Isolation And Identification Of Waterfowl Parvovirus,and Developme Nt Of A VP3-ELISA For The Detection Of Muscovy Duck Parvovirus Antibody

Waterfowl parvovirus disease is caused by goose parvovirus(GPV)and Muscovy duckparvovirus(MDPV),both of which belong to waterfowl parvovirus.GPV and MDPV mainly infect goslings and Muscovy ducklingwith high mortality and morbidity,which have restrictedthe development of the waterfowl industry.They are quite similar in etiology,epidemiologic featuresand clinicalsymptoms,and the serum antibody have some cross-protection.The conventional serology detection method hardly differentiates the two viruses,which brings very great difficultyin the differential diagnosis.To investigate the prevalence of waterfowl parvovirus,we collected a total of 150 samples of suspected infected with GPV or MDPV from 2014 to 2016 in 5 southern province,and a pair primers was designed to diagnosethesesuspected cases.The statistical result showsthat the MDPV infection occurs mostlyinspring and winter,and the GPV infection was sharply increaseespecially in Fujianprovince.The animal regression test showed that the GPV and MDPV caused similar clinical symptoms and pathological changes,and the GPV infection was earlier onset and shorter course than MDPV.Six pairs of primers were designed according to the sequences of GPV and MDPV available in GenBank respectively.Complete gene sequence of 11 waterfowlparvovirus strains was gained by a improved method of cloning.The sequence of recent isolates were used for multiple sequencealignments and phylogenetic analysis with representative waterfowl parvovirus strains available inGenBank using the C lustalX program.The results showed that: 1)The recent parvovirus strains was divided into 3lineages: classical MDPV,SAAS-SHNHcluster and GPV.2)We detected that LHX and YM strains occurredarecombinationevent between MDPV and GPV isolatesmostly within the VP3 region.3)The conservative of different isolates of the same lineage: VP3> VP1> NS1;The identity of different lineages: NS1> VP1>VP3.4)The SYG50,YM and LHX strains have the deletion within the 80-110 ntregion;the DHN,M and YFL strains lost the 110-140 ntregion;further workneed to define wether there existed common variations among vaccine strains,or a recombination event ocurred between vaccine strains and field strains.6)The MSY,WZH and DQF strains,which isolated in 2015,with significant sequencehomologyto classical MDPV cluster;the 2016 isolates YFL,LHX and WZQ strains,contained some mutation similar to GPV;these revealed that the recombination between MDPV and GPV have become increasingly common.The BALB/cmice wereintraperitoneallyimmunized with the recombinant protein MDPV-VP3,two hybridoma cell linesagainst the VP3 protein were obtained,named 2G1 and 2G5.These hybridoma cell lineswere identified to be IgG1.Western blot and indirect immunofluorescence(IFA)showed that the two Mc Abs could combine with the natural MDPV.Indirect-enzyme-linked immunosorbent assay(Indirect-ELISA)was developed for the detection of antibodies against MDPV using recombinant MDPV His-VP3 as coating antigen.The indirect-ELISA assay was confirmed to have good reproducibility and specificity,and have no cross reaction with positive se rum of GPV,promising for the diagnosis of MDPV infection.