Molecular Characterisation Of Blue Fox Parvovirus Tai'an Strain And Establishment Of A TaqMan-based Real-time PCR Method For Its Detection

A parvovirus strain was isolated from the feces of blue foxes in Tai'an, which was blue fox parvovirus by a series of physicochemical assay,viral hemagglutination test and examining animals attacked with it.It was named BFPV-TA.The VP1,VP2 and NS1 gene of BFPV-TA was sequenced and analyzed.Then the application of a TaqMan-based real-time PCR method for detection of BFPV was established.The result is reported as follows:1.A parvovirus strain was isolated from the feces of blue foxes in Tai'an,produced specific cytopathic effects in F81 cells,which was blue fox parvovirus by a series of physicochemical assay,viral hemagglutination test and examining animals attacked with it.It was named BFPV-TA.2.The primers were designed based on the VP1,NS1 genes of the strains of CPV and FPV in Genbank.The VP1,NS1 gene was amplified by PCR,cloned into pMD18-T vector,sequenced and analyzed.As a result,the ORF of the VP1 gene of BFPV-TA was 2256 bp and encoded 727 amino acids.The nucleotide homologies of VP1 genes between BFPV-TA between the CPV,FPV references are from 98.7%to 99.5%.According to phylogenetic analysis,it showed that BFPV-TA and FPV had the nearer inherit distance.The 375^th amino acids of the VP1protein of BFPV-TA were identical to those of CPV,but the 223^th,236^th,246^th,466^th,707^th and 711^th of the VP1 protein of BFPV-TA were consistent with those of FPV.According to phylogenetic analysis,it showed that BFPV-TA and FPV had the nearer inherit distance;The ORF of the VP2 gene of BFPV-TA was 1755 bp and encoded 584 amino acids.The VP2 protein possessed three substitutions, 219I→V,300G→P and 411E→A.The nucleotide homologies of VP2 genes between BFPV-TA and the CPV,FPV references are from 98.5%to 99.4%, According to phylogenetic analysis,it showed that BFPV-TA and FPV had the nearer inherit distance;The ORF of the NS1 gene of BFPV-TA was 2007 bp and encoded 668 amino acids.The NS1 protein had eight substitutions, 43R→H,135H→Y,172K→R,179A→E,350D→N,409I→V,574I→V, 616D→V.The nucleotide homologies of NS1 genes between BFPV-TA and the CPV,FPV references are from 98.6%to 99.2%.According to phylogenetic analysis,the clear cluster was observed based on NS1 gene phylogenetic analysis.Some animals in the family Canidae that are susceptible to FPLV-like viruses,suchas blue fox,may play a role as reservoirs for the ancestors of CPV.3.The probes and primers were designed and synthesized according to the conserved gene VP2 of BFPV available in GenBank,and then reaction parameters were optimized to develop a specific,sensitive method of TaqMan-based real-time PCR assay for the rapid detection of BFPV.The samples from foxs on farms in LIAO NING and SHAN DONG Province were detected by using the established quantitative PCR,Conventional PCR and HA.The sensitivity of the TaqMan-based real-time PCR was higher than the Conventional PCR and HA.while the results of the quantitative PCR were the same as that of the Conventional PCR,HA and VI.The results indicated that the real-time quantitative PCR could be used for the diagnosis of BFPV infection.