CN1768140B - High throughput diagnostic assay for human virus causing Severe Acute Respiratory Syndrome (SARS) - Google Patents

Abstract

The present invention relates to high throughput diagnostic assays for the virus that causes Severe Acute Respiratory Syndrome (SARS) ("hSARS virus") in humans. In particular, the invention relates to high throughput reverse transcription-PCR diagnostic detection of SARS-associated coronavirus (SARS-CoV). The test method of the present invention is a rapid and reliable test method, which can be used for diagnosing and monitoring SARS propagation, and is based on the nucleotide sequence of the N (nucleocapsid) -gene of hSARS virus. The method of the invention eliminates false negative structures and provides higher sensitivity for the test. The present invention also discloses the S (spike) -gene of hSARS virus. The invention also relates to the deduced amino acid sequences of the N-gene and S-gene products of hSARS virus and the use of the N-gene and S-gene products in diagnostic methods. The invention also includes diagnostic test methods and kits comprising antibodies against the N-gene or S-gene products.

Description

High-throughput diagnostic assay for the human virus causing severe acute respiratory syndrome (SARS)

This application claims priority to U.S. Provisional Application 60/457,031, filed March 24, 2003, U.S. Provisional Application 60/457,730, filed March 26, 2003, U.S. Provisional Application, filed Apr. 2, 2003 60/459,931, U.S. Provisional Application 60/460,357 filed April 3, 2003, U.S. Provisional Application 60/461,265, filed April 8, 2003, U.S. Provisional Application 60/462,805, filed April 14, 2003, 2003 U.S. Provisional Application 60/464,886, filed April 23, 2003, U.S. Provisional Application 60/465,738, filed April 25, 2003, and U.S. Provisional Application 60/470,935, filed May 14, 2003, each of which is incorporated by reference in its entirety to this article.

This application contains a long sequence listing, which is hereby incorporated by reference in its entirety, on CD-R in triplicate instead of printed paper. Said CD-Rs were burnt on March 22, 2004, respectively labeled "CRF", "Copy 1" and "Copy 2", each containing only one identical 1.6MB file (V9661077.APP).

1 Introduction

The present invention relates to a high-throughput diagnostic assay for the virus that causes severe acute respiratory syndrome (SARS) in humans ("hSARS virus"). In particular, the invention relates to high-throughput reverse transcription-PCR diagnostic assays for SARS-associated coronavirus (SARS-CoV). The test method of the present invention is a fast and reliable detection method, and can be used for diagnosing and monitoring the spread of SARS. The method of the invention eliminates false negative results and provides high sensitivity for the assay. The present invention further relates to nucleotide sequences and parts thereof for diagnosing SARS. The present invention also relates to nucleotide sequences and parts thereof for evaluating SARS genetic polymorphisms. The nucleotide sequence of the present invention includes the (nucleocapsid) N-gene and S-gene sequences of hSARS virus. The invention relates to a diagnostic kit for detecting hSARS virus N-gene or S-gene comprising nucleic acid molecules. The invention also relates to the deduced amino acid sequences of the hSARS virus N-gene and S-gene products. The invention also relates to the use of N-gene and S-gene products in diagnostic methods. The invention also includes diagnostic assay methods and kits comprising antibodies raised against N-gene or S-gene products.

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 Epidemiol Rec. 2003; 78:86). Patients suffering from SARS exhibited a variety of clinical symptoms, including fever (38 degrees Celsius or above for more than 24 hours), malaise, chills, headache, and body aches. Chest X-rays show changes compatible with pneumonia. Other symptoms include coughing, shortness of breath, or difficulty breathing. As of May 3, 2003, a total of 1621 cases and 179 deaths had occurred in Hong Kong, accounting for 26% and 41% of the reported cases (6234) and deaths (435) in the world respectively. Due to the highly contagious nature of the disease and its spread during daily activities, there is a great need to develop a rapid and reliable diagnostic test to monitor and control the disease. 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 groups of health care workers were identified as having the disease. In addition, multiple clusters of pneumonia cases have emerged among persons 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 (referred to as the present inventor) was one of the groups involved in investigating these patients. In these patients, all identification tests for common viruses and bacteria were negative. In addition, diagnostic assays that detect other genes in the hSARS virus, such as the 1b-gene, cannot be used to accurately diagnose SARS. The disease is named after the acronym for Severe Acute Respiratory Syndrome ("SARS"). The virus mutates and changes rapidly, so it was very difficult to diagnose SARS until the unique regions of the virus, the N-gene and S-gene of the hSARS virus, were isolated from SARS patients by the present inventors as disclosed herein. That is, the present invention discloses a diagnostic test method for fast, accurate, reliable and specific identification of hSARS virus using a special region in the virus genome. The present invention can be used for clinical and scientific research purposes. In addition, the present invention provides a high-throughput assay that can be used as a sensitive method for diagnosing and monitoring the spread of SARS.

3. Outline of the invention

The present invention is based on the inventor's identification of the unique region of hSARS virus, specifically, the 3' region of the hSARS virus genome, especially the hSARS virus (nucleocapsid) N-gene that can be used for diagnostic tests to detect hSARS. Specifically, the N-gene was used to diagnose SARS because compared with other genes in the hSARS virus, the N-gene had the highest copy number during virus infection, especially when cells were lysed. Therefore, the nucleotide sequence of the hSARS virus N-gene is especially useful for a rapid and reliable diagnostic test for hSARS virus. In addition, the method of the present invention eliminates false negative results and increases the sensitivity of the assay.

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. The hSARS virus is a very large RNA virus consisting of approximately 29,742 nucleotides. The complete genome sequence of the hSARS virus is deposited at Genbank, NCBI, under accession number AY278491 (SEQ ID NO: 15), which is incorporated herein by reference. The isolated hSARS virus was deposited at the China Center for Type Culture Collection (CCTCC) on April 2, 2003, given accession number CCTCC-V200303, as described in Section 7 below, which is incorporated herein by reference. Likewise, the complete genome sequence of the hSARS virus CCTCC-V200303 and its characterization are disclosed in U.S. Patent Application Attorney Docket No. V9661.0069 filed concurrently with this document on March 24, 2004, which is incorporated herein by reference in its entirety. The virus mutates and changes rapidly, making SARS very difficult to diagnose. The present inventors have designed a diagnostic test method for detecting the presence of N-gene nucleotide sequence or protein to quickly, accurately and specifically identify hSARS virus. In addition, the present inventors designed a test method for diagnostically detecting the presence of S-gene nucleotide sequence or protein to determine the genetic polymorphism of hSARS virus. Therefore, the present invention relates to the method for detecting hSARS virus N-gene and S-gene nucleotide sequence.

The invention provides a fast and reliable test method for detecting hSARS virus. In preferred embodiments, the detection of hSARS virus comprises the use of nucleic acid molecules of the invention in polymerase chain reaction, reverse transcription-polymerase chain reaction (RT-PCR), DNA analysis, RNA analysis or other nucleic acid hybridization to detect hSARS nucleic acid. 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, nasopharyngeal aspirates, sputum, blood, saliva, urine, feces, and the like. In preferred embodiments, the biological material is nasopharyngeal aspirate or feces. An increase or decrease in hSARS virus activity or expression in a sample relative to a control sample can be determined by contacting the biological material with a detection reagent that can directly or indirectly detect the presence, activity or expression of hSARS virus. In a specific embodiment, the detection reagent is a nucleic acid molecule of the invention.

The present invention also relates to a method for identifying a hSARS virus-infected subject, the method comprising obtaining total RNA from a biological sample of the subject; reverse transcribing the total RNA to obtain cDNA; and performing cDNA with a set of primers derived from hSARS nucleotide sequence PCR assay. In a preferred embodiment, the primers are derived from the (nucleocapsid) N-gene. In the most preferred embodiment, primer comprises SEQ ID NO:2475 and/or 2476 or SEQ ID NO:2480 and/or 2481 nucleotide sequence. In another preferred embodiment, the primers are derived from the (spike) S-gene. In a more preferred embodiment, the primer comprises SEQ ID NO: 2477 and/or 2478 nucleotide sequences.

The invention also relates to the use of the sequence information of the isolated virus in diagnostic and therapeutic methods. In a specific embodiment, the invention provides a nucleic acid molecule suitable as a primer, said nucleic acid molecule comprising or consisting of SEQ ID NO: 1, 11, 13, 15, 2471 or 2473 nucleotide sequence or its complementary sequence or at least part of its nucleotide sequence. In the most preferred embodiment, primer comprises SEQ ID NO:2475 and/or 2476 or SEQ ID NO:2480 and/or 2481 nucleotide sequence to detect N-gene. In another most preferred embodiment, primer comprises SEQ ID NO:2477 and/or 2478 nucleotide sequence to detect S-gene. In another specific embodiment, the present invention provides nucleic acid molecules suitable for hybridization with hSARS nucleic acids, including but not limited to PCR primers, reverse transcriptase primers, DNA analysis probes or other nucleic acid hybridization assays to detect hSARS nucleic acids, for example comprising or Consists of the nucleotide sequence of SEQ ID NO: 1, 11, 13, 15, 2471, 2473, 2475, 2476, 2477, 2478, 2480 or 2481 or a complementary sequence or a portion thereof. In a preferred embodiment, the amplification comprises (18057-18222 nucleotides of SEQ ID NO: 15 or part thereof) 1b gene, (21920-22107 nucleotides of SEQ ID NO: 15 or part thereof) M- The primers of the gene and (28658-28883 nucleotides of SEQ ID NO: 15 or part thereof) fragments of the N-gene can be used to synthesize probes for detecting hSARS nucleic acid. In a specific embodiment, the present invention provides a diagnostic kit comprising nucleic acid molecules suitable for detecting the N-gene of hSARS. In a specific embodiment, the N-gene comprises the nucleotide sequence of SEQ ID NO:2471. In a specific embodiment, the nucleic acid molecule comprises SEQ ID NO: 2475 and/or 2476 or SEQ ID NO: 2480 and/or 2481 nucleotide sequence. In a preferred embodiment, the diagnostic kit further comprises a control for the amplification of the 1b gene. In a specific embodiment, the primers used to amplify the 1b gene are SEQ ID NO: 3 and/or 4. In another specific embodiment, the diagnostic kit further comprises an internal control using the porcine β-actin gene. In a specific embodiment, the primers used to amplify the β-actin gene are SEQ ID NO: 2482 and/or 2483.

In a specific embodiment, the present invention provides a diagnostic kit comprising a nucleic acid molecule suitable for detecting the S-gene of hSARS. In a specific embodiment, the S-gene comprises the nucleotide sequence of SEQ ID NO: 2473. In a specific embodiment, the nucleic acid molecule comprises SEQ ID NO: 2477 and/or 2478 nucleotide sequences. The present invention also includes chimeric or recombinant viruses wholly or partly encoded by said nucleotide sequence.

