ABSTRACT
Objective To identify the infection and the replication of Tick-borne encephalitis virus(TBEV) in human neuroblastoma cells.Methods After being inffected with TBEV,the cell culture supernatant of human neuroblastoma cell line SK-N-SH was collected and assayed at different time points.Byusing real-time RT-PCR and plaque assay to measure the titer of virus in the supernatant,the replication andproliferation of TBEV in human neuroblastoma cell was identified.Meanwhile,the morphological change of SK-N-SH after TBEV infection was also visualized by observation under microscope and immunmquorescenceassay.Results Real-time RT-PCR and plaque assay both demonstrated that TBEV could replicate effectively in SK-N-SH cells,the peak titer could reach 2.92× 107 PFU/ml on 3 days post-inoculation.And significant morphological change occured on infected SK-N-SH cells after 2 days post inoculation.By immunofluorescence assay,the virus particles could be detected and visualized.Conclusion TBEV can replicate andproliferate effcctively and cause significant cell morphological changes in human neuroblastoma cell SK-N-SH,which demonstrated that SK-N-SH could be a suitable cell model for TBEV culture.
ABSTRACT
We report a complete genomic sequence of rare isolates (minor genotype) of the SARS-CoV from SARS patients in Guangdong, China, where the first few cases emerged. The most striking discovery from the isolate is an extra 29-nucleotide sequence located at the nucleotide positions between 27,863 and 27,864 (referred to the complete sequence of BJ01) within an overlapped region composed of BGI-PUP5 (BGI-postulated uncharacterized protein 5) and BGI-PUP6 upstream of the N (nucleocapsid) protein. The discovery of this minor genotype, GD-Ins29, suggests a significant genetic event and differentiates it from the previously reported genotype, the dominant form among all sequenced SARS-CoV isolates. A 17-nt segment of this extra sequence is identical to a segment of the same size in two human mRNA sequences that may interfere with viral replication and transcription in the cytosol of the infected cells. It provides a new avenue for the exploration of the virus-host interaction in viral evolution, host pathogenesis, and vaccine development.
Subject(s)
Base Sequence , China , Cluster Analysis , Gene Components , Genetic Variation , Genome, Viral , Genotype , Molecular Sequence Data , Phylogeny , Reverse Transcriptase Polymerase Chain Reaction , Severe acute respiratory syndrome-related coronavirus , Genetics , Sequence Analysis, DNA , Severe Acute Respiratory Syndrome , 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.