consisting of a SEQ ID NO: 15 nucleic acid sequence or a fragment thereof. In addition, the present invention provides a method for treating, improving, controlling or preventing SARS by administering the vaccine product or antibody of the present invention alone or in combination with: antiviral agents [such as amantadine, rimantadine, ganciclovir, Cyclovir, ribavirin, penciclovir, oseltamivir, foscarnet, zidovudine (AZT), didanosine (ddI), lamivudine (3TC), zalcitabine (ddC), stavudine (d4T), nevirapine, posvirdine, indinavir, ritonavir, vidarabine, nelfinavir, saquinavir, zanamivir, oxalate Semevir, pleconaril, interferon, etc.], steroids and corticosteroids (eg, prednisone, cortisone, fluticasone) and glucocorticoids, antibiotics, analgesics, bronchodilators, or other medications for respiratory and and/or drugs for the treatment of viral infections.

Furthermore, the present invention provides a pharmaceutical composition comprising an antiviral agent of the present invention and a pharmaceutically acceptable carrier. The present invention also provides kits comprising the pharmaceutical compositions of the present invention.

In another aspect, the present invention provides methods of screening for antiviral agents capable of inhibiting infectivity or replication of hSARS virus or natural or artificial variants, analogs or derivatives thereof.

In one embodiment, the present invention provides detection of the hSARS virus of the present invention or its natural or artificial variants, similar The method by which a substance or derivative exists, is active or expressed. The presence of hSARS virus or its natural or artificial variants, analogs or derivatives can be determined in a sample by contacting the biological material with a reagent that can directly or indirectly detect the presence of hSARS virus or its natural or artificial variants, analogs or derivatives exist. In a specific embodiment, the detection reagent is an antibody of the invention. In another embodiment, the detection reagent is a nucleic acid of the invention.

5.2. hSARS virus

5.2.1. Natural variants of hSARS virus

The present invention is based on the isolation and identification of novel viruses from SARS-affected subjects by the present inventors. The isolated hSARS virus was deposited in the China Center for Type Cultures (CCTCC) on April 2, 2003, and was given accession number CCTCC-V200303. The present invention also relates to the natural variant of the hSARS virus deposited with accession number CCTCC-V200303.

The natural variant of hSARS virus has a sequence different from the genome sequence of hSARS virus, which is due to one or more spontaneous mutations in the genome sequence, including but not limited to point mutations, rearrangements, insertions, deletions, etc., which will or No phenotypic changes occur. Preferably the variant comprises less than 25, 20, 15, 10, 5, 4, 3, or 2 amino acid substitutions, rearrangements, insertions and/or deletions relative to the hSARS virus.

Conservative or non-conservative amino acid substitutions may be made at one or more amino acid residues. In preferred embodiments, the variant is at one or more predicted non-essential amino acid residues (i.e., amino acids that are not critical for the expression of viral biological activities such as infectivity, replication capacity, protein synthesis capacity, assembly capacity, and cytopathic effects. residues) with conservative amino acid substitutions. In other embodiments, the variant is at one or more predicted non-essential amino acid residues (i.e., amino acids that are not critical for the expression of viral biological activities such as infectivity, replication capacity, protein synthesis capacity, assembly capacity, and cytopathic effects. residue) with non-conservative amino acid substitutions.

A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue of similar side chain charge. A "non-conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue with an oppositely charged side chain. Families of amino acid residues with similar side chain charges are well defined in the art. Genetically encoded amino acids can be divided into four families: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; (4) uncharged polarity = glycine, asparagine, Glutamine, cysteine, serine, threonine, tyrosine. In a similar manner, all amino acids can be classified as (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) aliphatic = glycine , alanine, valine, leucine, isoleucine, serine, threonine, serine and threonine are optionally classified separately as aliphatic-hydroxyl; (4) aromatic = phenylalanine , tyrosine, tryptophan; (5) amides = asparagine, glutamine; (6) sulfur = cysteine and methionine. (See e.g. Biochemistry, 4th Edition, L. Stryer, WH Freeman and Co eds.: 1995).

The present invention further relates to hSARS virus mutants. In one embodiment, mutations can be randomly introduced along all or part of the coding sequence of the hSARS virus or its variants, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. Mutagenesis techniques known in the art may also be used, including but not limited to site-directed mutagenesis, chemical mutagenesis, in vitro site-directed mutagenesis using, for example, the QuikChange site-directed mutagenesis kit (Stratagene) and the like. Non-limiting examples of such modifications include amino acid substitutions to cysteines to form disulfide bonds; amino acid substitutions to tyrosines followed by chemical treatment of the polypeptide to form dityrosine bonds, as disclosed in detail herein; one or Multiple amino acid substitutions and/or biological or chemical modifications to create small molecule (substrate or inhibitor) binding pockets; and/or introduction of side chain specific tags (e.g. to identify molecular interactions or capture protein-protein interaction partners ( protein-protein interaction partner)). In a specific embodiment, the biological modification comprises alkylation, phosphorylation, sulfation, oxidation or reduction, ADP-ribosylation, hydroxylation, glycosylation, addition of glucophosphatidylinositol, ubiquitination. In another specific embodiment, the chemical modification comprises altering the charge of the recombinant virus. In yet another embodiment, a positive or negative charge is chemically added to the amino acid residues, wherein the charged amino acid residues are modified to uncharged residues.

5.2.2. Recombinant and chimeric hSARS virus

The present invention also includes recombinant or chimeric viruses encoded by viral vectors derived from the genome of hSARS virus or natural variants thereof. In a specific embodiment, the recombinant virus is derived from the hSARS virus with the deposit number CCTCC-V200303. In a specific embodiment, virus has SEQ ID NO: 15 nucleotide sequence. In another specific embodiment, the recombinant virus is derived from a natural variant of hSARS virus. The natural variant of hSARS virus has a sequence different from the genome sequence (SEQ ID NO: 15) of hSARS virus CCTCC-V200303, which is due to one or more spontaneous mutations in the genome sequence, including but not limited to point mutations, rearrangements, Insertions, deletions, substitutions, etc., which may or may not result in a phenotypic change. According to the present invention, the viral vector derived from the genome of hSARS virus CCTCC-V200303 contains a nucleic acid sequence encoding at least a part of one ORF of hSARS virus. In a specific embodiment, the OFR comprises or consists of a SEQ ID NO: 1, 11 or 13 nucleic acid sequence or a fragment thereof. In a specific embodiment, there is more than one OFR in the nucleotide sequence of SEQ ID NO: 15 or a fragment thereof, as shown in Figure 11 (SEQ ID NO: 16, 240 and 737) and Figure 12 (SEQ ID NO: 1108, 1590 and 1965). In another embodiment, the polypeptide encoded by the ORF comprises or consists of SEQ ID NO: 2, 12 or 14 amino acid sequence or a fragment thereof, or as shown in Figure 11 (see SEQ ID NO: 17-239, 241-736 or 738-1107) and Figure 12 (see SEQ ID NO: 1109-1589, 1591-1964 or 1966-2470) shown in the polypeptide or its fragment composition. According to the invention, these viral vectors may or may not contain nucleic acid which is not the genome of the native virus.

In another specific embodiment, the chimeric virus of the present invention is a recombinant hSARS virus further comprising a heterologous nucleotide sequence. According to the present invention, a chimeric virus may be encoded by a nucleotide sequence in which a heterologous nucleotide sequence has been added to the genome or in which an endogenous or native nucleotide sequence has been replaced by a heterologous nucleotide sequence.

According to the present invention, the chimeric virus is encoded by the viral vector of the present invention further comprising a heterologous nucleotide sequence. According to the invention, chimeric viruses are encoded by viral vectors which may or may not contain nucleic acid other than the native viral genome. According to the invention, a chimeric virus is encoded by a viral vector in which a heterologous nucleotide sequence has been added, inserted or a natural or non-natural sequence has been substituted. According to the present invention, chimeric viruses may be encoded by nucleotide sequences derived from different strains or variants of hSARS virus. Specifically, chimeric viruses are encoded by nucleotide sequences encoding antigenic polypeptides derived from different strains or variants of hSARS virus.

Chimeric viruses are particularly useful for the production of recombinant vaccines against two or more viruses (Tao et al., J.Virol.72, 2955-2961; Durbin et al., 2000, J.Virol.74, 6821-6831; Skiadopoulos et al. , 1998, J. Virol. 72, 1762-1768; Teng et al., 2000, J. Virol. 74, 9317-9321). For example, it is conceivable that a viral vector derived from the hSARS virus expressing one or more proteins of a variant of the hSARS virus (and vice versa) would protect a subject vaccinated with such a vector from infection by the native hSARS virus and its variants . For the purpose of vaccination with live attenuated and replication deficient viruses can be used like other viruses. (See pages 6 and 23 of PCTWO02/057302, which is incorporated herein by reference).

According to the present invention, heterologous sequences to be incorporated into viral vectors encoding recombinant or chimeric viruses of the present invention include sequences obtained or derived from different strains or variants of hSARS.

In certain embodiments, the chimeric or recombinant virus of the present invention is encoded by a viral vector derived from the viral genome, wherein one or more sequences, intergenic regions, terminal sequences or ORFs in whole or in part have been heterologous or non-native sequence substitution. In certain embodiments of the invention, the chimeric virus of the invention is encoded by a viral vector derived from the viral genome, wherein one or more heterologous sequences have been inserted or added to the vector.

The choice of viral vector may depend on the species of viral infection to be treated or protected against. If the subject is a human, an attenuated hSARS virus can be used to provide the antigenic sequence.

According to the present invention, the viral vector can be artificially modified to provide antigenic sequences that can protect against infection by hSARS virus, its natural or artificial variants, analogs or derivatives. Viral vectors can be engineered to provide one, two, three or more antigenic sequences. According to the present invention, the antigenic sequences may be derived from the same virus, different strains or variants of the same type of virus, or different viruses.

The expression products and/or recombinant or chimeric virus particles obtained according to the present invention can be advantageously used in vaccine preparations. The expression products and chimeric virus particles of the invention can be engineered to produce vaccines against a variety of pathogens, including viral and bacterial antigens, tumor antigens, allergen antigens, and autoantigens associated with autoimmune diseases. Specifically, the chimeric virus particles of the present invention can be artificially modified to produce a vaccine capable of protecting subjects from infection by hSARS virus or its natural or artificial variants, analogs or derivatives.

In some embodiments, the expression products of the present invention and recombinant or chimeric virus particles can be artificially modified to produce vaccines against various pathogens, including viral antigens, tumor antigens and autoantigens related to autoimmune diseases . One way to achieve this goal involves modifying existing hSARS genes to include foreign sequences in their respective ectodomains. Where the heterologous sequences are epitopes or antigens of pathogens, these chimeric viruses can be used to induce a protective immune response against the disease agents from which these determinants are derived.

Accordingly, the present invention relates to the use of viral vectors and recombinant or chimeric viruses to produce vaccines against multiple viruses and/or antigens. The present invention also includes recombinant viruses comprising viral vectors derived from hSARS virus or natural or artificial variants, analogs or derivatives thereof, containing phenotypes (such as attenuated virulent phenotype or enhanced antigenicity). Variations and modifications can occur in the coding regions, intergenic regions, and leader and trailer sequences of the virus.

The invention provides host cells comprising a nucleic acid or vector of the invention. Plasmid or viral vectors containing hSARS viral polymerase components are produced in prokaryotic cells to express the components in relevant cell types (bacteria, insect cells, eukaryotic cells). Plasmid or viral vectors containing full-length or partial copies of the hSARS genome are produced in prokaryotic cells to express viral nucleic acids in vitro or in vivo. The latter vectors may contain other viral sequences to produce chimeric viruses or chimeric viral proteins, may lack portions of the viral genome to produce replication-defective viruses, and may contain mutations, deletions, substitutions or insertions to produce attenuated viruses.

The invention also provides host cells comprising a nucleic acid molecule of the invention. In addition, the present invention provides a host cell infected with hSARS virus, such as hSARS virus with deposit number CCTCC-V200303 or its natural or artificial variant, analog or derivative. In a specific embodiment, the invention includes hSARS virus-infected passage cell lines. Preferably the cell line is a primate cell line. These cell lines can be cultivated and maintained using known cell culture techniques, such as those described in Celis, Julio ed., 1994, Cell Biology Laboratory Handbook, Academic Press, N.Y. Various culture conditions for these cells, including media formulations with respect to specific nutrients, oxygen, tonicity, carbon dioxide, and reduced serum levels, can be selected and optimized by those skilled in the art.

Preferably, the cell line of the present invention is a eukaryotic cell line, preferably a primate cell line, more preferably a monkey cell line, most preferably a fetal rhesus monkey kidney cell line (eg FRhK-4), which transiently or stably expresses one or more Full-length or partial hSARS protein. Such cells can be produced by transfection (protein or nucleic acid vectors), infection (viral vectors) or transduction (viral vectors) and can be used in combination with the wild-type, attenuated, replication-deficient or chimeric Viruses complement each other. The cell lines used in the present invention can be cloned using known cell culture techniques familiar to those skilled in the art. Cells can be grown and expanded from single cells using commercially available media under known conditions suitable for cell proliferation.

For example, cell lines of the invention that are kept frozen until use can be warmed at a temperature of about 37°C before adding a suitable growth medium, such as DMEM/F-12 (Life Technologies, Inc.) containing 3% fetal bovine serum (FBS). . The cells can be incubated in a humidified incubator at about 5% CO ₂ at a temperature of 37° C. until confluent. To passage the cells, the growth medium was removed and 0.05% trypsin and 0.53 mM EDTA were added to the cells. The cells were separated, and the cell suspension was collected into a centrifuge tube and centrifuged to obtain a cell pellet. The trypsin solution can be removed and the cell pellet resuspended in new growth medium. The cells can then be further propagated to the desired density in additional growth flasks.

According to the invention, continuous cell lines include immortalized cells which can be maintained in vitro for at least 5, 10, 15, 20, 25 or 50 passages.

Infectious copies of hSARS (wild-type, attenuated, replication-defective or chimeric) can be produced upon co-expression of the polymerase component according to the prior art described above.

In addition, eukaryotic cells transiently or stably expressing one or more full-length or partial hSARS proteins may be used. Such cells can be produced by transfection (protein or nucleic acid vectors), infection (viral vectors) or transduction (viral vectors) and can be used in combination with the wild-type, attenuated, replication-deficient or chimeric Viruses complement each other.