In another specific embodiment, the invention provides a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 11, 13, 2471 and/or 2473. In a specific embodiment, the invention provides an isolated nucleic acid molecule comprising or consisting of a SEQ ID NO: 1 nucleotide sequence or a complementary sequence or a part thereof, preferably a SEQ ID NO: 1 nucleotide sequence or At least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600 or more consecutive nucleotides of its complementary sequence. In another specific embodiment, the present invention provides an isolated nucleic acid molecule comprising or consisting of a SEQ ID NO: 11 nucleotide sequence or a complementary sequence or a part thereof, preferably a SEQ ID NO: 11 nucleotide sequence or 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, 1000, 1050, 1100, 1150, 1200 or more contiguous nucleotides. In yet another specific embodiment, the present invention provides an isolated nucleic acid molecule comprising or consisting of a SEQ ID NO: 13 nucleotide sequence or a complementary sequence or a part thereof, preferably 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 another 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 part thereof, preferably a SEQ ID NO: 15 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, 750, 800 of its complement , 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 180000, , 19000, 20000, 21000, 22000, 23000, 24000, 25000, 26000, 27000, 28000, 29000 or more consecutive nucleotides. In another specific embodiment, the present invention provides an isolated nucleic acid molecule comprising or consisting of a SEQ ID NO: 2471 nucleotide sequence or its complementary sequence or a part thereof, preferably a SEQ ID NO: 2471 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, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 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: 2473 nucleotide sequence or its complementary sequence or a part thereof, preferably a SEQ ID NO: 2473 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, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 2000, 3000 or more contiguous nucleotides. Furthermore, in another specific embodiment, the present invention provides an isolated nucleic acid molecule which, under stringent conditions as defined herein, is compatible with a sequence having SEQ ID NO: 1, 11, 13, 15, 2471 or 2473 or its complement. hybridization of 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 present invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule comprising or consisting of at least 5, 10, 15, 20, A nucleotide sequence consisting of 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600 or more contiguous nucleotides. In yet another specific embodiment, the present invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule comprising or consisting of at least 5, 10, 15, 20 of the nucleotide sequence of SEQ ID NO: 11 or its complementary sequence. ,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000,1050,1100 , 1150, 1200 or more contiguous nucleotide sequences. In yet another specific embodiment, the present invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule comprising or consisting of at least 5, 10, 15, 20 of the nucleotide sequence of SEQ ID NO: 13 or its complementary sequence. , 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 present invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule comprising or consisting of at least 5, 10, 15, 20 of the nucleotide sequence of SEQ ID NO: 15 or its complementary sequence. ,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000,1050,1100 , 1150, 1200, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 21000, 2300 , 25000, 26000, 27000, 28000, 29000 or more contiguous nucleotide sequences. In yet another specific embodiment, the present invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule comprising or consisting of at least 5, 10, 15, 20 of the nucleotide sequence of SEQ ID NO: 2471 or its complementary sequence. ,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000,1050,1100 , 1150, 1200 or more contiguous nucleotide sequences. In yet another specific embodiment, the present invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule comprising or consisting of at least 5, 10, 15, 20 of the nucleotide sequence of SEQ ID NO: 2473 or its complementary sequence. ,25,30,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000,1050,1100 , 1150, 1200, 2000, 3000 or more contiguous nucleotide sequences. The present invention also provides proteins or polypeptides isolated from hSARS virus, including viral proteins isolated from virus-infected cells but not present in corresponding uninfected cells. The present invention also provides the proteins or polypeptides shown in Figures 11 (SEQ ID NOs: 17-239, 241-736 and 738-1107) and 12 (SEQ ID NOs: 1109-1589, 1591-1964 and 1966-2470). The present invention also provides a protein or polypeptide having the amino acid sequence of SEQ ID NO: 2472 or 2474. 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, 2471 or 2473. 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, the nucleotide sequence is the nucleotide sequence of SEQ ID NO: 15 or 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 of its complement , 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 180000, , 19000, 20000, 21000, 22000, 23000, 24000, 25000, 26000, 27000, 28000, 29000 or more consecutive nucleotides. In other embodiments, the polypeptide or protein of the present invention has the polypeptides of FIGS. biological activity (including antigenicity and/or immunogenicity) of the protein. The present invention also provides protein or polypeptide, the protein or polypeptide has the biological activity of the protein of SEQ ID NO: 2472 or 2474 amino acid sequence.

In one aspect, the present invention provides a method for propagating hSARS virus in a host cell, the method comprising infecting the host cell with an isolated hSARS virus, culturing the host cell to replicate the virus, and harvesting the resulting virion. The present invention also provides host cells infected by hSARS virus.

In one aspect, the invention relates to the use of isolated hSARS virus in methods of diagnosis and treatment. In a specific embodiment, the present invention provides a method for detecting hSARS virus immune-specific antibodies in a biological sample using the 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 and neutralize hSARS. This antibody is used for passive immunization or immunotherapy of hSARS-infected subjects.

The present invention also provides an antibody that specifically binds to the polypeptide of the present invention encoded by the nucleotide sequence of SEQ ID NO: 1, 11, 13, 2471 or 2473 or a fragment thereof, or comprises a combination with SEQ ID NO: 1 under stringent conditions. , 11, 13, 2471 or 2473 nucleotide sequence hybrid nucleic acid encoding polypeptide of the present invention, and/or any hSARS epitope having one or more biological activities of the polypeptide of the present invention. The present invention also provides an antibody that specifically binds to the polypeptide of the present invention encoded by the nucleotide sequence of SEQ ID NO: 15 or a fragment thereof. These polypeptides include those shown in Figures 11 (SEQ ID NOs: 17-239, 241-736, and 738-1107) and 12 (SEQ ID NOs: 1109-1589, 1591-1964, and 1966-2470). In another embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 2472 or 2474. The present invention also provides antibodies that specifically bind 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 have one or more Any hSARS epitope of the biological activity of the polypeptide of the present invention. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, Fab fragments, F(ab') ₂ Fragments, disulfide-linked Fvs, intrabodies, and fragments containing VL or VH domains or even complementarity determining regions (CDRs) that specifically bind a polypeptide of the invention.

In another embodiment, the invention provides vaccine preparations comprising hSARS virus (including recombinant and chimeric forms of said virus) or protein subunits of said 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 passages of the virus in host cells or by making recombinant or chimeric forms of the virus. Accordingly, the present invention also provides methods for producing recombinant or chimeric forms of hSARS. In another specific embodiment, the vaccine product of the present invention comprises a nucleic acid or fragment of hSARS virus (for example, the virus whose retrieval number is CCTCC-V200303), or its sequence is SEQ ID NO: 1, 11, 13, 15, 2471 or 2473 Nucleic acid molecules or fragments thereof. In another embodiment, the present invention provides a vaccine preparation comprising one or more polypeptides isolated from hSARS virus or produced from hSARS virus nucleic acid, such as the virus with deposit accession number CCTCC-V200303. In a specific embodiment, the vaccine preparation comprises the polypeptide of the present invention encoded by the nucleotide sequence of SEQ ID NO: 1, 11, 13, 2471 or 2473 or a fragment thereof. In a specific embodiment, the vaccine preparation comprises the compounds shown in Figures 11 (SEQ ID NOs: 17-239, 241-736, and 738-1107) and 12 (SEQ ID NOs: 1109-1589, 1591-1964, and 1966-2470). The polypeptide of the present invention described above or the polypeptide of the present invention encoded by the nucleotide sequence of SEQ ID NO: 15 or a fragment thereof. In a specific embodiment, the vaccine preparation comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 2472 or 2474. In addition, the present invention provides methods for treating, improving, controlling or preventing SARS by administering the vaccine product or antibody of the present invention alone or in combination with adjuvants 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 agent of the present invention is an antibody that immunospecifically binds hSARS virus or any hSARS epitope. In preferred embodiments, such antibodies neutralize hSARS virus. In a specific embodiment, the antiviral agent of the present invention binds to fragments, variants, homologues of hSARS virus N-gene or S-gene. In a specific embodiment, the antiviral agent of the present invention binds to fragments, variants, homologues of polypeptides comprising the amino acid sequence of SEQ ID NO: 2472 or 2474. 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 an antibody or antibody fragment that immunospecifically binds to the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1, 11, 13, 15, 2471 or 2473 or in Figure 11 or 12 The indicated polypeptides or fragments thereof, and antibodies or fragments thereof that do not non-specifically bind to other polypeptides. Antibodies or fragments thereof that immunospecifically bind a polypeptide of the invention may cross-react with other antigens. Preferably, 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, 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 library member nucleic acids that have not been separated from other library clones containing other nucleic acid molecules.

The term "portion" or "fragment" as used herein refers to a nucleic acid molecule comprising a nucleic acid molecule of at least about 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, 29,000 or more functional molecules of at least one contiguous related nucleic acid, and characteristic (or its encoded protein has a functional characteristic of the protein encoded by the related nucleic acid molecule); or refers to 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 have at least one associated A protein or polypeptide fragment that is a functional feature of a protein or polypeptide.

The term "the 3' region of the hSAR viral genome" refers to about 18000-29742 nucleotides of SEQ ID NO:15.

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, 1965, 2471 or 2473 nucleotide sequence coded polypeptides have similar or identical domains and/or have sufficient amino acid identity. 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, ElsevierScience 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, 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. Brief description of the drawings

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

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

Figure 3 shows 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.

Figure 13 shows the N-gene primer sequence (it amplifies nucleotides at 29247-29410 positions of SEQ ID NO: 2471); 150# (SEQ ID NO: 2475); 200# (SEQ ID NO: 2476); and S - gene primer sequence (it amplifies nucleotides at 24751-25049 positions of SEQ ID NO: 2473); 131# (SEQ ID NO: 2477); 132# (SEQ ID NO: 2478).

Figure 14A shows the nucleotide sequence of the N-gene (SEQ ID NO: 2471). Figure 14B shows the amino acid sequence of the N-gene (SEQ ID NO: 2472).

Figure 15A shows the nucleotide sequence of the S-gene (SEQ ID NO: 2473). Figure 15B shows the amino acid sequence of the S-gene (SEQ ID NO: 2474).

Figure 16 shows the genome organization and transcription strategy of SARS-CoV HK-39. Genomic and mRNA transcripts are capped (black circles), carry a leader sequence proximal to the 5' end (vertical line), and are polyadenylated (A ¹⁵ ). Arrows indicate the position of the intergenic sequence 5'-CTAAACGAAC-3' (SEQ ID NO: 2479). Following release of the positive-sense genomic RNA into the host cell cytoplasm, the viral RNA-dependent RNA polymerase encoded by ORF 1a and 1b is synthesized. It performs transcription of full-length complementary (antisense) RNA from which new genomic RNA, overlapping sets of subgenomic mRNA transcripts, and leader RNA are synthesized. It should be noted that all transcripts are preceded by a common 5' leader sequence and a common 3' end. ORF1a and 1b - RNA-dependent RNA polymerase; S - major envelope protrusion glycoprotein; M - transmembrane glycoprotein; N - nucleocapsid; X1, X2, X3 - putative proteins.

Figure 17 shows the construction diagram of pSARSCoV-ORF1b-N. PCR products amplified from the ORF1b (1b) and N genes of SARS-CoV were co-ligated into the cloning vector pCR2.1-TOPO (Invitrogen). The number of nucleotides (nt) corresponds to the position in the sequence of HK-39 strain SARS-CoV (AY278491). Shaded areas indicate amplicons amplified by primers used in the diagnostic assay (ie, SEQ ID NO: 2480 and 2481).

Figure 18 shows the agarose gel photo after electrophoresis of the total RNA extracted from SARS patients with the SV total RNA isolation system. The extracted RNA was then subjected to a reverse transcription polymerase chain reaction (RT-PCR) test to detect the coronavirus in the patient.

