Department of Clinical Microbiology, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain.
Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.
Microbiol Spectr. 2022 Apr 27;10(2):e0022122. doi: 10.1128/spectrum.00221-22. Epub 2022 Mar 29.
Mutant spectra of RNA viruses are important to understand viral pathogenesis and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultradeep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of 30 nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low-frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype, and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three-dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19. The study shows that mutant spectra of SARS-CoV-2 from diagnostic samples differ in point mutation abundance and complexity and that significantly larger values were observed in virus from patients who developed mild COVID-19 symptoms. Mutant spectrum complexity is not a uniform trait among isolates. The nature and location of low-frequency amino acid substitutions present in mutant spectra anticipate great potential for phenotypic diversification of SARS-CoV-2.
RNA 病毒的突变谱对于理解病毒发病机制和对选择压力的反应很重要。需要对从感染患者中采样的冠状病毒的突变谱复杂性进行特征描述。特别是，SARS-CoV-2 突变谱复杂性与疾病相关性之间的可能关系尚未建立。在本研究中，我们报告了对来自西班牙马德里第一波 COVID-19 大流行期间（2020 年 4 月）的 30 例鼻咽部 SARS-CoV-2 感染患者的 nsp12（聚合酶）和 Spike（S）编码区扩增子的突变谱进行超深度测序（UDS）分析，这些患者根据随后 COVID-19 的严重程度进行了分类。与相应分离株的共识序列相比，低频突变和缺失的数量非常多。我们表明，与中度或重度疾病（死亡）相关的 SARS-CoV-2 分离株相比，轻症患者分离株中的不同点突变、每个单倍型的突变数和几个多样性指数的平均值显著更高。然而，RNA 缺失没有观察到这种偏差。在 nsp12（聚合酶）和 S 的三维结构中氨基酸取代的位置表明存在重大的结构或功能影响。因此，与中度或重度 COVID-19 患者相比，发展为轻度症状的患者可能是 SARS-CoV-2 遗传变异的更丰富来源。该研究表明，来自诊断样本的 SARS-CoV-2 突变谱在点突变丰度和复杂性上存在差异，在轻症 COVID-19 患者的病毒中观察到显著更高的值。突变谱复杂性不是分离株的统一特征。在突变谱中存在的低频氨基酸取代的性质和位置预示着 SARS-CoV-2 表型多样化的巨大潜力。
