Genetic And Evolutionary Analysis Of Canine Bufavirus In Anhui Partial Areas And Establishment Of Quantitative PCR Detection Method

Bufavirus(CBuV),a member of the genus Parvovirus,which are associated with Canine diarrhea.CBuV was first detected in puppies in Italy in 2018,and has since been reported in China,including Shanghai,Guangxi and Henan provinces.CBuV has not been reported in Anhui Province,and its prevalence and evolutionary status are unknown,and there is a lack of effective detection methods.In this study,120 feces were collected from animal hospitals in Anhui province.3 strains of CBuV were detected.The complete genome sequences were amplified and uploaded to Genebank.In addition,the SYBR GreenⅠ-based real-time qPCR detection method for the detection and quantification of CBuV was established.The three strains showed high homology,and the homology of complete sequences,NS1 and VP2 with other reference strains was 93.5-99.5%,96.5%-100%and 98.5%-99.8%,respectively.Phylogenetic analysis based on complete sequences showed the strains formed three clads and Anhui strain showed a unique genetic relationship.Moreover,in the phylogenetic tree of VP2 indicated the potential differentiation in the VP2 region under different geographical states.Selective pressure analysis of the VP2 region indicated that the CBuV was mainly subject to negative selection during evolution.The negative selection site was located on the residue of B-cell epitopes,indicating minimal change to the immunogenicity of the virus.In the sensitivity experiment,the detection limit of real-time qPCR was 4.676×10~1 copies/μL,and the detection limit of conventional PCR(c PCR)was 4.676×10~3copies/μL.Furthermore,the qPCR method does not react with the other virus in dogs.The intra-and inter-assay coefficient of variation was 0.07%–0.55% and 0.03%–0.11%,indicating good repeatability.In clinical sample testing,the detection rate of qPCR was 5.0%(6/120)higher than that of c PCR(2.5%,3/120).In summary,This study is the first to report the prevalence of CBuV in Anhui Province and provide reference for the prevention of CBuV.And the SYBR GreenⅠ qPCR method established in this study has good sensitivity,specificity,and reproducibility for clinical sample detection and can also assist in diagnosis of CBuV.