CN1761745A - Novel Human Virus Causing Severe Acute Respiratory Syndrome (SARS) and Its Application - Google Patents

Abstract

The present invention relates to an isolated novel virus that causes Severe Acute Respiratory Syndrome (SARS) in humans ("hSARS virus"). The hSARS virus was identified to be morphologically and phylogenetically similar to known members of the family Coronaviridae (Coronaviridae). The present invention provides the complete genome sequence of hSARS virus. In addition, the invention provides nucleic acids and peptides encoded by and/or derived from the hSARS virus and their use in diagnostic and therapeutic methods, including vaccines. In addition, the invention provides chimeric or recombinant viruses encoded by the nucleotide sequences and antibodies immunospecific for the polypeptides encoded by the nucleotide sequences.

Description

Novel Human Virus Causing Severe Acute Respiratory Syndrome (SARS) and Its Application

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 US Provisional Application 60/464,886, filed April 23, 2010, each of which is hereby incorporated by reference in its entirety.

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 16, 2004, respectively labeled "CRF", "Copy 1" and "Copy 2", each containing only one identical 1.58MB file (V9661069.APP).

1 Introduction

The present invention relates to the isolation of novel viruses ("hSARS viruses") that cause severe acute respiratory syndrome (SARS) in humans. It was identified that the hSARS virus was morphologically and phylogenetically similar to known members of the Coronaviridae family. The present invention relates to a nucleotide sequence comprising the whole genome sequence of hSARS virus. The present invention further relates to a nucleotide sequence comprising a part of the genome sequence of hSARS virus. The present invention also relates to the deduced amino acid sequence of the whole genome sequence of hSARS virus. The invention still further relates to nucleic acids and peptides encoded and/or derived from these sequences and their use in diagnostic and therapeutic methods (eg as immunogens). The present invention further includes the chimeric virus or recombinant virus encoded by the nucleotide sequence and the antibody against the polypeptide encoded by the nucleotide sequence. Furthermore, the invention relates to vaccine preparations comprising hSARS virus, including recombinant and chimeric forms of said virus and protein extracts and subunits of said virus.

2. Background of the invention

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 Epidenziol 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 inventors of the present invention isolated the hSARS virus disclosed herein from a SARS patient, the pathogenic factor of the disease was known. That is, the present invention discloses a novel human virus isolated and identified from a SARS patient. The present invention can be used for clinical and scientific research purposes.

3. Outline of the invention

The present invention is based on the isolation and identification by the present inventors of a novel virus ("hSARS virus") causing severe acute respiratory syndrome in humans. The virus was isolated from a patient suffering from SARS in the recent severe atypical pneumonia outbreak in China. The isolated virus is an enveloped single-stranded RNA virus of positive polarity and belongs to the family Coronaviridae of the order Nidovirales. Accordingly, the present invention relates to isolated hSARS viruses that are morphologically and phylogenetically related to known members of the Coronaviridae family. In a specific embodiment, the isolated hSARS virus is the virus described in Section 7 below that was deposited at the China Center for Type Culture Culture (CCTCC) on April 2, 2003, and assigned the deposit number CCTCC-V200303. In another specific embodiment, the present invention provides the whole genome sequence of hSARS virus. In a preferred embodiment, the virus comprises the nucleotide sequence of SEQ ID NO:15. In another specific embodiment, the invention provides nucleic acids isolated from viruses. The virus preferably comprises the nucleotide sequence of SEQ ID NO: 1, 11 and/or 13 in its genome. In a specific embodiment, the present invention provides an isolated nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 1 or a complementary sequence or a part thereof, preferably a core of SEQ ID NO: 1 At least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600 or more of the nucleotide sequence or its complement consecutive nucleotides. In another specific embodiment, the present invention provides an isolated nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 11 or its complementary sequence or a part thereof, preferably SEQ ID NO: 11 At least 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, 1,000, 1,050, 1,100, 1,150, 1,200 or more contiguous nucleotides. In yet another specific embodiment, the present invention provides an isolated nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 13 or its complementary sequence or a part thereof, preferably SEQ ID NO: 13 At least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more contiguous nucleotides. In another specific embodiment, the present invention provides an isolated nucleic acid molecule comprising or consisting of a SEQ ID NO: 15 nucleotide sequence or a complementary sequence or a portion thereof, preferably a SEQ ID NO: 15 core At least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700 of the nucleotide sequence or its complement . , 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000 or more consecutive nucleotides. In addition, in another specific embodiment, the present invention provides a compound capable of combining with SEQ ID NO: 1, 11, 13, 15, 16, 240, 737, 1108, 1590 or 1965 sequence or its complement under the stringent conditions defined herein. nucleic acid molecules hybridized isolated nucleic acid molecules. In one embodiment, the invention provides isolated nucleic acid molecules that are antisense to the coding strand of a nucleic acid of the invention. In another specific embodiment, the invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 1 nucleotides At least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600 or more contiguous cores of the sequence or its complement glycosides. In yet another specific embodiment, the invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 11 nucleoside At least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200 or more contiguous nucleotides. In yet another specific embodiment, the present invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule consisting of a nucleotide sequence, said nucleotide sequence being a SEQ ID NO: 13 nucleotide sequence or at least 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. In yet another specific embodiment, the invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 15 nucleosides At least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 0 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000 or more contiguous nucleotides. The invention further provides proteins or polypeptides isolated from hSARS virus, including viral proteins isolated from virus-infected cells but absent from comparable uninfected cells. The present invention further provides the proteins or polypeptides of SEQ ID NOs: 2, 12 and 14 and the proteins or polypeptides of Fig. 11 (SEQ ID NOs: 17-239, 241-736 and 738-1107) and Fig. -1964, 1966-2470) proteins or polypeptides. The polypeptide or protein of the present invention preferably has the biological activity (including antigenicity and/or immunogenicity) of the protein encoded by the sequence of SEQ ID NO: 1, 11, 13, 16, 240, 737, 1108, 1590 or 1965. In other embodiments, the polypeptide or protein of the present invention has the biological activity (including antigenicity and/or immunogenicity) of the protein encoded by the nucleotide sequence of SEQ ID NO: 15 nucleotides sequence or its complementary , 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, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 17,000 , 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000 or more contiguous nucleotides. In other embodiments, the polypeptide or protein of the present invention has the ) biological activity (including antigenicity and/or immunogenicity) of the protein.

In one aspect, the invention provides a method of propagating hSARS virus in a host cell, the method comprising infecting the host cell with an isolated hSARS virus, culturing the host cell to propagate the virus, and harvesting the resulting virus particles. The present invention also provides host cells infected by hSARS virus. In another aspect, the invention relates to the use of the isolated hSARS virus in methods of diagnosis and methods of treatment. In a specific embodiment, the present invention provides a method for detecting antibodies immunospecific to hSARS virus in a biological sample using isolated hSARS virus or any protein or polypeptide thereof. In another specific embodiment, the present invention provides a method for screening antibodies that immunospecifically bind or neutralize hSARS. This antibody can be used for passive immunization or immunotherapy of patients infected with hSARS.

The invention further relates to the use of the sequence information of the isolated virus in diagnostic and therapeutic methods. In a specific embodiment, the present invention provides a nucleic acid molecule suitable for use as a primer comprising or consisting of a nucleotide sequence of SEQ ID NO: 1, 11, 13 or 15 or its complementary sequence or at least the nucleotide sequence thereof part of the composition. In another specific embodiment, the present invention provides nucleic acid molecules suitable for hybridization with hSARS nucleic acid, said nucleic acid molecules include but not limited to PCR primers, reverse transcriptase primers, for Southern analysis or other nucleic acid hybridization analysis to detect hSARS Nucleic acid probes, for example comprising or consisting of the nucleotide sequence of SEQ ID NO: 1, 11, 13 or 15 or a complementary sequence or part thereof. The present invention further includes chimeric viruses or recombinant viruses encoded by said nucleotide sequence in whole or in part.