The viral vectors and chimeric viruses of the invention can be used to modulate a subject's immune system by stimulating a humoral immune response, a cellular immune response, or by stimulating tolerance to an antigen. As used herein, subject refers to: humans, primates, horses, cattle, sheep, pigs, goats, dogs, cats, birds and rodents.

5.3. Vaccines and antiviral agents

In a preferred embodiment, the present invention provides protein molecules encoded by nucleic acids of the present invention or hSARS virus-specific viral proteins and functional fragments thereof. Useful protein molecules are for example derived from any gene or genome fragment that can be derived from the virus of the present invention, including envelope protein (E protein), membrane intrinsic protein (M protein), spike protein (S protein), nucleocapsid protein (N protein), hemagglutinin esterase (HE protein), and RNA-dependent RNA polymerase. Such molecules or antigenic fragments thereof provided herein are useful, for example, in diagnostic methods or kits, and in pharmaceutical compositions such as subunit vaccines. Particularly useful is the polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 1, 11, 13, 15, 2471, 2472, 2473, 2474, 2475 or 2476, Figure 11 (SEQ ID NO: 17-239, 241-736 or 738- 1107) and the polypeptide shown in Figure 12 (SEQ ID NO: 1109-1589, 1591-1964 or 1966-2470) or antigenic fragment thereof, for mixing as antigen or subunit immunogen, but inactivated whole Virus. Also particularly useful are proteinaceous substances encoded by recombinant nucleic acid fragments of the hSARS genome, more preferably within the preferred limits of the ORF, especially in vivo (e.g. for protective or therapeutic purposes, or for the provision of diagnostic antibodies) or in vitro (for example, by phage display technology or other techniques for producing synthetic antibodies) all elicit hSARS-specific antibody or protein substances that respond to T cells.

5.3.1 Attenuation of hSARS virus and its variants

The hSARS virus of the present invention or its variants can be genetically engineered to show an attenuated phenotype. In particular, the virus of the invention exhibits an attenuated phenotype in subjects to whom said virus is administered as a vaccine. Attenuation can be accomplished by any method known to those of ordinary skill. Without wishing to be bound by theory, an attenuated phenotype of a virus of the invention can be produced, for example, by using a virus that does not inherently replicate well in the intended host species, e.g. by reducing the Replication, either by reducing the ability of the virus to infect host cells, or by reducing the ability of viral proteins to assemble into infectious virus particles.

In one embodiment, the infectivity of the virus is reduced by a factor of 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,500, 2,000, 1,500, 1,250, 1,000 , 900 times, 800 times, 700 times, 600 times, 500 times, 400 times, 300 times, 200 times, 100 times, 50 times, 25 times, 10 times, 5 times, 1 times or 90%, 80%, 70 times %, 60%, 50%, 40%, 30%, 20% or 10%. The term "infectiousness" as used herein refers to the ability of a virus to enter, survive and replicate in a susceptible host. In a specific embodiment, when growing in a human host, if the growth of the hSARS virus or a variant thereof in a human host is reduced compared with a non-attenuated hSARS virus or a variant thereof, the infectivity of the hSARS virus is reduced. called reduced or weakened. Viral infectivity can be determined using various methods such as, but not limited to, Western blot (protein), northern blot (RNA), southern blot (DNA), plaque formation assays, colorimetry, microscopy, and chemiluminescence techniques. Viral infectivity can be determined in animal cells, preferably primate cells, more preferably monkey cells, most preferably human cells.

In another embodiment, the ability of the virus to replicate is reduced by a factor of 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2500, 2000, 1500, 1250, 1000 times, 900 times, 800 times, 700 times, 600 times, 500 times, 400 times, 300 times, 200 times, 100 times, 50 times, 25 times, 10 times, 5 times, 1 times or 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%. The term "replication competent" as used herein refers to the ability of a virus to replicate, reproduce and/or reproduce. Replication capacity can be determined using the doubling time, replication rate, growth rate and/or half-life of the virus. In a specific embodiment, when growing in a human host, if the growth of the hSARS virus or a variant thereof in a human host is reduced compared with a non-attenuated hSARS virus or a variant thereof, the replication ability of the hSARS virus is reduced called reduced or weakened. The replication capacity of a virus can be determined using various methods such as, but not limited to, Western blot (protein), northern blot (RNA), southern blot (DNA), plaque formation assay, colorimetry, microscopy, and chemiluminescence techniques. In some instances, copying and transcription can be synonymous. The replication capacity of the virus can be determined in animal cells, preferably primate cells, more preferably monkey cells, most preferably human cells.

In another embodiment, the protein synthesis capacity of the virus is reduced by a factor of 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2500, 2000, 1500, 1250, 1000 times, 900 times, 800 times, 700 times, 600 times, 500 times, 400 times, 300 times, 200 times, 100 times, 50 times, 25 times, 10 times, 5 times, 1 times or 90%, 80% , 70%, 60%, 50%, 40%, 30%, 20%, or 10%. The term "protein synthesis capacity" as used herein refers to the ability of a virus to synthesize proteins such as, but not limited to, envelope protein (E protein), membrane intrinsic protein (M protein), spike protein (S protein), nucleocapsid protein (N protein), hemagglutinin esterase (HE protein), and RNA-dependent RNA polymerase. Protein synthesis capacity can be determined by the rate of protein synthesis (eg, transcription level, translation level) and the type and amount of protein synthesized by the virus. In a specific embodiment, when growing in a human host, if the growth of the hSARS virus or a variant thereof in a human host is reduced compared with a non-attenuated hSARS virus or a variant thereof, the protein synthesis ability of the hSARS virus It is said to be lowered or weakened. The protein synthesis capacity of a virus can be determined using various methods such as, but not limited to, Western blot (protein), northern blot (RNA), southern blot (DNA), plaque formation assay, colorimetry, microscopy, and chemiluminescence techniques. The protein synthesis ability of viruses can be assayed in animal cells, preferably primate cells, more preferably monkey cells, most preferably human cells.

In another embodiment, the ability of the virus to assemble is reduced by a factor of 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,500, 2,000, 1,500, 1,250, 1,000 times, 900 times, 800 times, 700 times, 600 times, 500 times, 400 times, 300 times, 200 times, 100 times, 50 times, 25 times, 10 times, 5 times, 1 times or 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%. As used herein, the term "assembly ability" refers to the ability of a virus to assemble essential proteins or protein assemblies into virus particles. In a specific embodiment, when growing in a human host, if the growth of the hSARS virus or a variant thereof in a human host is reduced compared with a non-attenuated hSARS virus or a variant thereof, the assembly ability of the hSARS virus is inhibited. called reduced or weakened. The ability to assemble a virus can be determined using various methods such as, but not limited to, Western blot (protein), northern blot (RNA), southern blot (DNA), plaque formation assays, colorimetry, microscopy, and chemiluminescence techniques. The ability to assemble a virus can be determined in animal cells, preferably primate cells, more preferably monkey cells, most preferably human cells.

In another embodiment, the cytopathic effect of the virus is reduced 10000-fold, 9000-fold, 8000-fold, 7000-fold, 6000-fold, 5000-fold, 4000-fold, 3000-fold, 2500-fold, 2000-fold, 1500-fold, 1250-fold , 1000 times, 900 times, 800 times, 700 times, 600 times, 500 times, 400 times, 300 times, 200 times, 100 times, 50 times, 25 times, 10 times, 5 times, 1 times or 90%, 80 %, 70%, 60%, 50%, 40%, 30%, 20% or 10%. The term "cytopathic effect" as used herein refers to the damage to infected host cells caused by infection with a virus. Viral infection can lead to cellular abnormalities (biochemical and morphological) and/or cell death (eg, lysis). In a specific embodiment, when grown in a human host, if the growth of the hSARS virus or a variant thereof in a human host is reduced compared to a non-attenuated hSARS virus or a variant thereof, the cytopathic effect of the hSARS virus The effect is said to be reduced or attenuated. The cytopathic effect of a virus can be determined using various methods such as, but not limited to, Western blot (protein), northern blot (RNA), southern blot (DNA), plaque formation assay, colorimetry, microscopy, and chemiluminescent techniques. The cytopathic effect of viruses can be assayed in animal cells, preferably primate cells, more preferably monkey cells, most preferably human cells.

A virus of the invention may be attenuated such that one or more functional characteristics of the virus are impaired. The attenuated phenotype of hSARS virus and variants thereof can be detected by any method known to those of ordinary skill. Candidate viruses can be tested, for example, for their ability to infect a host or their rate of replication in a cell culture system. In certain embodiments, growth curves at different temperatures are used to detect the attenuated phenotype of the virus. For example, an attenuated virus can grow at 35°C, but not at 39°C or 40°C. In certain embodiments, different cell lines can be used to assess the attenuation phenotype of the virus. For example, the attenuated virus may only grow in monkey cell lines but not human cell lines, or the attenuated virus may achieve different viral titers in different cell lines. In certain embodiments, virus replication in the respiratory tract of small animal models (including, but not limited to, hamsters, cotton rats, mice, guinea pigs) is used to assess the attenuated phenotype of the virus. In other embodiments, virus-induced immune responses, including but not limited to antibody titers (as assayed for example by plaque reduction neutralization assay or ELISA), are used to assess the attenuated phenotype of the virus. In a specific embodiment, the plaque reduction neutralization assay or ELISA is performed at low doses. In certain embodiments, the hSARS virus can be tested for its ability to cause pathological symptoms in an animal model. A reduced ability of a virus to cause pathological symptoms in animal models is indicative of its attenuated phenotype. In a specific embodiment, the candidate virus is tested in a monkey model for nasal infection, as indicated by mucus production.

In certain other embodiments, attenuation is determined by comparison to a wild-type strain of the virus from which the attenuated virus is derived. In other embodiments, attenuation is determined by comparing the growth of attenuated viruses in different host systems. Therefore, as a non-limiting example, when grown in a human host, if the growth of the hSARS virus or its variant in the human host is reduced compared to non-attenuated hSARS or its variant, the hSARS virus or its variant is eliminated. called attenuated.

In certain embodiments, the attenuated virus of the invention is capable of infecting a host, or is capable of replicating in a host, thereby producing infectious virus particles. However, the attenuated strains grew to lower titers, or grew more slowly, compared to the wild-type strain. The growth curve of the attenuated virus can be determined and compared to the growth curve of the wild-type virus by any method known to those of ordinary skill.

In certain embodiments, the attenuated virus of the invention does not replicate as well as wild-type virus in human cells. However, attenuated viruses replicate well in cell lines lacking interferon function such as Vero cells.

In other embodiments, the attenuated virus of the present invention is capable of infecting a host, replicating in a host, and enabling the insertion of proteins of the virus of the present invention into the plasma membrane, but the attenuated virus does not cause the host to produce new infectious virus particles. In certain embodiments, the attenuated virus infects the host, replicates in the host, and causes viral protein insertion into the host's plasma membrane with the same efficiency as wild-type hSARS. In other embodiments, the attenuated virus results in a reduced ability of viral proteins to intercalate into the plasma membrane of the host cell relative to the wild-type virus. In certain embodiments, the attenuated hSARS virus has reduced ability to replicate in the host relative to the wild-type virus. Any technique known to those of ordinary skill can be used to determine whether a virus is capable of infecting a mammalian cell, replicating in the host, and causing insertion of viral proteins into the plasma membrane of the host.

In certain embodiments, the attenuated viruses of the invention are capable of infecting a host. But in contrast to the wild-type hSARS virus, the attenuated hSARS virus cannot replicate in the host. In a specific embodiment, the attenuated hSARS virus is capable of infecting a host, causing the host to embed viral proteins into its cytoplasmic membrane, but the attenuated virus cannot replicate in the host. Any method known to those of ordinary skill can be used to detect whether the attenuated hSARS virus has infected a host and has caused the host to embed viral proteins into its cytoplasmic membrane.

In certain embodiments, the ability of the attenuated virus to infect a host is reduced compared to the ability of the wild-type virus to infect the same host. Whether a virus is capable of infecting a host can be determined using any technique known to those of ordinary skill.

In certain embodiments, a mutation (e.g., a missense mutation) is introduced into the genome of the virus, e.g., into SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590, 1965, 2471, 2472, 2473, 2474, 2475 or 2476 nucleic acid sequence to produce a virus with an attenuated phenotype. Mutations (eg missense mutations) can be introduced into the structural and/or regulatory genes of the hSARS virus. Mutations can be additions, substitutions, deletions or combinations thereof. Such hSARS virus variants can be screened for expected functionality, such as infectivity in cell culture, replication ability, protein synthesis ability, assembly ability, and cytopathic effects. In a specific embodiment, the missense mutation is a cold-sensitive mutation. In another embodiment, the missense mutation is a thermosensitive mutation. In another embodiment, missense mutations prevent normal processing or splicing of viral proteins.

In other embodiments, deletions are introduced into the genome of the hSARS virus, resulting in attenuation of the virus.

In certain embodiments, attenuation of the virus is achieved by replacing genes of the wild-type virus with genes of a different species, subgroup, or variant of the virus. In another aspect, attenuation of a virus is achieved by replacing one or more specific domains of a protein of a wild-type virus with a domain derived from a corresponding protein of a different species of virus. In certain other embodiments, the virus is attenuated by deleting one or more specific domains of wild-type viral proteins.

When live attenuated vaccines are used, their safety must also be considered. The vaccine must not cause disease. Any technique known in the art to make vaccines safe may be used in the present invention. In addition to attenuation techniques, other techniques may also be used. A non-limiting example is the use of soluble heterologous genes that cannot be incorporated into the virion membrane. For example, a single copy of a soluble form of the viral transmembrane protein lacking the transmembrane and cytoplasmic domains can be used.

Vaccine safety is tested using various tests. For example, a sucrose gradient test and a neutralization test can be used to test safety. A sucrose gradient assay can be used to determine whether heterologous proteins are incorporated into viral particles. If heterologous proteins are inserted into virions, the virions should be tested for their ability to cause symptoms in appropriate animal models, as the virus may have acquired new, potentially pathogenic properties.

5.3.2 Preparation of vaccine

The invention provides a vaccine product for preventing or treating hSARS virus infection. In certain embodiments, the vaccines of the invention comprise recombinant and chimeric viruses of hSARS virus. In certain embodiments, the virus is attenuated, inactivated or killed.

In another embodiment of this aspect of the invention, an inactivated vaccine preparation may be prepared by "killing" the chimeric virus using conventional techniques. Inactivated vaccines are "dead" in the sense that their infectivity has been destroyed. Ideally, the infectivity of the virus is destroyed without affecting its immunogenicity. To prepare inactivated vaccines, chimeric viruses can be grown in cell culture or in the allantois of chicken embryos, purified by zonal ultracentrifugation, inactivated with formaldehyde or β-propiolactone, and harvested. The resulting vaccine is usually administered intramuscularly.

