ABSTRACT
Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.
Subject(s)
Humans , Genome, Viral , Haplotypes , Mutation , Open Reading Frames , Phylogeny , Severe acute respiratory syndrome-related coronavirus , GeneticsABSTRACT
Objective To develop a diagnostic test based on indirect immunofluorescence assay(IFA) to detect special antibodies in the serum of SARS patients, thus to provide a reference material for confirmation of the clinical diagnosis of SARS. Methods SARS coronavirus GZ01 and BJ01 strains isolated in our laboratory were used to infect Vero E6 cells. When CPE reached 25%, cells were trypsinized and transferred to 10 well slides in a quantity of 40?l with a cell density of 2?10 7 /ml. After 4 hour incubation at 37℃,the slides were fixed with acetone, and IFA was used to detect antibodies in serum samples, which were obtained from 154 SARS patients and 14 non SARS patients with respiratory disease, as well as 100 healthy volunteers. Results IFA method for detecting antibodies of SARS coronavirus was developed. Sera from one hundred and forty two out of 154 clinically diagnosed patients were IFA positive, with a positive rate of 92 3%. Sera from 14 non SARS patients with respiratory disease and 100 healthy persons were all IFA negative. Conclusion The IFA method we developed was sensitive and specific in detecting SARS antibodies in serum, and was a reliable test for laboratory diagnosis of SARS coronavirus.
ABSTRACT
Objective To establish a multiplex suspension array for simultaneously detecting five species of important arbovirus: dengue virus (including four serotypes), japanese B encephalitis virus, west nile virus, tick-borne encephalitis virus and yellow fever virus. Method All published genomic sequences of the above arboviruses were collected from GeneBank, and then, the genus universal primers and specific probes were designed according to the conservative and specific NS5 region of the genus Flavivirus. The amine-modified probes were covalently coupled to different carboxylated microspheres by carbodiimide methods. By combining general RT-PCR with virus-specific probe hybridization, a Luminex xMAP system-based suspension array, which could simultaneously differentiate five arboviruses and discriminate 1～4 serotypes of dengue virus, was successfully established. Results A cutoff value twice higher than that of the background fluorescence was judged as positive reaction. RT-PCR products of many arboviruses were used to evaluate the specificity and repeatability of the array, the results showed that the assay was highly specific and had good repeatability. Meanwhile, the coefficient of variability was less than 8% from inter- and intra- assay, implying that both the repeatability and stability of the array were good. Furthermore, several viruses which had been quantitated by plaque assay were used to determine the array’s sensitivity. The results showed that the array’s detection limits were about 1.4 PFU for JBEV and YFV, 14 PFU for WNV respectively. 55 clinical and cultured samples were detected by the established assay, 16 out of the 55 samples were confirmed to be positive. There was only one sample which was not detected contrasted with conventional RT-PCR. However, it had advantages in detecting complex samples rapidly. Conclusion A multiplex suspension array for simultaneously detecting and serotyping five species of important arboviruses has been successfully developed, and it may play an important role in diseases diagnosis and prevention as well as epidemiological studies.