The present invention further provides antibodies capable of specifically binding to the polypeptide of the present invention encoded by the nucleotide sequence of SEQ ID NO: 1, 11, 13, 16, 240, 737, 1108, 1590 or 1965 or a fragment thereof, or by A polypeptide of the present invention encoded by a nucleic acid comprising a nucleotide sequence that hybridizes to a nucleotide sequence of SEQ ID NO: 1, 11 or 13 under stringent conditions, and/or any hSARS having one or more biological activities of the polypeptide of the present invention gauge. The present invention further provides an antibody capable of specifically binding to the polypeptide of the present invention encoded by the nucleotide sequence of SEQ ID NO: 15 or its complementary sequence or a fragment thereof. These polypeptides include those shown in Figure 11 (SEQ ID NOs: 17-239, 241-736, and 738-1107) and Figure 12 (SEQ ID NOs: 1109-1589, 1591-1964, and 1966-2470). The present invention further provides antibodies capable of specifically binding to the polypeptide of the present invention encoded by a nucleic acid comprising a nucleotide sequence that hybridizes to the nucleotide sequence of SEQ ID NO: 15 under stringent conditions, and/or having Any hSARS epitope of one or more biological activities of the polypeptide. 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 are capable of specifically binding to a polypeptide of the invention.

In one embodiment, the present invention provides methods for detecting the presence, activity or expression of the hSARS virus of the present invention in biological materials such as cells, blood, saliva, urine, and the like. An increase or decrease in the activity or expression of hSARS virus in a sample relative to a control sample is determined by contacting the biological material with an agent capable of directly or indirectly detecting the presence, activity or expression of hSARS virus. In a specific embodiment, the detection reagent is an antibody or nucleic acid molecule of the invention. The antibody of the present invention can also be used to treat SARS.

In another embodiment, the invention provides vaccine preparations comprising hSARS virus (including recombinant and chimeric forms of said virus) or protein subunits of the virus. In a specific embodiment, the vaccine preparation of the invention comprises live but attenuated hSARS virus, with or without adjuvant. In another specific embodiment, the vaccine preparation of the invention comprises inactivated or killed hSARS virus. Such attenuated or inactivated viruses can be prepared by serial passage of the virus in host cells or by making recombinant or chimeric forms of the virus. Accordingly, the present invention further provides methods for producing recombinant or chimeric forms of hSARS. In another specific embodiment, the vaccine product of the present invention comprises the nucleic acid or fragment of hSARS virus (such as the virus whose deposit number is CCTCC-V200303), or has a sequence of SEQ ID NO: 1, 11, 13 or 15 or a fragment thereof. nucleic acid molecule. In another embodiment, the present invention provides a vaccine product comprising one or more polypeptides isolated from hSARS virus (such as the virus with deposit number CCTCC-V200303) or produced from its nucleic acid. In a specific embodiment, the vaccine preparation comprises a polypeptide of the invention encoded by the nucleotide sequence of SEQ ID NO: 1, 11, 13, 16, 240, 737, 1108, 1590 or 1965 or a fragment thereof. In a specific embodiment, the vaccine preparation comprises the compounds shown in Figure 11 (SEQ ID NO: 17-239, 241-736 and 738-1107) and Figure 12 (SEQ ID NO: 1109-1589, 1591-1964 and 1966-2470). The polypeptide of the present invention shown or the polypeptide of the present invention encoded by the nucleotide sequence of SEQ ID NO: 15 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 an adjuvant or other pharmaceutically acceptable excipients.

In another aspect, the invention provides a pharmaceutical composition comprising an antiviral agent of the invention and a pharmaceutically acceptable carrier. In a specific embodiment, the antiviral drug of the present invention is an antibody that immunospecifically binds hSARS virus or any hSARS epitope. In another specific embodiment, the antiviral agent is a polypeptide or protein of the invention or a nucleic acid molecule of the invention. The invention also provides kits comprising the pharmaceutical compositions of the invention.

3.1 Definition

The term "antibody or antibody fragment that immunospecifically binds to the polypeptide of the present invention" as used herein refers to immunospecifically binding to a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 1, 11, 13 or 15 or a fragment thereof, and does not nonspecifically Antibodies or fragments thereof that bind other polypeptides. Antibodies or fragments thereof that immunospecifically bind a polypeptide of the invention may cross-react with other antigens. Preferably, antibodies or fragments thereof that immunospecifically bind a polypeptide of the invention do 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 well known to those skilled in the art.

"Isolated" or "purified" peptides or proteins are substantially free of cellular material or other contaminating proteins from the cellular or tissue source 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 involved in 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.

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, nucleic acid molecules encoding polypeptides/proteins of the invention are 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 "portion" or "fragment" as used herein refers to a fragment comprising at least about 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650 . , 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000 or more contiguous nucleic acids, and have at least one functional feature (or its encoding The protein has a functional characteristic of the protein encoded by the related nucleic acid molecule); or refers to a nucleic acid molecule fragment containing 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, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,100, 4,200, 4,300, 4,350, 4,360, 4,370, 4,380 amino acid residues in length and having at least one functional feature of the relevant protein or polypeptide Protein or polypeptide fragments.

The term "having the biological activity of the protein of the present invention" or "having the biological activity of the polypeptide of the present invention" refers to the characteristics of a polypeptide or protein having a common biological activity, which is identical to SEQ ID NO: 1, 11, 13, 15, 16, 240, 737 , 1108, 1590 or 1965 nucleotide sequences have similar or identical structural domains and/or have sufficient amino acid identity compared to polypeptides encoded by nucleotide sequences. Such common biological activities of the polypeptides of the invention include antigenicity and immunogenicity.

The term "under stringent conditions" refers to hybridization and washing conditions under which nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to each other remain hybridized to each other . Such hybridization conditions are 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), described in pages 387-389, are well known to those skilled in the art. A non-limiting example of preferred stringent hybridization conditions is hybridization at about 68°C in 6X sodium chloride/sodium citrate (SSC), 0.5% SDS, followed by one or more washes in 2X SSC, 0.5% SDS at room temperature. Second-rate. Another non-limiting example of preferred 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.

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

4. Description of 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 exhibits RNA-dependent RNA aggregation with known coronaviruses (Coronaviruses) The enzyme proteins have 57% homology.

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

Figure 3 shows immunofluorescent staining of IgG antibodies specifically bound to FrHK-4 cells infected with novel coronaviruses (Coronaviridae) human respiratory virus.

Figure 4 shows an electron micrograph of an ultracentrifuge sediment of hSARS virus grown in cell culture and 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-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) over 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 SARS virus.

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

Figure 10 shows the entire genome DNA sequence (SEQ ID NO: 15) of SARS 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.

5. Detailed Description of the Invention

The present invention relates to isolated hSARS viruses that are morphologically and phylogenetically related to known coronaviruses. In a specific embodiment, the isolated hSARS virus is CCTCC-V200303 virus. In another specific embodiment, said virus comprises the nucleotide sequence of SEQ ID NO: 1, 11, 13 and/or 15. In a specific embodiment, the present invention provides an isolated nucleic acid molecule of hSARS virus or its complement or part thereof, the nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 1, 11, 13 and/or 15 . In another specific embodiment, the present invention provides that under stringent conditions as defined herein, with a nucleic acid molecule having a sequence of SEQ ID NO: 1, 11, 13 or 15, or a specific gene of a known member of the Coronaviridae family, An isolated nucleic acid molecule that hybridizes to its complement. 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, 15, 15, 15, 15, 15, 15, 15, 14, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, A nucleotide sequence of 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, 15, 15, 15, 15, 15, 15, 15, 15, 1, 1, 1, 1, 1, 1, 1, nucleotide sequence of a SEQ ID NO: 11 nucleotide sequence, or its complement, encoded by a nucleic acid molecule. 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, A nucleotide sequence of 1,150, 1,200 or more contiguous nucleotides. In yet another specific embodiment, the invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule comprising at least about 5, 10, 15, 15, A nucleotide sequence of 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more contiguous nucleotides. In yet another specific embodiment, the invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule comprising at least 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, 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, 19,000, 20,000, 22,000, 223,00 A nucleotide sequence of 25,000, 26,000, 27,000, 28,000, 29,000 or more contiguous nucleotides. These polypeptides include those shown in Figure 11 (SEQ ID NO: 17-239, 241-736 and 738-1107) and Figure 12 (SEQ ID NO: 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: the protein encoded by the sequence of SEQ ID NO: 1, 11, 13, 15; or containing the sequence of SEQ ID NO: 1, 11, 13 or 15 The native viral protein of the encoded amino acid sequence; or Figure 11 (SEQ ID NO:17-239,241-736 and 738-1107) and Figure 12 (SEQ ID NO:1109-1589,1591-1964 and 1966-2470) protein shown.

The present invention also relates to methods for propagating hSARS virus in host cells.

