Construction Of The Full-length CDNA Of SARS-CoV BJ01 Strain And Influence Of The Spike Gene Mutagenesis On Infectivity

SARS-CoV is a newly discovered human coronavirus, which belongs to the positive-strand RNA viruses. The genome size of the SARS-CoV is around 30 kb, which is the largest one found in any of the RNA viruses. The genomic organization is typical of coronaviruses, having the characteristic gene order, 5'-replicase(rep) -spike(S)-envelope(E)-membrane(M)-nucleocapsid(N)-3', and short untranslated regions at both termini. They encode four structural proteins S, E, M, N and other nonstructural proteins as well. The S protein is the most important structural protein for the virus, which role is participating the binding of the SARS-CoV to its cellular membrane receptor and the entry process of the virus into its target cells through membrane fusion, so S protein determine tissue tropism and virulence of the virus.Because SARS-CoV is a kind of human coronavirus which had been found lately, its genomic structure and function, molecular pathogenesis, etc are not known to mankind. Virulence correlate locus was mostly predicted by bioinformatics, and the defined sites related with the virulence of the viruse are just not verified at the bench. For the large SARS-CoV genome, it's difficult to produce the infectious cDNA clone and mutation in vitro, therefore constructing the full-length genomic cDNA in vitro will resolve the problem and offer a well platform for the investigation of the genomic characteristics and pathogenesis of the SARS-CoV BJ01 strain.For this goal, we constructed the full-length genomic cDNA of the SARS-CoV BJ01 strain applying fragments cloning and in vitro ligation strategy, and procured the rescued virus through in vitro transcription method, observing the infectivity and passage stability in the sensitive cell lines. Meanwhile, the possible virulence-related sites of the S protein were predicted applying bioinformatics method and substituted...