Isolation And Identification Of Duck Parvovirus SD Strain And Analysis Of Genomic Characteristics

Beak atrophy and dwarfism syndrome(BADS)was an infectious disease characterized by strong growth retardation,beak atrophy,protruding tongue with swelling and osteoporosis.Since November 2014,the disease has been prevalent in commercial duck farms in many southeastern provinces of China,which has caused serious economic losses to the duck breeding industry in China.From the perspective of epidemiology and etiology,the purpose of this study is to isolate the pathogen and analyze its pathogenicity,ascertain the cause of BADS,devolope laboratory diagnostic methods and explore the related characteristics of the pathogen,which would provide theoretical basis and technical support for the prevention and control of the disease.This article is mainly discussed from the following three aspects:Firstly,in order to ascertain the cause of BADS,this study detected the liver and spleen tissue samples collected from the BADS duck groups by PCR/RT-PCR.After excluding other common diseases in ducks,the pathogen of BADS was named as duck parvovirus(DPV).The grinding fluid of DPV positive tissues was inoculated into duck embryos.After 5 generations blind passage,allantoic liquid was collected.Through the sequencing and identification,homology and evolution analysis and animal regression test,a strain of DPV(designated DPV SD)was confirmed successfully isolated.In addition,this study also isolated the virus using duck embryo fibroblasts(DEF).The identification results of PCR and indirect immunofluorescence assay(IFA)showed that the SD strain was successfully isolated by DEF.The successful isolation of DPV SD strain laid the foundation for further research of DPV.Secondly,in order to explore the characteristics of DPV in molecular level,5 pairs of overlapping primers were designed based on the genomic characteristics of waterfowl parvovirus.Then the whole genome sequence of SD strain was amplified.The structural characteristics,homology and evolution relationship of the gene sequences were analyzed.The full genome of SD strain was found to be 5053 bp in size,which consisted of inverted terminal repeat(ITR)and open reading frames(ORFs).The left ORF was 1844 bp and encoded non-structural protein NS.The right ORF was 2199 bp and encoded capsid protein VP.SD strain is closer to other BADS strains and GPV European strains than MDPV strains.In addition,compared with most of the waterfowl parvovirus,the ITR region of SD strain has two 14 bases deletions in the location of 100~114 nt and 337~350 nt.Finally,the potential glycosylation sites were predicted and analyzed.The results showed that there were 6 potential glycosylation sites in the NS and VP proteins of SD strain.This study laid a foundation for further research on molecular pathogenesis and molecular epidemiology of DPV.At last,the VP3 gene of SD strain was amplified by PCR,and the recombinant plasmid pMD19-SD-VP3 was constructed for the preparation of standard plasmids.A pair of fluorescent quantitative PCR primers and one probe were designed based on the conserved region between DPV strains of VP3 gene.After optimizing reaction system and the creation of the standard curve,the TaqMan-based real-time fluorescent quantitative PCR detection method for DPV was established.After that,its specificity,sensitivity and repeatability were verified.The results indicated that the established fluorescent quantitative PCR method was specific,sensitive and reproducible.The development of this detection method could provide powerful technical support for laboratory diagnosis of DPV.