The invention further relates to the use of the sequence information of the isolated virus in diagnostic and therapeutic methods. In a specific embodiment, the present invention provides the entire nucleotide sequence SEQ ID NO of hSARS virus CCTCC-V200303: 15 or its fragment or complement. In addition, the present invention relates to nucleic acid molecules capable of hybridizing to any part of the genome SEQ ID NO: 15 of hSARS virus CCTCC-V200303 under stringent conditions. In a specific embodiment, the invention provides a nucleic acid molecule suitable for use as a primer comprising or consisting of a nucleotide sequence of SEQ ID NO: 1, 11, 13 or 15 or a complementary sequence or a portion thereof. In a non-limiting embodiment, the invention provides a primer comprising or consisting of the nucleotide sequence of SEQ ID NO: 3 and/or 4. In another specific embodiment, the present invention provides a nucleic acid molecule suitable for use as a hybridization probe for detecting a sequence comprising or consisting of a nucleotide sequence of SEQ ID NO: 1, 11, 13 or 15 or its complementary sequence or a portion thereof. Constituent, nucleic acid encoding a polypeptide of the present invention. The present invention further includes chimeric or recombinant viruses or viral proteins encoded by said nucleotide sequences.

The present invention further provides antibodies capable of specifically binding to the polypeptide of the present invention or any hSARS epitope encoded by the nucleotide sequence of SEQ ID NO: 1, 11, 13, 16, 240, 737, 1108, 1590 or 1965 or a fragment thereof. The present invention further provides an antibody capable of specifically binding to the polypeptide of the present invention or any hSARS epitope encoded by the nucleotide 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 are capable of specifically binding to a polypeptide of the invention.

In one embodiment, the present invention provides methods for detecting the presence, activity or expression of the hSARS virus of the present invention in biological materials such as cells, blood, saliva, urine, sputum, nasopharyngeal aspirates, and the like. The presence of hSARS virus in a sample can be determined by contacting the biological material with a reagent capable of directly or indirectly detecting the presence of hSARS virus. In a specific embodiment, the detection reagent is an antibody of the invention. In another embodiment, the detection reagent is a nucleic acid molecule of the invention.

In another embodiment, the invention provides vaccine preparations comprising hSARS virus (including recombinant and chimeric forms of said virus) or viral subunits. 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 according to the invention comprises inactivated or killed hSARS virus, with or without pharmaceutically acceptable excipients, including adjuvants.

The present invention further provides methods for producing recombinant or chimeric forms of hSARS. In another specific embodiment, the vaccine preparation according to the invention comprises one or more nucleic acid molecules comprising or consisting of the sequence of SEQ ID NO: 1, 11, 13 and/or 15 or fragments 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 and /or 1965 nucleotide sequence or a nucleotide sequence code composed of fragments thereof. In another embodiment, the invention provides a vaccine preparation comprising one or more polypeptides of the invention encoded by a nucleotide sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 15 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 drugs [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 therapy for viral infections.

In addition, the present invention provides a pharmaceutical composition comprising the antiviral drug 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 for screening antiviral drugs that inhibit the infectivity or replication of hSARS virus or variants thereof.

5.1 Recombinant and Chimeric hSARS Viruses

The present invention includes host 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, the virus has the nucleotide sequence of SEQ ID NO: 15. 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 , insertion, deletion, etc., said mutation 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, said OFR comprises or consists of the nucleotide sequence of SEQ ID NO: 1, 11 or 13 or a fragment thereof. In a specific embodiment, there is more than one OFR in the nucleotide sequence of SEQ ID NO: 15 or its complement or a fragment thereof, as shown in Figure 11 (SEQ ID NO: 16, 240 and 737) and Figure 12 ( Shown in SEQ ID NO: 1108, 1590 and 1965). In another embodiment, the polypeptide encoded by ORF comprises or consists of SEQ ID NO: 2, 12 or 14 amino acid sequence or fragment thereof, or as shown in Figure 11 (SEQ ID NO: 17-239, 241-736 and 738-1107) and Shown in Figure 12 (SEQ ID NO: 1109-1589, 1591-1964 and 1966-2470) or its fragment composition. According to the invention, these viral vectors may or may not contain nucleic acids that do not belong to the native viral genome.

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, a chimeric virus is encoded by a viral vector that may or may not contain nucleic acid that is not part of 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 replaced by a native or non-native sequence. 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 SARS 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 (1998); Teng et al., 2000, J. Virol. 74, 9317-9321). For example, it is conceivable that a viral vector derived from hSARS virus expressing one or more proteins of a hSARS virus variant (or vice versa) will protect a subject vaccinated with such a vector from infection by 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 PCT WO 02/057302, 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 and its natural variants. 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 viral particle of the present invention can be artificially modified to produce a vaccine capable of protecting subjects from infection by hSARS virus or its variants.

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 variants thereof, containing phenotypes (such as attenuated phenotypes or enhanced antigenicity) that make the virus more suitable for use in vaccine preparations the sequence of. 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 the polymerase component of hSARS virus are produced in prokaryotic cells to express the component 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 or insertions to produce attenuated viruses. In addition, the present invention provides a host cell infected with hSARS virus (such as the hSARS virus with the deposit number CCTCC-V200303).

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.

5.2 Preparation of vaccines and antiviral drugs

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 are those encoded by the nucleotide sequence of SEQ ID NO: 1, 11, 13 or 15 or Figure 11 (SEQ ID NO: 17-239, 241-736 and 738-1107) and Figure 12 (SEQ ID NO: 1109-1589, 1591-1964 and 1966-2470) polypeptides or antigenic fragments thereof, for mixing as antigens or subunit immunogens, but inactivated whole viruses can also be used. Also particularly useful are proteinaceous substances encoded by recombinant nucleic acid fragments of the hSARS genome, preferably within the preferred limits of the ORF, especially in vivo (e.g. for protective or therapeutic purposes, or for providing 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.

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.

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.

In another aspect, the present invention also provides DNA vaccine preparations, which comprise nucleic acid or fragments of hSARS virus (for example, a virus with a deposit number of CCTCC-V200303) or a nucleic acid molecule with a sequence of SEQID NO: 1, 11, 13 or 15 or its fragment. 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 (such as derived from the hSARS virus), a bacterial plasmid or other expression vector with an insert comprising the present invention operatively linked to one or more control elements. A nucleic acid molecule such that the vaccinating protein encoded by said nucleic acid molecule can be expressed in a vaccinated 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 type 1, Proc.Natl.Acad.Sci.USA 90:4156 4160; Lu, S. et al., 1996, Simian immunodeficiency virus DNA vaccine trial in 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 vaccination, 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-91: 957 Ulmer, J.B. et al., Heterologous protection agaainst 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 agaainst tuberculosis by DNA injection, Nature88Med.2 -892; Huygen, K. et al., 1996, Immunogenicity and protective efficacy of tuberculosis DNA vaccine, Nature Med., 2:893-898) and parasitic infections such as malaria (Sedegah, M., 1994, Protection against malaria by immunization with plasmamid DNA encoding circumsporozoite protein, Proc.Natl.Acad.Sci.USA 91:9866-9870; Doolan, D.L., etc., 1996, Circumventing genetic restriction of protection agaainst malaria with multigene DNAimmunization: CD8+T cell-interferon oxid ric end δ, and , J.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. 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, Direct gene transfer into mouse muscle in vivo, Science 247:1465-1468; Raz, E., 1994 , Intradermal geneimmunization: The possible role of DNA uptake 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 approaches to DNA vaccines 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 vaccines, Seminars in Immunology 9(5):269-270; and Robinson, H.L. et al., 1997, DNA vaccines, Seminars in Immunology 9(5):271-283.

5.3 Attenuation of hSARS virus or 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.

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 measured, 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.

A virus of the invention may be attenuated such that one or more functional characteristics of the virus are impaired. In certain 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 present invention is capable of infecting a host and replicating in the 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, an attenuated virus of the invention (eg, a recombinant or chimeric hSARS virus) does not replicate as well as a wild-type virus (eg, wild-type hSARS) 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 wild-type hSARS, 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 the sequence of SEQ ID NO: 1, 11, 13, or 15, to produce a virus with an attenuated phenotype. Mutations (eg, missense mutations) can be introduced into the structural and/or regulatory genes of hSARS. Mutations can be additions, substitutions, deletions or combinations thereof. Such hSARS 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 a protein of the wild-type virus.

When live attenuated vaccines are used, their safety must 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.

