Assessment of SARS-Coronavirus-2 Levels and Genetics in Vivo

Division Of Basic Sciences - Nci

Investigators

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Abstract

We investigated whole blood, plasma, and peripheral blood mononuclear cells from patients at the University of Pittsburgh Medical Center. In collaboration with investigators at the University of Pittsburgh, including Drs. John Mellors and Jana Jacobs, we detected CoV-2 RNA in the majority of samples from hospitalized donors and in few of the samples from outpatients. We found that levels of CoV-2 RNA in blood plasma were highly associated with disease pathogenesis and that CoV-2 RNA can be packaged in virus particles in the blood. We also characterized the population genetics of CoV-2 in the U.S. to establish a prevaccination baseline for viral genetic diversity and to profile the emergence of mutations prior to the introduction of the vaccines to the general public. Such a baseline allowed for the identification of mutations that were selected for by the introduction of the vaccine. My role in these studies was to advise on the development of a quantitative CoV-2 RNA assay, the development of CoV-2 sequencing assays, the selection of donor samples, and the interpretation of the results. I advised extramural investigators to modify our HIV Single-Copy Assay to quantify levels of CoV-2 RNA in the plasma of donors with COVID-19. My lab also assisted in modifying our HIV single-genome sequencing assays to CoV-2 for studies of intrapatient viral diversity and possible emergence of drug resistance. A CoV-2 SGS assay will allow for the detection of minor variants in vivo that may harbor mutations rendering resistance to therapies and preventives, including current vaccines. The CoV-2-adapted assays were carried out in the labs of my collaborators at the University of Pittsburgh, Drs. Mellors, Jana Jacobs, and Kevin McCormick. With the exception of a limited scope of experiments, such as cloning some fragments of the CoV-2 genome, testing primers for the CoV-2 SCA and uSGS assays, and validating the assays on CoV-2 RNA, we do not anticipate performing these studies in my lab. However, we assisted in the analyses of data obtained by our collaborators, as requested, to aid in our understanding CoV-2 pathogenesis, the efficacy of future antivirals, and the potential emergence of resistance to future treatments and vaccines. My commitment in time and resources were limited, as the goal is to transfer our experience in assay development and with RNA viruses to those focused on studies of CoV-2. _____ACCOMPLISHMENTS: ____Using our SARS-CoV-2 plasma viral load assay we found that plasma SARS-CoV-2 RNA levels are a biomarker of lower respiratory tract infection in critically ill patients with COVID-19 (Jacobs, et al. JID 2022) ____ We also found that SARS-COV-2 viremia is associated with disease severity and predicts clinical outcomes (Jacobs, et al. CID 2022) ____By analyzing publicly available sequences, we demonstrated that the genetic diversity of SARS-CoV-2 tripled in the U.S. in 2020 and included the emergence and persistence of 54 amino acid changes in the population (Capoferri et al., Viruses, 2021). ____ In a review article, we described the Tterapeutic implications of ongoing alveolar viral replication in COVID-19 (McGonagle D, et al. Lancet Rheumatol. 4: e135-e144, 2022).