West China School of Public Health, & No. 4 West China Teaching Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China; Chengdu Center for Disease Control and Prevention, Chengdu 610041, Sichuan Province, China.
Chengdu Center for Disease Control and Prevention, Chengdu 610041, Sichuan Province, China.
Infect Genet Evol. 2019 Jan;67:44-50. doi: 10.1016/j.meegid.2018.10.012. Epub 2018 Oct 19.
Enterovirus 71 is the main pathogen that causes severe and fatal hand-foot-mouth-disease (HFMD) cases. As the enterovirus virus mutation has implications for pathogenesis, vaccine development, antiviral therapy, and epidemiological disease management of the virus. In this study, we investigated the variations of enterovirus 71 in thermal adaption, using the method of adaptive laboratory evolution. The sibling virus strains were isolated from a 2-year-old severe case of HFMD (#100) and her symptomless close contact (#101). Both strains were cultured in Vero cells by serial passage of 36 generations at the temperatures of 28.0 °C, 33.0 °C and 39.5 °C to construct adaptive lineages. According to the comparative analysis of phenotypes between adapted strains and parental strains, differences in growth rate were observed in the sibling lineages and a larger plaque was found mainly in the hot adapted strains for lineage #101. Two sets of adaptive strains from six time points (parental, 12th 17th, 31st, 35th passage and endpoint) were sequenced and analyzed by both Sanger sequencing and Next Generation Sequencing. Several variations in most coding genes and one reverse mutation in 5'UTR was observed, along with the identity of 99.8% for complete genome for both lineages. Notably, thermal specific non-synonymous mutations were found in the gene of VP1\VP3\3A\2C\3C. Moreover, the concurrent mutations A292G, A434G and A355C/T of sibling lineages in VP1 showed quantificational trace with distinguishing patterns for different temperatures, which were suspected to be the thermo-sensitive mutation hotspots. These results highlight the possible rules of thermal adaption in enterovirus 71, produce a novel picture of genome evolution of the virus, and shed light on viral variation and evolution.
肠道病毒 71 型是引起重症手足口病（HFMD）的主要病原体。由于肠道病毒病毒突变对病毒的发病机制、疫苗开发、抗病毒治疗和流行病学疾病管理有影响。在本研究中，我们使用适应性实验室进化的方法研究了肠道病毒 71 型在热适应方面的变异。从一名 2 岁重症 HFMD 患儿（#100）及其无症状密切接触者（#101）中分离出的姐妹病毒株。两种病毒株均在 Vero 细胞中通过在 28.0°C、33.0°C 和 39.5°C 下连续传代 36 代进行培养，构建适应株系。根据适应株与亲代株之间表型的比较分析，在姐妹株系中观察到生长速度的差异，并且在主要适应热的株系中发现了更大的斑块。从六个时间点（亲代、第 12 代、第 17 代、第 31 代、第 35 代和终点）获得的两组适应株系进行测序，并通过 Sanger 测序和下一代测序进行分析。观察到大多数编码基因中的几个变异和 5'UTR 中的一个反向突变，两种株系的完整基因组的同一性为 99.8%。值得注意的是，在 VP1\VP3\3A\2C\3C 基因中发现了热特异性非同义突变。此外，VP1 中姐妹株系的 A292G、A434G 和 A355C/T 并发突变显示出与不同温度区分模式的定量痕迹，这些突变可能是热敏感突变热点。这些结果突出了肠道病毒 71 型热适应的可能规律，产生了该病毒基因组进化的新图景，并阐明了病毒变异和进化。
