Division of Livestock Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
J Virol. 2019 Jun 14;93(13). doi: 10.1128/JVI.00576-19. Print 2019 Jul 1.
Senecavirus A (SVA) is a reemerging virus, and recent evidence has emphasized the importance of SVA recombination on virus evolution. In this study, we report the development of an infectious cDNA clone for the SVA/HLJ/CHA/2016 strain. We used this strain to develop a reporter virus expressing enhanced green fluorescent protein (eGFP), which we then used to screen for a recombination-deficient SVA by an eGFP retention assay. Sequencing of the virus that retained the eGFP following passage allowed us to identify the nonsynonymous mutations (S460L alone and I212V-S460L in combination) in the RNA-dependent RNA polymerase (RdRp) region of the genome. We developed a Senecavirus-specific cell culture-based recombination assay, which we used to elucidate the role of RdRp in SVA recombination. Our results demonstrate that these two polymerase variants (S460L and I212/S460L) have reduced recombination capacity. These results indicate that the RdRp plays a central role in SVA replicative recombination. Notably, our results showed that the two recombination-deficient variants have higher replication fidelity than the wild type (WT) and display decreased ribavirin sensitivity compared to the WT. In addition, these two mutants exhibited significantly increased fitness compared to the WT. These results demonstrate that recombination and mutation rates are intimately linked. Our results have important implications for understanding the crucial role of the RdRp in virus recombination and fitness, especially in the molecular mechanisms of SVA evolution and pathogenicity. Recent evidence has emphasized the importance of SVA recombination on virus evolution We describe the first assays to study Senecavirus A recombination. The results show that the RNA-dependent RNA polymerase plays a crucial role in recombination and that recombination can impact the fitness of SVA in cell culture. Further, SVA polymerase fidelity is closely related to recombination efficiency. The results provide key insights into the role of recombination in positive-strand RNA viruses.
塞尼卡病毒 A（SVA）是一种重新出现的病毒，最近的证据强调了 SVA 重组在病毒进化中的重要性。在本研究中，我们报告了 SVA/HLJ/CHA/2016 株的传染性 cDNA 克隆的开发。我们使用该毒株开发了表达增强型绿色荧光蛋白（eGFP）的报告病毒，然后使用 eGFP 保留测定法筛选重组缺陷 SVA。在传代后保留 eGFP 的病毒的测序使我们能够鉴定基因组 RNA 依赖性 RNA 聚合酶（RdRp）区域中的非同义突变（单独的 S460L 和 I212V-S460L 的组合）。我们开发了一种基于塞尼卡病毒的细胞培养重组测定法，用于阐明 RdRp 在 SVA 重组中的作用。我们的结果表明，这两种聚合酶变体（S460L 和 I212/S460L）的重组能力降低。这些结果表明 RdRp 在 SVA 复制性重组中起核心作用。值得注意的是，我们的结果表明，与野生型（WT）相比，这两种重组缺陷变体具有更高的复制保真度，并且与 WT 相比，显示出降低的利巴韦林敏感性。此外，与 WT 相比，这两种突变体的适应性明显提高。这些结果表明重组和突变率密切相关。我们的结果对于理解 RdRp 在病毒重组和适应性中的关键作用具有重要意义，特别是在 SVA 进化和致病性的分子机制方面。最近的证据强调了 SVA 重组在病毒进化中的重要性。我们描述了用于研究塞尼卡病毒 A 重组的第一个测定法。结果表明，RNA 依赖性 RNA 聚合酶在重组中起关键作用，并且重组可以影响 SVA 在细胞培养中的适应性。此外，SVA 聚合酶保真度与重组效率密切相关。这些结果为正链 RNA 病毒中重组的作用提供了关键见解。
