Isolation And Phylogenetic Analysis Of Porcine Epidemic Diarrhea Virus From Partical Region Of China

From the winter of 2013 to the spring of 2014, large number of pigs were suspected to be infected by PEDV in farms of Jilin and Shaanxi provinces. In order to determine the pathogen origin and genetic property with home and abroad, feces and intestinal tissue samples were collected for virus isolation and key genes were sequenced and analyzed. The main results are as follows:1. Processed samples were inoculated with Vero cells. After three round passages, one strain of SP from Jilin Province, and three strains of YL9099, YL9140 and YL9234 from Shaanxi Province were isolated.2. All of S genes of the strains and M, ORF3 genes of strain SP were obtained by reverse transcription-polymerase chain reaction(RT-PCR) method. Then their sequences, genetic evolutionary relationship, antigenic epitopes and glycosylation sites were compared with S, M, ORF3 genes published in GenBank by DNAstar, MEGA6.0 and NetNGlyc 1.0 Server software.First, four strains had nucleotide homology from 94% to 99.4% with the vaccine strains CV777 and DR13. For the encoded amino acids, there were about 79 to 84 mutations compared with the vaccine strain CV777 and DR13, among which more than 70% mutations located in S1 zone which further supports that S1 is the most hypervariable region of PEDV. According to glycosylation sites and antigenicity analysis, core neutralizing epitope(COE), 2C10 and S1D6 had several mutations compared with vaccine strain CV777 and DR13, while none of the mutations were located in S1D5.Second, nucleotide sequence analysis of S gene with 68 strains showed that the S sequence of strain SP, YL9099, YL9140 and YL9234 had the highest nucleotide homology of 99.5%-100%with AJ1102、CH/JLCC、CH18,and had lower homology with the vaccine strains.Third, nucleotide sequence analysis of ORF3 gene showed that the ORF3 sequence of strain SP had the highest nucleotide homology of 99.7% with JS-HZ2012 China(Hangzhou) and GD-B China(Guangdong), while had the lowest homology of 95.6% with LZC China(Lanzhou). Nucleotide sequence analysis of M gene showed that the M sequence of strain SP had the highest nucleotide homology of 100% with 13-019349 American, USA Colorado 2013 American, BJ-2011-3 China, GD-B China, HB-2012-1 China, HBMC2012 China and JS-HZ2012 China. And it had the lowest nucleotide homology of 97.2% with LZC China.Analysis of the results indicated that PEDV show rapid variation and evolutionary trends at present with increasingly obvious regional and temporal difference. Therefore we may need to select new vaccine strains to control the outbreak of PEDV. Strain SP, YL9099, YL9140, YL9234 in this study represent the genetic characters of the new epidemic PEDVs and have potential to be used as candidate strains for vaccine R&D.