Inactivated virus can be formulated with suitable adjuvants to enhance the immune response. Such adjuvants may include, but are not limited to, inorganic gels such as aluminum hydroxide; surface active substances such as lysolecithin, pluronic polyols, polyanions; peptides; oil emulsions; and potentially useful human adjuvants Agents such as BCG and Corynebacterium parvum.

Vaccines of the invention may be multivalent or monovalent. Multivalent vaccines are prepared from recombinant viruses expressing more than one antigen.

In another aspect, the present invention also provides a DNA vaccine product, which comprises nucleic acid or fragments thereof of hSARS virus (for example, a virus whose deposit number is CCTCC-V200303), or has SEQ ID NO: 1, 11, 13, 15, 16, A nucleic acid molecule of the 240, 737, 1108, 1590, 1965, 2471, 2472, 2473, 2474, 2475 or 2476 sequence, or a complement, analog, derivative, fragment or portion thereof. In another specific embodiment, the DNA vaccine preparation of the present invention comprises a nucleic acid or a fragment thereof encoding an antibody that immunospecifically binds hSARS virus. In DNA vaccine preparations, the DNA vaccine comprises a viral vector (for example derived from hSARS virus, bacterial plasmid or other expression vector) with an insert comprising the The nucleic acid molecule of the present invention enables the expression of the vaccination protein encoded by the nucleic acid molecule in the vaccination subject. Such vectors can be prepared by recombinant DNA techniques as recombinant or chimeric viral vectors carrying the nucleic acid molecules of the invention (see Section 5.1 above).

Various heterologous vectors have been described for DNA vaccination against viral infection. For example, the vectors described in the following references can be used to express hSARS sequences rather than the sequences of the viruses or other pathogens described; especially the vectors described for: Hepatitis B virus (Michel, M.L. et al., 1995, DAN-mediated immunization to the hepatitis B surface antigen in mice: Aspects of the humoral response mimic hepatitis B viral infection in humans, Proc.Natl.Aca Sci.USA 92:5307-5311; Davis, H.L. et al., 1993, DNA-based immunization induces continuous en seretion of hepatitis high levels of circulating antibody, HumanMolec.Genetics 2:1847-1851), HIV virus (Wang, B. et al., 1993, Geneinoculation generates immune responses against human immunodeficiency virus type1, Proc.Natl.Acad.Sci.USA 90:416056- ; Lu, S. et al., 1996, Simian immunodeficiency virus DNA vaccine trialin macques, J.Virol.70: 3978-3991; Letvin, N.L. et al., 1997, Potent, protective anti-HIV immune responses generated by bimodal HIV envelope DNA plus protein va Proc Natl Acad Sci USA.94(17):9378-83) and influenza virus (Robinson, HL et al., 1993, Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmadDNA, Vaccine 11:957-960; Ulmer, J.B. et al., Heterologous protection against influenza by injection of DNA encoding a viral protein, Science259: 1745-1749), and bacterial infections such as tuberculosis (Tascon, R.E. et al., 1996, Vaccination against tuberculosis by DNA injection, Nature Med. 9: 288-8 ; Huygen, K. et al., 1996, Immunogenicity and protectiVe efficacy of a tuberculosis DNA vaccine, Nature Med., 2:893-898) and parasitic infections such as malaria (Sedegah, M., 1994, Protection against malaria by immunizationwith plasmamid DNA encoding circumsporozoite protein, Proc.Natl.Acad.Sci.USA 91: 9866-9870; Doolan, D.L. et al., 1996, Circumventing genetic restriction of protection against malaria with multigene DNAimmunization: CD8+T cell-interferonδ, and nitricend.demmunization- Exper. Med., 1183:1739-1746).

Various methods can be used to deliver the vaccine preparations described above. These methods include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and intranasal routes, as well as by incisional methods (eg, by scraping the superficial layer of the skin using a bifurcated needle).

In addition, chimeric virus vaccine preparations are preferably delivered via the natural route of infection of the pathogen for which the vaccine is designed. The DNA vaccine of the present invention can be administered in the form of a saline solution by injecting it into the muscle or skin with a syringe and a needle (Wolff J.A. et al., 1990, Directgene transfer into mouse muscle in vivo, Science 247:1465-1468; Raz, E., 1994 , Intradermal gene immunization: The possible role of DNAuptake in the induction of cellular immunity to viruses, Proc. Natl. Acd. Sci. USA 91: 9519-9523). Another way of administering a DNA vaccine is called the "gene gun" method, whereby tiny gold beads coated with the DNA molecule of interest are shot into cells (Tang, D. et al., 1992, Genetic immunization is a simple method for eliciting an immune response, Nature 356: 152-154). For a comprehensive review of DNA vaccine methods see Robinson, H.L., 1999, DNA vaccines: basic mechanism and immune responses (review), Int.J.Mol.Med.4(5):549-555; Barber, B., 1997, Introduction: Emerging vaccine strategies, Seminars in Immunology 9(5):269-270; and Robinson, H.L. et al., 1997, DNA vaccines, Seminars in Immunology 9(5):271-283.

The patient to be administered the vaccine is preferably a mammal, most preferably a human, but may also be a non-human animal including, but not limited to, cattle, horses, sheep, pigs, poultry (e.g. chickens), goats, cats, dogs, hamsters, mice and rats.

5.3.3. Adjuvants and carrier molecules

In certain embodiments, hSARS-associated antigens are administered with one or more adjuvants. In one embodiment, the hSARS-associated antigen is administered together with an inorganic salt adjuvant or an inorganic salt gel adjuvant. Such inorganic salt adjuvants and inorganic salt gel adjuvants include, but are not limited to, aluminum hydroxide (ALHYDROGEL, REHYDRAGEL), aluminum phosphate gel, aluminum hydroxyphosphate (ADJU-PHOS) and calcium phosphate.

In another embodiment, the hSARS-associated antigen is administered with an immunostimulatory adjuvant. Such adjuvants include, but are not limited to, cytokines (e.g., interleukin-2, interleukin-7, interleukin-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ, interleukin-1β (IL- 1β) and IL-1β peptide or Sclavo peptide), cytokine-containing liposomes, triterpenoid glycosides or saponins (such as QuilA and QS-21, also sold under the trademark STIMULON, ISCOPREP), muramyl dipeptide ( MDP) derivatives such as N-acetyl-muramoyl-L-threonyl-D-isoglutamine (threonyl-MDP, sold under the trademark TERMURTDE), GMDP, N-acetyl-demuramoyl -L-alanyl-D-isoglutamine, N-acetylmuramoyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1′-2′- Dipalmitoyl-sn-glyceryl-3-hydroxyphosphoryloxy)ethylamine, muramyl tripeptide phosphatidylethanolamine (MTP-PE), unmethylated CpG dinucleotides and oligonucleotides such as Bacterial DNA and fragments thereof, LPS, monophosphoryl lipid A (3D-MLA, sold under the trademark MPL) and polyphosphazene.

In another embodiment, the adjuvant used is a special adjuvant, including but not limited to emulsions, such as Freund's complete adjuvant, Freund's incomplete adjuvant, for example with block copolymers such as L-121 (polyoxygen Propylene/polyoxyethylene, sold under the trademark PLURONIC L-121) prepared squalene or squalane oil-in-water adjuvant formulations such as SAF and MF59, liposomes, virosomes, liposome rolls (cochleate) and An immunostimulatory complex sold under the trademark ISCOM.

In another embodiment, particulate adjuvants are used. Particulate adjuvants include, but are not limited to, biodegradable and biocompatible polyesters, homopolymers of lactic acid (PLA) and glycolic acid (PGA) and their copolymers, lactide-co-glycolide (PLGA) microparticles, polymers that self-associate into microparticles (poloxamer particles), soluble polymers (polyphosphazenes) and virus-like particles (VLP) such as recombinant protein particles, such as hepatitis B surface antigen (HbsAg).

Yet another class of adjuvants that can be used includes mucosal adjuvants including, but not limited to, heat labile enterotoxin (LT) from Escherichia coli, cholera holotoxin (CT) from Vibriocholerae, and cholera Toxin B subunits, mutant toxins (eg LTK63 and LTR72), microparticles and polymeric liposomes.

In other embodiments, any of the types of adjuvants described above may be used in combination with each other or with other adjuvants. For example, non-limiting examples of combination adjuvant formulations that can be used to administer hSARS-associated antigens of the invention include liposomes containing immunostimulatory proteins, cytokines, T-cell and/or B-cell peptides; or with or without entrapped IL-2 microbes or enterotoxin-containing microparticles. Other adjuvants well known in the art are also included within the scope of the present invention (see Vaccine Design: The Subunit and Adjuvant Appoach, Chapter 7, Michael F. Powell and Mark J. Newman (eds.), Plenum Press, New York, 1995, which is hereby incorporated by reference in its entirety).

The effectiveness of an adjuvant can be determined by measuring the induction of antibodies against an immunogenic polypeptide (containing hSARS polypeptide epitopes) produced by administering such polypeptide in a vaccine that also includes various adjuvants.

The polypeptides can be formulated as vaccines in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed through the free amino groups of the peptide) and salts with inorganic acids such as hydrochloric acid or phosphoric acid or organic acids such as acetic acid, oxalic acid, tartaric acid, maleic acid and the like. Salts formed via the free carbonyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol , histidine, procaine, etc.

5.4. Antibody preparation

Antibodies can be isolated from the sera of subjects infected with SARS. Specifically recognize the polypeptide of the present invention, such as but not limited to a polypeptide comprising a sequence of SEQ ID NO: 2, 12 or 14, or as shown in Figure 11 (SEQ ID NO: 17-239, 241-736 and 738-1107) and Figure 12 ( The polypeptide shown in SEQ ID NO: 1109-1589, 1591-1964, 1966-2470), or the antibody of hSARS epitope or its antigen-binding fragment, can be used to detect, screen and isolate the polypeptide of the present invention or its fragment or may encode other biological Similar sequences for similar enzymes. For example, in a specific embodiment, antibodies or fragments thereof that immunospecifically bind hSARS epitopes can be used in various in vitro detection assays, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, Western blot, etc., to detect in vitro Detect the polypeptide of the present invention or the polypeptide of the preferred hSARS virus in a sample such as biological material, said biological material including cells, cell culture medium (such as bacterial cell culture medium, mammalian cell culture medium, insect cell culture medium, yeast cell culture medium, etc.) , blood, serum, plasma, saliva, urine, feces, tissue, sputum, nasopharyngeal aspirate, etc.

Antibodies specific to any epitope of the polypeptide of the present invention or hSARS virus can be generated by any suitable method well known in the art. The polyclonal antibody against the target antigen (for example, the preservation number is CCTCC-V200303 or the hSARS virus comprising the nucleic acid sequence of SEQ ID NO: 15) can be produced by various methods well known in the art. For example, antigens can be administered to various host animals, including but not limited to rabbits, mice, rats, etc., to induce the production of antisera containing polyclonal antibodies specific for the antigen. Various adjuvants can be used to enhance the immune response, depending on the host species, including but not limited to Freund's (complete and incomplete) adjuvants, inorganic gels such as aluminum hydroxide, surface active substances such as lysolecithin, poly Ether polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenols and adjuvants with potential use in humans such as BCG (Bacillus Calmette-Guérin) and Corynebacterium smallis. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a variety of techniques well known in the art, including the use of hybridoma technology, recombinant technology, and phage display technology, or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including those known in the art and described, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammerling et al., Monoclonal Antibodies and T. - Hybridoma technology taught in Cell Hybridomas, pp. 563-681 (Elsevier, N.Y., 1981) (both of which are hereby incorporated by reference in their entirety). The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, and phage clones, and does not refer to the method of antibody production.

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with an antigen of interest or cells expressing such an antigen. Once an immune response is detected, eg, antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes are isolated. The spleen cells are then fused with any suitable myeloma cells by well known techniques. Hybridomas were selected and cloned by limiting dilution. The hybridoma clones are then tested for cells secreting antibodies capable of binding the antigen by methods well known in the art. Ascites fluid, which usually contains high levels of antibodies, can be produced by intraperitoneally inoculating mice with positive hybridoma clones.

Antibody fragments that recognize specific epitopes can be produced by known techniques. For example, Fab and F(ab') ₂ fragments can be produced by proteolytic hydrolysis of immunoglobulin molecules using enzymes such as papain (for production of Fab fragments) or pepsin (for production of F(ab') ₂ fragments). Production. The F(ab') ₂ fragment contains the entire light chain as well as the variable, CH1 and hinge regions of the heavy chain.

The antibodies or fragments thereof of the present invention can also be produced by any antibody synthesis method well known in the art, specifically by chemical synthesis, or preferably by recombinant expression techniques.

Nucleotide sequences encoding antibodies can be obtained from any information available to one of ordinary skill in the art (ie, from Genbank, literature or by routine cloning and sequence analysis). If a clone containing nucleic acid encoding a particular antibody or epitope-binding fragment thereof is not available, but the sequence of the antibody molecule or epitope-binding fragment thereof is known, the immunoglobulin-encoding nucleic acid can be chemically synthesized, or obtained from A suitable source (e.g., an antibody cDNA library, or a cDNA library produced from any tissue or cell that expresses an antibody, such as a hybridoma cell selected to express an antibody, or a nucleic acid, preferably poly A+ RNA, isolated therefrom), by using PCR amplification with synthetic primers that hybridize to the 3' and 5' ends of the sequence, or by using oligonucleotide probes specific for a particular gene sequence, such as for identifying cDNA clones from antibody-encoding cDNA libraries Get it by cloning. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence of the antibody is determined, the nucleotide sequence of the antibody can be manipulated using methods well known in the art for manipulating nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, e.g., Sambrook et al. and Ausubel et al., supra. eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are hereby incorporated by reference in their entirety), to be manipulated by introducing, for example, in the epitope-binding domain of an antibody or in any part of the antibody that enhances or reduces the biological activity of the antibody. Amino acid substitutions, deletions and/or insertions to generate antibodies with different amino acid sequences.

