关键词: 猪流行性腹泻病毒, 分离, 鉴定, 序列分析, 哺乳仔猪, 致病性

Abstract:

To understand the pathogenic characteristics and pathogenicity of the latest epidemic strain of porcine epidemic diarrhea virus (PEDV), isolation and identification of PEDV were conducted this study. PEDV positive samples were inoculated into Vero cells for continuous transmission for isolating PEDV epidemic strains. Then the culture characteristics, whole genome sequence of the isolated strains were analyzed, and their pathogenicity in piglets were evaluated. Results showed that a PEDV strain (named CHN-SC2021) was isolated from the 3rd passage (P3) Vero cells of continuous inoculation of PEDV positive sample. Under the electron microscope, the diameter of the virus was about 60-110 nm, forming classic "syncytium" on the cell; The virus titer from P10 to P35 generations increased from 10^4.7 TCID₅₀ ·mL^-1 to 10^6.43 TCID₅₀ ·mL^-1; The whole genome sequence of CHN-SC2021 (P5) (GenBank accession number: OM505025.1) is 28 014 bp, and its nucleotide sequence homology about 99.16%-96.32%, 99.01%-96.57% and 98.94%-96.33% compared with the reference strains from China, Europe and America, and other Asian countries, respectively; The phylogenetic analysis showed that the CHN-SC2021 strain is closely related to Jiangxi CH/JXJA/2017 (MF375374.1), Henan CH/HNAY/2015 (KT199103.1), and CH/HNKF-16 (KY649107.1) strains, belonging to the GⅡ a genotype type. The similarity between the S gene sequence and other strains ranges from 98.7% to 93.4%. Oral infection of 6-day-old suckling piglets with 3 mL of 10⁵ TCID₅₀ ·mL^-1 per pig of CHN-SC2021 virus solution resulted in loss of appetite at 12 hours, vomiting at 20 hours, and severe vomiting and yellow watery diarrhea at 24 hours; The main pathological changes during dissection are gastrointestinal distension, transparent jejunum and ileum; Histopathological changes showed that each segment of the intestine had varying degrees of intestinal villus lesions, shedding, loose connective tissue, and widened space between the muscularis mucosae and the muscle layer; Immunohistochemistry showed that viruses were present in all segments of the small intestine, with the most viruses present in the ileum. In conclusion, a genotype GⅡ a PEDV virulent strain was isolated, which will contribute to enriching the information of PEDV circulating strains in China and be helpful for clinical prevention and control of PED.

Key words: porcine epidemic diarrhea virus, isolation, identification, sequence analysis, sucking piglets, pathogenicity