Persistence and Evolution of SARS-CoV-2

SARS-CoV-2在体内的复制水平，由这种复制引起的病毒遗传变异，以及病毒在不同细胞类型和组织中的分布，可能有助于决定急性新冠肺炎的临床结果，以及病毒特异性记忆B和T细胞反应的长期保护能力。新冠肺炎患者的呼吸道和其他样本中SARS-CoV-2RNA的持续时间长达数周，一些患者的严重病程持续时间较长，这引发了人们对初级免疫反应无效的担忧。我们正在调查源于这一担忧的关键假设，如下所示。 假设1)复制SARS-CoV-2会积累基因突变，使其能够逃避主要宿主B和T细胞的反应。我们将通过对来自上呼吸道、下呼吸道、血液和组织部位的SARS-CoV-2基因组进行高通量、单分子深度测序来测试这一点。我们将确定体内病毒群体中同义和非同义遗传变异的流行率和纵向遗传变化。 假设2)SARS-CoV-2持续存在于解剖学或免疫学亚群特异性感染的细胞库中。PBMC和组织样本将用于特定淋巴和髓系细胞类型中(+)正义和(-)正义SARS-CoV-2RNA分子的量化和序列分析。这一方法将包括通过流式细胞仪进行细胞分选，通过成像细胞术和共聚焦显微镜进行联合成像/分子分析，以及通过RT-PCR进行常规分子分析。

The levels of SARS-CoV-2 replication in vivo, the virus genetic variation engendered by this replication, and the distribution of the virus among distinct cell types and tissues may help determine the clinical outcome of acute COVID-19 as well as the long-term protective capacity of virus-specific memory B and T cell responses. The weeks-long persistence of SARS-CoV-2 RNA in respiratory and other samples from people with COVID-19, as well as the protracted course of severe illness in some people with the disease, raise concern about ineffective primary immune responses. We are investigating key hypotheses stemming from this concern, as follows. Hypothesis #1) Replicating SARS-CoV-2 accumulates genetic mutations that allow escape from primary host B and T cell responses. We will test this by performing high-throughput, single-molecule deep sequencing of SARS-CoV-2 genomes from upper respiratory tract, lower respiratory tract, blood, and tissue sites. We will determine the prevalence and longitudinal genetic changes of synonymous and non-synonymous genetic variation within in vivo virus populations. Hypothesis #2) SARS-CoV-2 persists within anatomic or immunological subset-specific infected cellular reservoirs. PBMC and tissue samples will used for the quantification and sequence analysis of (+)-sense and (-)-sense SARS-CoV-2 RNA molecules within specific lymphoid and myeloid cell types. This approach will involve cell sorting by FACS, combined imaging/molecular analysis by imaging cytometry and confocal microscopy, and conventional molecular analysis by RT-PCR.