Construction Of Full-length CDNA Of SARS-CoV BJ01 Strain And Study On Infectivity Of In Vitro Transcripts

SARS-CoV is the largest single-stranded, positive-polarity RNA virus, containing 29700nt in the genome. Phylogenetic and sequence analysis have shown that SARS-CoV differ from all other already sequenced CoVs, and very little is known on it's genomic structure and molecular pathogenecity. Reverse genetics of the SARS associated coronavirus provides technical assistance for further research in this field.Reverse genetics is also known as infectious cDNA clone technology, through which RNA virus could be rescued from cDNA clone. Reverse genetics contains two stage: construction of the virus genomic full-length cDNA clone and preparation of the infectious transcripts using the recombinant DNA as templates. Reverse genetics not only provides a technology platform for the research on virus genomic structure, function and molecular pathogenecity, but also lay a solid foundation for development of chimeric vaccine, attenuated vaccine and construction of new virus vector.Because of the large size of the coronavirus genome, it is difficult to amplify the full-length cDNA directly by PCR with expand long TAQ polymerase, and find large capacity vector to construct genomic full-length cDNA clone. So, in this research we subcloned cDNAs that span the entire genome of SARS-CoV, then assembled them into full-length cDNA, and finally rescued SARS-CoV through in vitro transcripts.First, the condations and parameters for the RT-PCR to get long cDNA fragments was optimized. Total RNA was prepared from SARS-infected Vero cells by RNA extraction kit , reverse transcription was performed by using SuperscriptⅡ, and the first strand cDNA was synthesized. Then the cDNA fragments were amplified with LA Taq DNA polymerase.Diffrent amplification parameters, concluding Mg2+, dNTP and PCR recycle conditions, were compared and the best reaction system and parameters was set up: 55μL 10×Buffer (Mg2+, 37.5mmol/L), 2μL dNTP (10mmol/L), 1μL LA Taq Polymerase (2.5U), 1.5μL upstream and downstream primer each, 2μL template, and add nucleas-free water to 50μL. The PCR cycle parameters were following: 94℃for 2min, 28 cycles at 94℃for 30sec, 55℃45sec, 72℃ for 3.5～7min (which depend on the fragment size, about 1min for 1kb, and the extension time increase 1 sec every cycle ), finally at 72℃for 7min, and storage at 4℃. Good repeatability could be acquired under above amplification conditions.Second, seven pairs of primers were designed, by which specific restriction sites (BglI) were introduced into SARS-CoV BJ01 strain genome sequence by silent mutation. Seven amplicons (1.5kb, 2.8kb, 4.3kb, 3.3kb, 6.8kb, 4.5kb and 6.3kb) were obtained. Meanwhile, the T7 promoter core sequence was introduced at the 5′-end of the genome for in vitro transcription of the full-length cDNA. Seven cDNA fragments covering the whole virus genome were prepared and purified, then cloned into pGEM-T Easy, pCR-XL-TOPO or pWSK29 vectors. The subclones were screened and sequenced. Finally, seven recombinant plasmids named F1-3-Teasy, F2-6-Topo, F3-17-Topo, F4-10-Topo, F5-1-Topo, F6-7-Topo and F7-pWSK29 containing desired SARS sequences were obtained successfully.Large amount of 7 recombinant plasmids were prepared, purified and then digested with restriction endonuclease. The F1~F4 and F5~F7 fragments were ligated separately in vitro, and the 5′and 3′semi-cDNA of genome were obtained. Furthermore, the junctions of the cDNAs were sequenced. Then both semi-cDNAs were ligated in vitro and the full-length genomic cDNA was prepared. The full-length cDNA was used as template for in vitro transcription with the T7 RNA polymerase, and the transcripts were tested and electroporated into Vero-E6 cells. Eighty-four hours after transfection, the cells became toroid, aggregating, and gradually detaching. The culture medium was harvested and reinfected the normal Vero-E6 cells, and the same cytopathic effect was observed in 72 hours. RNAs was extracted from the cells with typical CPE, the sequence of 13 038～14 170nt and 27 229～28 332nt were amplified respectively. The length of the products was the same as expected. This demonstrate the genomic full-length cNDA and infectious transcripts of SARS-CoV BJ01 strain were obtained.This is the first report in China on construction of full-length cDNA of SARS-CoV by reverse genetics. The availability of the full-length cDNA and infectious transcripts (rescued virus) will provide a genetic approach to manipulate the SARS-CoV genome for the further identifying the SARS-CoV virulence sites, revealing the molecular pathogeneticity, exploring the complexity of the genome, and developing candidate vaccines.