Panova Natalya, Allan Nina P, Rubas Noelle C, Lee Rosa H, Kunihiro Braden P, Umeda Lesley, Peres Rafael, Juarez Ruben, Maunakea Alika K
University of Hawaii, USA.
Eur J Biomed Res. 2023;2(3):17-23. doi: 10.24018/ejbiomed.2023.2.3.66. Epub 2023 Jul 13.
Whole-genome SARS-CoV-2 sequencing tools are crucial for tracking the COVID-19 pandemic. However, current techniques require sampling of actively infectious patients following COVID-19 testing to recover enough SARS-CoV-2 RNA from the nasopharyngeal passage, which rapidly clears during the first few weeks of infection. A prospective assessment of the viral genome sourced from recovered non-infectious patients would greatly facilitate epidemiological tracking. Thus, we developed a protocol to isolate and sequence the genome of SARS-CoV-2 from stool samples of post-acute SARS-CoV-2 patients, at timepoints ranging from 10-120 days after onset of symptoms. Stool samples were collected from patients at varying timepoints post-convalescence, and viral DNA was isolated and sequenced using the QIAamp Viral RNA Mini Kit (Qiagen Inc.) and Ion Ampliseq Library Kit Plus (Life Technologies Corporation). Capacity of neutralizing antibodies in patient plasma was tested using a Luminex panel (Coronavirus Ig Total Human 11-Plex ProcartaPlex Panel, ThermoFisher). Of 64 samples obtained from post-acute patients, 21 (32.8%) yielded sufficient material for whole-genome sequencing. This allowed us to identify widely divergent phylogenetic relativity of the SARS-CoV-2 genome from post-acute patients living in the same households and infected around the same time. Additionally, we observed that individuals who recovered from infection expressed varying degrees of antibodies against SARS-CoV-2 structural proteins that corresponded to distinct variants. Interestingly, we identified a novel point mutation in the viral genome where infected patients expressed antibodies with a significantly reduced capacity to neutralize the virus relative to that of those infected with the wild-type strain. Altogether, we demonstrate a protocol to successfully sequence the SARS-CoV-2 genome from stool samples from patients up to 4 months post-infection, which can be applied to studies that assess the relationship between variants and immune response and safe monitoring of the SARS-CoV-2 genome during the pandemic.
全基因组SARS-CoV-2测序工具对于追踪新冠疫情至关重要。然而，目前的技术要求在新冠检测后对具有传染性的患者进行采样，以便从鼻咽通道中获取足够的SARS-CoV-2 RNA，而该通道在感染后的最初几周内会迅速清除。对康复的非传染性患者的病毒基因组进行前瞻性评估将极大地促进流行病学追踪。因此，我们制定了一项方案，用于从急性感染后SARS-CoV-2患者的粪便样本中分离并测序SARS-CoV-2基因组，时间点为症状出现后的10至120天。在康复后的不同时间点收集患者的粪便样本，并使用QIAamp Viral RNA Mini试剂盒（Qiagen公司）和Ion Ampliseq Library Kit Plus试剂盒（Life Technologies公司）分离病毒DNA并进行测序。使用Luminex检测板（ThermoFisher公司的冠状病毒Ig总人11联检ProcartaPlex检测板）检测患者血浆中的中和抗体能力。在从急性感染后患者获得的64份样本中，有21份（32.8%）产生了足够用于全基因组测序的材料。这使我们能够确定来自同一家庭且在同一时间左右感染的急性感染后患者的SARS-CoV-2基因组具有广泛不同的系统发育相关性。此外，我们观察到从感染中康复的个体表达了不同程度的针对SARS-CoV-2结构蛋白的抗体，这些抗体对应于不同的变体。有趣的是，我们在病毒基因组中发现了一个新的点突变，在该突变处，感染患者表达的抗体中和病毒的能力相对于感染野生型毒株的患者显著降低。总之，我们展示了一种方案，可成功对感染后长达4个月的患者粪便样本中的SARS-CoV-2基因组进行测序，该方案可应用于评估变体与免疫反应之间关系的研究以及疫情期间对SARS-CoV-2基因组的安全监测。
