Isolation， identification， and pathogenicity analysis of the G2c recombinant mutant strains of porcine epidemic diarrhoea virus

【Objective】 This study aimed to elucidate the pathogenicity and genetic variation of prevalent strains of porcine epidemic diarrhoea virus （PEDV） in the Jiangxi region， so as to provide a reference for the research and development of PEDV vaccines and the scientific prevention and control of porcine epidemic diarrhoea （PED）. 【Method】 Treated jejunal/ileal tissues from diarrhoeic piglets were inoculated into Vero81 cells for PEDV isolation. After identification via indirect immunofluorescence assay （IFA）， TCID₅₀ values of the isolated strain were calculated using the Reed-Muench method to plot an in vitro viral growth curve was plotted. The whole genome amplification was performed using RT-PCR， and after SeqMan assembly， genetic evolution analysis and recombination analysis were performed. The pathogenicity of the isolated strain was evaluated using the animal regression test. 【Result】 A PEDV strain of was isolated using Vero81 cells and designated CH-JX-2024， which could be stably propagated to the 50^th generation in Vero81 cells. When inoculated into Vero81 cells， the strain CH-JX-2024 proliferated rapidly 6-18 h post-infection， and the titer peaked at 24 h post-infection， after which a decline phase was observed with a gradually decreased viral titer. The whole genome sequence similarity between CH-JX-2024 and PEDV reference strains was 96.3%-99.0%； the highest similarity （99.0%） was observed in the strain CH/JSXZ/2015 （MT625963）， while the lowest similarity （96.3%） was in strain SM98 （GU937797）. The phylogenetic tree based on similarity of S amino acid sequence indicated that CH-JX-2024 belonged to the G2c subtype， exhibiting a closest relationship with strain LS-23 （OR089086） and distant relationships with classical strains such as AH-M （KJ15S152） and Attenuated CV777 （KT323979）. The 13730-22341 nt segment of the strain CH-JX-2024 may originated from recombinati gene sequences between JSHA2013 （KR818833） and ZJCZ4 （JX524137）； the strain CH-JX-2024 exhibited one amino acid insertion （¹¹⁹⁸H¹²⁰⁰） and 29 amino acid mutations compared to the strain AJ1102 （JX188454） of subtype G2b， with mutant loci predominantly concentrated within the neutralising epitope regions in the S1⁰. Healthy 5-day-old newborn piglets were orally inoculated with the 10^th-generation viral suspension （10⁶ TCID₅₀/mL） of strain CH-JX-2024. Watery diarrhoea and vomiting started at 12 h post-challenge， with all piglets succumbing to the disease by 72 h post-challenge. The stomachs of challenged piglets contained copious amounts of chyle， while the small intestines were filled with fluid. The intestinal walls appeared thin and translucent， with extensive loss of jejunal villi， necrosis of mucosal epithelial cells， and a reduction in goblet cell number. 【Conclusion】 The isolated strain CH-JX-2024 is the variant strain of PEDV G2c subtype， exhibiting high pathogenicity. Compared to the widely used variant strain vaccine， it has multiple amino acid mutations in epitope sequences of the S protein， therefore， cross-protective immunity could not be realized through commercialised inactivated vaccines of PEDV variant strain.