Isolation And Identification Of PEDV And Its Sequence Analysis Of Complete Gene

Porcine epidemic diarrhea (PED) is an acutent and highly contagious disease caused by porcine epidemic diarrhea virus (PEDV). It can cause acute diarrhea to rapid dehydration then death, and most harmful to the piglets. In recent years, PED in the Sichuan region broke out frequently, and studies indicated that the pathogen of PEDV has been changed, while it's tissue culture characteristics, pathological characteristics, molecular biological characteristics are unknown.This study collected intestinal and contents from piglets which suspected infected PEDV came from the farm in Sichuan Emei, detected by RT-PCR, PEDV positive samples were selected to be treated by conventional method and inoculated with Vero cells. At the same time, added 5 ug/mL trypsin in cell maintains liquid, continuous subculture. In the 5th generation, Vero cells appearred of cytopathetic effect (CPE), as to the 25th generation, Vero cells can appear of CPE in 20h hdistribution, as cells became round and bright, particulate matter changed, fusion made between cells, pull netcells formed after cells death and fell off. RT-PCR detection and S gene sequencing confirmed that the PEDV strain was successfully isolated in the study, and named as EM-P strain. And the TCID50 is 1�10-6.5/mL.Took the isolated inoculated in a week piglets, successfully replicated the PED case. Histopathological observation showed that the infection of EM-P PEDV strain was persistent and multiple of organs, and major injuried intestinal, lymph node, lung and spleen. Autopsy pathology manifestations of intestinal got thin and congested, intestinal epithelial fell off, mesenteric lymph became congested, lymphopenia cell reduced and vacuolar degeneration, pulmonary marginal anemia, local congestion and edema, alveolar fusion, the edge of the spleen became thin and translucent, and white pulp atrophy. Took PEDV N gene and GAPDH gene built qRT-PCR, detection of viral load by this method. The highest viral load was found in intestine, mesenteric lymph node, lung and spleen, and infected early, duration was long. The results of the above showed that the pathological damage of infected piglets was positively correlated with the viral load.According to the PEDV CV777 vaccine strains of gene sequence strain published in genebank, designed 18 pairs of primers, cloning complete gene of EM-P strain in fragments. EM-P strain was inoculated with Vero cells to emergenced more than 75% CPE, total RNA was extracted as template,18 fragments were amplified by RT-PCR. After cloning, sequencing and splicing, the whole genome sequence (cDNA) of EM-P strain was obtained. EM-P strain genome length 28029 bp, compared with 6 representative strains which have been reported, EM-P strain has typical characteristics of PEDV genome, and the homology of the reference strain was 97.3%-98.2%. Taking vaccine strain CV777 as a reference, EM-P strain S1 gene, ORF1 gene and other significant variation, which speculated may cause the change of the biological characteristics such as the immunogenicity of the virus and the ability of virus replication. ORF3 gene with complete open reading frame, was characteristic the strain was virulent. Referenced of CV777 PEDV strain and other 18 strains, the S gene sequence of EM-P strain was analyzed systematically and the genetic evolution tree was constructed:EM-P strain is located in G1-1 subgroup, but independenced in a separate branch, the EM-P strain could be classified as a new epidemic strain. While EM-P strain was in the same subgroup with the isolates about CV777 and from the South Korea, Japan, Canada and the China before 2010, so, it may be a new strain from abroad or the old strain in our country has experienced a certain degree of variation.