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.
J Virol. 2014 Apr;88(8):4008-20. doi: 10.1128/JVI.03594-13. Epub 2014 Jan 22.
Mutagenic nucleoside analogues can be used to isolate RNA virus high-fidelity RNA-dependent RNA polymerase (RdRp) variants, the majority of which are attenuated in vivo. However, attenuated foot-and-mouth disease virus (FMDV) high-fidelity RdRp variants have not been isolated, and the correlations between RdRp fidelity and virulence remain unclear. Here, the mutagen ribavirin was used to select a ribavirin-resistant population of FMDV, and 4 amino acid substitutions (D5N, A38V, M194I, and M296V) were identified in the RdRp-coding region of the population. Through single or combined mutagenesis using a reverse genetics system, we generated direct experimental evidence that the rescued D5N, A38V, and DAMM mutants but not the M194I and M296V mutants are high-fidelity RdRp variants. Mutagen resistance assays revealed that the higher replication fidelity was associated with higher-level resistance to ribavirin. In addition, significantly attenuated fitness and virulence phenotypes were observed for the D5N, A38V, and DAMM mutants. Based on a systematic quantitative analysis of fidelity and virulence, we concluded that higher replication fidelity is associated with a more attenuated virus. These data suggest that the resulting restricted quasispecies diversity compromises the adaptability and virulence of an RNA virus population. The modulation of replication fidelity to attenuate virulence may represent a general strategy for the rational design of new types of live, attenuated vaccine strains.
The ribavirin-isolated poliovirus (PV) RdRp G64S variant, the polymerases of which were of high replication fidelity, was attenuated in vivo. It has been proposed (M. Vignuzzi, E. Wendt, and R. Andino, Nat. Med. 14:154-161, http://dx.doi.org/10.1038/nm1726) that modulation of replication fidelity is a promising approach for engineering attenuated virus vaccines. The subsequently mutagen-isolated RdRp variants also expressed the high-fidelity polymerase, but not all of them were attenuated. Few studies have shown the exact correlation between fidelity and virulence. The present study investigates the effect of restricted quasispecies diversity on viral virulence via several attenuated FMDV high-fidelity RdRp variants. Our findings may aid in the rational design of a new type of vaccine strain.
诱变核苷类似物可用于分离 RNA 病毒高保真 RNA 依赖性 RNA 聚合酶 (RdRp) 变体，其中大多数在体内是减毒的。然而，尚未分离到具有低感染力的口蹄疫病毒 (FMDV) 高保真 RdRp 变体，并且 RdRp 保真度与毒力之间的相关性尚不清楚。在这里，诱变剂利巴韦林被用于选择 FMDV 的利巴韦林抗性群体，并且在该群体的 RdRp 编码区中鉴定出 4 个氨基酸取代 (D5N、A38V、M194I 和 M296V)。通过使用反向遗传学系统进行单个或组合诱变，我们生成了直接的实验证据，表明挽救的 D5N、A38V 和 DAMM 突变体但不是 M194I 和 M296V 突变体是高保真 RdRp 变体。诱变抗性测定表明，更高的复制保真度与更高水平的利巴韦林抗性相关。此外，D5N、A38V 和 DAMM 突变体的复制适应性和毒力表型明显减弱。基于对保真度和毒力的系统定量分析，我们得出结论，更高的复制保真度与更弱的病毒相关。这些数据表明，由此产生的受限准种多样性会损害 RNA 病毒群体的适应性和毒力。调节复制保真度以减弱毒力可能代表了合理设计新型活减毒疫苗株的一般策略。
