Isolation And Identification Of Goose-Derived Egg Drop Syndrome Virus And Genomic Evolutionary Analysis

Egg Drop Syndrome was a virulent infectious disease caused by EDSV with the main characteristics of laying shelless or soft shelled eggs and decreasing egg laying,and its occurrence and epidemic in the world caused enormous economy lose in poultry industry.Goose is one of the main natural hosts of EDSV,and adult goose infected with EDSV carry the virus without causing disease,it is one of the infection sources of chicken-derived EDS,but gosling infection cause acute respiratory diseases.EDSV has only one serotype,pathogenicity varies greatly among different isolates strain.Therefore,the isolation,identification and genetic variation analysis of geese-derived EDSV provide a theoretical basis and material basis for effective prevention of geese EDSV infection,at the same time,it played a positive role in preventing and controlling chicken EDS in the future.In this study,small intestine samples collected from goose suspected d ied from GPV in Ji Lin Province.The mixed infection of GP and EDS was confirmed by PCR.The tissue material was homogenized,then inoculated SPF duck embryos by allantoic cavity,and duck embryos died at 5th generation until passaged 20 th generations.PCR detection results showed that EDSV was purified preliminarily for no other virus proliferated.Next,allantoic fluid inoculated on DEL cells,and the typical EDSV cytopathic effect appeared after 72 hours.Hemagglutination test showed that the isolated virus only agglutinate erythrocytes from the bird such as chicken,duck and goose,but not agglutinate erythrocytes from mammals such as cattle,rabbit and sheep.The electron microscopy demonstrated of purified virus particles presented the typical morphology of adenovirus particles.The results above showed that the isolated virus was EDSV,which named JL-EDSV-629 strain.According to the EDSV reference sequence from Gene Bank,15 pairs of overlapping specific primers were designed to amplify the genomic sequence of the JL-EDSV-629 strain except the 5'UTR and 3'UTR.The complete sequence were assembled and analyzed.The genome of JL-EDSV-629 strain were 33215 bp in length,and A+T content was 56.98%,which consistented with Atadenovirus characteristics.The genome encoded 29 ORFs which were the main structural proteins including hexon,penton,fiber,p VIII,p IIIa,p VII,p X,TP and non structural proteins including DBP,IVa II,52 K,EP,100 K.Compared with EDSV reference strains AV-127?EDS and D11-JW-032,JL-EDSV-629 strain had 49 nucleotide mutations,and 16 of them were missense mutations.Amino acid homology alignment between JL-EDSV-629 strain and EDSV reference strain gene,among them,there are 19 genes with 100% homology such as penton,p X,hexon,DBP,and 10 genes with 99.3% to 99.8% homology such as TP,p VII,fiber.Bioinformatics analysis showed that the genomic structure and sequence of the isolate strain were completely consistent withl those of the EDSV reference strain.Nucleotide sequence homology and phylogenetic tree analysis showed that JL-EDSV-629 strain had high homology and close relationship with Atadenovirus reference strain,and the nucleotide homology of the EDSV reference strain was 99.8%,and is closely related to EDSV reference strain;And the nucleotide homology of the Atadenovirus reference strain BAd V-4,OAV-287 and Sn Ad V-1 was 54.6% to 60.4%.The homology with other Adenovirus reference strains was only 25.8% to 36.1%.At the same time,duck anti-JL-EDSV-629 strain polyclonal antibody were acquired,the HI titer of polyclonal antibody was 13log2.The recombinant penton protein was prepared by prokaryotic expression system,the ELISA results showed that the specific antibody titer of penton was 1:16000,Western Blot showed that the prepared antibody had good reaction with recombinant penton,which provides a material basis for deeply research on serological diagnosis method and structural protein biological function of JL-EDSV-629 strain in the future.