Characterization Of The Cloned Full Length Genome Of Severe Acute Respiratory Syndrome Coronavirus From A Sars Patient's Lung Tissue And The Assembly Of Viral-Like Particle

An infectious disease, characteristic of high fever, chills, myalgia, dry cough, hard breath and highly contagious, broken out mainly in Guangdong province and Beijing district in spring 2003,which is formally named after Severe Acute Respiratory Syndrome (SARS). Etiologic study showed that the etiologic agent for worldwiide prevalence of SARS is a new coronavirus species which later was called SARS-associated Coronavirus (SARS-CoV) by WHO. Little is known about the genetics, replication and assembly mechanism of the newly discovered virus after the outbreake of SARS, thus it is of great value for studying these aspects of the SARS-CoV.The author of the paper took part in the etiologic study of SARS. Isolation of etiologic virus using the clinical specimens from SARS patient was firstly carried out for the further elucidation of the etiologic characteristics of SARS-CoV prevailing in Beijing district. Vero cell cultures were inoculated with said clinical specimens such as laryngopharynx swab, sputum and lung tissue respectively and the distinct cytopathic effect (CPE) was observed after successive passage of inoculated Vero cell cultures, so it is indicated the possible existence of virus in clinical specimens. A virion-like particle with morphologically similar appearance of the typical coronavirus was observed in negative-stained specimens from the supernatant of the cell culture under the electroscope. Special fluorescence was also shown in the cellular plasma of the inoculated Vero cell culture by indirect immune-fluorescence method with a convalescent-phase serum from SARS patients. This meant the existence of the SARS-associated antigen in the inoculated Vero cell culture. A special fragment was amplified by RT-PCR using total RNA extracted from the inoculated Vero cell culture as template and a pair of oligonucleotides with sequence specificity of coronavirus genome as primers. The amplicon was identified to be partial sequence of coronavirus replication enzyme gene after sequencing and analysis of the clone of amplicon. The collected experimental data above supported that the isolated virus was SASR-CoV.In order to identify the diversity of SARS-CoV genomes between the virus directly from human tissue of SARS patient and strains isolated a, to characterize the genetics and mutation of the virus for further elucidation of pathogenic mechanism,...