Recombinant expression of antibodies requires construction of expression vectors containing nucleotide sequences encoding antibodies. Once the nucleotide sequence encoding the antibody molecule or the heavy or light chain of the antibody or a portion thereof is obtained, vectors for production of the antibody molecule can be produced by recombinant DNA techniques using techniques well known in the art as discussed in the preceding sections. Expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals can be constructed using methods well known to those of ordinary skill in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. An epitope-binding fragment encoding a heavy chain variable region, a light chain variable region, a heavy and light chain variable region, a heavy chain and/or a light chain variable region, or one or more complementarity determining regions of an antibody may be (CDR) nucleotide sequences were cloned into this vector for expression. The expression vector thus prepared can then be introduced into a suitable host cell to express the antibody. Accordingly, the invention includes host cells comprising a polynucleotide encoding an antibody specific for a polypeptide of the invention or a fragment thereof.

Host cells can be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain-derived polypeptide and the second vector encoding a light chain-derived polypeptide. The two vectors may contain the same selectable marker to allow equal expression of heavy and light chain polypeptides, or different selectable markers to ensure maintenance of both plasmids. Alternatively, a single vector encoding and capable of expressing a heavy chain polypeptide and a light chain polypeptide may also be used. In this case, the light chain should be located in front of the heavy chain to avoid excess toxic free heavy chain (Proudfoot, 1986, Nature, 322:52 and Kohler, 1980, Proc.Natl.Acad.Sci.USA, 77 :2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

In another embodiment, antibodies can also be produced using various phage display methods well known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying their encoding polynucleotide sequences. In a specific embodiment, such phage can be used to display antigen binding domains, such as Fab and Fv or disulfide bond stabilized Fv, expressed from repertoire or combinatorial antibody libraries (eg, human or murine). Phage expressing an antigen binding domain that binds an antigen of interest can be selected or identified with antigen, for example using labeled antigen or antigen bound or captured to a solid surface or bead. The phages used in these methods are generally filamentous phages, including fd and M13. The antigen binding domain is expressed as a recombinant fusion protein with the phage gene III or gene VIII protein. Examples of phage display methods that can be used to prepare the immunoglobulins of the present invention or fragments thereof include methods disclosed in: Brinkman et al., 1995, J. Immunol. Methods, 182:41-50; Ames et al., 1995, J. Immunol.Methods, 184:177-186; Kettleborough et al., 1994, Eur.J.Immunol., 24:952-958; Persic et al., 1997, Gene, 187:9-18; Burton et al., 1994, Advances in Immunology, 57:191-280; PCT Application No. PCT/GB91/01134; PCT Publication No. WO90/02809; WO91/10737; WO92/01047; WO92/18619; WO93/11236; WO95/15982; 5,698,426、5,223,409、5,403,484、5,580,717、5,427,908、5,750,753、5,821,047、5,571,698、5,427,908、5,516,637、5,780,225、5,658,727、5,733,743和5,969,108；以上各文献通过引用整体结合到本文中。

Following phage selection, as described in the above references, antibody coding regions from the phage can be isolated and used to generate all antibodies, including human antibodies or any other desired fragments, and can be expressed in any desired host, including mammals Animal cells, insect cells, plant cells, yeast and bacteria, such as those described in detail below. For example, techniques for the recombinant production of Fab, Fab' and F(ab') ₂ fragments can also be applied, using methods well known in the art such as disclosed in: PCT Publication No. WO92/22324; Mullinax et al., 1992, BioTechniques, 12(6):864-869; and Sawai et al., AJRI, 34:26-34, 1995; and Better et al., 1988, Science, 240:1041-1043 (each of which is hereby incorporated by reference in its entirety). Examples of techniques that can be used to produce single-chain Fv and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods in Enzymology, 203:46-88; Shu et al., 1993, PNAS, 90:7995-7999; and Skerra et al., 1988, Science, 240:1038-1040.

Once an antibody molecule of the invention has been produced by any of the methods described above, it can subsequently be purified by any method well known in the art for the purification of immunoglobulin molecules, such as chromatography (e.g. ion exchange chromatography, affinity chromatography, especially Purification of Protein A and Protein G followed by affinity chromatography for specific antigens, as well as size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. In addition, antibodies of the invention or fragments thereof may be fused to heterologous polypeptide sequences described herein or well known in the art to facilitate purification.

For certain applications, including the use of antibodies in humans and in vitro detection assays, the use of chimeric, humanized or human antibodies is preferred. Chimeric antibodies are antibody molecules in which different portions of the antibody are derived from different animal species, eg, antibodies having variable regions derived from murine monoclonal antibodies and constant regions derived from human immunoglobulins. Methods for producing chimeric antibodies are well known in the art. See, e.g., Morrison, 1985, Science, 229:1202; Oi et al., 1986, BioTechniques, 4:214; Gillies et al., 1989, J. Immunol. Methods, 125:191-202; U.S. Pat. Each document is hereby incorporated by reference in its entirety. Humanized antibodies are antibody molecules from a non-human species that bind the desired antigen and that have one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Typically, framework residues in the human framework regions will be substituted by corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions can be identified by methods well known in the art, such as by modeling the interactions of CDRs and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify substitutions at specific positions. Unusual framework residues. See, eg, Queen et al., US Patent No. 5,585,089; Riechmann et al., 1988, Nature, 332:323, which are hereby incorporated by reference in their entirety. Antibodies can be humanized using a variety of techniques well known in the art, including, for example, CDR grafting (EP239,400; PCT Publication No. WO91/09967; U.S. Patent Nos. 5,225,539; 5,530,101 and 5,585,089), surface veneering or surface gluing. Resurfacing (EP592,106; EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5): 489-498; Studnicka et al., 1994, Protein Engineering, 7(6): 805-814; Roguska et al., 1994, Proc Natl. Acad. Sci. USA, 91:969-973) and chain shuffling (US Patent No. 5,565,332), each of which is incorporated herein by reference in its entirety.

Fully human antibodies are particularly suitable for use in the therapeutic treatment of human patients. Human antibodies can be prepared by a variety of methods well known in the art, including phage display techniques described above using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publication Nos. WO98/46645; WO98/50433; WO98/24893; WO98/16654; WO96/34096; .

Human antibodies can also be produced using transgenic mice that do not express functional endogenous immunoglobulins, but instead express human immunoglobulin genes. For a review of this technology for producing human antibodies, see Lonberg and Huszar, 1995, Int. Rev. Immunol., 13:65-93. For a detailed discussion of such techniques for producing human antibodies and human monoclonal antibodies, and protocols for producing such antibodies, see, e.g., PCT Publication Nos. WO98/24893; WO92/01047; WO96/34096; WO96/33735; European Patent No. 0598877; US Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; Additionally, companies such as Abgenix, Inc. (Fremont, CA), Medarex (NJ), and Genpharm (San Jose, CA) may be contracted to provide human antibodies to selected antigens produced using techniques similar to those described above.

Fully human antibodies that recognize selected epitopes can be generated using a technique known as "directed selection." In this approach, selected non-human monoclonal antibodies such as mouse antibodies are used to guide the selection of fully human antibodies that recognize the same epitope. (Jespers et al., 1988, Bio/technology, 12:899-903).

Antibodies fused or conjugated to heterologous polypeptides can be used in in vitro immunoassays or purification methods (eg, affinity chromatography) well known in the art. See, eg, PCT Publication No. WO93/21232; EP439,095; Naramura et al., 1994, Immunol. Lett., 39:91-99; U.S. Patent No. 5,474,981; 1991, J. Immunol., 146:2446-2452, each of which is hereby incorporated by reference in its entirety.

Antibodies can also be adsorbed to solid supports, which is particularly useful for immunoassays and purification of the polypeptides of the invention, or fragments, derivatives, analogs or variants thereof, or similar molecules having enzymatic activity similar to the polypeptides of the invention. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

5.5 Pharmaceutical compositions and kits

The invention includes pharmaceutical compositions comprising an antiviral agent of the invention. In a specific embodiment, the antiviral agent is an antibody that immunospecifically binds and neutralizes hSARS virus or its natural or artificial variant, analog or derivative or any derived protein thereof. The virus-neutralizing antibodies neutralize the infectivity of the virus and protect the animal against disease when wild-type virus is subsequently administered to the animal.

In another specific embodiment, said antiviral agent is a polypeptide or nucleic acid molecule of the invention. The pharmaceutical composition is useful as a prophylactic antiviral agent and can be administered to a subject who has been or is expected to be exposed to the virus.

Various delivery systems are known for administering the pharmaceutical composition of the present invention, such as liposome encapsulation, microparticles, microcapsules, recombinant cells expressing mutant viruses, receptor-mediated endocytosis (See eg Wu and Wu, 1987, J. Biol. Chem. 262:44294432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, incisional and oral routes. The compounds may be administered by any convenient route, such as by infusion or bolus injection, absorption through epithelial or mucocutaneous linings (eg, oral mucosa, rectal mucosa, and intestinal mucosa, etc.), or in combination with other biologically active agents. Administration can be systemic or local. In a preferred embodiment, it is desired to introduce the pharmaceutical composition of the invention into the lungs by any suitable route. Pulmonary administration can also be effected, for example, by use of an inhaler or nebulizer, formulated with an aerosolized formulation.

In a particular embodiment, it is desired to administer the pharmaceutical composition of the invention topically to the area in need of treatment; this may be for example (without limitation) by intraoperative local infusion, topical application (e.g. with post-operative wound dressing ), by injection, by catheter, by suppository, or by implant, which is a porous, non-porous or gel-like material, including thin films such as sialastic membranes or fibers. In one embodiment, administration may be by direct injection at the site (or former site) of the infected tissue.

In another embodiment, the pharmaceutical composition can be delivered in vesicles, especially liposomes (see Langer, 1990, Science 249:1527-1533; Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 53-365 (1989); Lopez-Berestein, supra, pp. 317-327; see generally id.).

In yet another embodiment, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; and Saudek et al., 1989, N Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al. , 1989, Ann. Neturol. 25: 351; Howard et al., 1989, J. Neurosurg. 71: 105). In yet another embodiment, a controlled release system can be placed near the target of the composition, the lungs, so that only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, supra, Vol. 2, No. 115 -138 pages (1984)).

Other controlled release systems are discussed in the review by Langer (1990, Science 249: 1527-1533).

The pharmaceutical composition of the present invention comprises a therapeutically effective amount of live but attenuated, inactivated or dead hSARS virus, or recombinant or chimeric hSARS virus and a pharmaceutically acceptable carrier. In a particular embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government, or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeia, for use in animals, more particularly in humans. used in . The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, limestone, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, Propylene, ethylene glycol, water, ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral dosage forms can contain standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The dosage form should be compatible with the way of administration.

In a preferred embodiment, the composition is formulated according to conventional procedures into a pharmaceutical composition suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. The composition may also include a solubilizer and a local anesthetic such as lidocaine, if necessary, to relieve pain at the injection site. Typically, the ingredients are supplied alone or in admixture in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet, marked on the container with the quantity of active drug. Where the composition is administered by infusion, it may be dispensed with an infusion bottle filled with sterile pharmaceutical grade water or saline. Where the composition is to be administered by injection, an ampoule of sterile water for injection or saline may be provided for mixing the ingredients prior to administration.

The pharmaceutical composition of the present invention can be formulated as a neutral form or a salt form. Pharmaceutically acceptable salts include salts formed with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with free carboxyl groups, such as those derived from sodium hydroxide, potassium hydroxide, hydroxide Salts of ammonium, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of a pharmaceutical composition of the invention effective to treat a particular condition or disorder will depend on the nature of the condition or disorder and can be determined by standard clinical techniques. In addition, in vitro assays can optionally be employed to help determine optimum dosage ranges. The precise dosage to be employed in the formulation will also depend on the route of administration and the severity of the disease or condition, and should be decided according to the judgment of the practicing physician and each patient's circumstances. However, a suitable dosage range for intravenous administration is generally about 20-500 micrograms of active compound per kilogram of body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Suppositories generally contain 0.5%-10% active ingredient by weight; oral dosage forms preferably contain 10%-95% active ingredient.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions of the invention. This container may optionally be accompanied by a notice in the prescribed form of a governmental agency regulating the manufacture, use, or sale of a drug or biological Product approval. In a preferred embodiment, the kit comprises an antiviral agent of the present invention, for example, a polypeptide encoded by a nucleic acid sequence of SEQ ID NO: 1, 11, 13, 15, 2471, 2472, 2473, 2474, 2475 or 2476, or the polypeptide shown in Figure 11 (SEQ ID NO: 17-239, 241-736 or 738-1107) and the polypeptide shown in Figure 12 (SEQ ID NO: 1109-1589, 1591-1964 or 1966-2470), or any hSARS epitope, or this The polypeptide or protein of the invention, or the antibody specific for the nucleic acid molecule of the invention, can exist alone or in combination with adjuvants, antiviral drugs, antibiotics, analgesics, bronchodilators or other pharmaceutically acceptable excipients.

The invention further includes such kits comprising a container containing a pharmaceutical composition of the invention and instructions for use.

5.6 Detection test

The present invention provides a method for detecting antibodies that immunospecifically bind hSARS virus in biological samples from SARS patients, such as cells, blood, serum, plasma, saliva, urine, feces, sputum, nasopharyngeal aspirates, and the like. In a specific embodiment, the method comprises contacting the sample with the hSARS virus directly immobilized on the substrate, for example, the hSARS virus with the preservation number CCTCC-V200303 or the hSARS virus with the genome nucleic acid sequence of SEQ ID NO: 15, Virus-bound antibodies are then detected directly or indirectly through labeled heterologous anti-isotype antibodies. In another specific embodiment, the sample is contacted with host cells infected by hSARS virus, for example, the hSARS virus with the preservation number CCTCC-V200303 or the hSARS virus with SEQ ID NO: 15 genomic nucleotide sequence, and then the bound antibody can be passed through the following The immunofluorescence assay described in Section 6.5 was used for detection.

Exemplary methods for detecting the presence of a polypeptide of the present invention or a nucleic acid in a biological sample include obtaining a biological sample from various sources, contacting the sample with a compound or reagent capable of detecting an epitope or nucleic acid (such as mRNA, genomic RNA) of hSARS virus, This detects the presence of hSARS virus in the sample. A preferred reagent for detecting hSARS mRNA or genomic RNA of the present invention is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic RNA encoding a polypeptide of the present invention. This nucleic acid probe can for example comprise or consist of SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590, 1965, 2471, 2472, 2473, 2474, 2475 or 2476 nucleic acid sequence or its complement Nucleic acid molecules consisting of analogues, derivatives, fragments or parts, such as oligonucleotides of at least 15, 20, 25, 30, 50, 100, 250, 500, 750, 1000 or more contiguous nucleotides in length acid, which is sufficient under stringent conditions to specifically hybridize to hSARS mRNA or genomic RNA.

