Development Of A TaqMan QPCR Assay And Molecular Epidemiological Investigation Of Amdoparvoviruses In Fur Animals

Amdoparvoviruses is defined Amdovirus genus in the family Parvoviridae,this genus contains Aleutian mink disease virus(AMDV),Gray fox amdoparvovirus(GFAV),Raccoon dog and arctic fox amdoparvovirus(RFAV),Skunk amdoparvovirus(SKAV),Red fox fecal amdovirus(RFFAV),and Red panda amdoparvovirus(RpAPV).In order to establish a direct TaqMan qPCR assay for detection and quantification of carnivore amdoparvoviruses by using primers and probe based on the conserved VP2gene,and employed the direct alkaline lysis method to obtain viral DNA.The detection limit for AMDV and RFAV were 4.06×10~1 copies/?l and 2.93×10~1 copies/?l,respectively.Both intra-and inter-assay variability were less than 2%.Among 74 carnivore samples,the positive rates for amdoparvoviruses were 62.2%(46/74)by direct TaqMan qPCR,while only 40.5%(30/74)by SYBR Green I qPCR.The direct TaqMan qPCR assay can be used as a powerful tool for detection and quantification of various carnivore amdoparvoviruses except GFAV in epidemiological and pathogenesis studies.The aim of the current study was to detect and evaluate the genetic characteristics and diversity of amdoparvoviruses in China in order to improve our understanding of the molecular epidemiology of this virus.Here,125 blood samples of raccoon dog,80 blood samples of arctic fox,45 blood samples of silver fox,and 70 feces of martes zibellina from 3 different regions were collected for PCR detection,the positive rates were 62.4%(78/125),1.3%(1/80),0(0/45),and 0(0/70).Then PCR products were cloned and sequenced,we obtained 4 327 bp RFAV genomic sequence in length,710 bp arctic fox amdoparvovirus VP2 genome.In terms of nucleotide sequence identity,the sequence of arctic fox showed a similarity of 97.45%with the VP2 gene of RFAV strain(Accession no.KJ396350.1),the VP2nucleotide sequence of 6 raccoon dog showed nucleotide and amino acid similarity of VP2 and NS1were higher than 90%with RFAV published on GenBank,,indicating the newly RFAV may be mutant strains of RFAV.The VP2 nucleotide similarity of RFAV with AMDV,SKAV,RpAPV,and GFAV reference sequence was 75.7%?90.3%,amino acid compatibility was 77%?91%;NS1 nucleotide similarity was 67.8%?83.6%,NS1 amino acid similaritywas 62.3%?76.5%,less than 80%.The phylogenetic trees were constructed based on the complete amino acid sequence of VP2 and NS1,the nucleotide sequence of VP2 hypervariable region and the partial VP2 nucleotide sequence of newly arctic fox amdoparvovirus.The results showed that the newly raccoon dog,arctic fox amdoparvovirus and other RFAV strains from GenBank were included in group RFAV.SKAV,RpAPV,AMDV,and GFAV strains from GenBank were included in groups SKAV,RpAPV,AMDV,and GFAV.Taking the amino acid similarity of NS1 less than 85%as the classification criterion of parvovirus subfamily virus species,the phylogenetic trees showed that RFAV forms a branch,indicating that RFAV is a new species of Amdoparvovirus genus,which is similar to AMDV,SKAV,RpAPV and GFAV are different species within Amdoparvovirus genus.Molecular epidemiological investigation of VP2 hypervariable region showed that RFAV only infected raccoon dog and arctic fox,raccoon dog is naturally susceptible to RFAV infection,while AMDV only infected mink,we did not find cross-infection in natural condition.Functional domains of AMDV analysis was performed and showed that the functional domains about virus replication,virulence,and host have similar mutation in RFAV,indicating that amdoparvoviruses have various hosts may be induced by the mutation of certain major functional domains.In order to analyze the epitope characteristics of VP2 and NS1 proteins of RFAV,and further select the conserved B-and T-cell epitopes on VP2 and NS1 proteins of RFAV in Amdoparvovirus.The bioinformatics programs were used to predict the physical and chemical properties,secondary structure,post-translational modifications and potential B-and T-cell epitopes of VP2 and NS1 proteins of RFAV.Both VP2 and NS1 proteins are hydrophilic proteins and the secondary structure were mainly composed of random coils.We predicted there were 3 conserved B-cell epitopes,3 conserved T-cell epitopes and 8conserved post-translational modification sites on VP2 protein within the Amdoparvovirus genus;while NS1 protein had 1,2 and 7 in the respective conserved items.This study provides fundamental basis for the development of a candidate vaccine and disease diagnosis of amdoparvoviruses.