Dr. Ashwin Balagopal has conducted single-cell translational studies to examine the persistence and spread of viruses in human tissues. He led studies using single-cell laser capture microdissection (scLCM) to quantify hepatitis C virus RNA in individual hepatocytes during chronic infection, reporting for the first time these quantities and the proportion of hepatocytes that are infected. Pivoting to chronic hepatitis B virus (HBV) infection, he combined scLCM with droplet digital PCR (ddPCR) to report quantities of covalently closed circular DNA (cccDNA) in single hepatocytes in concert with pregenomic RNA (pgRNA). In doing so, Dr. Balagopal uncovered a subpopulation of infected hepatocytes that did not produce pgRNA. These transcriptionally silenced infected cells are enriched during antiviral therapy with nucleos(t)ide analogues (NA). This finding has therapeutic implications since NA are not known to inhibit viral transcription. Indeed, silencing of viral transcription in infected cells may result in a state that resembles a functional cure. Dr. Balagopal’s team was the first to use molecular approaches to quantify the proportion of HBV-infected hepatocytes and to estimate the decline in this proportion during NA treatment. In doing so, the group extrapolated that it could take up to ten decades for NA treatment alone to result in viral eradication, thus underscoring the need for novel cure strategies. Previously, Dr. Balagopal conducted studies to characterize HIV-1 tissue reservoirs: the team was the first to adapt a viral culture outgrowth assay to tissue resident macrophages (TRM) from people with HIV-1 who were virologically suppressed with antiretroviral therapy. Dr. Balagopal focused on liver macrophages (Kupffer cells) because they are the most abundant TRM in the body. He found that despite the presence of HIV-1 proviral DNA, there was no evidence of propagating virus that resulted in outgrowth. Thus, the team concluded, liver macrophages are not a likely long-lived HIV-1 reservoir.
Ashwin Balagopal, MD