5.4 Adjuvants and carrier molecules

The hSARS-associated antigen is 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 TERMURTIDE), 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 PLUROMC L-121) prepared squalene or squalane oil-in-water adjuvant formulations such as SAF and MF59, liposomes, virosomes, liposome rolls (cochleates) 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 subunit (CTB), 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 carboxyl 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.

The vaccines of the present invention may be polyvalent or monovalent. Multivalent vaccines are made from recombinant viruses that direct the expression of more than one antigen.

A variety of methods can be used to introduce the vaccine preparations of the invention; these methods include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal routes and by scarification (e.g., by scratching with a bifurcated needle). upper layer of the skin).

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

5.5 Antibody Preparation

Specific recognition of polypeptides of the present invention, such as but not limited to polypeptides comprising SEQ ID NO: 2, 12 and 14 sequences and Fig. 11 (SEQ ID NO: 17-239, 241-736 and 738-1107) and Fig. 12 (SEQ ID NO: 1109-1589, 1591-1964, 1966-2470), or antibodies to hSARS epitopes or antigen-binding fragments thereof, can be used to detect, screen and isolate polypeptides or fragments thereof of the present invention, or may encode other biological Similar sequences of body-like 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 The polypeptide of the present invention or preferably hSARS is detected in samples such as biological materials, which include cells, cell culture media (such as bacterial cell culture media, mammalian cell culture media, insect cell culture media, yeast cell culture media, etc.), blood, Plasma, serum, tissue, sputum, nasopharyngeal aspirates, etc.

Antibodies specific for a polypeptide of the invention or any epitope of hSARS 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 nucleotide 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 digestion 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 antibody or fragment 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 technology.

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 (such as an antibody cDNA library, or a cDNA library produced from any tissue or cell expressing an antibody, such as a hybridoma selected to express an antibody, or a nucleic acid isolated therefrom, preferably poly A+ RNA), can be obtained 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 used to identify cDNA clones from antibody-encoding cDNA libraries cloned to obtain. 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 incorporated herein by reference in their entirety), to manipulate, for example, in the epitope-binding domain of an antibody or in any portion that enhances or reduces the biological activity of an antibody. Amino acid substitutions, deletions and/or insertions are introduced 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, Nature, 322:52, 1986 and Kohler, Proc.Natl.Acad.Sci.USA, 77:2197 , 1980). 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 fused to 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., J. Immunol. Methods, 182:41-50, 1995; Ames et al., J. Immunol. Methods, 184:177-186, 1995; Kettleborough et al., Eur.J.Immunol., 24:952-958, 1994; Persic et al., Gene, 187:9-18, 1997; Burton et al., Advances in Immunology, 57: 191-280, 1994; PCT Application No. PCT/GB91/01134; PCT Publication No. WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; 20401；及美国专利号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 whole antibodies, including human antibodies, or any other desired fragment, 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') _fragments can also be applied, using methods well known in the art such as disclosed in: PCT Publication No. WO 92/22324; Mullinax et al., BioTechniques, 12 (6): 864-869, 1992; and Sawai et al., AJRI, 34:26-34, 1995; and Better et al., Science, 240:1041-1043, 1988 (each of which is hereby incorporated by reference in its entirety). Examples of techniques that can be used to produce single chain Fvs and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88, 1991; Sbu et al., PNAS, 90: 7995-7999, 1993; and Skerra et al., Science, 240:1038-1040, 1988.

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 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, for example, Morrison, Science, 229:1202, 1985; Oi et al., BioTechniques, 4:214 1986; Gillies et al., J. Immunol. Methods, 125:191-202, 1989; U.S. Pat. The literature 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. Rare framework residues. See, eg, Queen et al., US Patent No. 5,585,089; Riechmann et al., Nature, 332:323, 1988, 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 (EP 239,400; PCT Publication No. WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (resurfacing) (EP592,106; EP 519,596; Padlan, Molecular Immunology, 28(4/5): 489-498, 1991; Studnicka et al., Protein Engineering, 7(6): 805-814, 1994; Roguska et al., Proc. USA, 91:969-973, 1994) and chain shuffling (US Patent No. 5,565,332), each of which is hereby incorporated 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 the phage display technique 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. WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; integrated into this article as a whole.

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, Int. Rev. Immunol., 13:65-93,1995. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies, and protocols for producing such antibodies, see, e.g., PCT Publication Nos. WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent 0 598 877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 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., Bio/technology, 12:899-903, 1988).

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, e.g., PCT Publication No. WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett., 39:91-99, 1994; U.S. Patent No. 5,474,981; Gillies et al., PNAS, 89:1428-1432, 1992 and Fell et al., J. Immunol., 146:2446-2452, 1991, 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.

5.6 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 a variant thereof or any derived protein thereof (see section 5.5). In another specific embodiment, the antiviral agent is a polypeptide or nucleic acid molecule of the invention (see, eg, Sections 5.1 and 5.2). 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 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 desirable 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. in combination with post-operative wound dressings). ), by injection, by catheter, by suppository, by intranasal spray, or by implants that are porous, non-porous, or gel-like materials, including thin films such as sialastic membranes or fiber. In one embodiment, administration may be by direct injection at the site of the affected tissue (or from the aforementioned site).

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, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); 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 (Science 249:1527-1533 (1990)).

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. Generally, 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, such as a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 1, 11, 13 or 15, or a polypeptide of FIG. 11 (SEQ ID NO: 17-239, 241 -736 and 738-1107) and the polypeptide shown in Figure 12 (SEQ ID NO:1109-1589, 1591-1964 and 1966-2470), or any hSARS epitope, or polypeptide or protein of the present invention, or nucleic acid molecule of the present invention Specific antibodies, which may be present 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.

5.7 Detection test

The present invention provides a method for detecting antibodies that immunospecifically bind to hSARS virus in biological samples such as blood, serum, plasma, saliva, urine, etc. from SARS patients. 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 nucleic acid sequence, and then the bound antibody can pass through Use the immunofluorescence assay described in Section 6.5 below to detect.

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 epitopes or nucleic acids (such as mRNA, genomic DNA) 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. The nucleic acid probe can be, for example, a nucleic acid molecule comprising or consisting of a SEQ ID NO: 1, 11, 13 or 15 nucleotide sequence or a portion thereof, such as a length of at least 15, 20, 25, 30, 50, 100, 250 , 500, 750, 1,000 or more contiguous nucleotide oligonucleotides sufficient to specifically hybridize to hSARS mRNA or genomic RNA under stringent conditions.

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 or the hSARS virus with the genome nucleotide sequence of SEQ ID NO: 15), or based on the nucleotide sequence of SEQ ID NO: 1, 11, 13 or 15. In a non-limiting specific embodiment, preferred primers for the RT-PCR method are: 5'-TACACACCTCAGCGTTG-3' (SEQ ID NO: 3) and 5'-CACGAACGTG-ACGAAT-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, reverse transcribe the extracted total RNA to obtain cDNA, use specific primers such as Primers of ID NO: 3 and 4 nucleotide sequences and a fluorescent dye such as ^SYBR_Green I (which fluoresces when non-specifically bound to double-stranded DNA) were used to subject the cDNA to a PCR reaction. 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 profile (see section 6.7 below).

Preferably, the reagent for detecting hSARS is an antibody specifically binding to the polypeptide of the present invention or any epitope of hSARS, 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. Techniques for detecting hSARS epitopes in vitro 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 detecting hSARS in vivo 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 (such as the polypeptide of the present invention or mRNA or genomic RNA encoding the polypeptide of the present invention), thereby detecting The presence of hSARS or polypeptide or mRNA or genomic RNA encoding polypeptide in the sample, and the absence of hSARS or polypeptide or mRNA or genomic RNA encoding polypeptide in the control sample is compared with hSARS or polypeptide or mRNA or genomic RNA encoding polypeptide in the test sample compared to the presence of RNA.

The present invention also includes a kit for detecting the presence of hSARS or the polypeptide or nucleic acid of the present invention in a sample to be tested. The kit may, for example, comprise a labeled compound or reagent capable of detecting hSARS or a polypeptide or a nucleic acid molecule encoding a polypeptide in the sample to be tested, and in certain embodiments, means for determining the amount of the polypeptide or mRNA in the sample (e.g., binding Antibodies to polypeptides or oligonucleotide probes that bind to DNA or mRNA encoding polypeptides). 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 a polypeptide of the invention or an hSARS epitope; optionally (2) a different second antibody that The polypeptide or primary antibody is bound and conjugated to a detectable reagent.

