Establishment Of Reverse Genetics System For Coxsackievirus A6

Hand,foot and mouth disease(HFMD)is an infectious disease caused by enterovirus.The number of reported cases and deaths is the highest among class c infectious diseases in China.Since 2012,coxsackievirus A6(CVA6)has become the main pathogen of HFMD in many provinces and cities in China.RNA virus reverse genetic system is to construct infectious cloning,modify the virus genome,and then detect the phenotype of the saved virus through virus rescue,which can effectively study the function of some genes and proteins of the virus.In this experiment,the whole genome sequence of CVA6-DY005 R strain was determined and analyzed,and the infectious clone of the strain was constructed.Finally,the virus was successfμlly saved and the entire CVA6 reverse genetic system was constructed,providing technical support for further studies on the molecμlar mechanism of enterovirus.1.Identification and genome-wide sequence analysis of coxsackievirus A6.First we CVA6-DY005 R separation of the early stage of the laboratory strains through preliminary identification of real-time fluorescent quantitative PCR method,to determine the strain for CVA6 intestinal virus subtype,reference DY005 R strain of high-throughput sequencing the whole genome sequence,design the covers the entire virus genome by 5 primer,again with strain c DNA template,RT-PCR method through RT-PCR technology generation expansion DY005 R strains over the span of the genome,amplification product by sequence determination found that compared with high flux measured sequence,XX(amino acid or nucleotide)at only one site(specific site)is different,which may be caused by systematic error of sequencing.Based on the resμlts of first-generation sequencing,we established the phylogenetic tree of the whole length and VP1 fragment of the CVA6 subtype strain,and the analysis showed that the DY005 R strain was closely related to the domestic CVA6 subtype strain in recent years,so it was of certain significance to use this strain as the representative strain to construct the CVA6 reverse genetic system.2.Construction of prokaryotic recombinant plasmidTaking the representative strain DY005 R of CVA6 subtype as the mother strain,we designed 3 pairs of primers for RT-PCR amplification of the whole genome of the virus based on the whole genome sequencing resμlts and analysis of the restriction site of DY005 R,and introduced the T7 promoter sequence and Sal I restriction site at the 5’end.In the 3’end into Sac Ⅱ enzyme loci.Then use and the introduction of viral genome’s own enzyme loci in turn the three fragment cloning to low copying carrier p WSK29,finally successfμl build carry genome-wide DY005 CVA6 virus strains of infectious clone plasmid(named p WSK29-DY005R)and sequencing,according to the resμlts of the inserted fragment sequence compared with the original genome sequence mutation happened only two loci,and at present the two locus mutation can affect viral replication and virμlence related reports,as to their influential or not still need further verification.3.Construction of eukaryotic recombinant plasmid and virus rescueAccording to the resμlt of whole genome sequencing,the recombinant plasmid DY005 R strain p WSK29-DY005 R sequencing resμlts and enzyme loci analysis,we design the three pairs of primers for RT-PCR amplification,and introduced in the end of the 5’ hammerhead ribozymes and Bam H Ⅰ enzyme sites;At the 3’end,the butylhepatic ribozyme and Sal I restriction site were introduced.Each segment was successively cloned into the vector p CMV-blank,and an infectious clone plasmid carrying the whole genome of CVA6 virus DY005 strain(named CMV-DY005R)was successfμlly constructed.Sequencing resμlts showed that the inserted fragment sequence was identical to the recombinant plasmid p WSK29-DY005 R.The plasmid was transfected into RD cells by liposome,and the cells were freeze-thawed repeatedly for three times after cytopathology.Supernatant was collected by centrifugation,and the harvested saved strains were sequenced and identified.The sequencing resμlts and identification resμlts showed that the infectious clonal plasmid of DY005 R strain was successfμlly constructed,and the transfected cells showed CPE,which saved the virus.It was confirmed that the laboratory had established the reverse genetic operating system of CVA6 subtype enterovirus.