Figure 19 shows the effect of potential inhibitors in reverse transcription polymerase chain reaction (RT-PCR). To remove potential inhibitors, total RNA eluted from SV96 binding plates was precipitated with 95% ethanol and 3M sodium acetate and resuspended in 12 μl of nuclease-free water. RT-PCR was performed with actin-F (SEQ ID NO: 2482) and actin-R (SEQ ID NO: 2483) primers. The numbers shown are the number of porcine kidney epithelial cells (PK-15) added to the samples as an internal control. No DNA fragments were amplified with untreated RNA samples.

Figure 20 shows primers used to amplify various genes. SRS251 (SEQ ID NO: 2480) and SRS252 (SEQ ID NO: 2481) amplified a 225 base pair fragment from the N-gene region, which showed no homology to other coronaviruses. coro3 (SEQ ID NO:3) and coro4 (SEQ BD NO:4) amplify the RNA-dependent RNA polymerase (1b gene) as a control. Actin-F (SEQ ID NO: 2482) and actin-R (SEQ ID NO: 2483) amplified a 745 base pair fragment from the β-actin gene used as an internal control for PCR experiments.

Figure 21A shows an amplification plot of fluorescence intensity versus PCR cycle number. The black line indicates the dynamic range of N-gene-specific PCR with 10 ¹ -10 ⁶ copies of serially diluted plasmid constructs. Non-SARS patients, including those infected with adenovirus (n=5), respiratory syncytial virus (n=5), human metapneumovirus (n=5), influenza A virus (n=5), or influenza B NPA samples from patients with virus (n=5) are indicated by gray lines. Lines with triangles indicate SARS-CoV-positive NPA samples; NTC indicates no-template control; X-axis indicates cycle number of quantitative PCR performed, while Y-axis indicates fluorescence intensity (FAM-400) over background signal (ΔRn). Inset graph represents melting curve analysis of PCR products. Signals from positive (+ve) samples, negative (-ve) samples and no template control are shown. The X-axis represents temperature (°C), and the Y-axis represents fluorescence intensity (ΔRn). Figure 21B shows a comparison of the dynamic range of specific PCR for the N-gene and the 1b-gene. The dynamic range of both N-gene and 1b-gene PCR was obtained with the same plasmid construct, in which the corresponding amplicons were subcloned in a 1:1 ratio. Serially diluted plasmids with copy numbers ranging from 10 ⁻¹ to 10 ⁵ copies were used as templates in both PCRs. Lines with triangles indicate N-gene-specific PCR, while gray lines indicate 1b-gene-specific PCR. Inset graphs represent Ct values ± standard deviation in triplicate of two PCR experiments using different template copy numbers. NTC indicates no template control; the X-axis indicates the cycle number of quantitative PCR performed and the Y-axis indicates the fluorescence intensity.

22A and 22B show the amplification curves of real-time quantitative PCR specific to 1b (using primers with SEQ ID NO: 3 and 4) and N-gene (using primers with SEQ ID NO: 2480 and 2481) of SARS CoV, respectively and melting curve. Figure 22A: Amplification plot of fluorescence intensity versus PCR cycle number. 1 μl of cDNA from NPA, tracheal dispersion and lung biopsies of clinically symptomatic patients was used as template for each PCR. PCR was performed for 50 cycles to reach the saturation period of the reaction. The X-axis represents the number of cycles of quantitative PCR performed, while the Y-axis represents the fluorescence intensity (FAM-490) over the background signal. The horizontal gray line indicates the threshold calculated by the maximum curvature method, and the baseline cycle Ct is automatically calculated. The inset represents the half-maximal fluorescence values (1/2 max) and Ct of two PCRs using cDNA derived from various tissues isolated from key patients at three different time points (New Engl.J.Med.348 : Patient A) mentioned in 1967-76 (Drosten et al., 2003). NPA = nasopharyngeal aspirates; TW = tracheal washings; LW = lung washings. Figure 22B: Melting curves of PCR products. Analysis of melting curves was performed after a 10 min reaction further extension step. The temperature was raised in 76 increments from 56°C to 94°C in 0.5°C increments, and the temperature was held for 7 seconds at each set point for data collection and analysis. The melting temperatures of the 1b- and N-gene-specific PCR products were 80.5°C and 85.5°C, respectively. The X-axis is temperature in degrees Celsius and the Y-axis is fluorescence intensity (FAM-490) over background signal. 1 μl of water was used in the reaction as a no-template control.

Figure 23 shows the diagnostic results of 48 clinical samples, wherein primers with SEQ ID NO: 2480 and 2481 were used respectively, and β-actin PCR was used as an internal control. The band above each road represents the 745bp DNA fragment amplified with actin-F and actin-R, and the band below is the amplicon of the SARS coronavirus (225bp) N-gene-specific primer. The -ve control (water) and +ve control (cDNA from SARS-CoV-infected Vero cells) in the assay are indicated. 5 μl of PCR product from both reactions were mixed and added to the sample wells of a 2% agarose gel. M = 1 kb + molecular weight marker (Invitrogen).

Figure 24 shows Northern blot analysis of SARS-CoV total RNA. Total RNA of SARS-CoV was extracted from SARS-CoV-infected Vero E6 cells. RNA was separated on a 1% denaturing gel containing 6.29% formaldehyde. The RNA was then transferred to a positively charged nylon membrane and hybridized with digoxigenin-labeled fragments specific for the 1b, S, M, and N genes, respectively. Lane 1-1b; Lane 2-S; Lane 3-M; Lane 4-N. Vertical bars represent molecular weight references. Arrows indicate transcripts hybridized to N probes. Signals were analyzed by chemiluminescence.

Figure 25 shows DNA probes used for Northern blot analysis. The 1b gene (nucleotides 18057-18222; SEQ ID NO: 2484), the S gene (nucleotides 21920-22107; SEQ ID NO: 2485), the M gene (nucleotides 25867-26996; SEQ ID NO: 2486) and the N gene (nucleotides 28658-28883; SEQ ID NO: 2487).

5. Detailed Description of the Invention

The present inventors have developed a rapid, high-throughput reverse transcription-PCR diagnostic test for SARS-related coronavirus (SARS-CoV). The 3′ region of the hSARS viral genome including the nucleocapsid gene (N-gene) represents a sensitive molecular marker that can be used outside of the 1b gene to increase the sensitivity of the test. Use of PK-15 cells as an internal control can be used to ensure the integrity of the RNA during the extraction process and cDNA synthesis, thus eliminating false negative results.

In mouse hepatitis virus (MHV), a typical member of the Coronaviridae, genomic RNA and mRNA transcripts are capped with a common 3' end and a common 5' end leader sequence. According to this unique transcription strategy, the copy numbers of different viral genes are different when the virus propagates in the host (Fig. 19). The N gene encoding the nucleocapsid has the most abundant copy number during viral replication because all transcripts can carry nucleotide sequences from the N gene, although not all of them are in frame for translation of the gene product. The inventors have discovered that a diagnostic test based on the 3' region of the viral genome, including the N-gene, provides a more sensitive test than the rest of the viral genome. Therefore, in a preferred embodiment, the nucleic acid molecule that can be used for diagnostic tests comprises the nucleotide sequence of 18000-29742 nucleotides of SEQ ID NO: 15 or a part thereof. The part may comprise 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450 of the nucleic acid sequence from the 18000-29742 nucleotides of SEQ ID NO: 15 , 500, 550, 600, 650, 700, 750, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 nucleotides. In other preferred embodiments, the nucleic acid molecule used for the diagnostic test comprises the nucleic acid sequence of 28658-28883 or 29247-29410 nucleotides of SEQ ID NO:15.

Nasopharyngeal aspirates (NPA) and fecal samples were obtained from suspected SARS patients with major clinical symptoms and an obvious history of close contact with infected patients. Total RNA was extracted from patient samples, while PK-15 cells were used as an internal control. Samples were analyzed by reverse transcription PCR assay. Northern blot analysis was performed to reveal viral distinct subgenomic transcripts. Real-time quantitative PCR was used to compare the sensitivity of the two sites used in this diagnostic test. In a specific embodiment, PCR inhibitors are removed by ethanol precipitation after the RNA extraction process.

In a preferred embodiment, the present invention provides methods for detecting the presence or absence of an N-gene nucleic acid in a biological sample. The method includes obtaining a biological sample from various sources, contacting the sample with a compound or reagent that can detect hSARS virus N-gene nucleic acid (eg, mRNA, genomic RNA), thereby detecting the presence of the N-gene in the sample. In a preferred embodiment, the N-gene can be detected with a labeled nucleic acid probe comprising the nucleotide sequence of SEQ ID NO: 2471 or its complementary sequence or a portion thereof. The portion may be 10, 20, 30, 40, 50, 100, 200, 400, 500, 600, 800, 1000, 1200 nucleotides in length. In a preferred embodiment, primers comprising SEQ ID NO: 2475 and/or 2476 or SEQ ID NO: 2480 and/or 2481 nucleotide sequences can be used to amplify a portion of the N-gene for detection.

Preferably, the reagent used to detect hSARS mRNA or genomic RNA of the present invention is a labeled nucleic acid probe that can hybridize with the mRNA or genomic RNA encoding the polypeptide of the present invention. The nucleic acid probe may for example be a nucleic acid molecule comprising or consisting of a nucleotide sequence of SEQ ID NO: 1, 11, 13, 15, 2471 or 2473 or a complementary sequence or part thereof, e.g. Oligonucleotides that are at least 15, 20, 25, 30, 50, 100, 250, 500, 750, 1000 or more contiguous nucleotides in length and sufficient to specifically hybridize to hSARS mRNA or genomic RNA under stringent conditions .

In another preferred embodiment, using the N-gene partial nucleotide sequence or the genomic nucleotide sequence of SEQ ID NO: 15 or the nucleotide sequence based on SEQ ID NO: 1, 11, 13, 15, 2471 or 2473 The presence of the N-gene in the sample was detected by reverse transcription polymerase chain reaction (RT-PCR) using primers constructed from acid sequences. In a non-limiting specific embodiment, the primers preferably used in the RT-PCR method are: 5'-TACACACCTCAG-CGTTG-3' (SEQ ID NO: 3) and/or 5'-CACGAACGTGACGAAT-3' (SEQ ID NO :4), in the presence of 2.5mM MgCl ₂ , the thermal cycle is, for example 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 6.7 and 6.8 below. Festival). In a preferred embodiment, the primers comprise the nucleic acid sequences of SEQ ID NO: 2475 and 2476. In another preferred embodiment, the primer comprises the nucleic acid sequences of SEQ ID NO: 2480 and 2481. In a preferred embodiment, the thermal cycle is 94°C for 10 minutes, followed by 40 cycles of 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 30 seconds, and 72°C for 10 minutes. In another preferred embodiment, the thermal cycle is 94°C for 3 minutes, followed by 40 cycles of 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 30 seconds, and 72°C for 10 minutes. In more preferred embodiments, the present invention provides a real-time quantitative PCR assay to detect the presence of hSARS virus in a biological sample, by reverse-transcribing the extracted total RNA from the sample, and using the cDNA obtained with specific primers (for example, with SEQ ID NO: primers for 3 and 4 nucleotide sequences) and fluorescent dyes (such as Green I, which fluoresces upon non-specific binding to double-stranded DNA), was used for PCR reactions. As PCR products are generated over a certain number of thermal cycles, the fluorescent signal of these reactions is captured at the end of the elongation step, allowing quantification of the amount of virus in the sample based on the amplification plot (see 6.7 below).