For oligonucleotide-based kits, it can include, for example: (1) oligonucleotides, such as detectably labeled oligonucleotides, which can be associated with a nucleic acid sequence encoding a polypeptide of the present invention or a sequence within the hSARS genome Hybridization, or (2) a pair of primers, which 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.

5.8 Screening tests to identify antiviral agents

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, at least 5 minutes, at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours, at least 12 hours, or at least 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 to test 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

The Manual of Clinica 1 Virology by Wiedbrauk DL and Johnston SLG, Raven Press, New York, 1993 was used 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 is: (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. Paired acute and convalescent serum and stools were obtained 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 to become 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 the human coronaviruses identified to date are not known to cause similar diseases, the inventors believe that the virus isolates represent a novel virus that infects humans.

6.5 Antibody response to isolated virus

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 hSARS-infected FRhK-4 cells at 37°C for 45 min. 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 ^* 2b3a ^* 3b4a ^* 4b Acute recovery period Acute recovery period Acute recovery period Acute recovery period <50160050>160050>1600<50<50 5a ^* 5b6a ^* 6b Acute recovery period Acute recovery period <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. Using ^{Superscript_II} reverse transcriptase (Invitrogen) according to the manufacturer's recommendation in 20 μl of reaction mixture containing 10 pg of degenerate primer (5'-GCCGGAGCTCTGCAGAATTCNNNNNN-3': SEQ ID NO: 5; N=A, T, G or C) 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 AmpliTaq ^Gold® DNA polymerase (Applied Biosystems), 2.5 μCi [ α- ³² P]CTP (Amersham), 2 µl of 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 10x PCR buffer, 4 μl 25 mM MgCl ₂ , 0.5 μl 10 mM dNTPs, 0.25 μl AmpliTaq ^Gold® DNA polymerase (Applied Biosystems), 1 μl 10 μM primer (5'-GCCGGA GCTCTGCAGAATT-C-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 insert was 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, two other 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, see Figure 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 nucleotide sequence; see SEQ ID NO: 17-239, 241-736 for the amino acid sequence 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 nucleotide sequence; see SEQ ID NO: 1109-1589, 1591 for the amino acid sequence -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+Btest for rapid diagnosis of influenza A and B virus infections.J ClinMicrobiol.2002;40:1675- 1680), Culture of Nasopharyngeal Aspirates for Conventional Respiratory Pathogens in Mardin Darby Canine Kidney Cells, LLC-Mk2, RDE, Hep-2 and MRC-5 Cells (Wiedbrauk DL, Johnston SLG. Manual of clinical virology. RavenPress, 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 influenza A 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).

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 treated with primers 5′-TACACACCTCAGCGTTG-3′ (SEQ ID NO: 3) and 5′-CACGAACGTGACGAAT-3′ (SEQ ID NO: 4) at 2.5 mM Amplify in the presence _of MgCl (94°C for 8 minutes, then cycle 40 times at 94°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute). The two primers are based on the RNA-dependent RNA polymerase encoding of hSARS virus sequence (SEQ ID NO: 1) was constructed.

A typical RT-PCR protocol is outlined below:

1. RNA extraction

RNA was extracted from 140 μl NPA samples by QIAquick Viral RNA Extraction Kit and eluted in 50 μl elution buffer.

2. 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

94 ℃, 3 minutes

4℃

3. 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.

4. 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 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 eluted RNA samples were 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. The fluorescent signal of these reactions was captured at the end of the extension step of each cycle (see Figure 7A). 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; see Figure 7B) 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 median 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). The liver parenchymal enzyme alanine transaminase (ALT) and the 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 12.9(8.9-15.9)5.17(1.1-11.4) 9 (18%) 13 (26%) 11.5-16.5g/dl4-11×10 ⁹ /L Lymphocyte count Marked lymphopenia (<1.0×10 ⁹ /L) Platelet count Thrombocytopenia Alanine aminotransferase (ALT) Elevated albumin Low albumin globulin Elevated creatinine kinase Creatinine kinase 0.78(0.3-1.5)174(88-351)63(11-350)37(26-50)33(21-42)244(31-1379) 34 (68%) 20 (40%) 17 (34%) 34 (68%) 10 (20%) 13 (26%) 1.5-4.0×10 ⁹ /L150-400×10 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 concurrent cases were treated with ribavirin and steroids after admission to the intensive care unit, whereas all non-complicated cases were started on 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 ) mean (SD) initial total peripheral WBC count (×10 ⁹ /L) mean (SD) initial lymphocyte count (×10 ⁹ /L) 49.5±12.78/ ^115_151845.2 ±2.038.8±0.95.1±2.40.66±0.3 39.0±10.714/ ^{171_3945104.7} ±2.538.7±0.85.2±1.80.85±0.3 P<0.01N.SP<0.05NSNSNSP<0.05NSNSNSNSP<0.05 Presence of Thrombocytopenia (<150 x 10 ⁹ /L) Impaired Liver Function Test CXR Changes (Number of Affected Areas) Mean (SD) Days to Disease Deterioration from Symptom Onset Mean (SD) days of lin and steroids Ribavirin and steroids started after exacerbation Responses to ribavirin and steroids Results improved or healed No improvement‖ 8111.48.3±2.67.7±2.91211108 1261.2 Not applicable 5.7±2.6028310 NSP<0.01NSP<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 showed a lymphopenia of 0.7×10 ⁹ /L, and the total white blood cell and platelet counts were normal. Both ALT (41U/L) and CPK (405U/L) were damaged. He developed bilateral lung infiltrates and progressive oxygen desaturation despite oral azithromycin, amantadine, and intravenous ceftriaxone. 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. Examination also showed leukopenia (2.7×10 ⁹ /L), lymphopenia (0.6×10 ⁹ /L) and thrombocytopenia (104×10 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 from FRhK-4 cells from lung biopsies and nasopharyngeal aspirates from the above two patients, 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 HMPV, 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 ), whose 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 matching 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 results 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 6.48 x ¹⁰⁵ 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 the initial false-positive cases, the real-time quantitative PCR test had a sensitivity of 79% and a specificity of 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, 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.

discuss

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 (SEQ ID NO: 1) of the polymerase gene of the isolated hSARS virus and the whole genome, showed that the virus is related to the 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 the virus (Holmes KV.Coronaviruses.Eds Knipe DM, Howley PM Fields Virology, 4th edition, Lippincott Williams & Wilkins, Philadelphia, 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. 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. EdsKnipe DM, Howley PM Fields Virology, 4th ed. 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 in hospitalizied patients with “common cold” virus infections. Clin Infect Dis. 2000; 31: 96-100; and Foltz EJ, Elkordy MA. Coronavirus pneumonia 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 study 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. Human coronavirus OC43 and 229E persistence in neural cell cultures and human brains. AdvExp 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 the feces of 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 Arch Virol. 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, PoonLLM, 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 symptoms 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 above-mentioned combination therapy 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.

7. Preservation

The sample of isolated hSARS virus was deposited in the Chinese Center for Type Cultures (CCTCC) located at Wuhan University (Wuhan, China 430072) on April 2, 2003 according to the Budapest Treaty on the Deposit of Microorganisms, and the retrieval number given is CCTCC-V200303, which is adopted by reference 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 to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

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 (167)

1. isolating hSARS virus, described virus is CCTCC-V200303 at the preservation searching number at China typical culture center.

2. isolating hSARS virus, described virus comprises nucleic acid molecule, described nucleic acid molecule comprise SEQ ID NO:1 nucleotide sequence or under stringent condition with the nucleotide sequence of SEQ ID NO:1 hybridization.

3. isolating hSARS virus, described virus comprises nucleic acid molecule, described nucleic acid molecule comprise SEQ ID NO:11 nucleotide sequence or under stringent condition with the nucleotide sequence of SEQ ID NO:11 hybridization.

4. isolating hSARS virus, described virus comprises nucleic acid molecule, described nucleic acid molecule comprise SEQ ID NO:13 nucleotide sequence or under stringent condition with the nucleotide sequence of SEQ ID NO:13 hybridization.

5. each hSARS virus of claim 1-4, described virus is deactivation.

6. each hSARS virus of claim 1-4, described virus is attenuation.

7. the attenuation hSARS virus of claim 6, the infectivity of described virus reduces.

8. the attenuation hSARS virus of claim 7, the infectivity of described virus reduces at least 5 times, 10 times, 25 times, 50 times, 100 times, 250 times, 500 times or 10,000 times.

9. the attenuation hSARS virus of claim 6, the replication of described virus reduces.

