Etiological Study And Virus Isolation Identification Of Porcine Epidemic Diarrhea In 2021-2022

Swine epidemic diarrhea is a serious threat to pig health.Before 2010,the classical strain represented by CV777 was mainly popular,and the vaccine prepared with this strain could effectively prevent the occurrence of swine epidemic diarrhea.However,since 2010,swine epidemic diarrhea has again become prevalent on a large scale nationwide,and the emergence of a large number of mutated strains has led to insufficient protection of the vaccine,which has brought serious losses to the pig breeding industry.Therefore,it is important to understand the epidemic situation of porcine epidemic diarrhea for its prevention and control as well as vaccine development.1.Etiology study of porcine epidemic diarrheaIn this study,727 intestinal tissues suspected to be infected with porcine epidemic diarrhea were collected from 29 provinces,municipalities and autonomous regions（except Qinghai,Tibet and Taiwan）from December 2021 to November 2022.RT-q PCR was used to detect Po RV,TGEV and PDCo V at the same time,and the cases of mixed infection were counted.The results showed that 228 samples were positive for PEDV,with a positive rate of 31.36%,of which 140 were onset by PEDV infection alone,with a rate of 61.4%;68cases were mixed by PEDV and Po RV,with a rate of 29.8%;and 20 other mixed infections,with a rate of 8.8%.The correlation between the prevalence of PEDV and the seasons was analyzed,and it was found that the prevalence of PEDV mainly occurred in winter and spring.All 228 positive samples were classified by S1 gene sequencing,and 77 strains were successfully sequenced,including 68 strains of GⅡ-a,8 strains of GⅡ-b and 1 strain of Indel-like.The homology analysis revealed that the nucleotide and amino acid homologies of S1 gene among the 77 strains were 93.3%-100%and 92.1%-100%,respectively;the nucleotide and amino acid homologies with the classical strains were 90.4%-94.0%and86.0%-93.8%,respectively;the nucleotide and amino acid homologies with the popular strains in recent years were 93.4%-99.9%and 92.5%-99.5%,respectively.The nucleotide and amino acid homologies between these 77 strains and the popular strains in recent years were 93.4%-99.9%and 92.5%-99.9%,respectively.The S1 gene of these 77 strains was compared with the S1 gene of the classical vaccine strain CV777 by nucleotide and amino acid difference analysis,and base mutations,deletions and insertions were found.2.Isolation and identification of porcine epidemic diarrheaTo improve the isolation rate of PEDV,the ACE2 gene was inserted into a lentiviral expression vector with EGFP,and lentiviral packaging was performed after correct insertion,after which the collected lentivirus was inoculated onto Vero（ccl-81）cells and screened with puromycin and monoclonal,and finally the expression of the target gene was detected to confirm the successful cell line construction and to verify that the cell line could improve the the isolation rate of PEDV.The recombinant plasmid construction was verified by sequencing results;RT-PCR and Western Blot results showed that the ACE2overexpression cell line was successfully constructed;finally,the effect of the cell line on the viral copy value was measured by fluorescence quantification,and the results showed that the viral copy value of PEDV in ACE2 overexpression cells was significantly higher than that in normal Vero（ccl-81）cells with the same infection time cells,indicated that ACE2 overexpression cells could effectively improve the proliferation of PEDV.PEDV culture was performed on some positive samples,and RT-q PCR,purity detection,indirect immunofluorescence test,transmission electron microscopy test and TCID₅₀ test were performed on the isolated strains.The results showed that the isolated strains could be stably transmitted without contamination by exogenous viruses;further verified by indirect immunofluorescence test with PEDV antibody,the test group showed obvious red fluorescence;transmission electron microscopy observed that the virus particles were spherical,with vesicles and fibrils,which were consistent with the characteristics of PEDV;the viral virulence value of PEDV after purification of empty spots was detected,and the TCID₅₀ results were 10^4.2 The two isolated strains were tested for infection in piglets and were found to be significantly pathogenic,and the piglets showed obvious symptoms of PEDV infection.In this study,727 samples of diarrhea samples were tested for PEDV,and the positive rate was 31.36%.The sequencing analysis of S1 gene in positive samples showed that the main endemic mutant strain in China was Group GⅡ.Ace2-overexpressing Vero cell line Vero-Ace2 was constructed successfully.Two PEDV strains were isolated and found to have certain pathogenicity.These results revealed the genetic evolution of porcine epidemic diarrhea,provided a theoretical basis for better scientific prevention and control of porcine epidemic diarrhea virus,and laid a foundation for the efficient isolation of porcine epidemic diarrhea virus and the development of vaccine.