Abstract:

The aim of this study was to investigate the pathogen of piglet diarrhea and its genetic evolutionary characteristics as well as pathogenicity on a pig farm in Gansu Province, with a view to providing some references for the prevention and control of epidemics in this pig farm. In this study, the small intestine tissues of diseased pigs collected from the pig farm were processed, and then the common diarrhea pathogens of pigs were detected. Vero E6 cells were used to isolate the virus from the diseased materials with positive pathogen detection, and the identification, purification, genetic evolution analysis and piglet pathogenicity evaluation test of the isolated strains were carried out. The results showed that the samples collected by RT-PCR were positive for porcine epidemic diarrhea virus (PEDV). CPE observed that the cell contour disappeared, a large number of cells fell off, formed plaque, and the cells were drawn into a net. IFA observed specific green fluorescence in cells. Western blot showed the target band at 57 ku. The typical structural characteristics of the coronavirus were observed under the transmission electron microscope. The titer of purified virus was 10^6.0 TCID₅₀·mL^-1. Phylogenetic analysis showed that the isolate was closely related to SXSL and HK2021, and had a certain evolutionary distance from the traditional PEDV strain CV777. The experimental animals had severe diarrhea. Piglets were thin, emaciated, and had growth retardation. Anatomy showed that the small intestine wall became thinner, the small intestine was full, and the mesentery was congested. Pathological tissue sections showed that intestinal villi were shortened, broken and exfoliated, with scattered bleeding points and lymphocyte proliferation and necrosis. Immunohistochemical results showed that there were obvious positive signals in all segments of the small intestine, and the positive signal of PEDV in the ileum was the strongest. Genetic evolution analysis showed that the PEDV isolate belonged to the GⅡa type, and it was named LZ202401 strain. Pathogenicity analysis showed that it was a variant virulent strain, which was the main pathogen of diarrhea in piglets on the pig farm. This study provides a theoretical basis for further exploring the molecular characteristics and genetic evolution characteristics of PEDV, and provides a reference for the prevention and control of PEDV.

Key words: porcine epidemic diarrhea virus, isolation, genetic evolution analysis, pathogenicity analysis