Establishment Of The Full-length Genome Reverse Genetic Operating System Of Coxsackie B Virus Type 4

Coxsackievirus B4(CVB4)belongs to the B component of enterovirus.In China,CVB4 has been reported in severe cases of hand,foot and mouth disease,which can cause encephalitis and has a high mortality rate.CVB4 belongs to the genus Picotavirus Enterovirus,which is a single-stranded positive-strand RNA virus(+RNA).It uses direct-stranded RNA as a template to synthesize viral RNA during replication and translation,and assembles with proteins to form a virus.Particles,which do not pass through DNA intermediates during replication,increase the difficulty of studying RNA viruses.With the continuous development of reverse genetics technology,by obtaining the c DNA of RNA virus in vitro and integrating it into the vector,the recombinant plasmid can be used to obtain the virion carrying the virus and the infectious virus.Research on the genome of RNA viruses at the DNA level.At present,the reverse genetic system of CVB4 has not been constructed,and there is still no effective research program.Based on the laboratory-established enterovirus sample bank,the genetic development of the whole genome and VP1 fragments of the isolated CVB4 virus was selected,and SDLW1/CHN/2013(accession number: MF179587)(LW1)was selected as the representtative strain of CVB4.For the full-length genomic sequence of LW1 strain,a pair of specific primers were designed,and the full-length c DNA was amplified in 7 segments,and the T7 promoter sequence and Not I restriction site were introduced in the upstream of the5’UTR region.A 26-base Poly(A)tail and a Sal I restriction site were introduced downstream of the UTR.Three extension fragments were obtained by Overlap-PCR,ie5’UTR-F1(1-3336 bp),F2(3337 bp-6324 bp)and F3-3’UTR(6325 bp-7439 bp),and each fusion c DNA fragment was obtained.They were sequentially cloned into the p WSK29 vector.Full-length ligation was performed sequentially using Not I,Sal I introduced at both ends and internal self-cleavage sites Alw N I and Eco R V.The sequence identification and genomic sequence homology comparison showed that the prokaryotic clone plasmid p WSK29-LW1 containing the full-length genome of LW1 strain was successfully obtained,which had 99.98% homology with the wild-type CVB4 strain genome.Based on the constructed prokaryotic clone plasmid p WSK29-LW1,the hammerhead ribozyme(Ham Rz)and Not I restriction site recognition sequence,pc DNA3.1(-)vector were introduced by PCR amplification at the 5’UTR end of the CVB4 genome.a homologous region,introducing a hepatitis Dribozyme(Hdv Rz)sequence and a Bam H I restriction site recognition sequence,a pc DNA3.1(-)vector homologous region in the3’UTR,and adopting an Overlap-PCR(fusion PCR)method The LW1 genome was introduced into the pc DNA3.1(-)vector containing the CMV promoter to obtain an infectious clone of the eukaryotic recombinant plasmid pc DNA3.1-LW1,that is,the CVB4 epidemic strain LW1.By sequencing analysis,it was shown that the full length sequence of the CVB4 genome in the recombinant plasmid maintained 100% affinity with the CVB4 genome sequence in the p WSK29-LW1 recombinant plasmid.The constructed pc DNA3.1-LW1 infectious clone was added to Vero cells using liposome transfection to rescue the CVB4 virus.At 24 h after transfection,cells began to exhibit typical CPE characterizations of shrinkage,rounding,and enhanced refractive index.The rescued primary virus continued to be stably passaged in Vero cells,and the PCR-specific amplification of the VP1 fragment was determined in the cell supernatant of the three consecutive passages,indicating that a CVB4 recombinant virus was successfully rescued.After sequencing,the resulting strain sequence was identical to the expected genomic sequence.In summary,in this study,we have used the CVB4 representative strain isolated and identified in the laboratory,and constructed a full-length c DNA-containing eukaryotic infectious clone by means of prokaryotic cloning plasmid,and transfected with liposome to rescue the virus.The reverse genetic operating system of the CVB4 virus genome lays the foundation for further exploration of Coxsackie virus replication,infection,pathogenesis and immune molecular mechanisms and the development of a new vaccine for enterovirus CVB4.