Huang Weihong, Chen Yongjie, Xu Ting, Xiong Ting, Lv Yadi, Liu Dingxiang, Chen Ruiai
College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
Zhaoqing Branch of Guangdong Laboratory of Lingnan Modern Agricultural Science and Technology, Zhaoqing, China.
Front Microbiol. 2024 Sep 26;15:1443696. doi: 10.3389/fmicb.2024.1443696. eCollection 2024.
Senecavirus A (SVA), belonging to the genus in the family , is an emerging pathogen causing vesicular disease in pigs. The main clinical manifestations of SVA infection include high mortality in neonatal piglets, skin ulceration, and vesicular lesions. So far, there is no commercially available vaccines or drugs against SVA. Construction of SVA infectious clones carrying reporter genes will help understand the characteristics of SVA and promote vaccine development.
In this study, we established a reverse genetics system for a local SVA isolate and used it to rescue a recombinant SVA, rSVA-eGFP, expressing the enhanced green fluorescent protein (eGFP) by inserting eGFP, GSG linker and the P2A sequence between 2A and 2B genes.
We found that rSVA-eGFP exhibited a high replication efficiency comparable to the parental virus, was able to express the eGFP reporter efficiently and stable in maintaining the reporter gene up to six rounds of serial passages in BHK-21 cells. In mice, rSVA-eGFP also showed similar replication kinetics and pathogenicity to the parental virus, both causing mild lung lesions. In addition, a high-throughput viral neutralization assay was developed using eGFP as a surrogate readout in a fluorescence-based direct titration (FBT) assay based on rSVA-eGFP, facilitating rapid and accurate determination of the neutralizing antibody (nAb) titers.
The successful establishment of an SVA reverse genetics system and the rescue of rSVA-eGFP would create a powerful tool for future studies of SVA replication mechanisms and pathogenicity as well as for antiviral development.
A 型塞内卡病毒（SVA）属于 科 属，是一种新兴的可引起猪水疱病的病原体。SVA 感染的主要临床表现包括新生仔猪高死亡率、皮肤溃疡和水疱性病变。到目前为止，尚无针对 SVA 的商业可用疫苗或药物。构建携带报告基因的 SVA 感染性克隆将有助于了解 SVA 的特性并促进疫苗开发。
在本研究中，我们建立了一种针对本地 SVA 分离株的反向遗传系统，并利用该系统拯救了一种重组 SVA，即 rSVA-eGFP，通过在 2A 和 2B 基因之间插入 eGFP、GSG 接头和 P2A 序列来表达增强型绿色荧光蛋白（eGFP）。
我们发现 rSVA-eGFP 表现出与亲本病毒相当的高复制效率，能够有效表达 eGFP 报告基因，并在 BHK-21 细胞中连续传代六轮时稳定维持报告基因。在小鼠中，rSVA-eGFP 也表现出与亲本病毒相似的复制动力学和致病性，两者均引起轻度肺部病变。此外，基于 rSVA-eGFP，开发了一种高通量病毒中和试验，在基于荧光的直接滴定（FBT）试验中使用 eGFP 作为替代读数，有助于快速准确地测定中和抗体（nAb）滴度。
成功建立 SVA 反向遗传系统并拯救 rSVA-eGFP 将为未来研究 SVA 复制机制和致病性以及抗病毒开发创造一个强大的工具。
