The Establishment Of RT-PCR For Detection Of Porcine Enterovirus And The Complete Genome Sequencing And Preparation Of Egg Yolk Antibody Against PEDV Variant Strain

Porcine enteroviruses(PEVs)belong to porcine enteroviruses G(EV-G),a members of the genus of Enterovirus,the family Picornaviridae.Pig is the only natural host of EV-G,and it can infect all ages of pigs.Most of pigs infected with EV-G will generally not show significant clinical symptoms.To date,eleven EV-G types(EV-G1 through EV-G11)have been identified in pigs from North America,Asia and Europe.However,they have never been reported in Jiangxi Province in China.In this study,we designed a set of specific primers covering the conserved region of EV-G genome,and established a RT-PCR assay for a rapid detection of EV-G.A total of 43 fecal samples from diarrhea piglets collected in Jiujiang(25)and Yingtan(18)in Jiangxi Province,2015.The total fecal sample positive rate of EV-G was 11.6%(5/43)and the positive rate was 12.0%(3/25)in Jiujiang and 11.1%(2/18)in Yingtan.In addition,the complete genome of an EV-G strain identified which was collected from a diarrhea wild boart from Jiangxi,China was determined and then characterized.To amplify the complete genome sequence of a representative EV-G isolate identified in this study,designated as WB/2016/CH three pairs of primers were designed based on an EV-G reference strain(accession # Y14459,AF363453 and AF363455)By utilizing these primers,the whole genome of the EV-G was determined and analyzedWB/2016/CH.The total length of the sequence was 7,404 nt.And the whole genome of WB/2016/CH strain was align with the complete sequence of 20 reference sequences in DNAStar Lasergene7.10 software,then carried out homologous analysis with ClustalW method in MegAlign.The results showed that the WB/2016/CH strain had nucleotide identities ranging from69.8% to 78.4% compared to the reference strains at the complete genome level,and the deduced polyprotein amino acid sequence similarities were 71.8% ~ 77.9%.The highest homology of nucleotide and amino acid were both EV-G2 type(Gen Bank accession number AF363455.1).WB/2016/CH strain showed greater complete nucleotide and amino acid sequence similarity than any of the other strains to EV-G2(AF363455.1).The results of phylogenetic analysis demonstrated that the WB/2016/CH strain and EV-G2 had the closest genetic evolutionary relationship.Porcine epidemic diarrhea virus(PEDV)is one of the most important enteroviruses causing piglet diarrhea.The clinical symptoms are similar to porcine transmissible gastroenteritis virus(TGEV),and PEDV is mainly transmitted via the faecal-oral route.Variant PEDV had widely spread among the neonatal piglets since the end of 2010 in China and showed high morbidity and high mortality.At present,there is no specific treatment measures available.In this study,CH/JX/01 PEDV strain isolated in our lab was used an immunogen toimmunize the hens.After four times immunization,the hypoimmu-antisera against PEDV were obtaine,and the antisera was subject to purification.For this purpose,three different extraction methods were attempted to purify the egg yolk antibody.And this study established a fast and accurate indirect ELISA method to identify egg yolk antibody.And we used this indirect ELISA method to measure the heat resistance,acid and alkali resistant and three different protective agents for IgY.The results showed the IgY was denaturalization under high temperature(> 70℃)and stable under 70℃;in the pH 4 to 11.And IgY became more stable when added with 20% sucrose,glucose,and sucralfate at pH2.0 and 3.0.And the protective agent of 20% sucrose had the best protective effect when compared with the addition of sucrose and glucose.