In another preferred embodiment, the presence of the hSARS virus in the sample is detected by reverse transcription polymerase chain reaction (RT-PCR) using primers based on the hSARS virus (for example, deposit accession number CCTCC-V200303 The partial nucleotide sequence of the genome of the hSARS virus) is constructed, or constructed based on the nucleotide sequence of SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590 or 1965. In a non-limiting specific embodiment, preferred primers for the RT-PCR method are: 5'-TACACACCTCAGCGTTG-3' (SEQ ID NO: 3) and 5'-CACGAACGTGACGAAT-3' (SEQ ID NO: 4) , in the presence of 2.5 mM MgCl ₂ , thermal cycling such as but not limited to 94°C for 8 minutes followed by 40 cycles of 94°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute (see also section 6.7 below). In a more preferred embodiment, the present invention provides a real-time quantitative PCR assay to detect the presence of hSARS virus in a biological sample as follows: extract total RNA from the sample, perform reverse transcription on the extracted total RNA to obtain cDNA, and use specific primers such as Primers having the nucleic acid sequences of SEQ ID NO: 3 and 4 and a fluorescent dye such as ^SYBR_Green I (which fluoresces when non-specifically bound to double-stranded DNA) are used to subject the cDNA to a PCR reaction. In yet another preferred embodiment, the real-time quantitative PCR used in the present invention is a ^TaqMan® assay (see section 5 above). Specifically, for the real-time quantitative PCR test for detecting the presence of hSARS virus in biological samples, the primers used are preferably primers having the nucleic acid sequences of SEQ ID NO: 2471 and 2472. In this case, the amplification product is detected by a ^TaqMan® probe, preferably said probe has the nucleotide sequence of SEQ ID NO: 2473. Another preferred primer used in the ^TaqMan® assay is a primer having the nucleotide sequence of SEQ ID NO: 2474 and 2475, preferably the ^TaqMan® probe has the nucleotide sequence of SEQ ID NO: 2476. As PCR products are generated after a series of thermal cycles, the fluorescent signal of these reactions is captured at the end of each extension step, allowing quantification of the viral load in the sample based on the amplification plot (see Sections 6.7 and 6.8 below).

In another preferred embodiment, the presence of hSARS virus in the sample is determined using fluorescent cDNA microarray technology. A series of cDNA probes derived from hSARS virus (for example, the preservation number CCTCC-V200303 or the virus with SEQ ID NO: 15 genome nucleic acid sequence) are prepared by reverse transcription and amplification using suitable primers, which are based on, for example, the hSARS Partial nucleotide sequence of viral genome or SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590 or 1965 nucleic acid sequence construction. This amplified product is then purified and immobilized on a chip such as a poly-L-lysine-coated glass plate as a cDNA microarray. Total RNA is extracted from biological samples and reverse transcribed in the presence of fluorescently labeled nucleotides. Labeled cDNA representing mRNA in the sample is then contacted with immobilized cDNA probes on the microarray, and the fluorescent signal bound to the cDNA is detected and quantified. Various DNA microarray methods have been described, for example in Nucleic Acid Res.28(22): 4552-7 (Kane, M.D. et al., 2000); Science 2000 Sep 8; 289(5485): 1757-60 (Taton, T.A. et al. , 2000); Nature, 405(6788): 827-836 (Lockhart, D.J. et al., 2000).

Preferably another reagent for detecting hSARS virus is an antibody that specifically binds the polypeptide of the invention or any hSARS epitope, preferably an antibody with a detectable label. The antibodies may be polyclonal antibodies, or more preferably monoclonal antibodies. Whole antibodies or fragments thereof (eg Fab or F(ab') ₂ ) can be used.

The term "label" in reference to a probe or antibody includes direct labeling of the probe or antibody by conjugating (i.e., physically linking) a detectable substance to the probe or antibody, as well as direct labeling of the probe or antibody by reaction with another reagent that is directly labeled. Indirect labeling of probes or antibodies. Examples of indirect labeling include detection of a primary antibody with a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin so that it can be detected with fluorescently-labeled streptavidin. The detection method of the present invention can be used to detect mRNA, protein (or any epitope) or genomic RNA in a sample in vitro or in vivo. For example, techniques for detecting mRNA in vitro include Northern hybridization, in situ hybridization, RT-PCR, and RNase protection. In vitro techniques for detection of hSARS viral epitopes include enzyme-linked immunosorbent assay (ELISA), western blotting, immunoprecipitation, and immunofluorescence. Techniques for the detection of genomic RNA in vitro include Northern blot, RT-PCR, and RNase protection. In addition, techniques for in vivo detection of hSARS virus include introducing labeled antibodies against the polypeptide into the subject organism. For example, antibodies can be labeled with radioactive markers, the presence and location of which in the subject's organism can be detected by standard imaging techniques, including autoradiography.

In a specific embodiment, the method of the present invention further comprises obtaining a control sample from a control subject, contacting the control sample with a compound or reagent capable of detecting hSARS virus (such as the polypeptide of the present invention or mRNA or genomic RNA encoding the polypeptide of the present invention), thereby Detect the presence of hSARS virus or polypeptide or mRNA or genomic RNA encoding polypeptide in the sample, and compare the presence of hSARS virus or polypeptide or encoding polypeptide mRNA or genomic RNA in the control sample with the hSARS virus or polypeptide or encoding polypeptide in the test sample The presence of mRNA or genomic RNA was compared.

In a specific embodiment, the invention provides a diagnostic kit comprising a nucleic acid molecule suitable for the detection of hSARS virus or natural or artificial variants, analogs or derivatives thereof. In a specific embodiment, the nucleic acid molecule has SEQ ID NO: 2471 and 2472 nucleic acid sequences. In specific embodiments, nucleic acid molecule has SEQID NO:2473 nucleic acid sequence. In another specific embodiment, the nucleic acid molecule has SEQID NO: 2474 and 2475 nucleic acid sequences. In specific embodiments, nucleic acid molecule has SEQ ID NO:2476 nucleic acid sequence.

The present invention also includes a kit for detecting the presence of the hSARS virus or polypeptide or nucleic acid of the present invention in a sample to be tested. Said kit can, for example, comprise a labeled compound or reagent capable of detecting hSARS virus or polypeptide or a nucleic acid molecule encoding a polypeptide in the sample to be tested, and in some embodiments, also comprise a tool for determining the amount of polypeptide or mRNA in the sample (e.g. An antibody that binds a polypeptide or an oligonucleotide probe that binds to DNA or mRNA encoding a polypeptide). The kit can also include instructions for use.

For antibody-based kits, it may comprise, for example: (1) a primary antibody (e.g., attached to a solid support) that binds to a polypeptide of the invention or an epitope of the hSARS virus; optionally (2) a different secondary antibody, It binds the polypeptide or primary antibody and is conjugated to a detectable reagent.

For oligonucleotide-based kits, it may include, for example: (1) oligonucleotides, such as detectable marker oligonucleotides, which can hybridize to nucleic acid sequences encoding polypeptides of the present invention or sequences within the hSARS genome, Or (2) a pair of primers that can be used to amplify nucleic acid molecules containing hSARS sequences. The kit may also contain, for example, buffers, preservatives or protein stabilizers. The kit may also comprise components (eg, enzymes or substrates) required for detection of a detectable agent. The kit may also contain a control sample or series of control samples that can be used to perform the assay and compared to that contained in the sample to be tested. The components of the kit are usually enclosed in individual containers, all of the various containers being contained in a single package together with instructions for use.

5.7. Screening test

The present invention provides methods for identifying compounds that inhibit the ability of hSARS virus to infect a host or host cells. In certain embodiments, the invention provides methods for identifying compounds that reduce the ability of hSARS virus to replicate in a host or host cell. Compounds that disrupt or reduce the ability of the hSARS virus to infect a host and/or replicate in a host or host cells can be screened using any technique known to those of ordinary skill.

In certain embodiments, the present invention provides methods for identifying compounds that inhibit the ability of hSARS virus to replicate in a mammal or a mammalian cell. More specifically, the present invention provides methods for identifying compounds that inhibit the ability of hSARS virus to infect a mammal or mammalian cells. In certain embodiments, the present invention provides methods for identifying compounds that inhibit the ability of hSARS virus to replicate in mammalian cells. In a specific embodiment, the mammalian cells are human cells.

In another embodiment, cells are contacted with a test compound and infected with hSARS virus. In certain embodiments, a control culture is infected with hSARS virus in the absence of a test compound. Cells can be contacted with the test compound before, simultaneously with or after infection with hSARS virus. In a specific embodiment, the cells are mammalian cells. In an even more specific embodiment, the cells are human cells. In certain embodiments, the cells are incubated with the test compound for at least 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 5 hours, 12 hours, or 1 day. Virus titers can be measured at any time during the assay. In certain embodiments, the time course of viral growth in culture is determined. A test compound can be identified as effective in inhibiting or reducing growth or infection of hSARS virus if virus growth is inhibited or reduced in the presence of the test compound. In a specific embodiment, compounds that inhibit or reduce the growth of hSARS virus are tested for their ability to inhibit or reduce the growth rate of other viruses, and/or are tested for their specificity for hSARS virus.

In one embodiment, a test compound is administered to a model animal infected with hSARS virus. In certain embodiments, a control model animal is infected with hSARS virus but is not administered a test compound. Test compounds can be administered before, simultaneously with or after infection with hSARS virus. In a specific embodiment, the model animal is a mammal. In an even more specific embodiment, the model animal may be, but is not limited to, a cotton rat, a mouse or a monkey. Virus titers in model animals can be measured at any time of the experiment. In certain embodiments, the time course of viral growth in culture is determined. A test compound can be identified as effective in inhibiting or reducing growth or infection of hSARS virus if virus growth is inhibited or reduced in the presence of the test compound. In a specific embodiment, the ability of a compound that inhibits or reduces the growth of hSARS virus in a model animal to inhibit or reduce the growth rate of other viruses is tested to test its specificity for hSARS virus.

6. Example

The following examples illustrate the isolation and characterization of novel hSARS viruses. These examples should not be construed as limiting the invention.

Methods and Results

Use Wiedbrauk DL and Johnston SLG's Manual of Clinical Virology, Raven Press, New York, 1993 as a general reference.

6.1 Clinical objects

This study included a total of 50 patients meeting the revised World Health Organization (WHO) definition of SARS who were admitted to two acute disease regional hospitals in the Hong Kong Special Administrative Region (HKSAR) between February 26 and March 26, 2003 ( WHO. Severe acute respiratory syndrome (SARS) 2000, Weekly Epidemiol Rec 78:81-83). The study also included a lung biopsy from another patient who had typical SARS and was admitted to a third hospital. Briefly, the case definition of SARS was: (i) fever of 38°C or higher; (ii) cough or shortness of breath; (iii) new pulmonary infiltrates on chest X-ray; History of exposure or nonresponse to empirical antibiotic drug coverage (beta-lactam and macrolide drugs, fluoroquinolones, or tetracyclines) for typical and atypical pneumonia.

Nasopharyngeal aspirates and serum samples were collected from all patients. Both acute and convalescent serum and feces are available from some patients. A lung biopsy from one patient was processed for viral culture, RT-PCR, routine histopathological examination, and electron microscopy. Nasopharyngeal aspirates, feces and sera for microbiological testing of other diseases were included in this study under blinded conditions as controls.

Medical records were reviewed by the attending physician and clinical microbiologist. Routine hematological, biochemical, and microbiological examinations were performed, including bacterial cultures of blood and sputum, serological studies, and nasopharyngeal aspirates were collected for virological testing.

6.2 Cell lines

FRhK-4 (fetal macaque kidney) cells were maintained in minimal essential medium (MEM) containing 1% fetal bovine serum, 1% streptomycin and penicillin, 0.2% nystatin and 0.05% gentamicin sulfate.

6.3 Virus infection

FRhk-4 cells were infected with 200 μl of clinical (nasopharyngeal aspirates) samples from two patients (see "Results" section below) in viral transport medium. Inoculated cells were incubated at 37°C for 1 hour. Then 1 ml of MEM containing 1 μg of trypsin was added to the culture, and the infected cells were incubated in a 37°C incubator supplied with 5% carbon dioxide. After 2-4 days of incubation, the appearance of cytopathic effects in infected cells was observed. The infected cells were passaged into new FRhK-4 cells, and the cytopathic effect was observed within 1 day after inoculation. Infected cells tested by immunofluorescence for influenza A, influenza B, respiratory syncytial virus, parainfluenza types 1, 2, and 3, adenovirus, and human metapneumovirus (hMPV), test results for all cases Both are negative. Infected cells were also tested by RP-PCR for influenza virus A and human metapneumovirus and the results were negative.

6.4 Virus Morphology

The infected cells prepared as above were collected, centrifuged into pellets, and the cell pellets were processed for thin-section transmission electron microscopy. Virus particles were identified in cells infected with both clinical samples but not in control cells not infected with the virus. Virus particles isolated from infected cells were approximately 70-100 nm in size (Figure 2). Viral capsids are mainly found in vesicles of the Golgi apparatus and endoplasmic reticulum, but also in the cytoplasm. Viral particles are also found in cell membranes.

An aliquot of the virus isolate was subjected to ultracentrifugation and the resulting cell pellet was negatively stained with phosphotungstic acid. This revealed virus particles characteristic of the Coronaviridae family. Since hitherto recognized human coronaviruses are not known to cause similar diseases, the inventors believe that said virus isolate represents a novel virus infecting humans.

6.5 Antibody response

In order to further confirm that this new virus caused SARS in infected patients, serum samples were obtained from SARS patients for neutralization test. Typical dilutions of sera (x50, x200, x800 and x1600) were incubated with acetone-fixed FRhK-4 cells infected with hSARS virus at 37°C for 45 minutes. The incubated cells were then washed with phosphate-buffered saline and stained with an anti-human IgG-FITC-conjugated antibody. Cells were then washed and examined under a fluorescent microscope. In these experiments, a positive signal was found in eight SARS patients (Fig. 3), indicating that these patients had an IgG antibody response to this novel human respiratory virus of the Coronaviridae family. In contrast, no signal was detected in the 4 negative control paired sera. The serum titers of anti-hSARS antibodies of the tested patients are shown in Table 1.

