猪塞内卡病毒A SDWF/11/2024株的分离鉴定及其遗传演化特征分析

Isolation, identification, and genetic evolutionary analysis of the porcine Senecavirus A strain SDWF/11/2024

摘要:

目的 塞内卡病毒A (Senecavirus A, SVA)仍对我国养猪业构成严重危害，进一步了解最新流行毒株的基本生物学特性和遗传演化特征。方法 于2024年11月从华东地区某猪场采集疑似口蹄疫患病猪的水疱皮组织，经逆转录PCR (reverse transcription-PCR, RT-PCR)检测，将阳性样品按病毒分离的常规方法接种BHK-21细胞；利用间接免疫荧光试验初步验证病毒分离情况，并对其全基因组进行扩增测序和遗传演化分析，与重要流行节点的参考毒株VP1蛋白序列比对分析氨基酸变异特点；通过蚀斑形成试验和一步生长曲线评估分离毒株的增殖特性，再利用透射电镜观察病毒粒子形态。结果 经RT-PCR检测和病毒分离试验证实，该发病猪的病原为SVA，将分离获得的毒株命名为SDWF/11/2024株。其基因组全长为7 292 bp，整体结构与已报道的SVA株高度一致；系统发育树分析表明，SDWF/11/2024株归属于USA-like进化分支，与中国2015-2018年分离株存在一定分化，与智利2022年分离株亲缘关系最近；在体外复制特性方面，该毒株可在BHK-21细胞上稳定增殖，呈现出典型的细胞病变效应，其复制动力学特征与早期分离毒株HN/11/2017相似，但形成的蚀斑形态存在差异；通过透射电子显微镜观察，可见直径约为25-35 nm的颗粒，其大小特征与SVA病毒粒子相符。结论 本研究成功分离并表征了2024年我国猪群中流行的SVA毒株SDWF/11/2024，揭示了其分子演化特征。SDWF/11/2024株的分离为我国监测SVA流行提供了新的参考样本，提示该病毒仍可能以低水平存在于猪群中。这对认识SVA遗传多样性与流行趋势、完善其分子流行病学监测体系及制定科学防控策略具有重要意义。

Abstract:

Objective Senecavirus A (SVA) keeps posing a serious threat to the swine industry in China. This study aimed to characterize the biological properties and genetic evolutionary features of the latest circulating SVA strains.Methods In November 2024, vesicular lesion tissue samples were collected from pigs suspected of foot-and-mouth disease at a farm in eastern China. RT-PCR was performed, and positive samples were inoculated onto BHK-21 cells following standard virus isolation procedures. Indirect immunofluorescence assay was employed to preliminarily confirm viral isolation. The complete viral genome was amplified and sequenced, followed by genetic evolution analysis. Amino acid variations in the VP1 protein of the strain were identified by comparison with representative strains from key epidemic nodes. Viral replication characteristics were evaluated through plaque formation assay and one-step growth curve analysis. Viral particle morphology was observed via transmission electron microscopy (TEM).Results RT-PCR and virus isolation confirmed SVA as the causative agent of the disease, and the isolated strain was designated SDWF/11/2024. The viral genome of this strain was 7 292 bp in length, and its overall organization was highly consistent with that of previously reported SVA strains. Phylogenetic analysis revealed that SDWF/11/2024 belonged to the USA-like evolutionary clade, showing genetic divergence from Chinese strains circulating between 2015 and 2018, while exhibiting the closest relationship to a Chilean strain isolated in 2022. The isolate replicated efficiently in BHK-21 cells and induced typical cytopathic effects. Its replication kinetics was comparable to that of the early Chinese isolate HN/11/2017, although differences in plaque morphology were observed. TEM examination identified spherical viral particles with diameters of 25-35 nm, consistent with typical SVA virions.Conclusion This study successfully isolated and characterized the SVA strain SDWF/11/2024 circulating in China, 2024, and elucidated its molecular evolutionary features. The isolated SDWF/11/2024 provides a new reference strain for SVA surveillance in China and suggests that the virus may still persist at low levels in pig populations. These findings enhance our understanding about the genetic diversity and epidemic dynamics of SVA and support the improvement of molecular epidemiological monitoring and the development of prevention and control strategies.