10. the attenuation hSARS virus of claim 9, the replication of described virus reduces at least 5 times, 10 times, 25 times, 50 times, 100 times, 250 times, 500 times, 1,000 times or 10,000 times.

11. the attenuation hSARS virus of claim 6, the protein synthesis capacity of described virus reduces.

12. the attenuation hSARS virus of claim 11, the protein synthesis capacity of described virus reduces at least 5 times, 10 times, 25 times, 50 times, 100 times, 250 times, 500 times, 1,000 times or 10,000 times.

13. the attenuation hSARS virus of claim 6, the assemble ability of described virus reduces.

14. the attenuation hSARS virus of claim 13, the assemble ability of described virus reduces at least 5 times, 10 times, 25 times, 50 times, 100 times, 250 times, 500 times, 1,000 times or 10,000 times.

15. the attenuation hSARS virus of claim 6, the cytopathic effect of described virus reduces.

16. the attenuation hSARS virus of claim 15, the cytopathic effect of described virus reduces at least 5 times, 10 times, 25 times, 50 times, 100 times, 250 times, 500 times, 1,000 times or 10,000 times.

17. an isolated nucleic acid molecule, described nucleic acid molecule comprise nucleotide sequence or its complementary sequence of each hSARS virus of coding claim 1-4.

18. an isolated nucleic acid molecule, described nucleic acid molecule under stringent condition with nucleic acid molecule or its complementary sequence hybridization of claim 17.

19. an isolated nucleic acid molecule, described nucleic acid molecule comprise SEQ ID NO:1 nucleotide sequence or its complementary sequence.

20. an isolated nucleic acid molecule, described nucleic acid molecule comprise nucleotide sequence or its complementary sequence of at least 100,150,200,250,300,350,400,450,500,550 or 600 continuous nucleotides with SEQ ID NO:1 nucleotide sequence.

21. an isolated nucleic acid molecule, described nucleic acid molecule comprise the nucleotide sequence of coding SEQ ID NO:2 aminoacid sequence or the complementary sequence of described nucleotide sequence.

22. an isolated nucleic acid molecule, described nucleic acid molecule comprise SEQ ID NO:11 nucleotide sequence or its complementary sequence.

23. isolated nucleic acid molecule, described nucleic acid molecule comprises and has at least 45,50,60,70,80,90,100,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1 of SEQ ID NO:11 nucleotide sequence, 000,1,050,1,100,1, the nucleotide sequence of 150 or 1,200 continuous nucleotides or its complementary sequence.

24. an isolated nucleic acid molecule, described nucleic acid molecule comprise SEQ ID NO:13 nucleotide sequence or its complementary sequence.

25. an isolated nucleic acid molecule, described nucleic acid molecule comprise nucleotide sequence or its complementary sequence of at least 5,500,550,600,650 or 700 continuous nucleotides with SEQ ID NO:13 nucleotide sequence.

26. isolated nucleic acid molecule, described nucleic acid molecule under stringent condition with have the making nucleic acid molecular hybridization of SEQ ID NO:1,11 or 13 nucleotide sequences or its complementary sequence, the aminoacid sequence of wherein said nucleic acid molecule encoding biologically active, described biological activity is showed by SEQ ID NO:1,11 or 13 nucleotide sequence coded polypeptide.

27. the nucleic acid molecule of claim 17, wherein said molecule is RNA.

28. the nucleic acid molecule of claim 18, wherein said molecule is RNA.

29. each nucleic acid molecule of claim 19-26, wherein said molecule is RNA.

30. the nucleic acid molecule of claim 17, wherein said molecule is DNA.

31. the nucleic acid molecule of claim 18, wherein said molecule is DNA.

32. each nucleic acid molecule of claim 19-26, wherein said molecule is DNA.

33. an isolated polypeptide, described polypeptide is by the nucleic acid molecule encoding of claim 17.

34. an isolated polypeptide, described polypeptide is by the nucleic acid molecule encoding of claim 18.

35. an isolated polypeptide, described polypeptide is by each nucleic acid molecule encoding of claim 19-26.

36. an isolated polypeptide, described polypeptide comprise SEQ ID NO:2 aminoacid sequence.

37. an isolated polypeptide, described polypeptide comprise the aminoacid sequence of at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150 or 200 continuous amino acid residues with SEQ ID NO:2 aminoacid sequence.

38. an isolated polypeptide, described polypeptide comprise SEQ ID NO:12 aminoacid sequence.

39. an isolated polypeptide, described polypeptide comprise the aminoacid sequence of at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150,200,250,300,350 or 400 continuous amino acid residues with SEQ ID NO:12 aminoacid sequence.

40. an isolated polypeptide, described polypeptide comprise SEQ ID NO:14 aminoacid sequence.

41. an isolated polypeptide, described polypeptide comprise the aminoacid sequence of at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150 or 200 continuous amino acid residues with SEQ ID NO:14 aminoacid sequence.

42. an isolated antibody or its Fab, described antibody or its Fab immunologic opsonin are in conjunction with the hSARS virus of preservation searching number CCTCC-V200303.

43. the isolated antibody of claim 42 or its Fab are in described antibody or its Fab and hSARS virus.

44. an isolated antibody or its Fab, described antibody or its Fab immunologic opsonin are in conjunction with each hSARS virus of claim 2-4.

45. the isolated antibody of claim 44 or its Fab are in described antibody or its Fab and hSARS virus.

46. an isolated antibody or its Fab, described antibody or its Fab immunologic opsonin are in conjunction with the polypeptide of claim 33.

47. the isolated antibody of claim 46 or its Fab are in described antibody or its Fab and hSARS virus.

48. an isolated antibody or its Fab, described antibody or its Fab immunologic opsonin are in conjunction with the polypeptide of claim 34.

49. the isolated antibody of claim 48 or its Fab are in described antibody or its Fab and hSARS virus.

50. an isolated antibody or its Fab, described antibody or its Fab immunologic opsonin are in conjunction with the polypeptide of claim 35.

51. the isolated antibody of claim 50 or its Fab are in described antibody or its Fab and hSARS virus.

52. an isolated antibody or its Fab, described antibody or its Fab immunologic opsonin are in conjunction with each polypeptide of claim 36-41.

53. the isolated antibody of claim 52 or its Fab are in described antibody or its Fab and hSARS virus.

54. the method that test right requires each hSARS virus of 1-4 to exist in biological sample, described method comprises:

(a) compound of the described hSARS virus of sample and selective binding is contacted;

(b) whether detect described compound in conjunction with the described hSARS virus in the sample.

55. the method for claim 54, wherein biomaterial is selected from cell, blood, serum, blood plasma, saliva, urine, ight soil, phlegm and nasopharynx aspirate.

56. the method for claim 54, wherein the compound in conjunction with described virus is an antibody.

57. the method for claim 54, wherein the compound in conjunction with described virus is the nucleic acid molecule that comprises SEQID NO:1 nucleotide sequence or its complementary sequence.

58. the method for claim 54, wherein the compound in conjunction with described virus is the nucleic acid molecule that comprises nucleotide sequence or its complementary sequence, and described nucleotide sequence has at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150,200,250,300,350,400,450,500,550 or 600 continuous nucleotides of SEQ ID NO:1 nucleotide sequence.

59. the method for claim 54, wherein the compound in conjunction with described virus is the nucleic acid molecule that comprises SEQID NO:11 nucleotide sequence or its complementary sequence.

60. the method for claim 54, wherein the compound in conjunction with described virus is the nucleic acid molecule that comprises nucleotide sequence or its complementary sequence, described nucleotide sequence has at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1 of SEQ ID NO:11 nucleotide sequence, 000,1,050,1,100,1,150 or 1,200 continuous nucleotides.

61. the method for claim 54, wherein the compound in conjunction with described virus is the nucleic acid molecule that comprises SEQID NO:13 nucleotide sequence or its complementary sequence.

62. the method for claim 54, wherein the compound in conjunction with described virus is the nucleic acid molecule that comprises nucleotide sequence or its complementary sequence, and described nucleotide sequence has at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150,200,250,300,350,400,450,500,550,600,650 or 700 continuous nucleotides of SEQ ID NO:13 nucleotide sequence.

63. one kind in biological sample test right require the method for the existence of 33 polypeptide, described method comprises:

(a) biological sample is contacted with the compound of the described polypeptide of selective binding;

(b) whether detect described compound in conjunction with the described polypeptide in the sample.