Table 1 Name date experiment number Anti-SARS Patient A Patient B Patient C Patient D Patient E Control F Patient G February 25, 03, March 6, 03, February 26, 03, March 3, 03, March 4, 03, March 14, 03, March 6, 03, March 11, 03 March 4, 03 March 18, 03 February 13, 03 March 1, 03 March 3, 03 March 7, 03 S2728S2728S2441S2441S3279S3279M41045MB943703M38953KWH03/3601M27124MB942968M38685KWH03/2900 <501600502002001600<50800<50800<50<50<50 suspicious Blind test samples: 1a ^* 1b2a ^* Acute phase Rehabilitation phase Acute phase <50160050

2b3a ^* 3b4a ^* 4b5a ^* 5b6a ^* 6b Rehabilitation Acute Rehabilitation Acute Rehabilitation Acute Rehabilitation Acute Rehabilitation ＞160050＞1600＜50＜50＜50＜50＜50＜50

Note: ^* SARS patients

These results suggest that this new member of the Coronaviridae family is the key pathogen of SARS.

Sequence of 6.6hSARS virus

Total RNA was harvested from infected or uninfected FrHK-4 cells two days after infection. ^{Superscript_II} reverse transcriptase (Invitrogen) was used in 20 μl of reaction mixture containing 10 pg of degenerate primer (5′-GCCGGAGCTCTGCAGAATTCNNNNNNN-3′, SEQ ID NO: 5; N=A, T, G or C) according to the manufacturer’s recommendation. 100ng of purified RNA was reverse transcribed. The reverse transcription product was then purified by ^QIAquick_PCR Purification Kit according to the manufacturer's instructions, and eluted into 30 μl of 10 mM Tris-HCl (pH 8.0). 3 μl of the purified cDNA product was added to a 25 μl reaction mixture containing: 2.5 μl 10x PCR buffer, 4 μl 25 mM MgCl ₂ , 0.5 μl 10 mM dNTP, 0.25 μl ^{AmpliTaqGold_DNA} polymerase (Applied Biosystems), 2.5 μCi [α- ³² P] CTP (Amersham), 2 μl 10 μM primer (5′-GCCGGAGCTCTGCAGAATTC-3′: SEQ ID NO: 6). The reaction was thermally cycled as follows: 94°C for 8 minutes, followed by 2 cycles of 94°C for 1 minute, 40°C for 1 minute, and 72°C for 2 minutes. After this thermal cycle, a cycle of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute was performed 35 times. 6 μl of PCR product was taken for 5% denaturing polyacrylamide gel electrophoresis analysis. The gel was exposed to X-ray film, which was developed overnight after exposure to X-ray film. Unique PCR products identified only in infected cell samples were separated from the gel and eluted with 50 μl of 1x TE buffer. The eluted PCR products were then reamplified in a 25 μl reaction mixture containing: 2.5 μl 10xPCR buffer, 4 μl 25 mM MgCl ₂ , 0.5 μl 10 mM dNTPs, 0.25 μl ^{AmpliTaqGold®} DNA polymerase (Applied Biosystems), 1 μl 10 μM Primer (5'-GCCGGAGCTCTGCAGAATTC-3': SEQ ID NO: 6). The reaction was thermally cycled as follows: 94°C for 8 minutes, followed by 35 cycles of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute. The PCR product was cloned using the TOPO TA ^Cloning® Kit (Invitrogen), and the ligated plasmid was transformed into TOP 10 E. coli competent cells (Invitrogen). The PCR inserts were sequenced by ^BigDye_Cycle Sequencing Kit according to the manufacturer's (Applied Biosystems) recommendation, and the sequencing products were analyzed by an automatic sequencer (Applied Biosystems, model 3770). The obtained sequence (SEQ ID NO: 1) is shown in FIG. 1 . The amino acid sequence (SEQ ID NO: 2) deduced from the obtained DNA sequence showed 57% homology with the polymerase protein of the identified coronavirus.

Similar to this, other two partial sequences (SEQ ID NOs: 11 and 13) and deduced amino acid sequences (respectively SEQ ID NOs: 12 and 14) were obtained from hSARS virus, as shown in Fig. 8 (SEQ ID NOs: 11 and 12 ) and Figure 9 (SEQ ID NO: 13 and 14).

The entire genome sequence of hSARS virus is shown in Figure 10 (SEQ ID NO: 15). The deduced amino acid sequence of SEQ ID NO: 15 obtained with all three reading frames is shown in Figure 11 (see SEQ ID NO: 16, 240 and 737 for the DNA sequence; see SEQ ID NO: 17-239, 241-736 and 738-1107). The deduced amino acid sequence of the complement of SEQ ID NO: 15 obtained with all three reading frames is shown in Figure 12 (see SEQ NO: 1108, 1590 and 1965 for the DNA sequence; see SEQ ID NO: 1109-1589, 1591- 1964 and 1966-2470).

6.7 Detection of hSARS virus in nasopharyngeal aspirates

First, nasopharyngeal aspirates (NPA) were examined for influenza A and B, parainfluenza types 1, 2, and 3, respiratory syncytial virus, and adenovirus by rapid immunofluorescent antigen assay (Chan KH, Maldeis N, Pope W, Yup A, Ozinskas A. Gill J, Seto WH, Shortridge KF, Peiris JSM. Evaluation of Directigen Fly A+B test for rapid diagnosis of influenza A and B virus infections. J Clin Microbiol. 2002; 40: 1675-1680 ), routine respiratory pathogens of nasopharyngeal aspirates cultured in Mardin Darby canine kidney cells, LLC-Mk2, RDE, Hep-2 and MRC-5 cells (Wiedbrauk DL, Johnston SLG. Manual of clinical virology. Raven Press, New York. 1993). Subsequently, fetal rhesus monkey kidney cells (FRhk-4) and A-549 cells were added to the array of cell lines used. Reverse transcription polymerase chain reaction (RT-PCR) directly on clinical samples for the detection of influenza virus A (Fouchier RA, Bestebroer TM, Herfst S, Van Der Kemp L, Rimmelzwan GF, Osterhaus AD. Detection of influenzaA virus from different species by PCR amplification of conserved sequences in the matrix gene. J Clin Microbiol. 2000; 38: 4096-101) and human metapneumovirus (HMPV). Primers used for HMPV were: first round, 5'-AARGTSAATGCATCAGC-3' (SEQ ID NO: 7) and 5'-CAKATTYTGCTTATGCTTTC-3' (SEQ ID NO: 8); and nested primer: 5'-ACACCTGTTACAATACCAGC -3' (SEQ ID NO: 9) and 5'-GACTTGAGTCCCAGCTCCA-3' (SEQ ID NO: 10). The size of the nested PCR product is 201 bp. Cell cultures were screened by ELISA against Mycoplasma (Roche Diagnostics GmbH, Roche, Indianapolis, USA).

6.7.1. RT-PCR test

After culturing and genetic sequencing of hSARS virus from two patients (see section 6.6 above), RT-PCR was developed for the detection of hSARS virus from NPA samples. Total RNA from clinical samples was reverse-transcribed with random hexamers, and cDNA was reverse-transcribed with primers 5′-TACACACCCTCAGCGTTG-3′ (SEQ ID NO: 3) and 5′-CACGAACGTG-ACGAAT-3′ (SEQ ID NO: 4) in Amplified in the presence of 2.5mM _MgCl2 (94°C for 8 minutes, then 40 cycles of 94°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute), the two primers were constructed based on the hSARS virus genome.

A typical RT-PCR protocol is outlined below:

RNA extraction

RNA was extracted from 140 μl NPA samples by ^{QIAquick_Viral} RNA Extraction Kit and eluted in 50 μl elution buffer.

reverse transcription

RNA 11.5μl

0.1M DTT 2μl

5x buffer 4 μl

10mM dNTP 1μl

Superscript II, 200U/μl (Invitrogen) 1μl

Random hexamer, 0.3μg/μl 0.5μl

Reaction Conditions 42°C, 50 minutes

                                                                                                                           

4℃

PCR

cDNA generated by random primers was amplified in a 50ul reaction as follows:

cDNA 2 μl

10mM dNTP 0.5μl

10x buffer 5 μl

25mM _MgCl2 5μl

25μM forward primer 0.5μl

25μM reverse primer 0.5μl

AmpliTaq ^{Gold_Polymerase} , 5U/μl (Applied Biosystems) 0.25μl

Water 36.25μl

Thermal cycle conditions: 95°C for 10 minutes, then 40 cycles of 95°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute.

Primer sequence

Primers were designed based on the RNA-dependent RNA polymerase coding sequence (SEQ ID NO: 1) of hSARS virus.

Forward primer: 5'TACACACCCTCAGCGTTG 3' (SEQ ID NO: 3)

Reverse primer: 5'CACGAACGTGACGAAT 3' (SEQ ID NO: 4)

Product (amplicon) size: 182bp

real-time quantitative PCR assay

Total RNA was extracted from 140 μl nasopharyngeal aspirate (NPA) by ^QIAamp® Viral RNA Mini Kit (Qiagen) according to the manufacturer's instructions. In a 20 μl reaction mixture containing 0.15 μg random hexamers, 10 mmol/L DTT and 0.5 mmol/L dNTP, 10 μl of the eluted RNA sample was reverse transcribed with 200 U ^{Superscript_II} reverse transcriptase (Invitrogen) as described . Complementary DNA was then amplified in ^SYBR_Green I fluorescent reaction mix (Roche). Briefly, 2 μl of cDNA, 3.5 mmol/L MgCl ₂ , 0.25 μmol/L forward primer (5′-TACACACCTCAGCGTTG-3′; SEQ ID NO: 3) and 0.25 μmol/L reverse primer (5′- CACGAACGTGACGAAT-3'; SEQ ID NO: 4) in 20 μl reaction mixture, use Light-Cycler (Roche) according to the PCR program [95 ° C for 10 minutes, then 95 ° C for 10 minutes; 57 ° C for 5 seconds; 72 ° C for 9 seconds cycle 50 times] for thermal cycling. A plasmid containing the sequence of interest was used as a positive control. Fluorescent signals from these reactants are captured at the end of the extension step of each cycle. To determine the specificity of the assay, the PCR product (184 base pairs) was subjected to melting curve analysis (65°C to 95°C, 0.1°C per second) at the end of the assay.

clinical outcome

Clinical findings:

All 50 SARS patients were of Chinese ethnicity. They represent 5 different epidemiologically related clusters as well as other sporadic cases meeting the case definition. They were hospitalized an average of 5 days after symptoms started. Their average age was 42 years (23 to 74 years), and the ratio of females to males was 1.3. Among them, 14 (28%) were medical workers, and 5 (10%) had a history of visiting hospitals where severe SARS broke out. Thirteen people (26%) had contact with SARS patients at home, and another 12 people (24%) had contact with SARS patients in society. Four (8%) had recent travel history to mainland China.

The main complaints of most patients were fever (90%) and shortness of breath. More than half of the patients developed cough and myalgia (Table 2). A minority of patients experienced upper respiratory symptoms such as rhinorrhea (24%) and sore throat (20%). Diarrhea (10%) and decreased appetite (10%) were also reported. On initial auscultatory examination, only 38% of patients had crepitus and decreased air intake. 62% of patients reported a dry cough. All patients were found signs of consolidation by radiological examination, including 1 area (36 cases), 2 areas (13 cases) and 3 areas (1 case).

Table 2 Clinical symptoms Number of patients (percentage) Fever Chills or chills Cough Myalgia Malaise Runny nose Sore throat Shortness of breath Loss of appetite Diarrhea Headache Dizziness 50(100%) 37(74%) 31(62%) 27(54%) 25(50%) 12(24%) 10(20%) 10(20%) 10(20%) 5(10%) 10(20%)6(12%)

^* 1 patient had a maculopapular rash on the trunk.

Most patients (98%) had no evidence of leukocytosis despite high fever. Peripheral blood examination revealed lymphopenia (68%), leukopenia (26%), thrombocytopenia (40%), and anemia (18%) (Table 3). Levels of the liver parenchymal enzyme alanine transaminase (ALT) and muscle enzyme creatinine kinase (CPK) were elevated in 34% and 26% of cases, respectively.

table 3 Assay parameters mean (range) Abnormal percentage normal range

Hemoglobin anemia White blood cell count Leukopenia Lymphocyte count Significant lymphopenia (<1.0x10 ⁹ /L) Platelet count Thrombocytopenia Alanine transaminase (ALT) Elevated albumin Low albumin globulin Elevated creatinine kinase Creatinine kinase Elevated acid anhydride kinase 12.9(8.9-15.9)5.17(1.1-11.4)0.78(0.3-1.5)174(88-351)63(11-350)37(26-50)33(21-42)244(31-1379) 9 (18%) 13 (26%) 34 (68%) 20 (40%) 17 (34%) 34 (68%) 10 (20%) 13 (26%) 11.5-16.5g/dl4-11x 10 ⁹ /L1.5-4.0x 10 ⁹ /L150-400x 10 ⁹ /L6-53U/L42-54g/dl24-36g/dl34-138U/L

Routine microbiological investigations for known viruses and bacteria by culture, antigen detection, and PCR were negative in most cases. A blood culture of a 74-year-old man admitted to the intensive care unit was positive for E. coli because of a hospital-acquired urinary tract infection. Klebsiella pneumoniae and Hemophilus influenzae were isolated from sputum samples from two other patients on admission.

Levofloxacin 500 mg orally every 24 hours was given to 9 patients and amoxicillin-clavulanate was given intravenously (1.2 g every 8 hours)/po (375 mg three times daily) and intravenously/po every 12 hours to another 40 patients 500mg clarithromycin. Four patients were given 75 mg of oseltamivir orally twice daily. One patient was given ceftriaxone 2 gm intravenously every 24 hours, azithromycin 500 mg orally every 24 hours, and amantadine 100 mg orally twice daily for empirical coverage of typical and atypical pneumonia.

Nineteen patients developed severe disease with oxygen desaturation requiring intensive care and ventilatory support. The average number of days since the onset of symptoms was 8.3 days. A mean of 6.7 days after the onset of symptoms, 49 patients were given intravenous ribavirin 8 mg/kg every 8 hours along with steroids.

Risk factors associated with severe comorbidity requiring intensive care and ventilatory support were older age, lymphopenia, impaired ALT, and delayed administration of ribavirin and steroids (Table 4). All complicated cases were treated with ribavirin and steroids after admission to the intensive care unit, whereas all noncomplicated cases were treated with ribavirin and steroids in the general ward. As expected, 31 uncomplicated cases recovered or improved, while 8 concurrent cases deteriorated and 1 died at the time of writing. All 50 patients were monitored for an average of 12 days at the time of writing this specification.

