| The present study is to investigate the properties of SARS coronavirus (SARS-CoV) RNA and specific antibody detection profiles, SARS-CoV isolation, as well as S-gene and N-gene genetic variation from clinical specimens of patients with severe acute respiratory syndrome (SARS).1. SARS-CoV RNA and specific antibody detections from specimens of clinically diagnosed SARS patients. By using nested reverse transcription-polymerase chain reaction (nested RT-PCR), 486 various specimens collected from 54 patients with a clinical diagnosis of SARS were investigated for the presence of SARS-CoV RNA. Combination of our designed N-gene-targeting primers based on BJ01 strain of SARS-CoV and World Health Organization (WHO) recommended R-gene-targeting primers significantly increased the viral RNA detectable rate from clinical specimens, resulting the viral RNA detectable in the 28 patients. Higher positive rate of SARS-CoV RNA than anti-SARS-CoVantibody makes the viral RNA detection more valuable for early-stage diagnosis.2. Cultural isolation of SARS-CoV in urine and stool specimens from patients with SARS. Twenty-one stool and urine specimens collected from 12 SARS patients were employed for cultural isolation of SARS-CoV with VeroE6 cells. Nested RT-PCR showed that 16 out of the 21 specimens were positive for viral RNA. An improved blind passage assay was used to increase the sensitivity of the method. As a result, viable SARS-CoV was isolated from RNA-positive 10 specimens, including one urine and two stool specimens collected from three convalescent-phase patients more than 4 weeks after disease onset (29-36 days), suggesting that excretions of the convalescent patients may remain infectious.3. Cloning of SARS-CoV S gene from the first-cluster SARS patients in Beijing. The throat swab from one of the first-cluster SARS patients in Beijing was employed for cell inoculation. RNA exacted from culture supernatant was used for the template of nested RT-PCR, by which 6 overlapping fragments covering S-gene sequence of SARS-CoV was amplified. Intact S gene was linkedand cloned with these fragments by several rounds of overlapping PCR, using DNA polymerase of high-fidelity. The cloned S gene had identical sequence with that of BJOl stain of SARS-CoV.4. Genetic variation analysis of SARS-CoV S gene directly obtained from SARS patients. The full-length S-gene sequences of SARS-CoV were amplified as 6 overlapping fragments by nested RT-PCR, using the templates of 29 RNA samples directly extracted from specimens of 20 SARS patients. Both a TA-cloning assay and direct screening of PCR products were performed for sequence analysis. A total of 113 sequence variations with 9 variant sites were identified in analyzed sequences compared with the S gene of the BJO 1 strain. Among these, eight were first documented in humans, including 6 nonsynonymous variants. These newly identified variant S-gene sequences were registered in GenBank as BJ302Clonel-8, individually. Most importantly, the results suggested that SARS-CoV might consist of complex and dynamic distributions of mutants in vivo, the typical characteristic of quasispecies. Phylogenetic analysis based on the S gene showed that the variant sequences were closest to the isolates from the Beijing and Guangdong areas in China.5. Genetic variation analysis of SARS-CoV N gene directly obtained from SARS patients. The full-length N-gene sequences of SARS-CoV were amplified as 3 overlapping fragments by nested RT-PCR, using the templates of 12 RNA samples from 12 individual SARS patients. Three sequence variations with 2 variant sites, including 1 nonsynonymous one, were identified in analyzed sequences compared with the BJOl strain. In comparison with S gene, N gene had a much lower variation occurrence, suggesting that its stability is important.Taken together, the findings furthers the knowledge of improving virological diagnostic assay for SARS, understanding the viral profile and characterizing genetic variation and quasispecies of SARS-CoV in patients with SARS. |
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