Isolation,Identification And Genetic Evolution Analysis Of PEDV Variants

Porcine epidemic diarrhea virus(PEDV)is a single-stranded positive-stranded RNA virus belonging to alpha-coronavirus,which can cause porcine epidemic diarrhea(PED),with clinical manifestations of vomiting,diarrhea,and dehydration Wait.PEDV is the main pathogen causing viral diarrhea,with a wide range of epidemics.At present,most provinces and cities in China except Tibet have reported that the continuous emergence of PEDV mutants has greatly reduced the protection efficiency of the existing vaccines and caused serious damage to pig farms.Losses can be called the "reaper" of pig farm income.Therefore,it is of great significance for the prevention and control of PEDV to strengthen the research on new mutant strains.In this study,the small intestinal tissue samples of piglets with diarrhea in a pig farm in Shanxi Province were selected as samples.The PCR method was initially identified as PEDV positive.Vero cells were used to inoculate and isolate the virus,and then the target band of PEDV S gene was detected by RT-PCR;The indirect immunofluorescence detection of N protein antibody showed obvious fluorescence,and the virus was identified by electron microscopy and other methods.The results showed that there were typical spike protein virions around the virus.PEDV was successfully isolated and named as SXYC strain.In order to further track the genetic evolution of PEDV-SXYC strain,this study cloned and sequenced the genome of PEDV-SXYC strain by molecular biology method,and compared it with the sequence analysis of the reported strains.The phylogenetic analysis of the whole genome shows that the SXYC strain belongs to the G2 subgroup and has the closest relationship with the ZJU/G2/2013 strain;the homology data analysis shows that the genome-wide homology rate of the PEDV-SXYC strain and the classic strain CV777 is 96.5%,S,E,M,N,ORF1a,ORF1b amino acid homology rates were 93.2%,97.4%,98.1%,96.4%,99.1%,and 99.8%,respectively,indicating that the genes of SXYC strains have undergone great changes compared with the classic strains..In order to verify whether the virulence of SXYC is weakened after subculture,this study carried out continuous subculture of SXYC strains in vitro,and selected the tenth generation(SXYC-P10)and 100th generation(SXYC-P100)strains against Three-day-old healthy piglets were tested for infection,and the results showed that piglets infected with SXYC-P10 strain had obvious clinical symptoms of PED,with thinning of the intestinal wall of the small intestine,flatulence in the intestine,and thin content;The small intestinal villi of piglets infected with SXYC-P10 strain atrophied and fell off.IHC showed that the virus was mainly distributed on the surface of the small intestinal villi.The virus was detected by RT-qPCR in the jejunum,ileum and duodenum of piglets infected with SXYC-P10 strain per gram of sample.The contents were 108.2 copies,108.2 copies and 106.8 copies,respectively.However,the piglets in the P100 challenge group showed no abnormal performance,the small intestinal villi of the piglets were intact,and no virus was detected.In summary,the PEDV-SXYC strain was successfully isolated.After gene cloning and sequencing analysis,the PEDV-SXYC strain belonged to the G2 subtype,and the homology rate with the classic strain CV777 was 96.5%.Compared with the S gene of CV777,There are a large number of base mutations,and the amino acid homology rate of S protein is 93.2%,indicating that the PEDV-SXYC strain isolated in this study is a variant strain,and the virulence of SXYC is significantly weakened after subculture.It provides theoretical and experimental basis for PEDV prevention and vaccine development.