In a preferred embodiment, the present invention provides methods for detecting the presence or absence of S-gene nucleic acid in a biological sample. The method comprises obtaining a biological sample from various sources, contacting the sample with a compound or reagent capable of detecting hSARS virus S-gene nucleic acid (eg, mRNA, genomic RNA), thereby detecting the presence of the S-gene in the sample. Preferably, the reagent used to detect hSARS mRNA or genomic RNA of the present invention is a labeled nucleic acid probe that can hybridize with the mRNA or genomic RNA encoding the 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, 15, 2471 or 2473 nucleotide sequence or a complementary sequence or a part thereof, for example having a length of at least 15, 20, 25 , 30, 50, 100, 250, 500, 750, 1000 or more consecutive nucleotides, and oligonucleotides sufficient to specifically hybridize to hSARS mRNA or genomic RNA under stringent conditions.

In another preferred specific embodiment, use the primer that builds based on S-gene partial nucleotide sequence (SEQ ID NO:2473), detect the presence of S-gene in the sample by reverse transcription polymerase chain reaction (RT-PCR) .

Techniques for detecting mRNA in vitro include Northern hybridization, in situ hybridization, RT-PCR, and RNase protection. Techniques used to detect genomic RNA in vitro include Northern blot, RT-PCT, and RNase protection.

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

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

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

The primers used in the method of the present invention are complementary to each strand of the nucleotide sequence to be amplified. The term "complementary" indicates that the primers must hybridize to their respective strands under conditions that allow the reagents to polymerize. In other words, a primer complementary to the flanking sequence hybridizes to the flanking sequence, allowing the amplification of the nucleotide sequence. The 3' end of the continuation primer preferably has full base pairing complementarity to the side strand. Primers and probes for N-gene or S-gene encoding polynucleotides of the invention can be developed using known methods in conjunction with the present disclosure.

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

试验(Holland等，Proc Natl Acad Sci USA，88(16)：7276(1991)；也参见2004年3月24日提交的美国专利申请，代理人档案号V9661.0078，其通过引用整体结合到本文中)。本发明试验可在设计用于进行这种试验的仪器上进行，例如可获自Applied Biosystems(Foster City，CA)的仪器。用于这种试验的引物和探针可根据本领域技术人员已知的方法设计。The methods of the invention may include real-time quantitative PCR assays, such as

Test (Holland et al., Proc Natl Acad Sci USA, 88(16):7276(1991); see also U.S. Patent Application filed March 24, 2004, Attorney Docket No. V9661.0078, which is incorporated herein by reference in its entirety middle). The assays of the present invention can be performed on instruments designed to perform such assays, such as those available from Applied Biosystems (Foster City, CA). Primers and probes for such assays can be designed according to methods known to those skilled in the art.

Primers used to amplify N-gene or S-gene parts are at least 10, 15, 20, 25, 30 nucleotides in length. Specifically, primers that amplify the N-gene or the S-gene are most preferred. Preferably the GC ratio should be higher than 30%, 35%, 40%, 45%, 50%, 55% or 60% to prevent hairpin structures on the primers. In addition, amplicons should be of sufficient length to be detected by standard molecular biology methods. Preferably the amplicon is at least 40, 60, 100, 200, 300, 400, 500, 600, 800, 1000 base pairs in length.

In a specific embodiment, the method of the present invention further involves obtaining a control sample from a control subject, contacting the control sample with a compound or reagent capable of detecting the N-gene or S-gene, thereby detecting the N-gene or S-gene encoded in the sample The presence of mRNA or genomic RNA, and whether there is N-gene or S-gene or mRNA or genomic RNA encoding polypeptide in the control sample and the mRNA or genomic RNA of N-gene or S-gene or encoding polypeptide in the test sample exist for comparison.

The present invention also includes a kit for detecting the presence of N-gene nucleic acid in a test sample. The kit, for example, may include a labeled compound or reagent capable of detecting a polypeptide-encoding nucleic acid molecule in a sample to be tested, and in some embodiments, a method for detecting the amount of mRNA in a sample (oligonucleotides that bind to DNA or mRNA encoding a polypeptide acid probe).

For oligonucleotide-based kits, the kit may, for example, comprise: (1) an oligonucleotide, such as a detectably labeled oligonucleotide, associated with a nucleotide sequence encoding a polypeptide of the invention or an N-gene (2) primer pairs for amplifying nucleic acid molecules containing N-gene sequences. The kit may also contain, for example, buffering agents, preservatives, or protein stabilizing agents. The kit may also comprise components necessary to detect a detectable substance (eg, an enzyme or a substrate). The kit may also contain a control sample or series of control samples, which can be tested and compared to the samples to be tested. The various components of the kit are usually contained in individual containers, all of which are enclosed together in a single package along with instructions for use.

The present invention relates to the isolated N-gene and S-gene of hSARS virus. In a specific embodiment, the virus comprises SEQ ID NO: 1, 11, 13, 15, 2471 and/or 2473 nucleotide sequence. In a specific embodiment, the present invention provides an isolated nucleic acid molecule of hSARS virus, which nucleic acid molecule comprises or consists of SEQ ID NO: 1, 11, 13, 15, 2471 and/or 2473 nucleotide sequence or its complementary sequence or Partial composition. In another specific embodiment, the present invention provides a nucleic acid molecule with a sequence of SEQ ID NO: 1, 11, 13, 15, 2471 and/or 2473 or a known member of the Coronaviridae under stringent conditions defined herein. An isolated nucleic acid molecule to which a specific gene, or its complement, hybridizes. In another specific embodiment, the invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule comprising at least about 5, 10, 15, 20, 25 of the nucleotide sequence of SEQ ID NO: 1 or its complement. , 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, 20, 25 of the nucleotide sequence of SEQ ID NO: 11 or its complement. . or a nucleotide sequence of more contiguous nucleotides. In yet 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, 20, 20, A nucleotide sequence of 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 present invention provides an isolated polypeptide or protein encoded by a nucleic acid molecule comprising at least 5, 10, 15, 20, 25, 30 of the nucleotide sequence of SEQ ID NO: 2471 or its complementary sequence. ,35,40,45,100,150,200,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000,1050,1100,1150,1200 or more nucleotide sequences of contiguous nucleotides. In yet another specific embodiment, the present 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, 1000, 1050, 1, 100, A nucleotide sequence of 1150, 1200, 2000, 3000 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, 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 comprise the polypeptide 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) or have SEQ ID NO: a polypeptide of 2472 or 2474 amino acid sequence. 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, 2471 or 2473; or the polypeptide shown in Figure 11 and Figure 12 or a native viral protein comprising the amino acid sequence encoded by the sequence of SEQ ID NO: 1, 11, 13, 15, 2471 or 2473.

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 complementary sequence. 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 present invention provides a nucleic acid molecule suitable for use as a primer, the nucleic acid molecule comprising or consisting of a SEQ ID NO: 1, 11, 13, 15, 2471 or 2473 nucleotide sequence or a complementary sequence or a portion thereof composition. In a specific embodiment, the primer comprises a nucleotide sequence of SEQ ID NO: 2475, 2476, 2477, 2478, 2480 or 2481. In another specific embodiment, the present invention provides nucleic acid molecules suitable for use as hybridization probes for detecting sequences comprising or consisting of SEQ ID NO: 1, 11, 13, 15, 2471 or 2473 nucleotide sequences or their complementary sequences A nucleic acid encoding a polypeptide of the present invention, or a part thereof. The present invention also relates to a kit comprising primers having the nucleotide sequences of SEQ ID NO: 2475 and 2476 and SEQ ID NO: 2480 and 2481 to detect the N-gene. In another embodiment, the present invention relates to the test kit that comprises primer, and described primer has SEQ ID NO:2477 and/or 2478 nucleotide sequence, to detect S-gene. In a preferred embodiment, the kit further comprises reagents for detecting genes that are absent in the hSARS virus used as a negative control. The present invention further includes chimeric or recombinant viruses or viral proteins encoded by said nucleotide sequences.

The present invention 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: 1, 11, 13, 2471 or 2473 or a fragment thereof. The present invention also provides an antibody that specifically binds to the polypeptide whose amino acid sequence is SEQ ID NO: 2472 or 2474. The present invention further provides an antibody that specifically binds the polypeptide of the present invention encoded by the nucleotide sequence of SEQ ID NO: 15 or a fragment thereof, or the polypeptide shown in Figures 11 and 12 or a fragment thereof, or any hSARS epitope. 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 of the invention comprises inactivated or killed hSARS virus, with or without pharmaceutically acceptable carriers, including adjuvants. The vaccine preparations of the present invention may also contain adjuvants or vaccines. Accordingly, 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, 15, 2471 and/or 2473 or fragments thereof. In another embodiment, the present invention provides vaccine preparations comprising one or more polypeptides of the present invention comprising or consisting of SEQ ID NO: 1, 11, 13, 2471 and/or 2473 nucleotide sequences or The nucleotide sequence coded by its fragments, or the polypeptides shown in Figures 11 and 12 or 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 agents [such as amantadine, rimantadine, ganciclovir, Cyclovir, ribavirin, penciclovir, oseltamivir, foscarnet, zidovudine (AZT), didanosine (ddI), lamivudine (3TC), zalcitabine (ddC), stavudine (d4T), nevirapine, posvirdine, indinavir, ritonavir, vidarabine, nelfinavir, saquinavir, zanamivir, oxalate Semevir, pleconaril, interferon, etc.], steroids and corticosteroids (eg, prednisone, cortisone, fluticasone) and glucocorticoids, antibiotics, analgesics, bronchodilators, or other medications for respiratory and and/or therapy for viral infections.

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

In another aspect, the invention provides methods of screening for antiviral agents that inhibit the infectivity or replication of hSARS virus or variants thereof.

5.1 Recombinant and Chimeric hSARS Viruses

The present invention includes recombinant or chimeric viruses encoded by viral vectors derived from the genome of hSARS virus or natural variants thereof. In a specific embodiment, the recombinant virus is derived from the hSARS virus with the deposit number CCTCC-V200303. In a specific embodiment, 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 has a sequence different from the genome sequence (SEQ ID NO: 15) of hSARS virus CCTCC-V200303, which is due to one or more spontaneous mutations in the genome sequence, including but not limited to point mutations, rearrangements, Insertions, deletions, etc., which may or may not result in a phenotypic change. According to the present invention, the viral vector derived from the genome of hSARS virus CCTCC-V200303 contains a nucleic acid sequence encoding at least a part of one ORF of hSARS virus. In a specific embodiment, said OFR comprises or consists of SEQ ID NO: 1, 11, 13, 2471, 2473 nucleotide sequence or a fragment thereof. In a specific embodiment, there is more than one OFR in the nucleotide sequence of SEQ ID NO: 15 or a fragment thereof, as shown in Figure 11 (SEQ ID NO: 16, 240 and 737) and Figure 12 (SEQ ID NO: 1108, 1590 and 1965). In another embodiment, the polypeptide encoded by the ORF comprises or consists of the amino acid sequence of SEQ ID NO: 2, 12, 14, 2472 or 2474 or a 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 acid which is not the genome of the native virus.

