Genetic Evolution Analysis Of PEDV In Partial Area Of China From 2017 To 2018

Porcine epidemic diarrhea?PED?is a highly contacted porcine intestinal infectious disease caused by Porcine epidemic diarrhea virus?PEDV?.Its main features are diarrhea,dehydration and vomiting.Infection can occur in pigs of all ages,but the clinical symptoms are somewhat different.Among them,the clinical symptoms of suckling piglets are the most serious and the mortality is high.Since the PED broke out in the south in October 2010 and spread rapidly to the country,continuous monitoring of the prevalence of PEDV is necessary.In this study,22 pig diarrhea diseases were collected from different farms in some parts of China,and the disease was detected by RT-PCR.Among them,13 samples showing PEDV-positive disease.13samples of PEDV-positive disease were subjected to nucleic acid extraction,and the S gene was amplified and sequenced.Compared with the S gene sequence of CV777 strain,the nucleotide homology was 93.8%⁹4.9%,and the deduced amino acid sequence homology was 93.1%⁹4.1%.Phylogenetic tree analysis indicated that they belonged to the G2 subgroup and were non S-INDEL type,which was far from the North American strain.Further pathogen isolation,PEDV-positive disease material was treated and inoculated into Vero cells,and DMEM containing 4‰trypsin was used as a maintenance solution and observed the lesion.It was identified by RT-PCR test,electron microscopy and plaque purification test.Three PEDV strains were isolated and named HuB-WH-2017?HLJ-JMS-2018 and HLJ-2018,respectively.Stable passage to F25,detecting the TCID₅₀ of the virus,10^5.62 TCID₅₀/mL,10^5.6 TCID₅₀/mL and 10^6.33 TCID50/mL respectively.The S gene of the three strains was amplified and sequenced,and the nucleotides and amino acids of the S gene of CV777 strain were compared.It was found that all three strains had amino acid point mutations on the epitope.Then,the isolated three strains of virus HuB-WH-2017,HLJ-JMS-2018 and HLJ-2018 were separately cultured,and the virus was purified by sucrose density gradient centrifugation,and the mice were separately immunized and the change in serum neutralizing antibodies level were monitored.Neutralizing antibodies were not present in the mice before immunization.At 14 days after immunization,the levels of neutralizing antibodies of the three viruses increased to 1:478,1:489,and1:502.And as the number of immunizations increased,the level of neutralizing antibodies also increased.Finally,on the 7th day after strengthen immunization,the serum neutralizing titers reached1:614,1:614 and 1:631,respectively.The antigen-antibody neutralization test was carried out with the immunized sera of different strains including the 3 isolated strains and 1 vaccine strain CV777.The results showed that there was a difference in the neutralization activity between the three newly isolated strains and the CV777 vaccine strain,but the difference was not significant.