Biological Characters Of The Novel Duck Parvovirus DS-15 Strain And The Establishment Of An Indirect ELISA For The Detection Of Antibodies

Since 2014,a novel duck epidemic has been endemic in many duck farms of China.The mainly clinical symptoms of the duck disease were growth retardation,beak atrophy and tongue exposed,and limp paralysis.Based on the typical clinical symptoms,this new disease was called duck short beak dwarf syndrome(DSBDS)or duck big tongue disease.It has been identified that the pathogen of this disease is a novel duck parvovirus(NDPV).So far,the biological characteristics of NDPV has not been clear,and there is no effective vaccine or drug for controlling this disease.Moreover,the method or techniques for detecting the disease are lacking.In this paper,the biological characteristics of NDPV DS-15 strain was studied,and a reliable method(indirect ELISA)for detecting the specific antibody against NDPV was established..Firstly,the pathogen of DSBDS was isolated by duck embryo,named NDPV DS15 strain.After passaged three times,NDPV DS15 strain could killed 60%-70% duck embryo in 96-120 h.The titre(ELD50)of DS-15 strain was 10-5.5/0.2mL.Ducklings infected with the recovered DS-15 strain from duck embryo showed typical clinical symptoms of DSBDS.The VP3 gene of NDPV DS-15 strain was amplified and sequenced,and its sequence was compared with those sequences of Goose parvoviruses(GPV)and Muscovy duck parvoviruses(MDPV).The results showed that the identities of nucleotide sequence and amino acid sequence of VP3 gene between DS-15 strain and the referenced strains of GPV were 91.4%-98.4% and 96.6% ~ 98.1%,respectively.However,the homology of VP3 gene between DS-15 strain and MDPV isolates were 81.4% ~ 91.5%(nucleotide sequence)and 91% ~ 97%(amino acid sequence),respectively.The phylogenetic tree showed that NDPV DS-15 strain located in the same topology branch with GPVs,suggesting that their relationship is close.After NDPV VP3 gene was inserted into pGEX-4T-1,The recombinant plasmid(pGEX-4T-VP3)was transformed into DE3 competent cells and induced with IPTG.The results of SDS-PAGE showed that the recombinant GST-VP3 protein were successfully expressed and its molecular weight was 84 kDa.Western blot assay showed that the recombinant protein could react with the specific positive sera against NDPV,suggesting that the recombinant VP3 could be used as diagnostic antigen.Subsequently,the purified VP3 protein was used as antigen to coat ELISA plates,after optimized the concentration and conditions of the coated antigen,the conditions of the antigen-blocking condition,the incubation condition of the primary antibody,the incubation condition and the coloring condition of the enzyme-labeled secondary antibody,a sensitive and specific indirect ELISA method was successfully established.The positive threshold was determined to be 0.35.Besides,this method has good reproducibility.Finally,some clinical samples were collected and detected with this ELISA method,the results showed that this method could be used for epidemiological investigation of NDPV infection.In a word,the proliferation characteristics and genetic evolution of NDPV were studied in this study.We speculated that NDPV and GPV may be from the same viral ancestors.Besides,an indirect ELISA method for detecting NDPV antibody was successfully established.All of these results provide an effective method for clinical epidemiological investigation of NDPV and vaccine development.