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

According to the present invention, the chimeric virus is encoded by the viral vector of the present invention further comprising a heterologous nucleotide sequence. According to the invention, chimeric viruses are encoded by viral vectors which may or may not contain nucleic acid other than the native viral genome. According to the invention, a chimeric virus is encoded by a viral vector in which a heterologous nucleotide sequence has been added, inserted or a natural or non-natural sequence has been substituted. According to the present invention, chimeric viruses may be encoded by nucleotide sequences derived from different strains or variants of hSARS virus. Specifically, chimeric viruses are encoded by nucleotide sequences encoding antigenic polypeptides derived from different strains or variants of 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 the hSARS virus expressing one or more proteins of a variant of the hSARS virus (and vice versa) would protect a subject vaccinated with such a vector from infection by the native hSARS virus and its variants . For the purpose of vaccination with live, attenuated and replication-deficient viruses can be used like other viruses. (See pages 6 and 23 of 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 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 virions obtained according to the present invention can be advantageously used in vaccine preparations. The expression products and chimeric virions of the invention can be artificially 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 virion 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 virions 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. Mutations and modifications can occur in the coding regions, intergenic regions, and leader and trailer sequences of the virus.

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

In a preferred embodiment, the present invention provides protein molecules encoded by nucleic acids of the present invention or hSARS virus-specific viral proteins and functional fragments thereof. Useful protein molecules are for example derived from any gene or genome fragment that can be derived from the virus of the present invention, including envelope protein (E protein), membrane intrinsic protein (M protein), spike protein (S protein), nucleocapsid protein (N protein), hemagglutinin esterase (HE protein), and RNA-dependent RNA polymerase. Such molecules or antigenic fragments thereof provided herein are useful, for example, in diagnostic methods or kits, and in pharmaceutical compositions such as subunit vaccines. Particularly useful are the polypeptides encoded by the nucleotide sequences of SEQ ID NO: 1, 11, 13, 15, 2471, 2473, Fig. 11 (SEQ ID NO: 17-239, 241-736 and 738-1107) and Fig. 12 (SEQ ID NO: 17-239, 241-736 and 738-1107) and Fig. ID NOs: 1109-1589, 1591-1964 and 1966-2470), a polypeptide having an amino acid sequence of SEQ ID NO: 2472 or 2474 or an antigenic fragment thereof, for incorporation as an antigen or subunit immunogen, but also Inactivated whole virus can 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 a DNA vaccine preparation, which comprises nucleic acid or fragments thereof of hSARS virus (for example, a virus whose deposit number is CCTCC-V200303), or has SEQ ID NO: 1, 11, 13, 15, 2471, A nucleic acid molecule of sequence 2473 or a fragment thereof. In another specific embodiment, the DNA vaccine preparation of the present invention comprises a nucleic acid or a fragment thereof encoding an antibody that immunospecifically binds hSARS virus. In DNA vaccine preparations, the DNA vaccine comprises a viral vector (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 a 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., Heterolog ous protection against influenza by injection of DNA encoding a viral protein, Science259: 1745-1749), and bacterial infections such as tuberculosis (Tascon, R.E. et al., 1996, Vaccination against tuberculosis by DNA injection, Nature Med. 2: 888-892; Huygen, K. et al., 1996, Immunogenicity and protective efficacy of a tuberculosis DNA vaccine, Nature Med., 2:893-898) and parasitic infections such as malaria (Sedegah, M., 1994, Protection against malaria by immunizationwith plasmad DNA encoding circumspoinrozoite protein Proc.Natl.Acad.Sci.USA 91: 9866-9870; Doolan, D.L. et al., 1996, Circumventing genetic restriction of protection against malaria with multigene DNA immunization: CD8+T cell-interferonδ, and nitric oxide-dependent immunity, J.Exper. ., 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 saline solution by injecting into muscle or skin with a syringe and needle (Wolff J.A. et al., 1990, Direct gene transfer into mouse muscle in vivo, Science 247:1465-1468; Raz, E., 1994 , Intradermal gene immunization: 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 vaccine strategies, Seminars in Immunology 9(5):269-270; and Robinson, H.L. et al., 1997, DNA vaccines, Seminars in Immunology 9(5):271-283.

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 virions.

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

In certain embodiments, the attenuated virus of the invention (eg, recombinant or chimeric hSARS) does not replicate as well as 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 body. 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, attenuated hSARS 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, 15, 2471, or 2473, to produce a virus with an attenuated expression. type of virus. 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 wild-type viral proteins.

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 a heterologous protein is inserted into the virion. If the heterologous protein is inserted into the virion, the virion should be tested for its ability to cause symptoms in an appropriate animal model, 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 polyphosphazenes.

In another embodiment, the adjuvant used is a special adjuvant, including but not limited to emulsions, such as Freund's complete adjuvant, Freund's incomplete adjuvant, for example with block copolymers such as L-121 (polyoxygen Propylene/polyoxyethylene, sold under the trademark PLURONIC L-121) prepared squalene or squalane oil-in-water adjuvant formulations such as SAF and MF59, liposomes, virosomes, liposome rolls (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, 14, 2472, 2474 sequences and Figure 11 (SEQ ID NO: 17-239, 241-736 and 738-1107) and Figure 11 The polypeptide shown in 12 (SEQ ID NO: 1109-1589, 1591-1964, 1966-2470), or the hSARS epitope antibody or its antigen-binding fragment, can be used to detect, screen and isolate the polypeptide of the present invention or its fragment or possibly Similar sequences encoding other biologically similar enzymes. For example, in a specific embodiment, antibodies or fragments thereof that immunospecifically bind hSARS epitopes can be used in various in vitro detection assays, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, Western blot, etc., to detect in vitro 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 hSARS virus with the deposit number CCTCC-V200303 or 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, keyholes Hemocyanin, dinitrophenol and potential human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium smallis. Such adjuvants are also well known in the art.

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

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

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

The 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 (enabling 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. WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; 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 Fy 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; Shu 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) (EP 592,106; 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 Natl.Acad. Sci. USA, 91:969-973, 1994) and chain shuffling (US Patent No. 5,565,332), each of which is incorporated herein by reference in its entirety.

Fully human antibodies are particularly suitable for use in the therapeutic treatment of human patients. Human antibodies can be prepared by a variety of methods well known in the art, including 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; The references are incorporated herein in their entirety.

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 technique for producing human antibodies and human monoclonal antibodies, as well as protocols for producing such antibodies, see, e.g., PCT Publication Nos. WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; Patent No. 0 598 877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 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. WO93/21232; 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. In another specific embodiment, the antiviral agent is a polypeptide or nucleic acid molecule of the invention. The pharmaceutical composition is useful as a prophylactic antiviral agent and can be administered to a subject who has been or is expected to be exposed to the virus.

Various delivery systems are known for administering the pharmaceutical composition of the present invention, such as liposome encapsulation, microparticles, microcapsules, recombinant cells expressing mutant viruses, receptor-mediated endocytosis (See e.g. Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432). 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, or by implant, which is a porous, non-porous, or gel-like material, including thin films such as sialastic membranes or fibers. In one embodiment, administration may be by direct injection at the site 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, CRCCrit. 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. Typically, the ingredients are supplied alone or in admixture in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet, marked on the container with the quantity of active drug. Where the composition is administered by infusion, it may be dispensed with an infusion bottle filled with sterile pharmaceutical grade water or saline. Where the composition is to be administered by injection, an ampoule of sterile water for injection or saline may be provided for mixing the ingredients prior to administration.

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

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

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

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions of the invention. This container may optionally be accompanied by a notice in the prescribed form of a governmental agency regulating the manufacture, use, or sale of a drug or biological Product approval. In a preferred embodiment, the kit comprises an antiviral agent of the present invention, for example, a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 1, 11, 13, 15, 2471 or 2473, 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 the polypeptide or protein of the present invention, or The nucleic acid molecules of the present invention have specific antibodies, which may exist alone or in combination with adjuvants, antiviral drugs, antibiotics, analgesics, bronchodilators or other pharmaceutically acceptable excipients.

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

5.7 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 an epitope or nucleic acid (such as mRNA, genomic RNA) of hSARS virus, This detects the presence of hSARS virus in the sample. A preferred reagent for detecting hSARS mRNA or genomic RNA of the present invention is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic RNA encoding a polypeptide of the present invention. The nucleic acid probe can be, for example, a nucleic acid molecule comprising or consisting of a SEQ ID NO: 1, 11, 13, 15, 2471 or 2473 nucleotide sequence or a portion thereof, such as a length of at least 15, 20, 25, 30, 50 , 100, 250, 500, 750, 1000 or more contiguous nucleotide oligonucleotides, which are 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 hSARS virus with SEQ ID NO: 15 genome nucleotide sequence), or based on the nucleotide sequence of SEQ ID NO: 1, 11, 13, 2471 or 2473. In a non-limiting specific embodiment, preferred primers for the RT-PCR method are: 5'-TACACACCTCAGCGTTG-3' (SEQ ID NO: 3) and 5'-CACGAACGTGACGAAT-3' (SEQ ID NO: 4) , in the presence of 2.5 mM MgCl ₂ , thermal cycling such as but not limited to 94°C for 8 minutes followed by 40 cycles of 94°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute (see also Sections 6.7 and 6.8 below). In a preferred embodiment, the primer comprises the nucleotide sequence of SEQ ID NO: 2475 and 2476 or the nucleotide sequence of SEQ ID NO: 2480 and 2481. In a preferred embodiment, the thermal cycle is 94°C for 10 minutes, followed by 40 cycles of 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 30 seconds, and 72°C for 10 minutes. In a preferred embodiment, the primer comprises the nucleotide sequences of SEQ ID NO: 2477 and 2478. 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 IDNO: Primers for 3 and 4 nucleotide sequences and fluorescent dyes such as Green I, which fluoresces when non-specifically bound to double-stranded DNA, subjected 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 for in vitro or in vivo detection of mRNA, protein (or any epitope) or genomic RNA in a sample. 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 compare the presence of hSARS or polypeptide or mRNA or genomic RNA encoding polypeptide in the control sample with hSARS or polypeptide or mRNA or genomic RNA encoding polypeptide in the test sample existence in comparison.

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 for testing and compared to those contained in the samples 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 in a model animal to inhibit or reduce the growth rate of other viruses is tested to test its specificity for hSARS virus.

