Isolation,Identification And Main Gene Phylogenetic Analysis Of A Variant Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus?PEDV?,a member of the genera Alphacoronavirus in the family Coronaviridae,causes acute diarrhea,vomiting and other symptoms in pigs.One-week-old piglets have more severe morbidity with a 100%of mortality.In recent years,PEDV has caused epidemics again,also,it has been repoted in the worldwide.In the majority of provinces and cities in China,pigs have infected PEDV,which has caused huge losses to the pig breeding industry.The re-epidemic of PEDV,compared with the previous epidemic strains,has a large difference in genes.Current vaccines have poor protection against emerging variant PEDV strains.Therefore,the study of the novel strains is of great significance to the prevention and control of PEDV.In this study,we collected intestine samples which came from suspected PEDV-infected piglets in a farm in Dehui,Jilin Province.One PEDV strain was successfully isolated and cultured in Vero E6 cells with the additon of trypsin.Morphology,molecular biology and serology identification were performed,so were determination of its TCID50 and animal pathogenicity experiments.The complete genome of the isolated strain was amplified and sequenced.DNASTAR and MEGA 6.0 software were used to blast homology analysis with the strains which published on GenBank and performed phylogenetic analysis.Also,the variant characteristic of main gene S,M,ORF3 and N genes were analysized.The intestine samples were inoculated sterilely with Vero E6 cells.The cytopathic effect appeared in the fourth passage of cultured cells.The lesions were characterized by cell rounding,breaking and shedding.The isolated PEDV virus,provisionly named as CH/JLDH/2016 strain,was identified by morphological observation and serological analysis.The strain was proved to be a variant strain by molecular biological nano-nested PCR method.The TCID₅₀0 of the PEDV strain was relatively stable in the 16 passages and reached a titer of 10^-5.2/mL.The animal experiments confirmed that the strain had pathogenicity to the piglets.In order to know the variation characteristics of the isolated virus,complete genome amplification and sequencing were carried out on CH/JLDH/2016 strain,and the main gene S,ORF3,M and N genes were selected for analysis.The results manifests that the mutated of S gene was significant,which contained 16 nucleotide insertions and 10 nucleotide deletions.As a result,there was 5 amino acid insertions and 3 amino acid deletions.There were 10 amino acid changes in the COE epitope?499⁶38aa?,and the 766 amino acid position in the SS6epitope were changed from Y to S.Compared to reference sequences in NCBI,the homology of S,ORF3,M and N genes were 93.6%⁹9.1%,91.3%⁹9.4%,97.4%⁹9.7%and 95.7%⁹9.5%respectively.The phylogenetic analysis showed that S,M and N genes were not in the same group compared as the vaccine strain,and the relationship was far away,which was close to the genetic distance of the epidemic strains in recent years.Although the ORF3 gene was in the same group compared as the vaccine strain CV777,it belonged to different subgroups.Through phylogenetic analysis,the main structural genetic variation trends of isolated strains were consistent.The genetic analysis of isolated strains indicated that there was a prevalence of PEDV variant strain in Jilin Province.In this study,the novel PEDV strain was successfully isolated by adding trypsion to the culture cells.This virus can be passaged stably in Vero E6 cells and had pathogenicity to piglets.Also it provides PEDV candidate strains for research and development of vaccines.The genetic sequence analysis of the isolated PEDV contributes to understanding the epidemicity of variant PEDV strains and enriches the molecular epidemiological datas.