Isolation And Identification Of A Novel Goose Parvovirus And Development Of Monoclonal Antibody Against Goose Parvovirus

Gosling plague(GP)is an acute or subacute septicemia infectious disease caused by Goose Parvovirus(GPV)and characterized by intestinal embolism.The disease mainly infects goslings aged from 4 to 20 days,and causes rapid infection and high mortality,which results in serious economic losses to goose breeding industry.Short beak and dwarfism syndrome(SBDS)in meat duck is an infectious disease caused by Novel Goose Parvovirus(NGPV).Since the occurrence of SBDS,the incidence of disease has been increasing year by year.The growth and development of the diseased ducks is hindered because of the difficulty in feeding,which affects the slaughter rate of meat duck and brings great economic losses to the meat duck breeding industry.In this study,we isolated and identified a novel GPV,generated the recombiannt baculoviruses expressing VP1,VP2 and VP3 protein of GPV and developed four monoclonal antibodies against GPV.The results of this study provides a basis for further understanding the molecular epidemiology of the novel GPV in China and for developing effective immune prevention and control techniques for GPV1.Isolation,identification and sequence analysis of a novel GPVTo investigate the molecular characteristics of the novel goose parvovirus causing the SBDS in meat duck,in this study,the viral isolation and identification were carried out by inoculating 10-day-old duck embryos from typical samples of suspected SBDS.Results showed that a novel GPV,named NGPV-SDLQ21,was isolated and identified.Nucleotide alignment shows that the NS1 gene of NGPV-SDLQ21 has 98.8%-99.8%and 93.6%-96.9%homology with the that of other NGPV and GPV reference strains,and the VP1 gene of NGPV-SDLQ21 has 96.3%-99.8%and 92.8%-96.2%homology with that of other NGPV and GPV reference strains,respectively.Compared with GPV reference strains,NGPV-SDLQ21 and other NGPV reference strains shows nine amino acid mutations in the nonstructural protein NS1 and nine amino acid mutations in the structural protein VP1,The isolation and identification of the novel goose parvovirus NGPV-SDLQ21 and the analysis of its molecular characteristics enriches the data of the novel goose parvovirus Molecular epidemiology in China.2.Expression of VP1,VP2 and VP3 protein of GPV in baculovirus expression systemTo develope diagnostic antigen and potential subunit vaccine,the VP1,VP2 and VP3 genes of GPV cell-adapted strain CZM-142 were first cloned into the donor plasmid pFastBacHTA,and then transformed into DH10Bac to obtain recombinant rBacmid-VP1,rBacmid-VP2 and rBacmid-VP3,the recombinant baculoviruses rBac-VP1,rBac-VP2 and rBac-VP3 were rescued by transfection of the rBacmid into sf9 cells.The expression of VP1,VP2 and VP3 protein in rBac-VP1,rBac-VP2 and rBac-VP3 was identified by IFA and Western-blot by using the positive sera against GPV.In addition,using the VP3 protein expressed by rBac-VP3 as detection antigen,an indirect immunofluorescence assay(IFA)was preliminarily established for detection of antibodies against GPV.The specificity assay shows that the IFA only reacts with goose sera against GPV,but not with the sera against other pathogens.The construction of rBac-VP1,rBac-VP2 and rBac-VP3 expressing VP1,VP2 and VP3 protein of GPV,and the development of the IFA method based on the VP3 protein expressed by rBac-VP3 for detection of antibody against GPV provides efficient antigens and technical supports for further development of effective GPV serological diagnostics and subunit vaccine.3.Developemnet of monoclonal antibodies against GPVTo generate monoclonal antibody(mAb)against GPV,6 to 8 weeks old BALB/c mice were immunized with GPV cell-adapted strain CZM-142,and the mAb against GPV were developed using conventional hybridoma cell fusion techniques and indirect immunofluorescence screening methods.Results shows that four hybridoma cell lines,named GPV-Mab-2F4,GPV-Mab-2F9,GPV-Mab-4D9 and GPV-Mab-6E1 1,were obtained,which stably secreting mAb against GPV.Subclass identification shows that the subclass of all the four mAbs was IgG and all the light chain was kappa chain.The titer of IFA in mouse ascites for the four mAbs was between 1:3200 and 1:6400.The specificity assay shows that the four mAbs only reacts with GPV,but did not cross-react with other pathogens including goose astrovirus detected.Neutralization test shows that the four mAbs has virus neutralization.The four mAbs could react specifically with sf9 cells infected with rBac-VP1,rBac-VP2 and rBac-VP3.The results shows that the epitopes recognized by all the four mAbs were located in VP3 protein.In addition,the four mAbs could recognize specifically with the VP3 protein of NGPV-SDLQ21 expressed by LMH cells.The development of four mAbs specific to GPV provides materials for further elucidating the B cell epitopes of VP3 protein and the diagnosis of GPV.