6. Example

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

Methods and Results

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

6.1 Clinical objects

This study included a total of 50 patients meeting the revised World Health Organization (WHO) definition of SARS who were admitted to two acute disease regional hospitals in the Hong Kong Special Administrative Region (HKSAR) between February 26 and March 26, 2003 ( WHO. Severe acute respiratory syndrome (SARS) 2000, Weekly Epidemiol Rec. 78: 81-83). The study also included a lung biopsy from another patient who had typical SARS and was admitted to a third hospital. Briefly, the case definition of SARS 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 empiric antibiotic drugs (beta-lactam and macrolide drugs, fluoroquinolones, or tetracyclines) covering 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 serum for microbiological tests of other diseases were included in this study in a blinded condition 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. Virions were identified in cells infected with two clinical samples, but not in control cells not infected with the virus. Virions 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. Virions 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 a virion with characteristics 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

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

DNA聚合酶(Applied Biosystems)、2.5μCi[α-³²P]CTP(Amersham)、2μl10μM引物(5′-GCCGGAGCTCTGCAGAATTC-3′：SEQ ID NO：6)。按以下程序对反应进行热循环：94℃8分钟，然后94℃1分钟、40℃1分钟，72℃2分钟的循环2次。该热循环后，进行94℃1分钟、60℃1分钟、72℃1分钟的循环35次。取6μlPCR产物进行5％变性聚丙烯酰胺凝胶电泳分析。将凝胶对X光片曝光，X光片曝光过夜后显影。将只在感染细胞样品中鉴定出的独特PCR产物从凝胶中分离出来，用50μl 1x TE缓冲液洗脱。然后将洗脱的PCR产物在含有以下成分的25μl反应混合物中再扩增：2.5μl10xPCR缓冲液、4μl 25mM MgCl₂、0.5μl 10mM dNTP、0.25μl AmpliTaqDNA聚合酶(Applied Biosystems)、1μl10μM引物(5′-GCCGGAGCTCTGCAGAATT-C-3′：SEQ ID NO：6)。按以下程序对反应进行热循环：94℃8分钟，然后94℃1分钟、60℃1分钟、72℃1分钟的循环35次。用TOPO TA克隆试剂盒(Invitrogen)克隆PCR产物，将连接的质粒转化到TOP 10大肠杆菌(E.coli)感受态细胞(Invitrogen)中。PCR插入片段通过BigDye循环测序试剂盒按生产商(AppliedBiosystems)的建议进行测序，测序产物通过自动测序仪(AppliedBiosystems，型号3770)进行分析。获得的序列(SEQ ID NO：1)见图1。从获得的DNA序列推断的氨基酸序列(SEQ ID NO：2)显示，其与已鉴定冠状病毒的聚合酶蛋白有57％的同源性。Total RNA was harvested from infected or uninfected FrHK-4 cells two days after infection. Superscript II reverse transcriptase (Invitrogen) was used in a 20 μl reaction mixture containing 10 pg of a degenerate primer (5′-GCCGGAGCTCTGCAGAATTCNNNNNNN-3′, N=A, T, G or C; SEQ ID NO: 5) according to the manufacturer’s recommendation. 100 ng of purified RNA was reverse transcribed. The reverse transcript 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). Add 3 μl of purified cDNA product to 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

DNA polymerase (Applied Biosystems), 2.5 μCi [α- ³² P]CTP (Amersham), 2 μl 10 μM primer (5′-GCCGGAGCTCTGCAGAATTC-3′: SEQ ID NO: 6). The reaction was thermally cycled as follows: 94°C for 8 minutes, followed by 2 cycles of 94°C for 1 minute, 40°C for 1 minute, and 72°C for 2 minutes. After this thermal cycle, a cycle of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute was performed 35 times. 6 μl of PCR products were 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 product was 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 DNA polymerase (Applied Biosystems), 1 μl of 10 μM primer (5′-GCCGGAGCTCTGCAGAATT-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 NO: 11 and 13) and deduced amino acid sequences (respectively SEQ ID NO: 12 and 14) obtained from hSARS virus are shown in Figure 8 (SEQ ID NO: 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 in all three reading frames (SEQ ID NO: 16, 240 and 737) is shown in Figure 11 (SEQ ID NOs: 17-239, 241-736 and 738-1107). The deduced amino acid sequence of the complementary sequence of SEQ ID NO: 15 obtained in all three reading frames (SEQ NO: 1108, 1590 and 1965) is shown in Figure 12 (SEQ ID NO: 1109-1589, 1591-1964 and 1966-2470).

6.7 Detection of hSARS virus in nasopharyngeal aspirates

First, nasopharyngeal aspirates (NPA) were examined for influenza A and B, parainfluenza types 1, 2, and 3, respiratory syncytial virus, and adenovirus by rapid immunofluorescent antigen assay (Chan KH, Maldeis N, Pope W, Yup A, Ozinskas A.Gill J, Seto WH, Shortridge KF, Peiris JSM.Evaluation of Directigen Fly A+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. Raven Press, New York. 1993). Subsequently, fetal rhesus monkey kidney cells (FRhk-4) and A-549 cells were added to the array of cell lines used. Reverse transcription polymerase chain reaction (RT-PCR) directly on clinical samples for the detection of influenza virus A (Fouchier RA, BestebroerTM, Herfst S, Van Der Kemp L, Rimmelzwan GF, Osterhaus AD. Detection of influenza A virus from different species by PCRamplification 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 reverse-transcribed with primers 5′-TACACACCCTCAGCGTTG-3′ (SEQ ID NO: 3) and 5′-CACGAACGTG-ACGAAT-3′ (SEQ ID NO: 4) in Amplified in the presence of 2.5mM _MgCl2 (94°C for 8 minutes, then 40 cycles of 94°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute), the two primers were constructed based on the hSARS virus genome.

A typical RT-PCR protocol is outlined below:

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°C, 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 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

II逆转录酶(Invitrogen)按说明进行逆转录。然后互补DNA在

Green I荧光反应(Roche)混合物中进行扩增。简单的说，含有2μl的cDNA、3.5mmol/L MgCl₂、0.25μmol/L正向引物(5′-TACACACCTCAGCGTTG-3′；SEQ ID NO：3)和0.25μmol/L反向引物(5′-CACGAACGTGACGAAT-3′；SEQ ID NO：4)的20μl反应混合物中，用Light-Cycler(Roche)按PCR程序[95℃10分钟，然后95℃10分钟；57℃5秒；72℃9秒的循环50次]进行热循环。含有目标序列的质粒用作阳性对照。这些反应的荧光信号在每个循环的延伸步骤结束时捕捉。为确定试验的特异性，在试验结束时对PCR产物(184个碱基对)进行解链曲线分析(65℃至95℃，每秒0.1℃)。pass Total RNA was extracted from 140 μl nasopharyngeal aspirate (NPA) by viral RNA mini kit (Qiagen) according to the manufacturer's instructions. In a 20 μl reaction mixture containing 0.15 μg random hexamers, 10 mmol/L DTT and 0.5 mmol/L dNTP, 10 μl of the eluted RNA sample was washed with 200 U

II Reverse Transcriptase (Invitrogen) was used for reverse transcription as indicated. Complementary DNA is then

Amplification was performed in a Green I fluorescence 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 is captured at the end of the extension step of each cycle. To determine the specificity of the assay, the PCR product (184 base pairs) was subjected to melting curve analysis (65°C to 95°C, 0.1°C per second) at the end of the assay.

6.8 Detection of hSARS virus N-gene in patients

6.8.1 RT-PCR diagnostic method for coronavirus in SARS patients

Required equipment (96 samples):

1×SV Total RNA Isolation System

2×Mega titer plate

3×96-well PCR plate

1×0.5-10μl multichannel pipette

1×10-100μl multichannel pipette

1×20-200μl multichannel pipette

1×vacuum pump

1 x bucket rotor with micro plate bucket

2×PCR machine (compatible with 96-well plate)

1× gel electrophoresis apparatus

Phase 1* - Clinical Sample Processing (1 Medical Staff/Clinical Technician)

Samples from each subject in 500 μl Virus Transport Medium (2 g sodium bicarbonate, 5 g bovine serum albumin, 200 μg vancomycin, 18 μg amikacin, and 160 U nystatin per liter Earle's Balanced Salt Solution) Aliquot into wells of a 96-well mega titer plate with 500 μl of lysis buffer (1×) containing 100 μl of PK-15 cells in complete minimal essential medium (ATCC CCL-33; 5.0×10 ⁵ cells/ml), Earle's salts (EMEM, Invirtogen) were used as internal controls**.

• Mix lysate by pipetting 3 times.

• Go to Phase 2.

*Phase 1 should be performed in a Class III biological safety cabinet .

**At least two negative samples should be included in the 96-well platform as negative controls.

Phase 2 - Total RNA Extraction (1 lab technician)

●Set multi-head vacuuming device. Place the bonding plate on the Manifold Base.

• Transfer the lysate from the mega titer plate to the wells of the SV 96 binding plate (binding plate).

• Apply vacuum until lysate passes through binding plate. Remove the vacuum.

• Add 500 μl of SV RNA Wash Solution (Wash Solution) to each well of the Binding Plate.

• Apply vacuum until wash solution passes through binding plate. Remove vacuum.

● Prepare the DNase incubation mix for the entire 96-well plate as follows:

Yellow core buffer 2ml

0.09M _MnCl2 250μl

DNase I 250μl

• Apply 25 μl of freshly prepared DNase incubation mix directly to the membrane of the binding plate.

• Incubate for 10 minutes at 20-25°C.

●Add 200 μl of SV DNase Stop Solution to each well of the binding plate.

• Apply vacuum until SV DNase Stop Solution passes through binding plate. Remove vacuum.

• Add 500 [mu]l of wash solution to each well of the binding plate.

• Apply vacuum until wash solution passes through binding plate. Turn off the vacuum.

• Centrifuge binding plate at 3000 xg for 30 seconds to remove residual wash solution.

• Transfer the binding plate to the top of a 96-well RT plate.

• Add 50 μl of nuclease-free water to each well of the binding plate to elute the RNA.

• Incubate for 1 minute at room temperature.

• Centrifuge the binding plate at 3000 x g for 1 min at 4°C.

• Collect the eluted RNA into a 96-well RT plate.

• Add 5 μl 3M sodium acetate and 200 μl 95% ethanol to each well of the plate.

●Place RT plate on ice and incubate for 15 minutes.

• Centrifuge plate at 3000 xg for 15 minutes at 4°C.

• Discard the supernatant by inverting the plate and blot dry on a clean paper towel.

• Wash the pellet with 200 μl 70% ethanol.

• Centrifuge plate at 3000 xg for 10 minutes at 4°C.

• Discard the supernatant by inverting the plate and blot dry on a clean paper towel.

●Air dry the pellet for 5 minutes.

• Add 12 [mu]l nuclease-free water to each well.

• Vortex the plate briefly to dissolve the pellet (see Figure 18 for example results).

• Go to Phase 3.

Phase 3 - Reverse Transcription (1 Lab Technician)

● Prepare the RT universal mix for the entire 96-well plate (100 reactions) in 1.5-ml tubes as follows:

Each response ×100

Random hexamer, 3μg/μl 0.05μl 5μl

DNTP, 10mM 1μl 100μl

First strand buffer, 5× 4μl 400μl

DTT, 0.1M 2μl 200μl

Superscript II, 200U/μl 1μl 100μl

Total 8.05μl 805μl

• Aliquot 100 μl RT mix into 8 wells of a clean 96-well universal mix plate.

• Transfer 8.05 μl RT mix from the plate to each well of the RT plate containing 12 μl RNA, mix by pipetting 3 times with a multichannel pipette. Replace tips after each transfer.

• Incubate samples at 42°C for 50 minutes, then at 70°C for 15 minutes.

• Go to Phase 4.

Phase 4-N-gene-specific PCR (1 lab technician)

Prepare the PCR master mix for the entire 96-well plate (100 reactions) in two 2059 culture tubes as follows:

• N-gene specific PCR and control PCR were performed in two separate PCR plates.

• Aliquot 290 μl of PCR Master Mix into the first column of a 96-well PCR plate.

• Aliquot 24 μl of the universal mix from the first column into the wells of the PCR plate.

• Transfer 1 μl of cDNA template (from stage 4) to each well of the PCR plate.

• Mix by pipetting 3 times with a multichannel pipette. Replace tips after each transfer.

• Seal the plate with sealing tape.

● Perform the following reactions in two 96-well PCR machines:

N-gene-specific PCR control PCR

10 minutes at 94°C 10 minutes at 94°C

●Stage 5 - gel electrophoresis (1 lab technician)

● Mix 5 μl of N-gene-specific PCR product and 5 μl of control PCR product with 1 μl of bromophenol blue loading dye.

