Epidemiological Investigation And The Establishment Of Sandwich ELISA For Detection Of Goose Parvovirus

Goose parvovirus infection,also known as gosling plague,has posed serious harm on the waterfowl,such as geese and ducks.It was first found in Yangzhou,Jiangsu Province,in 1956.In 2015,a unique GPV virus,the novel goose paravovirus(Novel goose parvovirus,NGPV),were isolated from cherry valley ducks for the first time in northern China Compared with the early classical GPV,NGPV infection had high morbidity and low mortality.In recent years,GPV is widespread in many regions of China.GPV evolved in gene recombination when waterfowl mixed-infected with Muscovy duck parvovirus,(MDPV),GPV,and duck-origin parvovirus.Therefore,it is of great significance to surveil the prevalence of GPV and establish suitable detection methods for the prevention and control of gosling plague.1.Epidemiological investigation of GPV and the genetic evolution of VP gene of GPVFrom 2017?2019,we collected 50 clinical samples from waterfowl suspected of GPV infection in Jiangsu,Anhui,Guangdong and Shandong provinces.Virus isolation by goose embryo and identification by PCR.We successfully obtained 14 strains of GPV,among of which included 11 classic GPVs,2 MDGPVs and 1 NGPV.According to host distribution,classic GPVs were all isolated from goslings,while MDGPVs were both from muscovy ducks and the NGPV was isolated from ducklings.As for seasonal distribution,those GPVs presented a relatively higher isolation rate in spring and winter.Furthermore,VP gene sequencing was conducted for all 14 GPVs.Then we performed molecular evolution and phylogenetic analysis for the structural proteins VP1,VP2,and VP3 of GPV,respectively.The results showed that the 14 isolates all belonged to the GPV based on the phylogenetic trees of VP 1,VP2 and VP3 genes,but possessed certain genetic diversity as dispersed in different subclades according to host and geographical distribution.However,the isolates from Yangzhou in Jiangsu and Anhui provinces all belonged to the classic GPV subclade,while the two strains XI and X2 from Guangdong province clustered into the MDGPV subclade and X7 strain from Shandong province divided into the NGPV subclade.The recombination events were confirmed by RDP analysis constructed on the basis of VP gene.The results showed that recombinant events occurred in VP gene of X1 and X2 with the major parental strain GPV-Yan-2(classic GPV)and the minor parental strain MDPV-89384(MDPV)in the recombinant regions 1-702 nt and 1833-2199 nt,respectively.2.Preparation of monoclonal antibodies against GPV and establishment of sandwich ELISAWe selected one isolate from each branch in phylogenetic trees,such as classic GPV,MDGPV and NGPV.The viruses was purified by sucrose density gradient centrifugation,and used as antigen to inmmunize BALB/c mice at the dosage of 100ug/mL and prepare monoclonal antibodies.We obtained 7 strains of hybridoma cells,named as 1H3,1D4,2C8,3G10,2F11,6F12,6H3,respectively,secreting antibodies against GPV and screened by indirect ELISA test.Correspondingly,the antibody subclasses were determined as IgG2?,IgG2?,IgG2?,IgG2b,IgG2b,IgG2b,IgG2b,respectively.The VP3 antigen expressed by baculovirus was immunized,concentrated and purified,and immunized with formaldehyde inactivated mice.Four anti-GPV VP3 hybridoma cells were selected and named as 1A5?1H5,4H5,and 5C7,respectively,and the antibody subclasses were IgM,IgM,IgM,IgG1.Western-blot confirmatory test exhibited positive bands for Mabs 1H3 and 5C7 reaction with GPV.We selected Mabs 2F11 and 5C7 with high titers and good stability and purified by ProteinG affinity chromatography with the concentrations of 1.2 mg/ml and 6 mg/ml,respectively.In order to develop sandwich ELISA to detect GPV,2F11 was used as the capture antibody and GPV rabbit antisera as the detection antibody.Moreover,we also develop a sandwich ELISA to detect the VP3 antibodies in goose sera,and selected 5C7 as the capture antibody with quantitative GPV antigen.The sandwich ELISA method established in this study had lower OD values in the negative control samples and showed better specificity.In summary,this study successfully isolated 14 GPV strains,which belonged to the classic GPV,MDGPV or NGPV.Additionally,we prepared 11 monoclonal antibodies against GPV,and developed high-sensitivity sandwich ELISA methods for detecting GPV antigen and antibody,respectively.