64. the method for claim 63, wherein biomaterial is selected from cell, blood, serum, blood plasma, saliva, urine, ight soil, phlegm and nasopharynx aspirate.

65. the method for claim 63, wherein the compound in conjunction with described polypeptide is antibody or its Fab.

66. the method that the polypeptide of a test right requirement 34 in biological sample exists, described method comprises:

(a) biological sample is contacted with the compound of the described polypeptide of selective binding;

(b) whether detect described compound in conjunction with the described polypeptide in the sample.

67. the method for claim 66, wherein biomaterial is selected from cell, blood, serum, blood plasma, saliva, urine, ight soil, phlegm and nasopharynx aspirate.

68. the method for claim 66, wherein the compound in conjunction with described polypeptide is antibody or its Fab.

69. the method that the polypeptide of a test right requirement 35 in biological sample exists, described method comprises:

(a) biological sample is contacted with the compound of the described polypeptide of selective binding;

(b) whether detect described compound in conjunction with the described polypeptide in the sample.

70. the method for claim 69, wherein biomaterial is selected from cell, blood, serum, blood plasma, saliva, urine, ight soil, phlegm and nasopharynx aspirate.

71. the method for claim 69, wherein the compound in conjunction with described polypeptide is antibody or its Fab.

72. the method that test right requires each polypeptide of 36-41 to exist in biological sample, described method comprises:

(a) biological sample is contacted with the compound of the described polypeptide of selective binding;

(b) whether detect described compound in conjunction with the described polypeptide in the sample.

73. the method for claim 72, wherein biomaterial is selected from cell, blood, serum, blood plasma, saliva, urine, ight soil, phlegm and nasopharynx aspirate.

74. the method for claim 72, wherein the compound in conjunction with described polypeptide is antibody or its Fab.

75. a method that detects first nucleic acid molecule existence of the hSARS virus that derives from claim 1 in biological sample, described method comprises:

(a) compound of described first nucleic acid molecule of biological sample and selective binding is contacted;

(b) whether detect described compound in conjunction with described first nucleic acid molecule in the sample.

76. the method for claim 75, wherein the compound in conjunction with described first nucleic acid molecule is second nucleic acid molecule that comprises SEQ ID NO:1 nucleotide sequence or its complementary sequence.

77. the method for claim 75, wherein second nucleic acid molecule comprises at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150,200,250,300,350,400,450,500,550 or 600 continuous nucleotides of SEQ ID NO:1 nucleotide sequence or its complementary sequence.

78. the method for claim 75, wherein the compound in conjunction with described first nucleic acid molecule is second nucleic acid molecule that comprises SEQ ID NO:11 nucleotide sequence or its complementary sequence.

79. the method for claim 75, wherein second nucleic acid molecule comprises at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1 of SEQ ID NO:11 nucleotide sequence or its complementary sequence, 000,1,050,1,100,1,150 or 1,200 continuous nucleotides.

80. the method for claim 75, wherein the compound in conjunction with described first nucleic acid molecule is second nucleic acid molecule that comprises SEQ ID NO:13 nucleotide sequence or its complementary sequence.

81. the method for claim 75, wherein second nucleic acid molecule comprises at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150,200,250,300,350,400,450,500,550,600,650 or 700 continuous nucleotides of SEQ ID NO:13 nucleotide sequence or its complementary sequence.

82. a method that detects first nucleic acid molecule existence of the hSARS virus that derives from claim 2-4 in biological sample, described method comprises:

(a) compound of described first nucleic acid molecule of biological sample and selective binding is contacted;

(b) whether detect described compound in conjunction with described first nucleic acid molecule in the sample.

83. the method for claim 82, wherein the compound in conjunction with described first nucleic acid molecule is second nucleic acid molecule that comprises SEQ ID NO:1 nucleotide sequence or its complementary sequence.

84. the method for claim 82, wherein second nucleic acid molecule comprises at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150,200,250,300,350,400,450,500,550 or 600 continuous nucleotides of SEQ ID NO:1 nucleotide sequence or its complementary sequence.

85. the method for claim 82, wherein the compound in conjunction with described first nucleic acid molecule is second nucleic acid molecule that comprises SEQ ID NO:11 nucleotide sequence or its complementary sequence.

86. the method for claim 82, wherein second nucleic acid molecule comprises at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1 of SEQ ID NO:11 nucleotide sequence or its complementary sequence, 000,1,050,1,100,1,150 or 1,200 continuous nucleotides.

87. the method for claim 82, wherein the compound in conjunction with described first nucleic acid molecule is second nucleic acid molecule that comprises SEQ ID NO:13 nucleotide sequence or its complementary sequence.

88. the method for claim 82, wherein second nucleic acid molecule comprises at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150,200,250,300,350,400,450,500,550,600,650 or 700 continuous nucleotides of SEQ ID NO:13 nucleotide sequence or its complementary sequence.

89. a host cell, described cell is by the hSARS virus infection of preservation searching number CCTCC-V200303.

90. the host cell of claim 89, described cell are the primate cells.

91. the host cell of claim 90, described cell are FRhK-4 tire RhMK cells.

92. a host cell, described cell is by each hSARS virus infection of claim 2-4.

93. the host cell of claim 92, described cell are the primate cells.

94. the host cell of claim 93, described cell are FRhK-4 tire RhMK cells.

95. the method that immunologic opsonin exists in conjunction with the antibody of hSARS virus in the detection of biological sample, described method comprises:

(a) biological sample is contacted with the host cell of claim 89;

(b) detect and cell bonded antibody.

96. the method that immunologic opsonin exists in conjunction with the antibody of hSARS virus in the detection of biological sample, described method comprises:

(a) biological sample is contacted with the host cell of claim 92;

(b) detect and cell bonded antibody.

97. an immunogenic formulation, described preparation comprise the hSARS virus and the drug acceptable carrier of the claim 5 of immunogenicity significant quantity.

98. an immunogenic formulation, described preparation comprise the hSARS virus and the drug acceptable carrier of the claim 6 of immunogenicity significant quantity.

99. an immunogenic formulation, described preparation comprise protein extract or its subunit and the drug acceptable carrier of hSARS virus of the claim 5 of immunogenicity significant quantity.

100. an immunogenic formulation, described preparation comprise protein extract or its subunit and the drug acceptable carrier of hSARS virus of the claim 6 of immunogenicity significant quantity.

101. an immunogenic formulation, described preparation comprise the nucleic acid molecule and the drug acceptable carrier that comprise SEQ ID NO:1 nucleotide sequence or its complementary sequence of immunogenicity significant quantity.

102. an immunogenic formulation, described preparation comprise the nucleic acid molecule and the drug acceptable carrier that comprise SEQ ID NO:11 nucleotide sequence or its complementary sequence of immunogenicity significant quantity.

103. an immunogenic formulation, described preparation comprise the nucleic acid molecule and the drug acceptable carrier that comprise SEQ ID NO:13 nucleotide sequence or its complementary sequence of immunogenicity significant quantity.

104. an immunogenic formulation, described preparation comprises the polypeptide of the claim 33 of immunogenicity significant quantity.

105. an immunogenic formulation, described preparation comprises the polypeptide of the claim 34 of immunogenicity significant quantity.

106. an immunogenic formulation, described preparation comprises the polypeptide of the claim 35 of immunogenicity significant quantity.

107. an immunogenic formulation, described preparation comprise each the polypeptide of claim 36-41 of immunogenicity significant quantity.

108. a vaccine preparation, described preparation comprise the hSARS virus and the drug acceptable carrier of the claim 5 of treatment or prevention significant quantity.

109. a vaccine preparation, described preparation comprise the hSARS virus and the drug acceptable carrier of the claim 6 of treatment or prevention significant quantity.

110. a vaccine preparation, described preparation comprise protein extract or its subunit and the drug acceptable carrier of hSARS virus of the claim 5 of treatment or prevention significant quantity.

111. a vaccine preparation, described preparation comprise protein extract or its subunit and the drug acceptable carrier of hSARS virus of the claim 6 of treatment or prevention significant quantity.

112. a vaccine preparation, described preparation comprise the nucleic acid molecule that comprises SEQID NO:1 nucleotide sequence or its complementary sequence and the drug acceptable carrier of treatment or prevention significant quantity.

113. a vaccine preparation, described preparation comprise the nucleic acid molecule that comprises SEQID NO:11 nucleotide sequence or its complementary sequence and the drug acceptable carrier of treatment or prevention significant quantity.