• Load samples into wells of a 2% agarose gel.

• Electrophoresis of PCR product at 140V, 250mA for 30 minutes.

• Stain the gel with ethidium bromide.

• Visualize the product with UV and record the result.

6.8.2 Primers using SEQ ID NO: 2480 and 2481

The RT-PCR diagnostic method was performed as described in Section 6.8.1, but with certain modifications.

RNA isolation from clinical samples

Clinical samples including nasopharyngeal aspirates (NPA) and stool samples were provided by the Department of Microbiology, University of Hong Kong. In addition, tracheal dispersions and lung biopsies from pivotal Patient A at three time points as described in New Engl. J Med. 348: 1967-76 (Drosten CS et al., 2003) were also collected. Sample collection was performed from April 1 to April 28, 2003 in a local hospital. The method of sample collection was as described in the previous section (see also Poon et al., 2003, Clinical Chemistry 49:953-955). Extraction of total RNA from clinical samples was performed using the SV96 Total RNA Isolation System (Promega, WI, USA) with the following modifications to the manufacturer's protocol. NPA/feces samples in 500 μl of virus transport medium (containing 2 g sodium bicarbonate, 5 g bovine serum albumin, 200 μg vancomycin, 18 μg amikacin, and 160 U nystatin per liter of Earle’s balanced salt solution) were compared with et al. Volume SV RNA lysis buffer mixed with 100 μl of porcine kidney epithelial (PK-15) cells (ATCC CCL-33; 5.0×10 ⁵ cells/ml) in complete minimal essential medium in Earle's salt (EMEM , Invitrogen) as an internal control. The mixture was transferred to the wells of an SV 96 binding plate. After washing with 500 [mu]l of SVRNA wash solution, the plate was centrifuged at 3000 xg for 30 seconds to remove residual wash solution before the elution step. The RNA was then eluted with 50 μl of nuclease-free water, and the RNA was collected in a clean 96-well PCR plate by centrifuging the plate at 3000×g for 1 min. The eluted RNA was concentrated by incubation on ice for 15 minutes in the presence of 5 μl 3M sodium acetate and 200 μl 95% ethanol. After centrifugation at 3000×g for 15 min at 4°C, the RNA pellet was washed with 200 μl of 75% ethanol and dissolved in 12 μl of nuclease-free water. The extracted RNA is immediately reverse transcribed into first-strand cDNA.

Synthesis of first-strand cDNA

II逆转录酶(Invitrogen，USA)，在含有0.15μg随机六聚物、RT缓冲液(1×)、10mM二硫苏糖醇(DTT)和0.5mM三磷酸脱氧核苷酸(dNTP)的20μl反应液中进行逆转录。反应在Peltier Thermal Cycler(MJ Research)中以下述条件进行：42℃50分钟，然后70℃15分钟。Use 200U

II reverse transcriptase (Invitrogen, USA), in 20 μl containing 0.15 μg random hexamers, RT buffer (1×), 10 mM dithiothreitol (DTT) and 0.5 mM deoxynucleotide triphosphate (dNTP) Reverse transcription was carried out in the reaction solution. The reaction was carried out in a Peltier Thermal Cycler (MJ Research) under the following conditions: 42°C for 50 minutes, then 70°C for 15 minutes.

Polymerase Chain Reaction (PCR)

Primers were designed based on the whole SARSCoV genome sequence of the previously announced local sample HK-39 (Accession No. AY278491). Forward primer (SRS251: 5'-GCAGTCAAGCCTCTTCTCG-3'; SEQ ID NO: 2480, corresponding to HK-39SARS 28658-28676 nucleotides of the genome of CCTCC200303), reverse primer (SRS252: 5'-GCCTCAGCAGCAGATTTC-3 ', SEQ ID NO: 2481; corresponding to the 28866-28883 nucleotides of the HK-39 SARS genome) amplifies the 225bp fragment from the N-gene region, and this fragment does not show homology with other coronaviruses. Primers for amplifying RNA-dependent RNA polymerase (1b gene) were used as parallel controls (coro3: 5'-TACACACCCTCAGCGTTG-3' (SEQ ID NO: 3), corresponding to 18041-18057 nucleotides; and coro4: 5' -CACGAACGTGACGAAT-3' (SEQ ID NO: 4), corresponding to 18207-18222 nucleotides, Department of Microbiology, University of Hong Kong). Both amplicons were cloned into the same pCR2.1 cloning vector (Figure 17). Serially diluted plasmids were used to determine the dynamic range and optimal conditions for PCR (Figures 21A and 21B). Another set of primers for amplifying a 745bp fragment from the porcine β-actin gene was used as an internal control for the diagnostic PCR assay (actin-F: 5'TGAGACCTTCAACACGCC-3' (SEQ ID NO: 2482); and actin-R: 5 '-ATCTGCTGGAAGGTGGAC-3' (SEQ ID NO: 2483)).

Routine PCR and gel electrophoresis were performed as preliminary experiments. Briefly, 0.5 U recombinant Taq DNA polymerase (Invitrogen Life Technologies) was used in a 25 μl reaction containing PCR buffer (1×), 1.5 m MgCl ₂ , 0.1 mM dNTPs, and 0.5 pmol each of the forward and reverse primers 1 μl of cDNA from clinical samples was amplified. Reactions were performed in a Peltier Thermal Cycler (MJ Research) at 94°C for 3 min, followed by 50 cycles of 94°C for 10 s, 56°C for 10 s, 72°C for 10 s, and a final elongation step at 72°C for 10 min. Amplicons were analyzed by 2% agarose gel electrophoresis (Figure 23). For quantitative real-time PCR in the diagnosis of clinical samples A green fluorophore is performed. In a 25 μl reaction, 1 μl cDNA template was mixed with 12.5 μl (2×) Green PCR Universal Mix (Applied Biosystems) and 0.5 pmol each of forward and reverse primers. The volume of the reaction solution was adjusted to 25 μl with distilled water. The reaction was carried out in the iCycler iQ real-time PCR detection system (Bio-Rad) under the same conditions as conventional PCR. Fluorescence signals (FAM, Excitation = 490nm, Emission = 530nm) were collected at the end of each elongation step during PCR cycling (Fig. 22A). The threshold cycle number (Ct) was determined for each sample using the maximum curvature method. Melting curve analysis was performed after a final extension of 10 min (Figure 22B). Non-SARS patients, including those infected with adenovirus (n=5), respiratory syncytial virus (n=5), human metapneumovirus (n=5), influenza A virus (n=5), or influenza B virus (n=5) 5), the cDNA from them was used as a negative control for the experiment.

Northern blot analysis

试剂(Invitrogen Life Technologies)根据厂家的方法从细胞提取总RNA。通过在含3.7％甲醛的1％琼脂糖凝胶上电泳分离8μg总RNA。RNA通过毛细管印迹转移至正电性尼龙膜(Roche DiagnosticCorporation)上，通过UV交联固定。用相同RNA样品合成的cDNA用作探针合成的模板。四对从1b(核苷酸18057-18222；SEQ ID NO：2484)、S(核苷酸21920-22107；SEQ ID NO：2485)、M(核苷酸26867-26996；SEQ ID NO：2486)和N(核苷酸28658-28883；SEQ IDNO：2487)基因扩增片段的引物用于探针合成。探针的DIG-标记、杂交和条带的检测用地高辛系统(Roche Molecular Biochemcials)根据厂家的方法进行。信号用化学发光法分析(图24)。Vero cells infected by SARS-CoV HK-39 strain were provided by the Department of Microbiology, University of Hong Kong. use

Reagents (Invitrogen Life Technologies) were used to extract total RNA from cells according to the manufacturer's protocol. 8 μg of total RNA was separated by electrophoresis on a 1% agarose gel containing 3.7% formaldehyde. RNA was transferred to a positively charged nylon membrane (Roche Diagnostic Corporation) by capillary blotting, and fixed by UV crosslinking. cDNA synthesized from the same RNA sample was used as a template for probe synthesis. Four pairs from 1b (nucleotides 18057-18222; SEQ ID NO: 2484), S (nucleotides 21920-22107; SEQ ID NO: 2485), M (nucleotides 26867-26996; SEQ ID NO: 2486) Primers for the amplified fragment of the and N (nucleotides 28658-28883; SEQ ID NO: 2487) genes were used for probe synthesis. DIG-labeling of probes, hybridization and detection of bands were performed with the Digoxigenin system (Roche Molecular Biochemcials) according to the manufacturer's protocol. Signals were analyzed by chemiluminescence (Figure 24).

Results and discussion

Large-scale RT-PCR assays provide a rapid method for monitoring and screening patients suspected of SARS. The results can be used to supplement the clinical diagnostic assessment. For diagnostic purposes, the test should be reliable and its accuracy should be guaranteed to prevent false negative and false positive results. However, the accuracy of the test can be affected by certain factors. A common technical problem with PCR is the failure of amplification due to the presence of PCR inhibitors (see Figure 21).

These PCR inhibitors include heme compounds found in blood, aqueous and vitreous humor, heparin, EDTA, urine and polyamines (Fredricks et al., 1998, J Clin. Micro. 36:2810-16). Currently, NPA or fecal samples are collected in transport media to preserve virion viability. In the pilot experiment, when the extracted total RNA was directly used for first-strand cDNA synthesis without any treatment, RT-PCR was inhibited (25 out of 27 samples). However, the amplified DNA was retained after a simple ethanol precipitation step (Figure 19). The same results were obtained using the SV or SV96 total RNA isolation systems (data not shown). This suggests that certain components in the culture medium or NPA/fecal samples can affect downstream processes of diagnostic testing.

In addition, this sample collection method dilutes the viral titer in the sample, especially early in the infection, when the virus titer in the nose and throat swab samples is low (Drosten et al., 2003, New England Journal of Medicine, available on the Internet at http://content.nejm.org/cgi/reprint/NEJMoa030747v2). This suggests that the sensitivity of the PCR test for SARS depends on the quality of the sample and the timing of the test during the course of the disease. To increase the sensitivity of the test, total RNA isolated from clinical samples was concentrated prior to first-strand cDNA synthesis.

To avoid false-negative PCR results due to failures during RNA isolation and first-strand cDNA synthesis, total RNA was extracted in parallel from clinical samples and PK-15 mammalian cells. Figure 23 shows the results of RT-PCR screening of 48 clinical samples, including NPA and fecal samples. Diagnostic PCR was performed in parallel with β-actin PCR. All samples were positive in β-actin PCR. This result demonstrates that RNA and cDNA can be successfully extracted and synthesized from samples in the single-step method disclosed herein. Based on this internal control, isolation of total RNA and synthesis of cDNA from the sample is ensured, which eliminates false negatives resulting from failure of either of the above procedures. Moreover, the 96-well assay format developed in this paper can adopt a high-throughput screening method, so that we can obtain the diagnostic results of more than 90 clinical samples within 3 hours by 1 clinician, while existing methods process in individual tubes Samples, each technician can only process about 30-50 samples per day.

Real-time quantitative PCR assays are more sensitive than conventional agarose gel electrophoresis combined with PCR assays (Poon et al., J Clin. Virol. 28:233-8), and thus were used for SARS-CoV diagnostic purposes. Positive signals were detected in 38 of 136 randomly selected clinical samples in N-gene and 1b-gene specific PCR. Of these 38 positives, 3 were fecal samples (2.2%) and 35 were NPA samples (25.7%). The detection rates of the assays using N-gene-specific RT-PCR at different time points are shown in Table 2.

