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

#The instant application contains a lengthy Sequence listing which is being concurrently submitted via triplicate CD-R in lieu of a printed paper copy, and is hereby inconrporated by reference in its entirety. Said CD-R, recorded on Mar. 22, 2004, are labeled “CRF”, “Copy 1” and “Copy 2”, respectively, and each contains only identical 1.6 MB file (V9661077.APP).

#The present invention relates to a high-throughput diagnostic assay for the virus causing Severe Acute Respiratory Syndrome (SARS) in humans (“hSARS virus”). In particular, the invention relates to a high-throughput reverse transcription-PCR diagnostic test for SARS associated coronavirus (SARS-CoV). The present assay is a rapid, reliable assay which may be used for diagnosis and monitoring the spread of SARS. The present method eliminates false negative results and provides increased sensitivity for the assay. The invention further relates to nucleotide sequences and portions thereof, useful for the diagnosis of SARS. The invention further relates to nucleotide sequences and portions thereof, useful for assessing genetic diversity of SARS. The nucleotide sequences of the present invention comprise the (Nucleocapsid) N-gene and the S-gene sequences of the hSARS virus. The invention relates to a diagnostic kit that comprises nucleic acid molecules for the detection of the N-gene or S-gene of hSARS virus. The invention also relates to the deduced amino acid sequences of the N-gene and S-gene products of the hSARS virus. The invention further relates to the use of the N-gene and S-gene products in diagnostic methods. The invention further encompasses diagnostic assays and kits comprising antibodies generated against the N-gene or S-gene product.

#Recently, there has been an outbreak of atypical pneumonia in Guangdong province in mainland China. Between November 2002 and March 2003, there were 792 reported cases with 31 fatalities (WHO. Severe Acute Respiratory Syndrome (SARS) Weekly Epidemiol Rec. 2003; 78: 86). Patients with SARS show various clinical symptoms, including fever (of 38 degrees Celsius or above for over 24 hours), malaise, chills, headache and body ache. Chest X-rays show changes compatible with pneumonia. Other symptoms include coughing, shortness of breath or difficulty in breathing. By 3 May 2003, a cumulative total number of 1621 cases and 179 deaths had been occurred in Hong Kong, which contributed to 26% and 41% of the global reported cases (6234) and deaths (435) respectively. As the disease is highly contagious and spreads in daily-life activities, it is important to develop a rapid and reliable diagnosis test to monitor and control the disease. In response to this crisis, the Hospital Authority in Hong Kong has increased the surveillance on patients with severe atypical pneumonia. In the course of this investigation, a number of clusters of health care workers with the disease were identified. In addition, there were clusters of pneumonia incidents among persons in close contact with those infected. The disease was unusual in its severity and its progression in spite of the antibiotic treatment typical for the bacterial pathogens that are known to be commonly associated with atypical pneumonia. The present inventors were one of the groups involved in the investigation of these patients. All tests for identifying commonly recognized viruses and bacteria were negative in these patients. Furthermore, diagnostic tests for the detection of other genes in the hSARS virus, such as the 1b-gene are not useful to accurately diagnose SARS. The disease was given the acronym Severe Acute Respiratory Syndrome (“SARS”). This virus mutates and changes rapidly and hence the diagnostic of SARS was extremely difficult until the isolation of particular regions of the virus, the N-gene and S-gene, of the hSARS virus from the SARS patients by the present inventors as disclosed herein. Namely, the present invention discloses a diagnostic assay using particular regions in the genome of the virus for rapid, accurate, reliable and specific identification of the hSARS virus. The invention is useful in both clinical and scientific research applications. Furthermore, the present invention provides a high-throughput assay which can be used as a sensitive method for diagnosis and monitoring the spread of the SARS.

#FIG. 1 shows a partial DNA sequence (SEQ ID NO:1) and its deduced amino acid sequence (SEQ ID NO:2) obtained from the SARS virus that has 57% homology to the RNA-dependent RNA polymerase protein of known Coronaviruses.

#FIG. 2 shows an electron micrograph of the novel hSARS virus that has similar morphological characteristics of coronaviruses.

#FIG. 3 shows an immunofluorescent staining for IgG antibodies that are specifically bound to the FrHK-4 cells infected with the novel human respiratory virus of Coronaviridae.

#The present inventors developed a rapid, high-throughput reverse transcription-PCR diagnostic test for SARS associated coronavirus (SARS-CoV). 3′ region of the hSARS virus genome including the Nucleocapsid gene (N-gene) represents a sensitive molecular marker which can be used in addition to 1b gene to increase the sensitivity of the test. An internal control using PK-15 cells may be employed to ensure the integrity of RNA during its extraction process and cDNA synthesis, thus eliminating false negative results.

#In mouse hepatitis virus (MHV), a typical member of the genus Coronavirus, both genomic RNA and mRNA transcripts are capped and with common 3′ ends and common leader sequences on their 5′ ends. With this unique transcription strategy, the copy numbers of different viral genes during proliferation of virus in its host are different (FIG. 19). N gene that encodes for the nucleocapsid has the most abundant copy number during virus replication as all transcripts may carry nucleotide sequence from N gene, although they are not all in-frame for translation for this gene product. The present inventors have discovered a diagnostic assay that is based on the 3′ region, including the N-gene, of the viral genome provide a more sensitive assay than the rest of the viral genome. Accordingly, in preferred embodiments, nucleic acid molecules that may be used for a diagnostic assay comprise nucleic acid sequence of nucleotide position 18000 to 29742 of SEQ ID NO:15, or portions thereof. The portions may comprise 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 of nucleotides having nucleic acid sequence from nucleotide position 18000 to 29742 of SEQ ID NO:15. In other preferred embodiments, nucleic acid molecules that may be used for a diagnostic assay comprising nucleic acid sequence of nucleotide position 28658 to 28883 or 29247-29410 of SEQ ID NO:15.

#Nasopharyngeal aspirate (NPA) and stool samples were obtained from SARS suspected patients with major clinical symptoms and significant history of close contact with infected patients. Total RNA was extracted from the subject samples, together with PK-15 cell as an internal control. Samples were analyzed by the reverse-transcription-PCR assay. Northern blot analysis was performed to show different subgenomic transcripts of the virus. Real-time quantitative PCR was employed to compare the sensitivity of two loci used in this diagnostic assay. In specific embodiments, PCR inhibitor was removed with ethanol precipitation after RNA extraction process.