Table 4 Concurrent cases (n=19) No concurrent cases (n=31) P value Mean (SD) age (range) male/female ratio potential disease exposure mode travel to China medical workers visiting family in hospital exposure social contact to admission mean (SD) duration of symptoms (days) mean (SD) admission temperature (°C ) 49.5±12.78/ ^115_151845.2 ±2.038.8±0.9 39.0±10.714/ ^{171_3945104.7} ±2.538.7±0.8 P＜0.01N.SP＜0.05NSNSNSSP＜0.05NSNSNS

Mean (SD) initial total peripheral WBC count (x10 ⁹ /L) mean (SD) initial lymphocyte count (x10 ⁹ /L) presence of thrombocytopenia (<150x10 ⁹ /L) impaired liver function check CXR changes (affected Number of regions) Mean (SD) days from symptom onset to disease progression § Mean (SD) days from symptom onset to initiation of ribavirin and steroids Response to warin and steroids got better or cured didn't get better‖ 5.1±2.40.66±0.38111.48.3±2.67.7±2.91211108 5.2±1.80.85±0.31261.2 not applicable 5.7±2.6028310 N.S.P<0.05N.S.P<0.01N.S.P<0.05P<0.001P<0.05P<0.01P<0.01

^* Multivariate analysis was not performed due to the small number of cases;

^_ 2 patients had diabetes, 1 had hypertrophic obstructive cardiomyopathy, 1 had chronic active hepatitis B, and 1 had a brain tumor;

^_ 1 patient had essential hypertension;

§ Desaturation requires intensive care support;

‖1 patient died.

Two viral isolates were isolated from two patients and later identified as members of the Coronaviridae family (see below). One virus isolate was obtained from an incisional lung biopsy of a 53-year-old Hong Kong resident, and the other was obtained from a nasopharyngeal aspirate of a 42-year-old female with good past health. The 53-year-old male had 10 hours of family contact with a Chinese tourist from Guangzhou who later died of SARS. Two days after exposure, he developed fever, malaise, myalgia, and headache. There was crepitus in the lower right lung area, and chest radiographs showed corresponding alveolar shadows. Blood tests revealed a lymphopenia of 0.7x10 ⁹ /L, with normal total white blood cell and platelet counts. Both ALT (41U/L) and CPK (405U/L) were damaged. Despite oral azithromycin, amantadine, and intravenous ceftriaxone, he experienced increased bilateral lung infiltrates and progressive oxygen desaturation. Therefore, an open lung biopsy was performed 9 days after his admission. Histopathological examination revealed moderate interstitial inflammation, and scattered alveolar cells exhibited cytomegalomorphism, granular amphichromatic cytoplasm, enlarged nuclei, and prominent nucleoli. None of the cells showed inclusion bodies typical of herpesvirus or adenovirus infection. The patient underwent surgery and required ventilation and intensive care. He was given empiric intravenous ribavirin and hydrocortisone. But he still died 20 days after being admitted to the hospital. Coronavirus-like RNA was found in his nasopharyngeal aspirate, lung biopsy, and postmortem lungs at retrospective time. His antibody titer against his own hSARS isolate increased significantly from 1/200 to 1/1600.

The second patient from whom hSARS virus was isolated was a 42-year-old female with good previous health. She traveled to Guangzhou in mainland China for two days and developed fever and diarrhea five days after returning to Hong Kong. Physical examination of her revealed crepitus in the right lower lung region with corresponding alveolar opacities on chest radiographs. The examination also showed a decrease in leukocytes (2.7x10 ⁹ /L), lymphocytes (0.6x10 ⁹ /L) and thrombocytopenia (104x10 ⁹ /L). Despite her empiric antimicrobial coverage with amoxicillin-clavulanate, clarithromycin, and oseltamivir, she deteriorated five days after admission and required mechanical ventilation and intensive care for five days. She gradually improved and did not require ribavirin or steroids. Her nasopharyngeal aspirate was positive for the virus in a RT-PCR test, and she achieved seroconversion with antibody titers from <1/50 to <1/1600 against hSARS isolates.

Virological findings:

Viruses were isolated on FRhk-4 cells from lung biopsies and nasopharyngeal aspirates of the two patients mentioned above, respectively. The initial cytopathic effect appeared 2 to 4 days after inoculation, but upon subsequent passages, the cytopathic effect appeared within 24 hours. Neither virus isolate reacted with a range of reagents routinely used to identify virus isolates, including those used to identify influenza A, B, parainfluenza types 1, 2, and 3, adenovirus, and respiratory syncytial virus reagents (DAKO, Glostrup, Denmark). The two virus isolates also did not react in RT-PCR assays for influenza A and HMV, or in PCR assays for Mycoplasma. The virus is sensitive to ether, indicating that it is an enveloped virus. Electron microscopy of negative-stained (2% potassium phosphotungstate, pH 7.0) cell culture extracts obtained by ultracentrifugation revealed the presence of polytypic enveloped virus particles approximately 80-90 nm in diameter (70-130 nm range), and its surface morphology appeared to be comparable to members of the Coronaviridae family (Fig. 5A). Thin-section electron microscopy of infected cells revealed smooth-walled vesicles in the cytoplasm containing viral particles 55-90 nm in diameter (Figures 5A and 5B). Viral particles are also visible on the cell surface. The overall findings are consistent with cellular infection by a Coronaviridae virus.

Thin-section electron micrographs of the lung biopsy of the 53-year-old male showed desquamated cells containing 60-90 nm virus particles in the cytoplasm. These virus particles were similar in size and morphology to those observed in cell culture virus isolates from two patients (Figure 4).

The RT-PCR products generated in the random primer RT-PCR experiments were analyzed, and the unique bands found in the virus-infected samples were cloned and sequenced. Of the 30 clones examined, a clone containing 646 base pairs (SEQ ID NO: 1 ) of unknown origin was identified. Sequencing analysis of this DNA fragment showed that this sequence had weak homology with viruses of the Coronaviridae family (data not shown). However, the amino acid sequence (215 amino acids: SEQ ID NO: 2) deduced from this unknown sequence has a high degree of homology (57%) with the RNA polymerase of bovine coronavirus and murine hepatitis virus, confirming that this virus belongs to the Coronaviridae family . Phylogenetic analysis of protein sequences revealed that this virus, although most closely related to coronavirus type II, is a distinct virus (Figures 5A and 5B).

According to the 646 base pair sequence of this isolate, specific primers for detecting the new virus were designed for RT-PCR detection of the hSARS virus genome in clinical samples. Of 44 nasopharyngeal aspirate samples obtained from 50 SARS patients, 22 samples had evidence of hSARS RNA. Viral RNA was detectable in 10 of 18 stool samples tested. The specificity of the RT-PCR reaction was confirmed by sequencing selected positive RT-PCR amplification products. None of the 40 nasopharyngeal aspirates and stool samples from patients with unrelated diseases were reactive in the RT-PCR assay.

To determine the dynamic range of real-time quantitative PCR, serial dilutions of plasmid DNA containing the target sequence were prepared and subjected to real-time quantitative PCR experiments. As shown in Figure 7A, the assay was able to detect as few as 10 copies of the target sequence. In contrast, no signal was observed in the water control (Fig. 7A). Positive signals were observed in 23 of 29 serologically confirmed SARS patients. In all these positive cases, a unique PCR product ( _Tm = 82°C) corresponding to the signal of the positive control was observed (Fig. 7B, data not shown). These data indicate that the assay is highly specific for the target. The copy number of the target sequence in these reactions ranged from 4539 to less than 10. Thus, up to ^6.48x105 copies of this viral sequence could be found in 1 ml of NPA sample. In 5 of the above positive cases, NPA samples could be collected before seroconversion. Viral RNA was detected in 3 of these samples, indicating that the assay can detect virus even early in the onset of infection.

To further confirm the specificity of this test, recruit healthy people (n=11) and those infected with adenovirus (n=11), respiratory syncytial virus (n=11), human metapneumovirus (n=11), influenza virus NPA samples from patients with A (n=13) or influenza virus B (n=1) served as negative controls. All but one of these samples tested negative. False positive cases were subsequently tested negative. Including false positive cases, the sensitivity of the real-time quantitative PCR test was 79% and the specificity was 98%.

Epidemiological data show that droplet transmission is one of the main routes of transmission of this virus. This study detected live virus and high-copy virus sequences from NPA samples, clearly supporting that cough and sneeze droplets from SARS patients may be the main source of this infectious agent. Interestingly, 2 out of 4 stool samples from SARS patients in this study were positive in the test (data not shown). Detection of virus in feces indicates possible other routes of transmission. It is pointed out that some animal coronaviruses are transmitted by the fecal-oral route (McIntoshK., 1974, Coronaviruses: a comparative review. Current Top Microbiol Immunol. 63: 85-112). However, further research is needed to test whether the virus in stool is contagious.

In addition to this hSARS virus, there are currently two known human coronavirus serogroups (229E and OC43) (Hruskova J. et al., 1990, Antibodies to human coronaviruses 229E and OC43 in the population of C.R., Acta Virol.34: 346 -52). The primer pair used in this experiment has no homology with the 229E strain. Since the corresponding OC43 sequence is not available in Genebank, it is unknown whether these primers can cross-react with this strain. However, sequence analysis of sequences available in other regions of the OC43 polymerase gene revealed that the novel human virus associated with SARS is genetically distinct from OC43. Furthermore, the primers used in this study had no homology to any sequences of known coronaviruses. Therefore, it is unlikely that these primers will cross-react with the OC43 strain.

It has been reported that, in addition to the novel pathogen, metapneumoviruses were also identified in some SARS patients (Center for Disease Control and Prevention, 2003, Morbidity and Mortality Weekly Report 52:269-272). No signs of metapneumovirus infection were detected in any of the patients in this study (data not shown), suggesting that the novel hSARS virus of the present invention is a major player in the pathogenesis of SARS.

Immunofluorescence antibody assay:

Of the 50 most recent serum samples from SARS patients, 35 had evidence of anti-hSARS antibodies (see Figure 3). All 27 patients with paired acute and convalescent sera were seroconverted or their antiviral antibody titers increased >4-fold. Five other pairs of sera from other SARS patients from groups outside our study group were also tested to allow for a wider sampling of SARS patients in society, all of whom seroconverted. None of the 80 sera from patients with respiratory diseases or other diseases and 200 normal blood donors had detectable antibodies.

If seropositivity for HP-CV in a single serum or detection of viral RNA in NPA or stool were both considered evidence of infection with hSARS virus, 45 of 50 patients had evidence of infection. Of the 5 patients without any virological evidence of Coronaviridae virus infection, only 1 patient underwent serological testing >14 days after the onset of clinical symptoms.

6.8 Quantitative TaaMan for detection of hSARS virus ^_ test

6.8.1. Materials and methods

Patient and Sample Collection

Stored clinical samples from 50 patients fulfilling the SARS clinical WHO case definition (http://www.who.int/csr/sars/casedefinition/en/) whose diagnosis was subsequently confirmed by seroconversion were used in this study . NPA samples were collected from 1-3 days from the onset of symptoms as described above (Poon et al., 2003, Clin. Chem. 49:953-955). NPA samples from patients with unrelated diseases were used as controls.

RNA extraction and reverse transcription

RNA was extracted from clinical samples using the ^QIAamp® Viral RNA Mini Kit (Qiagen) according to the manufacturer's instructions. In the routine RT-PCR experiments described above, 140 μl of NPA was used for RNA extraction. In the modified RNA extraction protocol, 540 μNPA was used for RNA extraction. Extracted RNA was finally eluted into 30 μl of RNase-free water and stored at -20°C. Total RNA from clinical samples was then reverse transcribed using random hexamers.

Conventional PCR for SARS-CoV

Routine PCR assays were performed as described in Section 6.7.1.

Real-time quantitative PCR assay for SARS-CoV

Real-time quantitative PCR specific for SARS-CoV 1b region was used in this study. Complementary DNA was amplified in a 7000 Sequence Detection System (Applied Biosystems) by ^TaqMan_PCR Core Reagent Kit. Briefly, 4 μl of cDNA was amplified in a 25 μl reaction containing: 0.625 U AmpliTaq ^Gold® Polymerase (Applied Biosystems), 2.5 μl 10x ^TaqMan® Buffer A, 0.2 mM dNTP, 5.5 mM MgCl ₂ , 2.5 U ^AmpErase_UNG and 1x primer-probe mix (Assays by Design, Applied Biosystems). The primer sequences are 5'-CAGAACGCTGTAGCTTCAAAAATCT-3' (SEQ ID NO: 2471) and 5'-TCAGAACCCTGTGATGAATCAACAG-3' (SEQ ID NO: 2472), and the probe is 5'-(FAM)TCTGCGTAGGCAATCC(NFQ)-3'( SEQ ID NO: 2473; FAM, 6-carboxyfluorescein; NFQ, no fluorescence quencher). Reactions were incubated initially at 50°C for 2 minutes, then at 95°C for 10 minutes. The reactants were then thermally cycled 45 times (95°C for 15 seconds, 60°C for 1 minute). A plasmid containing the sequence of interest was used as a positive control.

6.8.2. Results

A total of 50 isolated NPA samples collected from serologically confirmed SARS patients during the first 3 days of their illness were studied. Of them, 11 (22%) were positive in our previously reported conventional RT-PCR test (see Section 6.7.1) (Table 5).

table 5 start days Sample size number of positives Routine RT-PCR test Routine RT-PCR assay with modified RNA extraction protocol ^* Real-time RT-PCR assay with modified RNA extraction protocol ^*+ 123 81626 0(0%)3(19%)8(31%) 2(25%)8(50%)12(46%) 5 (63%) 14 (88%) 21 (81%)

^* The total detection rate of the test is statistically different from that of the conventional RT-PCR test (McNemar test, P<0.001)

The total detection rate of the ⁺ test was statistically different from the conventional RT-PCR test with the improved RNA extraction protocol (McNemar test, P<0.0001)

We reasoned that the low sensitivity of SARS-CoV RT-PCR detection in the early stages of disease could be enhanced by increasing the extraction volume of the initial NPA samples from 140 μl to 560 μl. Using this modified RNA extraction protocol, the sensitivity of the conventional RT-PCR assay was doubled from 11/50 to 22/50 (Table 5). The total detection rate of the improved RT-PCR protocol was statistically different from our first-generation RT-PCR protocol (McNemar test, P<0.001, Table 5). For 30 negative control samples, one false positive result was observed. Using RNA extraction improvements, the sensitivity and specificity of conventional RT-PCR for samples collected during the first 3 days of illness were 44.0% and 96.6%, respectively.

