Isolation and identification of a PEDV stain and sequence analysis of its ORF3 and S genes

In order to investigate the etiology and genetic evolution of diarrhea in piglets from a pig farm in Shenyang City, Liaoning Province, abdominal dissection was performed on piglets with diarrhea and pathological changes of small intestine were recorded. The tissues with obvious pathological changes were collected for section and H.E. staining, and the results were observed and recorded. Virus nucleic acid was extracted from the samples, and RT-PCR was performed by PEDV, TGEV, PDCoV, PRoV and SADS-CoV detection primers. Then the virus was isolated, cytopathic effect（CPE） observation, passage, indirect immunofluorescence assay and titer determination were performed. Finally, the key genes of the virus were amplified by PCR and genetic evolution analysis. The results showed that the intestinal wall was thin, swollen and filled with watery contents; the mesentery was dendritic hyperemia, and the mesentery lymph node edema was observed. Duodenal and intestinal villi were broken, with cell necrosis, and lamina propria cell hyperemia; jejunal villi atrophied and shortened; epithelial cells vacuolated and a small amount of lymphocytes infiltrated, which were the same as the pathological changes of PEDV-infected pigs. The nucleic acid test results of PEDV were positive, and the test results of other 4 pathogens were negative. The 6-generation blind virus culture medium was inoculated into Vero cells, and obvious CPE appeared after 48 h of culture. The indirect immunofluorescence test using PEDV N-specific monoclonal antibody as the primary antibody showed specific green fluorescence, and the virus titer was measured to be 1×10^5.83TCID₅₀/mL. The isolated strain was named CH/XF1.C.3/2020. The genetic similarities of PEDV isolates CH/XF1.C.3/2020 ORF3 gene and S gene with domestic and foreign PEDV reference strains were 89.9%-99.6% and 92.7%-98.4%, respectively.This strain was closely related to PEDV variants isolated at home and abroad in recent years, but far related to the classic strain CV777（GenBank accession number is KT323979.1）, and belonged to the GⅡ/GⅡa type. These results indicated that a GⅡ/GⅡa PEDV strain was isolated successfully and it was the causative agent of diarrhea in piglets in this pig farm.