114. a vaccine preparation, described preparation comprise the nucleic acid molecule that comprises SEQID NO:13 nucleotide sequence or its complementary sequence and the drug acceptable carrier of treatment or prevention significant quantity.

115. a pharmaceutical composition, described composition comprise the anti-hSARS medicine and the drug acceptable carrier of treatment or prevention significant quantity.

116. the pharmaceutical composition of claim 115, wherein anti-hSARS medicine is antibody or its Fab, and described antibody or its Fab immunologic opsonin are in conjunction with hSARS virus or its derive polypeptide or protein of preservation searching number CCTCC-V200303.

117. the pharmaceutical composition of claim 115, wherein anti-hSARS medicine are to comprise SEQID NO:1 nucleotide sequence or its segmental nucleic acid molecule.

118. the pharmaceutical composition of claim 115, wherein anti-hSARS medicine are to comprise SEQID NO:11 or 13 nucleotide sequences or its segmental nucleic acid molecule.

119. the pharmaceutical composition of claim 115, wherein anti-hSARS medicine is the polypeptide that comprises the nucleic acid molecule encoding of SEQID NO:1 nucleotide sequence, or has the bioactive fragment of described polypeptide.

120. the pharmaceutical composition of claim 115, wherein anti-hSARS medicine is the polypeptide that comprises the nucleic acid molecule encoding of SEQID NO:11 or 13 nucleotide sequences, or has the bioactive fragment of described polypeptide.

121. a medicine box, described medicine box comprises the container of the immunogenic formulation that contains claim 97.

122. a medicine box, described medicine box comprises the container of the immunogenic formulation that contains claim 98.

123. a medicine box, described medicine box comprises the container of the immunogenic formulation that contains claim 99.

124. a medicine box, described medicine box comprises the container of the immunogenic formulation that contains claim 100.

125. comprising, a medicine box, described medicine box contain each the container of immunogenic formulation of claim 101-103.

126. a medicine box, described medicine box comprises the container of the immunogenic formulation that contains claim 104.

127. a medicine box, described medicine box comprises the container of the immunogenic formulation that contains claim 105.

128. a medicine box, described medicine box comprises the container of the immunogenic formulation that contains claim 106.

129. a medicine box, described medicine box comprises the container of the immunogenic formulation that contains claim 107.

130. a medicine box, described medicine box comprises the container of the vaccine preparation that contains claim 108.

131. a medicine box, described medicine box comprises the container of the vaccine preparation that contains claim 109.

132. a medicine box, described medicine box comprises the container of the vaccine preparation that contains claim 110.

133. a medicine box, described medicine box comprises the container of the vaccine preparation that contains claim 111.

134. comprising, a medicine box, described medicine box contain each the container of vaccine preparation of claim 112-114.

135. a medicine box, described medicine box comprises the container of the pharmaceutical composition that contains claim 115.

136. a method of identifying object, described object infects the hSARS virus of claim 1, and described method comprises:

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

(b) total RNA carries out reverse transcription acquisition cDNA;

(c) with one group of primer amplification cDNA that derives from the hSARS viral nucleotide sequences.

137. the method for claim 136, wherein said primer derive from the genome nucleotide sequence of the hSARS virus of preservation searching number CCTCC-V200303.

138. the method for claim 136, wherein said primer derive from SEQ ID NO:1,11 or 13 nucleotide sequences or its complementary sequence.

139. the method for claim 136, wherein said primer have SEQ ID NO:3 and 4 nucleotide sequences respectively.

140. a method of identifying object, described object infect each hSARS virus of claim 2-4, described method comprises:

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

(b) total RNA carries out reverse transcription acquisition cDNA;

(c) with one group of primer amplification cDNA that derives from the hSARS viral nucleotide sequences.

141. the method for claim 140, wherein said primer derive from the genome nucleotide sequence of the hSARS virus of preservation searching number CCTCC-V200303.

142. the method for claim 140, wherein said primer derive from SEQ ID NO:1,11 or 13 nucleotide sequences or its complementary sequence.

143. the method for claim 140, wherein said primer have SEQ ID NO:3 and 4 nucleotide sequences respectively.

144. the nucleotide sequence that an isolating hSARS virus, described virus have SEQ ID NO:15 nucleotide sequence or hybridize with SEQ ID NO:15 under stringent condition.

145. an isolated nucleic acid molecule, described nucleic acid molecule comprise SEQ ID NO:15 nucleotide sequence or its complementary sequence.

146. comprising, an isolated nucleic acid molecule, described nucleic acid molecule have at least 5 of SEQ ID NO:15 nucleotide sequence, 10,15,20,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1,000,1,050,1,100,1,150,1,200,2,000,3,000,4,000,5,000,6,000,7,000,8,000,9,000,10,000,11,000,12,000,13,000,14,000,15,000,16,000,17,000,18,000,19,000,20,000,21,000,22,000,23,000,24,000,25,000,26,000,27,000,28,000, the nucleotide sequence of 29,000 continuous nucleotides or its complementary sequence.

147. an isolated nucleic acid molecule, described nucleic acid molecule are included under the stringent condition nucleotide sequence with SEQ ID NO:15 nucleotide sequence or its complementary sequence hybridization.

148. an isolated polypeptide, described polypeptide is by the nucleic acid molecule of claim 145 or the fragment coding of described nucleic acid molecule.

149. an isolated antibody or its Fab, described antibody or its Fab immunologic opsonin are in conjunction with the polypeptide of claim 148.

150. the isolated antibody of claim 149 or its Fab are in described antibody or its Fab and hSARS virus.

151. the method that the hSARS virus of a test right requirement 144 in biological sample exists, described method comprises:

(a) compound of the described hSARS virus of sample and selective binding is contacted;

(b) whether detect described compound in conjunction with the described hSARS virus in the sample.

152. the method for claim 151, wherein said biological sample are selected from cell, blood, serum, blood plasma, saliva, urine, ight soil, phlegm and nasopharynx aspirate.

153. the method for claim 151, wherein the compound in conjunction with described virus is an antibody.

154. the method for claim 151, wherein the compound in conjunction with described virus is the nucleic acid molecule that comprises SEQ ID NO:1,11 or 13 nucleotide sequences or its complementary sequence.

155. the method that the polypeptide of a test right requirement 148 in biological sample exists, described method comprises:

(a) biological sample is contacted with the compound of the described polypeptide of selective binding;

(b) whether detect described compound in conjunction with the described polypeptide in the sample.

156. the method for claim 155, wherein said biological sample are selected from cell, blood, serum, blood plasma, saliva, urine, ight soil, phlegm and nasopharynx aspirate.

157. the method for claim 155, wherein the compound in conjunction with described polypeptide is antibody or its Fab.

158. one kind is detected in biological sample and comprises the nucleic acid molecule of SEQ ID NO:15 nucleotide sequence or the method for its segmental existence, described method comprises:

(a) biological sample is contacted with the compound of the described nucleic acid molecule of selective binding;

(b) whether detect described compound in conjunction with the described nucleic acid molecule in the sample.

159. the method for claim 158, wherein said biological sample are selected from cell, blood, serum, blood plasma, saliva, urine, ight soil, phlegm and nasopharynx aspirate.

160. a host cell, the hSARS virus of described cell infection claim 144.

161. a vaccine preparation, described preparation comprise the hSARS virus and the medicine acceptable carrier of the claim 144 of treatment or prevention significant quantity, wherein said hSARS virus is deactivation.

162. a vaccine preparation, described preparation comprise the hSARS virus and the medicine acceptable carrier of the claim 144 of treatment or prevention significant quantity, wherein said hSARS virus is attenuation.

163. a vaccine preparation, described preparation comprise the protein extract and the medicine acceptable carrier of hSARS virus of the claim 144 of treatment or prevention significant quantity.

164. a vaccine preparation, described preparation comprise the polypeptide and the medicine acceptable carrier of the claim 148 of treatment or prevention significant quantity.

165. a vaccine preparation, described preparation comprise the nucleic acid molecule that comprises SEQID NO:15 nucleotide sequence or its complementary sequence or the fragment of treatment or prevention significant quantity, and the medicine acceptable carrier.

166. a method of identifying object, described object infects the hSARS virus of claim 144, and described method comprises:

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

(b) total RNA carries out reverse transcription acquisition cDNA;

(c) with one group of primer amplification cDNA that derives from the hSARS viral nucleotide sequences.

167. the method for claim 136 or 166, wherein said primer derive from SEQ ID NO:15 nucleotide sequence or its complementary sequence.

Images