To further improve the detection of SARS-CoV in early-stage samples, we employed a highly sensitive real-time quantitative assay for SARS-CoV detection (Fig. 14). Using the modified RNA extraction protocol, 40 of 50 NPA samples were positive in the real-time assay (Figure 15 and Table 5). The total detection rate of the improved RT-PCR protocol was statistically different from the other two tests (McNemar test, P<0.0001, Table 5). Specifically, 63% of NPA samples isolated on day 1 of disease onset were positive in real-time quantitative RT-PCR assays. In contrast, samples isolated on day 1 were not positive in conventional RT-PCR assays. For samples isolated on days 2-3, more than 81% of the samples were positive in the quantitative assay (Table 5). Using an improved RNA extraction protocol and real-time PCR technology, the sensitivity and specificity of the quantitative test for early SARS samples were 80% and 100%, respectively.

The real-time assay also allowed quantification of the viral load of these clinical samples (1 copy/reaction = 27.8 copies/ml NPA sample). As shown in Figure 16, disease progression resulted in elevated viral load in NPA (open bar). In addition, we further investigated the viral load of negative clinical samples (N=39) in our first generation RT-PCR assay (Figure 16, gray bars). As expected, the viral loads of these clinical samples (gray bars) were much lower than the overall global viral loads (blank bars).

6.8.3. Discussion

Our goal in this study was to establish a highly sensitive RT-PCR assay for the detection of SARS-CoV. Specifically, we focused on detecting SARS-CoV RNA in samples isolated 1–3 days after symptom onset. Using our first-generation conventional RT-PCR assay, only 22% of these samples appeared to have SARS-CoV RNA. To establish a more sensitive assay, we improved the RNA extraction method and employed quantitative techniques in our current study. By increasing the initial volume used for RNA extraction from 140 μl to 540 μl, the proportion of positive cases increased to 44%. In addition, by further using real-time quantitative PCR technology in the improved test, 80% of early SARS samples became positive. More importantly, the use of 5' nuclease probes in real-time quantitation assays minimizes the false positive rate due to increased signal specificity. Taken together, the results of this study demonstrate that our improved RT-PCR assay allows early and accurate diagnosis of SARS.

The quantitative results of our modified RT-PCR assay provided further information on the viral load of SARS-CoV in these clinical samples. Our results show that viral load increases with disease progression. Of the samples that were negative in the first generation RT-PCR assay, all contained very little viral RNA (Figures 15 and 16). This phenomenon explains why most of these samples were negative using our first generation RT-PCR assay. Interestingly, of the samples that were positive in the first generation assay, some contained extremely high amounts of viral RNA (Figure 16).

In conclusion, by increasing the initial sample volume for RNA extraction and using real-time quantitative PCR technology, we established a sensitive and accurate RT-PCR assay for the immediate identification of SARS-CoV. It is expected that, with this rapid diagnostic method, immediate identification of such pathogens will facilitate disease control and immediate treatment regimes.

6.9. Clinical Observations and Discussion

The SARS outbreak was unusual in many respects, especially the concentration of patients with pneumonia among healthcare workers and household contacts. In this series of SARS patients, tests for conventional pathogens of atypical pneumonia proved negative. However, viruses belonging to the Coronaviridae family were isolated from lung biopsies and nasopharyngeal aspirates obtained separately from two SARS patients. The virus is not phylogenetically closely related to any known human or animal coronaviruses or orbiviruses. This analysis, based on a 646 base pair fragment of the polymerase gene (SEQ ID NO: 1), indicates that the virus is related to antigenic class 2 of coronaviruses, as well as murine hepatitis virus and bovine coronavirus. However, the Coronaviridae virus can carry out heterologous recombination within the virus family, so it is necessary to carry out genetic analysis to other parts of the genome of the novel virus, and then more conclusively define the nature of this virus (Holmes KV.Coronaviruses.EdsKnipe DM, Howley PM Fields Virology, 4th ed., Lippincott Williams & Wilkins, Philadelphia, pp. 1187-1203). The combination of biological, genetic, and clinical data suggests that this novel virus is not either of the two known human coronaviruses.

The majority (90%) of patients with clinically defined SARS had serological and RT-PCR evidence of infection with the virus. In contrast, no antibody or viral RNA was detectable in healthy human controls. All 27 patients for whom acute and convalescent sera were available showed elevated antibody titers against hSARS virus, which strengthens the argument that recent infection with this virus was a necessary factor in the development of SARS. In addition, all 5 pairs of acute and convalescent sera from patients from other hospitals in Hong Kong also showed seroconversion to the virus. The five patients who showed no serological or virological evidence of hSARS virus infection required subsequent testing of convalescent sera to determine whether they had also seroconverted. However, the concordance of the hSARS virus with the clinical definition of SARS remains striking, given that the definition of a clinical case has never been clarified.

None of these patients had detectable signs of HMPV infection, either by RT-PCR or by rising anti-HMPV antibody titers. No other pathogens were consistently detected in our SARS patient group. Therefore, it is likely that this hSARS virus is the cause of SARS or a necessary prerequisite for the development of the disease. The question of whether other microbial factors or additional cofactors play a role in the development of the disease remains to be investigated.

The Coronaviridae family includes the genera Coronaviridae and Orbiviridae. They are enveloped RNA viruses that can cause disease in humans and animals. Human coronavirus types 229E and OC43 were previously known to be the main cause of the common cold (Holmes KV. Coronaviruses. Eds Knipe DM, Howley PM Fields Virology, 4th Edition, Lippincott Williams & Wilkins, Philadelphia, 1187-1203). However, although coronaviruses can sometimes cause pneumonia in the elderly, newborn infants, or immunocompromised patients (El-Sahly HM, Atmar RL, Glezen WP, Greenberg SB. Spectrum of clinical illness inhospitalizied patients with “common cold” virus infections. Clin Infect Dis. 2000; 31: 96-100; and Foltz EJ, Elkordy MA. Coronavirus following autologous bone marrow transplantation for breast cancer. Chest 1999; 115: 901-905), which have been reported to be an important cause of pneumonia in military recruits, Accounted for up to 30% of cases in some studies (Wenzel RP, Hendley JO, Davies JA, Gwaltney JM, Coronavirus infections in military recruits: Three-year studv with coronavirus strains OC43 and 229E. Am Rev Respir Dis. 1974;109:621- 624). Human coronaviruses can infect neurons, and viral RNA has been detected in the brains of patients with multiple sclerosis (Talbot PJ, Cote G, Arbour N. Humancoronavirus OC43 and 229E persistence in neural cell cultures and humanbrains. Adv Exp Med Biol. Published middle). On the other hand, certain animal coronaviruses (e.g. porcine transmissible gastroenteritis virus, murine hepatitis virus, avian infectious bronchitis virus) can cause respiratory, gastrointestinal, neurological or hepatic disease in their respective hosts (McIntosh K . Coronaviruses: a comparative review. Current Top Microbiol Immunol. 1974; 63: 85-112).

For the first time, we describe the clinical manifestations and complications of SARS. Less than 25% of patients with COVID-19 have upper respiratory symptoms. As expected for atypical pneumonia, the respiratory symptoms and positive auscultation findings were quite disproportionate to the chest radiograph findings. Gastrointestinal symptoms occurred in 10% of patients. Of concern, coronavirus RNA can be detected in fecal samples from some patients, and coronaviruses are known to be associated with diarrhea in animals and humans (Caul EO, Egglestone SI. Further studies on human enteric coronaviruses ArchVirol. 1977;54:107 -17). The high incidence of liver dysfunction, leukopenia, marked lymphopenia, thrombocytopenia, and subsequent development of adult respiratory distress syndrome suggested that this hSARS virus caused severe systemic inflammatory damage. Therefore it is important to complement antiviral therapy with ribavirin by immunomodulation with steroids. In this regard, it is also appropriate to assume that severe human disease associated with avian influenza H5N1 subtype (another virus that has recently crossed from animals to humans) has an immunopathological component (Cheung CY, Poon LLM, Lau ASY et al , Induction of proinflammatory cytokines in human macrophages by influenza A(H5N1) viruses: a mechanism for the unusual severity of human disease. Lancet 2002; 360: 1831-1837). Like H5N1 disease, severe SARS patients are also adults with more pronounced lymphopenia and features of organ dysfunction other than the respiratory tract (Table 4) (Yuen KY, Chan PKS, Peiris JSM et al, Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. Lancet 1998; 351: 467-471). It is worth noting that there is a window of opportunity of about 8 days from the onset of the disease to respiratory failure. Severe complication cases were strongly associated with underlying disease and delayed use of ribavirin and steroid therapy. Based on our clinical experience from the initial case, we implemented the combination therapy described above very early in the subsequent case that was largely free of complications on admission. With this regimen, the overall mortality rate at the time of this writing was only 2%. Eight of the 19 complication cases did not show significant response. A detailed analysis of the response to this combination regimen was not possible due to inconsistencies in dose and timing of initiation of treatment.

Other factors associated with this severe illness are illness through household contact, which can be attributed to high doses or continued exposure to the virus and the presence of underlying disease.

The clinical descriptions given here are basically of the more severe cases admitted to the hospital. Currently we do not have any data on the full clinical extent of Coronaviridae infections occurring in society and outpatient settings. The availability of the diagnostic test described here will help to address the above issues. In addition, this also allows to address questions regarding the period of viral shedding (and infectivity) during convalescence, the presence of virus in other bodily fluids and excretions, and the occurrence of viral shedding during the incubation period.

Current epidemiological data seem to indicate that the virus is transmitted by droplets or direct and indirect contact, although airborne transmission cannot be ruled out in some cases. This contention is supported by the discovery of infectious viruses in the respiratory tract. Preliminary evidence also suggests that the virus may be shed in feces. However, it is worth noting that the detection of viral RNA does not prove that the virus is viable or infectious. If live virus is detected in faeces, this may be another potential route of transmission that needs to be considered. It may be pertinently noted that certain animal coronaviruses are transmitted by the fecal-oral route (McIntosh K., Coronaviruses: a comparative review. Current Top Microbiol Immunol. 1974, 63:85-112).

In conclusion, this report provides evidence that viruses of the Coronaviridae family are the causative agents of SARS. The invention discloses a quantitative diagnostic test for rapid, sensitive and specific identification of hSARS virus.

7. Preservation

The sample of isolated hSARS virus was deposited in the Chinese Type Culture Center (CCTCC) located at Wuhan University (Wuhan, China 430072) on April 2, 2003 according to the Budapest Treaty on the Deposit of Microorganisms. integrated into this article as a whole.

8. Market potential

Large-scale cultivation of hSARS virus is now possible, which allows the development of various diagnostic tests as described above and the development of vaccines and antiviral drugs that can effectively prevent, ameliorate or treat SARS. Given the severity of the disease and its rapid worldwide spread, the demand for diagnostic tests, therapies and vaccines to combat the disease is likely to rise significantly worldwide. In addition, this virus contains extremely important and valuable genetic information for clinical and scientific applications.

9. Equivalent scheme

Those of ordinary skill in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are covered by the following claims.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Citation or discussion of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.

Claims (18)

1. isolated nucleic acid molecule, described nucleic acid molecule is made up of SEQ ID NO:2471,2472 nucleotide sequences or its complementary sequence substantially.

2. isolated nucleic acid molecule, described nucleic acid molecule is made up of SEQ ID NO:2474,2475 nucleotide sequences or its complementary sequence substantially.

3. isolated nucleic acid molecule, described nucleic acid molecule is made up of SEQ ID NO:2473,2476 nucleotide sequences or its complementary sequence substantially.

4. isolated nucleic acid molecule, described nucleic acid molecule under stringent condition with the making nucleic acid molecular hybridization with SEQID NO:2471,2472,2473,2474,2475 or 2476 nucleotide sequences or its complementary sequence.

5. isolated polypeptide, described polypeptide is by each nucleic acid molecule encoding of claim 1-4.

6. an antibody or its Fab, described antibody or its Fab immunologic opsonin are in conjunction with the polypeptide of SEQ ID NO:2471,2472 or 2473 nucleic acid sequence encodings.

7. an antibody or its Fab, described antibody or its Fab immunologic opsonin are in conjunction with the polypeptide of SEQ ID NO:2474,2475 or 2476 nucleic acid sequence encodings.

8. one kind is detected the method that hSARS virus exists in biological sample, and described method comprises:

(a) use has the nucleic acid of the primer amplification hSARS virus of SEQ ID NO:2471 and/or 2472 nucleotide sequences;

(b) use probe in detecting nucleic acid with SEQ ID NO:2473 nucleotide sequence.

9. one kind is detected the method that hSARS virus exists in biological sample, and described method comprises:

(a) use has the nucleic acid of the primer amplification hSARS virus of SEQ ID NO:2474 and/or 2475 nucleotide sequences;

(b) use probe in detecting nucleic acid with SEQ ID NO:2476 nucleotide sequence.

10. method of identifying hSARS virus infection object, described method comprises:

(a) obtain total RNA from biological sample from object;

(b) the total RNA of reverse transcription is to obtain cDNA;

(c) overlap the primer that derives from the hSARS nucleotide sequence with one cDNA is carried out the PCR test.

11. a method of identifying hSARS virus infection object, described method comprises:

(a) obtain total RNA from biological sample from object;

(b) the total RNA of reverse transcription is to obtain cDNA;

(c) overlap primer with one cDNA is carried out the PCR test with SEQ ID NO:2471 and/or 2472 nucleotide sequences.

12. the method for claim 11, described method further comprise (d) product with probe in detecting PCR test.

13. the method for claim 12, wherein said probe are the nucleic acid molecule with SEQ ID NO:2473 nucleotide sequence.

14. a method of identifying hSARS virus infection object, described method comprises:

(a) obtain total RNA from biological sample from object;

(b) the total RNA of reverse transcription is to obtain cDNA;

(c) overlap primer Monday cDNA is carried out the PCR test with SEQ ID NO:2474 and/or 2475 nucleotide sequences.

15. the method for claim 14, described method further comprise (d) product with probe in detecting PCR test.

16. the method for claim 15, wherein said probe are the nucleic acid molecule with SEQ ID NO:2476 nucleotide sequence.

17. a test kit, described test kit comprise one or more isolated nucleic acid molecule in one or more containers, described nucleic acid molecule comprises the nucleotide sequence that is selected from SEQ ID NO:2471, SEQ IDNO:2472 and SEQ ID NO:2473.

18. a test kit, described test kit comprise one or more isolated nucleic acid molecule in one or more containers, described nucleic acid molecule comprises the nucleotide sequence that is selected from SEQ ID NO:2474, SEQ IDNO:2475 and SEQ ID NO:2476.

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