Isolation And Identification Of Porcine Epidemic Diarrhea Virus In Shanghai And Development Of Inactivated Vaccine

Porcine epidemic diarrhea（PED）is an infectious disease caused by Porcine epidemic diarrhea virus（PEDV）,which is characterized by severe diarrhea and high mortality in piglets.The protective effects of the existing domestic vaccines in different epidemic areas are different to varying degrees.At the same time,it is not clear about the genomic characteristics and genetic evolution information of the new PEDV epidemic strains.Therefore,in order to understand the genomic characteristics and genetic evolution information of the new PEDV epidemic strains and develop vaccines in line with the genotype of the epidemic strains,in the study,the small intestine tissues and contents of infected pigs in Shanghai were collected for isolation and identification of PEDV and developed the inactivated vaccine,which made a new exploration for the prevention and control of PED.The results are as follows:Isolation,identification and sequence analysis of PEDV:Small intestine tissues and their contents were collected from piglets who died of diarrhea at 7 days of age in Shanghai pig farms,and the virus was isolated and identified.Three strains of PEDV were isolated and named shxx-1902,shxx-1912 and SH1302 respectively.The whole gene sequence analysis and S gene sequence analysis showed that SH1302 belonged to GI gene group,and shxx1902 and shxx1912 belonged to GII-C gene group.Pathogenicity test of shxx1902 strain in piglets:Thirty piglets at the age of 5 days were selected and challenged with shxx1902-P5 strain after 10^-3～10^-7 gradient dilution.The diarrhea and death were observed.The PDD₅₀ and LD₅₀ were calculated by Reed Muench method.Twelve 3-day-old piglets were selected.After two days of adaptation,shxx1902-P5 and P35 virus solutions with virus titers of 10^5.84TCID50/m L were used to attack piglets respectively.The pathogenicity of different generations of virus strains was compared through diarrhea score,virus elimination,pathological dissection and histopathological changes.The results showed that when the virus titer was10⁴TCID₅₀/m L,all five piglets died on the 5th day after virus challenge.The virulence of shxx1902-P5 strain attenuated when it passed to the 35th generation（P35）.Preparation of inactivated vaccine:The titer with 2×10⁶TCID50/m L virus solution was inactivated with formaldehyde for 28h.After passing the inactivation test,the aluminum adjuvant inactivated vaccine was prepared by 1:1 mixing with aluminum adjuvant.The mice were immunized to observe the safety and effectiveness of the vaccine.The results showed that shxx1902-P5 aluminum adjuvant inactivated vaccine could effectively induce antibody production in mice and there was no significant difference with commercial vaccine on the 49th day after immunization.Clinical trial of inactivated vaccine:Pregnant sows were immunized with shxx1902-P5 strain of inactivated vaccine while serum and breast milk of sows were collected at a specific time for detection of antibody levels.The results showed that shxx1902-P5inactivated vaccine could make pregnant sows produce Ig G,Ig A antibodies and serum neutralizing antibodies,and there was no significant difference between Ig G antibodies,serum neutralizing antibodies and commercial vaccines.