Construction And Rescue Of Bovine Enterovirus Strain BJ101 Infectious Clone

Bovine enteroviruses?BEV?is a member of the enterovirus genus of Picornaviridae family.It contains a single-stranded,positive-sense and polyadenylated viral RNA genome of approximately 7 500 bp.It has a wide range of hosts such as cattle,sheep,possums and dolphins.A large range of natural hosts can be infected by BEV,such as cattle,sheep,possums,dolphins.Animals after being infected are typically asymptomatic or subclinical and the main clinical symptoms are diarrhea and trouble breathing.BEV is divided into two subtype:E?E1^E4?and F?F1^F6?.In this study,a strain named BJ101 of BEV was isolated.Based on the whole genome of BJ101,the full-length infectious cDNA clone of BJ101 was constructed.Then the virus was rescued successfully and the biological characteristics of the rescued virus was analyzed to provide conditions for the study of the pathogenesis of BEV and the development of a new genetic engineering vaccine.A strain of bovine enteroviruses was isolated from the fecal samples of cattle affected with diarrhea in Beijing and identified as a bovine enterovirus by RT-PCR,and then we performed the full-length genome sequencing and molecular characteristic analysis of the isolated strain?named BJ101?.The TCID₅₀ of the isolate was 1×10^-6.4/0.1mL and the virion was observed as non-enveloped particles about 25-30 nm in diameter.The full genomic length of BJ101 was 7 409 bp,which contained 5'UTR?812 bp?,the open reading frame?ORF,6 525 bp?and 3'UTR?72 bp?.The ORF encoded a polyprotein with 2 193 amino acid residues.The sequence alignments showed that the BJ101 strain shared the highest nucleotide identities to PS42?84.9%?and PS83?85.0%?in the 5'UTR,to K2577?91.5%?and SL305?91.5%?in the 3'UTR.Compared with other reference strains based on the VP1,VP2,VP3,and P1,BJ101 isolate shared the highest identity of nucleotide and amino acid sequence with the strains of E2 subgenotype.And the phylogenetic analysis revealed that they gathered with the same cluster.Howere based on the VP4,BJ101 isolate shared the highest identity of nucleotide and amino acid sequence with the strains of E1 subgenotype,which belonged to the same cluster.Since VP1 is the main criterion for the discrimination of genotypes of BEV,the BJ101 strain belonged to E2 subgenotype,and there may be recombination with the strains of E1subgenotype at VP4.According to the full-length nucleotide sequence of BJ101,the pBlueScript?-?vector was out of T7 promoter and there were only Sal?and EcoR?for MCS.Full-length sequence of BJ101 is divided into three gene fragments of A,B and C,T7 promoter was added to 5'end of A.Mutated EcoRV restriction site was introduced as molecule label in overlap portion of the B and C fragments?synonymous amino acid mutation?.After linearization by EcoR I,recombinant plasmid GpBSK-BEV was transfected into BSR cell line which can express T7 RNA polymerase stably.The recombinant virus rBEV was identified by sequencing of molecule label,indirect immunofluorescence and immune electron microscopy.By comparison tests,it was shown that the recombinant virus had similarity with the parent strain in host tropism,TCID50 and growth curves.The results of the study demonstrated that the infectious clone of bovine enterovirus was constructed successfully.To sum up,we have obtained the infectious cDNA clone of BJ101 strain and can successfully rescued the virus,which provides a technical platform for further research on the pathogenesis of BEV and the development of a new genetic engineering vaccine.