| Porcine epidemic diarrheas is an intestinal infectious disease caused by porcine epidemic diarrhea virus,which is characterized by vomiting,diarrhea and dehydration.Over the past 30 years,PEDV infection has caused significant economic losses in the pig industry in Europe and Asia.Therefore,a comprehensive understanding of the epidemic characteristics of epidemic or endemic PEDV strains is needed in order to prevent and control diseases in the affected areas and to develop effective vaccines.In order to have a better understanding of the epidemic trend,genetic variation and molecular evolution of PEDV in Henan Province,In this study,diarrhea samples collected from 2020 to the end of 2021 were detected and analyzed,and the genetic variation and phylogeny of S gene and whole genome sequencing of some strains were analyzed,On this basis,the isolation and identification of the virus,the development of inactivated vaccine and the preliminary evaluation of immune effect were carried out.1.Molecular Epidemiological investigation of PEDV in Henan Province499 clinical samples of piglets with diarrhea were collected in Henan from 2020 to 2021.RT-PCR was used for PEDV screening,and a total of 162 positive samples were detected,with a positive rate of 35.2%.The S genes of 21 PEDV strains were sequenced,homologous alignment and genetic evolution analysis.The results of homology comparison showed that the nucleotide homology and amino acid homology among 21 PEDV strains were 96.9%~100% and 95.2%-100%,respectively.The nucleotide homology and amino acid homology with 52 strains prevalent in China from 2011 to 2019 were 95.1%~99.5% and 91%~99.8% respectively.The results of genetic evolution showed that all the 21 PEDV strains belonged to GIIb group.Amino acid sequence alignment showed that there were 7 new and regular amino acid mutations in the S protein of the epidemic strain.In this study,the PEDV S genes prevalent in China were sequenced and analyzed,so as to provide reference for understanding the genetic variation trend of PEDV epidemic strains and developing new effective vaccines.2.Isolation and identification of PEDV epidemic strains.The identified PEDV positive samples were inoculated into Vero ATCCL-81 cells for continuous blind transmission,and CH-HNKF-03 and CH-HNPDS-01 strains were successfully isolated in 2020 and 2021,respectively.The whole genomes of the two strains were sequenced.the whole gene sequence homology and genetic evolution of the two strains were analyzed by using the sequence of PEDV strain published in Gen Bank.The results showed that the nucleotide homology between the two strains and the reference strain was 95.6%-98.7%,which belonged to the mutant strain GIIb group.CH-HNKF-03 and CH-HNPDS-01 isolates were artificially infected with 1-dayold piglets without colostrum,and the incidence of piglets was 100%,and the mortality rate was 100%,It showed that the isolates were virulent strains.The results of this study will provide a theoretical basis for grasping the genomic characteristics,evolutionary trends and improved vaccine prevention and control strategies of the latest epidemic strains of PEDV in Henan Province.3.Development of inactivated vaccines and preliminary evaluation of immune effectsDue to the virus genome is prone to mutation,recombination,vaccine replacement slowly and other factors,some areas still lack effective prevention measures.In order to better prevent and control the spread and epidemic of PED in Henan Province,this study used PEDV CH-HNKF-03 isolate to develop inactivated vaccines,studied the best culture conditions for viruses in this process,inactivated process,adjuvant screening,etc.,and screened out a water adjuvant with good effect,which was initially proved to have good effects by the immunization test of pregnant sows and the poison protection experiment of piglets,which provided a certain reference for the prevention and control and purification of PED in Henan Province.To sum up,the genetic variation of S gene and the characteristics of the whole genome of PEDV strains prevalent in Henan Province were clarified.The inactivated vaccine was prepared by PEDV CH-HNKF-03 isolates,and the good effect of the vaccine was preliminarily determined by animal experiments,which laid the foundation for the development of inactivated PEDV vaccine based on the latest epidemic strains. |