Table 2

The positivity of these 38 positive cases was confirmed by melting curve analysis of PCR products. The specific melting temperatures of the N gene and 1b gene PCR products (85.5°C and 80.5°C, respectively) indicated that the target fragment was amplified in the reaction. The specificity of the assay was also confirmed by samples from non-SARS patients, including patients with adenovirus (n=5), respiratory syncytial virus (n=5), human metapneumovirus (n=5), influenza A Virus (n=5) and influenza B virus (n=5) patients. The results showed that all of these samples were negative in the assay (Figure??). These results suggest that the N-gene-specific RT-PCR assay is specific for the diagnosis of SARS-CoV.

Furthermore, we also demonstrated that N-gene-specific PCR was more sensitive than PCR amplification of the 1b RNA polymerase gene. Amplification conditions for both PCR experiments were first optimized with plasmid constructs containing 1:1 1b- and N-gene fragments (see Figure 22) (see Figure 20). The dynamic range of the N-gene-specific PCR was obtained (Fig. ??), and its Ct value was found to be lower than that of the 1b-specific PCR. This shows that N-gene-specific PCR can achieve higher amplification efficiency than 1b-gene-specific PCR when using the same copy number of templates. PCR was then performed with cDNA from clinical samples or virus-infected Vero cells. Figure 22A shows the Ct and half maxima of the N gene and Ib gene specific PCR fluorescence signals generated from NPA, tracheal dispersion and lung biopsies from Patient A. The results showed that among all the positive samples, the fluorescent signal generated in the N gene-specific PCR was higher than that in the 1b-specific PCR (26.0% on average, varying within 6.3-60%). Moreover, the Ct values of N gene-specific PCR were lower than those of 1b-specific PCR (0.1–4.6 cycles) in most SARS-CoV-positive samples (Table 3).

table 3

ΔCt=1.49±0.47, 95% confidence interval=0.74-2.23 (F-test)

Statistical analysis showed that the Ct of the N-gene PCR assay was significantly lower than that of the ab-gene assay (95% confidence interval = 0.74-2.23, F-test). The stronger fluorescent signal and lower Ct value of N gene-specific PCR provide more sensitive diagnostic results and a large number of targets for the test.

Using cDNA from SARS-CoV-infected Vero cells, the amplification curves shown in Figure 21B demonstrate the difference between N gene and 1b gene-specific PCR. The Ct of N gene and 1b gene specific PCR were 35.3 and 37.8, respectively. This phenomenon has two main reasons: (1) the expression level of the N gene is higher than that of the 1b gene; (2) the copy number of the N gene is much higher than that of the 1b gene, because each transcript in SARS-CoV-infected cells is followed by a A copy of the N gene. Northern blot analysis supported this hypothesis (Figure 24). When N-gene specific PCR products were used as probes, at least 5 transcripts from viruses were hybridized and positive signals were obtained (Fig. 24). This result is consistent with the finding that five subgenomic mRNAs were detected by Northern hybridization of RNA from SARS-CoV-infected cells using a probe derived from the 3′ untranslated region (Rota et al., 2003, Science 300:1394 -99). On the other hand, when the 1b PCR product was used as a probe, only the 2 large molecular weight transcripts were hybridized, indicating that the copy number of the N gene was much higher than that of the 1b gene during transcription and gene expression in host cells. The results of Northern blot strongly support the conclusion that, as a target for diagnostic screening, the PCR-amplified region of the N gene of SARS-CoV is more sensitive than other regions. It is possible that amplification of more than one genomic region may increase the specificity of detection (Yam W.C. et al., 2003, J: Clin. Microbiol. 41:4521-24).

In conclusion, we have developed a new generation of RT-PCR diagnostic assays that are more sensitive for detecting SARS-associated coronaviruses than traditional diagnostic tests. The test method of the present invention provides a high-throughput, high-sensitivity screening platform, which enables us to expand our scale to detect tens of thousands of suspected SARS cases every day in a single working line. The addition of PK-15 cells to the assay as an internal control and the use of the N gene and 1b gene as diagnostic targets enhanced the sensitivity and accuracy of the test. We used a 96-well real-time quantitative PCR and sequencing format to shorten the time required for testing and obtain information on viral genotypic variation.

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 4). 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 examination, auscultation revealed crepitus and decreased air intake in only 38% of patients. 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 4

*1 patient developed a maculopapular rash on the trunk.

Most patients (98%) had no evidence of leukocytosis despite high fever. Peripheral blood examination revealed lymphopenia (68%), leucopenia (26%), thrombocytopenia (40%), and anemia (18%) (Table 5). The liver parenchymal enzyme alanine transaminase (ALT) and the muscle enzyme creatinine kinase (CPK) were elevated in 34% and 26% of cases, respectively.

table 5

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 6). All complicated cases were treated with ribavirin and steroids after admission to the intensive care unit, whereas all noncomplicated 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 6

*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. Despite oral azithromycin, amantadine, and intravenous ceftriaxone, he experienced increased bilateral lung infiltrates and progressive oxygen desaturation. Therefore, an open lung biopsy was performed 9 days after his admission. Histopathological examination revealed moderate interstitial inflammation, and scattered alveolar cells exhibited cytomegalomorphism, granular amphichromatic cytoplasm, enlarged nuclei, and prominent nucleoli. None of the cells showed inclusion bodies typical of herpesvirus or adenovirus infection. The patient underwent surgery and required ventilation and intensive care. He was given empiric intravenous ribavirin and hydrocortisone. But he still died 20 days after being admitted to the hospital. Coronavirus-like RNA was found in his nasopharyngeal aspirate, lung biopsy, and postmortem lungs at retrospective time. His antibody titer against his own hSARS isolate increased significantly from 1/200 to 1/1600.

The second patient from whom hSARS virus was isolated was a 42-year-old female with good previous health. She traveled to Guangzhou in mainland China for two days and developed fever and diarrhea five days after returning to Hong Kong. Physical examination of her revealed crepitus in the right lower lung region with corresponding alveolar opacities on chest radiographs. 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 on FRhk-4 cells from lung biopsies and nasopharyngeal aspirates of the two patients mentioned above, respectively. The initial cytopathic effect appeared 2 to 4 days after inoculation, but upon subsequent passages, the cytopathic effect appeared within 24 hours. Neither virus isolate reacted with a range of reagents routinely used to identify virus isolates, including those used to identify influenza A, B, parainfluenza types 1, 2, and 3, adenovirus, and respiratory syncytial virus reagents (DAKO, Glostrup, Denmark). The two virus isolates also did not react in RT-PCR assays for influenza A and HMPV, or in PCR assays for Mycoplasma. The virus is sensitive to ether, indicating that it is an enveloped virus. Electron microscopy of negative-staining (2% potassium phosphotungstate, pH 7.0) cell culture extracts obtained by ultracentrifugation revealed the presence of polytypic enveloped virions, 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 virions with a diameter of 55-90 nm inside slippery-walled vesicles in the cytoplasm (Fig. 5B). Virions are also seen 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 60-90 nm virions in the cytoplasm of desquamated cells. These virions 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 the protein sequences revealed that this virus, although most closely related to class II coronaviruses, is a distinct virus (Figures 5a and 5b).

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

To determine the dynamic range of real-time quantitative PCR, serial dilutions of plasmid DNA containing the target sequence were prepared and subjected to real-time quantitative PCR experiments. As shown in Figure 7A, the assay was able to detect as few as 10 copies of the target sequence. In contrast, no signal was observed in the water control (Fig. 7A). Positive signals were observed in 23 of 29 serologically confirmed SARS patients. In all these positive cases, a unique PCR product ( _Tm = 82°C) corresponding to the signal of the positive control was observed (Fig. 7B, data not shown). These 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 very unlikely that these primers will cross-react with the OC43 strain.

It has been reported that, in addition to the novel pathogen, metapneumoviruses were identified in some SARS patients (Center for Disease Control and Prevention, 2003, Morbidity and Mortality Weekly Report 52269-272). 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.

When seropositivity for HP-CV in a single serum or detection of viral RNA in NPA or stool was 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 groups 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 is based on a 646 base pair fragment of the polymerase gene (SEQ ID NO: 1), suggesting that the virus is related to antigenic class 2 of coronaviruses, as well as murine hepatitis virus and bovine coronavirus. However, the Coronaviridae virus can carry out heterologous recombination within the virus family, so it is necessary to carry out genetic analysis to other parts of the genome of the novel virus, and then more conclusively define the nature of 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. Eds Knipe 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, 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. Humancoronavirus OC43 and 229E persistence in neural cell cultures and humanbrains. Adv Exp Med Biol. To be published). 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 ArchVirol. 1977;54:107- 17). The high incidence of liver dysfunction, leukopenia, marked lymphopenia, thrombocytopenia, and subsequent development of adult respiratory distress syndrome suggested that this hSARS virus caused severe systemic inflammatory damage. Therefore it is important to complement antiviral therapy with ribavirin by immunomodulation with steroids. In this regard, it is also appropriate to assume that severe human disease associated with avian influenza H5N1 subtype (another virus that has recently crossed from animals to humans) has an immunopathological component (Cheung CY, Poon LLM, Lau ASY et al , Induction of proinflammatory cytokines in human macrophages by influenza A(H5N1) viruses: a mechanism for the unusual severity of human disease. Lancet 2002; 360: 1831-1837). Like H5N1 disease, severe SARS patients are also adults with more pronounced lymphopenia and features of organ dysfunction other than the respiratory tract (Table 4) (Yuen KY, ChanPKS, 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).

7. Preservation

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

8. Market potential

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

9. Equivalent scheme

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

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference 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 (13)

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

2. the nucleic acid molecule of claim 1, wherein said molecule is RNA.

3. the nucleic acid molecule of claim 1, wherein said molecule is DNA.

4. isolated polypeptide, described polypeptide is by the nucleic acid molecule encoding of claim 1.

5. an antibody or its Fab, described antibody or its Fab immunologic opsonin are in conjunction with the polypeptide of claim 4.

6. by SEQ ID NO:2471 nucleotide sequence or its complementary sequence or comprise SEQID NO:2480 and/or nucleic acid probe that the nucleic acid molecule of 2481 nucleotide sequences is formed is used for detecting purposes in the test kit of hSARS virus at biological sample in preparation.

7. the purposes of claim 6, wherein said nucleic acid probe is the nucleic acid molecule that comprises SEQ ID NO:2480 and/or 2481 nucleotide sequences.

8. immunologic opsonin is used for detecting purposes in the test kit of hSARS virus at sample in preparation in conjunction with the antibody of the polypeptide of claim 4 or Fab.

9. the purposes of claim 8, wherein the compound in conjunction with polypeptide is an antibody.

10. the primer of nucleotide sequence of nucleic acid molecule that derives from claim 1 is to being used for identifying by the test of reverse transcription and PCR in real time the purposes of the test kit of hSARS virus infection object in preparation.

11. the purposes of claim 10, wherein said primer is to being made up of SEQ ID NO:2480 and 2481 nucleotide sequences respectively.

12. test kit, described test kit comprises one or more and is applicable to the isolated nucleic acid molecule that detects hSARS N gene in one or more containers, wherein said nucleic acid molecule is made up of the nucleotide sequence of SEQ ID NO:2480 and/or SEQ ID NO:2481.

13. a test kit, described test kit comprise the antibody of one or more claims 5 in one or more containers.

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