Isolation And Identification Of An Variant Subtype Giib Porcine Epidemic Diarrhea Virus And Development Of An Inactivated Vaccine

Porcine epidemic diarrhea virus(PEDV)is the causative agent of porcine epidemic diarrhea(PED).with highly pathogenic and infectious.Since December 2010,a large-scale outbreak of diarrhea has occurred in swine herds around China,characterized by water-based diarrhea,vomiting,dehydration and high mortality in suckling pigs.Studies have shown that the disease is caused by the virulent gene Ⅱb subtype PEDV(GⅡb subtype PEDV)mutant strain,there is an urgent need to develop the appropriate vaccine for the prevention and control of the disease.In this study,a PEDV strain(JSX2014 strain)was isolated from the small intestine samples of suckling piglets with acute diarrhea in Jiangsu province,which could proliferate on Vero cells and produce cytopathic effect(CPE).The virus could be stable in Vero cells by measuring the virus titer of passaged cytotoxicity.The whole gene of JSX2014 strain was amplified and sequenced by RT-PCR,the complete genomic sequence and the nucleotide sequence of S gene of JSX2014 strain and other PEDV strains were analyzed by MEGA5.05 software,which indicated that JSX2014 strain belonged to GⅡb subtype PEDV.In this study,Determination the proliferation of cell adaptation strain by examining the virus titer on Vero cells at different time points and different time points.The specific primers were designed for the N gene of PEDV,and the real-time fluorescence quantitative method was established to quantify the proliferated JSX2014 strain.According to the requirements of"Chinese Veterinary Pharmacopoeia" and "the People’s Republic of China veterinary biological products quality standards" to establish the seed bank of JSX2014 strain,which has no external virus contamination by RT-PCR.The inactivated vaccine was prepared by proliferating JSX2014 strain,formaldehyde inactivated virus and aluminum hydroxide as adjuvant by Vero cell culture.In this study,prenatal sows and 50 days old weaned piglets were immunized with GⅡb subtype PEDV JSX2014 inactivated vaccine.The immunoprotective efficacy of the inacti,vated vaccine was detected by indirect ELISA method and serum neutralization test.In the immunoprotection experiment,indirect ELISA test showed that weaned piglets had begun to produce immune protection at 14 days after immunization,and 100%serum antibody positive at 42 days after immunization.Neutralize experiment using JSX2014 as the indicator virus,it was shown that the weaned piglets inoculated with the inactivated JSX2014 vaccine showed the highest serum antibody level at 42 days after immunization,which was consistent with the indirect ELISA test results.The neutralizing antibody titer of serum and colostrum was significantly higher than that of inactivated CV777 immunized group.The traditional inactivated vaccine made by CV777 could not protect sow from JSX2014 Challenge.All the experimental results of JSX2014 inactivated vaccine can provide high efficiency immune protection for PEDV GⅡb strain.