Isolation and Identification of Bovine Enterovirus Types E and F in Guangxi
[Objective]By isolating Bovine enterovirus(BEV),this study was aimed to understand its transmission routes and pathogenic characteristics in Guangxi,provide data support for epidemic monitoring and vaccine development,and further promoting the prevention and control of the virus in Guangxi.[Method]The BEV-positive samples were inoculated into fetal bovine kidney cells(MDBK).After continuous passage,the isolated strains were identified through cytopathic effects(CPE),RT-PCR and indirect immunofluorescence assay(IFA).The identified strains were subjected to plaque purification,virus titer determination,and multi-step growth curve experiments.Specific primers were designed to amplify the full-length genome sequence of the virus.The nucleotide and amino acid similarity between the isolated strains and reference strains were analyzed using MegAlign software.Phylogenetic trees of the viral whole genome and VP1 gene sequences were constructed.[Result]The third passage of inoculated cells showed significant CPE.RT-PCR results indicated the presence of target bands(239 bp),and IFA results showed that specific green fluorescence could be observed in MDBK cells inoculated with the isolated strains,confirming the isolation of two BEV strains,named GXHC2317 and GXWZ2317,respectively.50％tissue culture infective dose(TCID50)results showed that the TCID50 of strains GXHC2317 and GXWZ2317 were 107.47 and 105 75/0.1 mL,respectively.Multi-step growth curve experiments demonstrated that strains GXHC2317 and GXWZ2317 replicated well in MDBK cells,reaching peak viral replication capacity within 24 h.Similarity analysis showed that the nucleotide and amino acid sequence similarities between strain GXHC2317 and the reference strain were 69.5％to 80.1％and 81.1％to 96.5％,respectively.The amino acid sequence similarity between strain GXHC2317 and strain NX-FY40 isolated from Ningxia region was the highest.The nucleotide and amino acid sequence similarities between strain GXWZ2317 and reference strain were 69.3％to 87.3％and 80.3％to 96.4％,respectively,with the highest amino acid sequence similarity between GXWZ2317 strain and SD-S67 strain isolated from Shandong region.Phylogenetic analysis of the viral whole genome and VP1 gene sequences indicated that the GXHC2317 strain belonged to the BEV E3 subtype,and the GXWZ2317 strain belonged to the BEV F1 subtype.[Conclusion]This study isolated BEV E3 and F1 subtypes from Guangxi,enriching the investigation of BEV epidemiology and pathogenesis in China.It laid a virological foundation for basic and applied research related to BEV in Guangxi.
Bovine enterovirus(BEV)isolation and identificationgenetic typing
万方数据
维普
