猪流行性腹泻病毒G2c重组变异毒株分离鉴定及致病性分析

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

  • 摘要: 【目的】 明确江西地区猪流行性腹泻病毒(PEDV)流行株的致病性和遗传变异情况,为研发PEDV疫苗及科学防控猪流行性腹泻(PED)提供参考依据。【方法】 将腹泻仔猪的空/回肠组织处理后接种至Vero81细胞进行PEDV分离,经间接免疫荧光(IFA)鉴定后通过Reed-Muench法计算分离毒株的TCID50,并绘制病毒体外生长曲线;使用RT-PCR进行全基因组扩增,经SeqMan拼接后进行遗传进化分析和重组分析,并通过动物回归试验评价分离毒株的致病性。【结果】 利用Vero81细胞成功分离出1株PEDV,命名为CH-JX-2024,可在Vero81细胞中稳定传至第50代。以CH-JX-2024株接种Vero81细胞,在感染后6~18 h快速增殖,于感染后24 h达最高滴度,随后进入衰减期,病毒滴度缓慢下降。CH-JX-2024株全基因组序列与PEDV参考株全基因组序列的相似性为96.3%~99.0%,其中,与CH/JSXZ/2015(MT625963)株的相似性最高(99.0%),与SM98(GU937797)株的相似性最低(96.3%)。基于S氨基酸序列相似性构建的系统发育树显示,CH-JX-2024株属于G2c亚型,与LS-23(OR089086)株的亲缘关系最近,与AH-M(KJ158152)、Attenuated CV777(KT323979)等经典毒株的亲缘关系较远。CH-JX-2024株的13730~22341 nt片段可能来源于JSHA2013(KR818833)株与ZJCZ4(JX524137)株的重组基因序列;CH-JX-2024株与G2b亚型AJ1102(JX188454)株存在1个氨基酸插入(1198H1200)和29个氨基酸突变,且其突变位点主要集中在S10中和表位区域。5日龄健康新生仔猪口服接种CH-JX-2024株第10代病毒液(106 TCID50/mL),在攻毒后12 h开始出现水样腹泻和呕吐,至攻毒后72 h全部发病死亡;攻毒组仔猪的胃内含有大量乳糜,小肠内充满液体,肠壁薄而透明,空肠肠绒毛大量脱落,黏膜上皮细胞坏死,杯状细胞数量减少。【结论】 分离获得的CH-JX-2024株属于PEDV G2c亚型变异株,具有较高的致病性,与当前广泛使用的变异疫苗株相比,在S蛋白抗原表位序列上存在多处氨基酸变异,因此无法通过接种PEDV商品化变异株灭活疫苗获得免疫交叉保护。

     

    Abstract: 【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 50th 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 10th-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.

     

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