Isolation And Identification And Complete Genomic Sequence Analysis Of Duck-derived New Goose Parvovirus

Called as short beak and dwarfism syndrome,a disease mainly characterized as short beak and inhibition of the sticking out of tongues as well as the growth and development of ducks appeared in breeding areas of the cherry valley duck in Shandong,Anhui,Jiangsu and Henan in early 2015.The incidence of the disease has been increasing since the emergence of the disease.Due to its influence on meat-type duck products,huge economic losses have been caused on duck breeding households and enterprises.The study isolated and identified the pathogen of the disease and measured and analyzed the whole genome sequence of one isolated strain.At the same time,the detection method of the digoxin labeled nucleic acid probe was established to provide a theoretical basis for the epidemiological investigation of the disease.In order to identify the pathogen causing the disease,diseased duck liver tissues were collected from different duck breeding areas in the paper for respective isolation and identification as well as the conventional PCR detection of the bacteria.The results showed that Escherichia coli was isolated from a few tissues.The PCR detection results showed that all the samples were GPV positive.After the grinding of liver tissues,the duck embryo and goose embryo were inoculated through the chorioallantoic membrane to observe the thickening and opacification of the chorioallantoic membrane and embryo hemorrhage caused by known viruses,which however had no obvious pathogenicity on goose embryo.The allantoic fluid of the duck embryo was collected and the identification result showed that the allantoic fluid was GPV positive.A virus named SDLC01 strain isolated from a duck farm in Liaocheng,Shandong was selected.The results of the coagulation inhibition test showed that the virus had no agglutination on red blood cells of three kinds of poultry including chicken,ducks and geese.The animal regression test showed that the SDLC01 strain could cause short beak and inhibition of the sticking out of tongue as well as growth and development of ducks after the inoculation in meat-type ducks.The pathogeny of short beak and dwarfism syndrome was confirmed to be the duck-derived new goose parvovirus.According to the known whole genome sequence of gosling plague,five pairs of specific primers were designed and synthesized to cover the whole genome.The PCR method was used to amplify and obtain the whole genome sequence of the SDLC01 strain.The analysis showed that the full length of the SDLC01 strain was 5006 bp and the two sides of the genome were ITR with the length of 366 bp.The middle part contains the left and right ORFs to respectively encode NS proteins(NS1 and NS2)and VP proteins(VP1,VP2 and VP3).The lengths of NS1 and NS2 were respectively 1884 bp and 1356 bp.And the lengths of VP1,VP2 and VP3 were respectively 2199 bp,1764bp and 1605 bp.The analysis of whole genome sequence showed that the SDLC01 strain had a homology of 92.3%-97.2% with GPV,and 81.4%-85.5% with MDPV.The strain of virus has a closer genetic relationship with Europe-source GPV in evolution and may have the same ancestor.The homology of VP3 gene of the isolated strain between GPV was 93.1%-98.5% and the homology of NS2 gene with GPV was 93.1%-98.2%,which were all higher.Compared with the ITR regions of the GPV pathogenic strain,vaccine strain and MDPV pathogenic strain,the isolated strain showed a marked loss in the ITR region of the isolated strain.The online software was used to predict the glycosylation sites of the isolated strain.The results showed that there were 6 potential glycosylation sites in the isolated strain of SDLC01,which may have an effect on the host range and virulence of the virus.A specific primer with a length of 301 bp was designed according to the nucleoti de sequence of the VP3 gene of SDLC01 strain.Through the PCR amplification,thetarget fragment was recovered,purified and labeled with digoxin to establish the de tection method of the digoxin labeled nucleic acid probe of the duck-derived new go ose parvovirus.The specificity test results showed that the probe only had a specificreaction with the duck-derived new goose parvovirus but had no hybridization reacti on with other common duck-derive viruses.The sensitivity test results showed that t he minimum detection amount of the probe was 25 pg.The probe was used to detect the diseased ducks collected from different areas,which had a coincidence rate of 98% compared with the results of the virus isolation and identification.