Persistence and Evolution of SARS-CoV-2

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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.