ABSTRACT
Background: Knowing the true incidence of HIV-1 infections (recent infections) among people newly diagnosed is pivotal to monitoring the course of the epidemic. We have developed a Primer ID Next Gen Sequencing (PID-NGS) assay to identify recent infection by measuring within-host viral diversity over multiple regions of the HIV-1 genome. We implemented a state-wide project to identify recent infections and transmitted drug resistance mutations (DRMs) in diagnostic samples in near real time. Methods: Serum samples from individuals with newly HIV-1 diagnoses (diagnostic sample collected within 30 days of diagnosis) were sequenced. PID-NGS libraries were constructed covering the coding regions for protease, a portion of reverse transcriptase, integrase, and the env gene. The use of the PID-NGS strategy allows for significant error correction and also a definition of the sampling depth of the viral population. Recent infection was defined as within 9-month of infection. DRMs were summarized at detection sensitivities of 30%, 10% and 1% based on viral population sampling depth. Results: From Jan 2018 to Jun 2021, we successfully sequenced partial genomes from 743 individuals with new diagnoses. Year 2020 had the lowest number of new diagnoses (Fig 1a, red bar). Overall, 39.2% of samples were inferred to have represented infection within the previous 9 months. Percent of recent infection varied significantly over the years, increasing from 29.6% in late 2018 to 50.9% in early 2020, but decreasing significantly to 32.7% in 2021 (Fig 1a, blue lines). Individuals younger than 30 y/o were more likely to be identified with recent infection (p<0.01). NNRTI DRMs, especially K103N, were the most abundant DRMs. Fig 1b shows the trend of DRMs over the four years. We observed a trend of decrease in the overall NNRTI DRMs and an increase in the NRTI DRMs in the population. Further analysis suggests that the increase in NRTI DRMs were from TAMs and their revertants, while clinically important NRTI DRMs (K65R and M184) were low (<1%). Conclusion: We have demonstrated a state-wide, all-in-one platform to monitor HIV-1 recency and DRMs in new diagnoses. The number of new diagnoses decreased significantly in 2020 in concert with the COVID-19 pandemic which suggests a decrease in overall HIV testing. The decline in the percentage of recent infections in early 2021 signals a return to broader HIV-1 testing and diagnosis. The increase of other NRTI DRMs suggests ongoing evolution at these sites within the viral population.
ABSTRACT
Background: We previously showed that β-D-N4-hydroxycytidine (rNHC) and its orally bioavailable prodrug, molnupiravir, acts as a broad-spectrum antiviral against coronaviruses in vitro and in vivo through lethal mutagenesis. Molnupiravir is currently in clinical trials for the treatment of SARS-CoV-2 infection. However, there are concerns that rNHC could be metabolized to dNHC and cause mutations in host cells. We examined the in vitro antiviral and mammalian cell mutagenic activity of three different nucleoside/base analogs, rNHC, favipiravir, and ribavirin, on SARS-CoV-2. We further examined the in vitro genotoxicity of a panel of antiviral nucleotide/nucleoside analogs, including rNHC, using a modified HPRT gene mutation assay. Methods: A549-hACE2 cells were infected with SARS-CoV-2 in the presence of nucleoside analogs. After 48 hours, the supernatants were collected and viral RNA was extracted. We constructed multiplexed-Primer ID libraries from viral RNA and sequenced them using MiSeq. HPRT knockout assays were performed using CHO-K1 cells treated with a panel of nucleotide/nucleoside analogs for 32 days. After 6-thioguanine selection, resistant cell colonies were counted as a measure of HPRT knockout mutations in host cells, and HPRT mRNA was sequenced from selected colonies. Results: rNHC showed dose-dependent antiviral and mutagenic effects against SAR-CoV-2 in vitro. In the 10 μM group, we found 7-fold and 14-fold increases in the overall substitution rate and the C to U mutation rate, respectively. The HPRT assay showed an rNHC dose-dependent increase in the number of resistant colonies with HPRT gene mutations. Other analogs showed no significant increase in the number of 6-thioG resistant colonies except for a slight increase with favipiravir (Fig 1a). Most colonies had missense substitutions or frame-shift deletions within HPRT mRNA, with most being distinct. Conclusion: rNHC showed a dose-dependent inhibition and mutagenic effect of SAR-CoV-2 in vitro. However, rNHC would be expected to be metabolized into the deoxynucleotide pool (by host RNR), resulting in DNA mutation of dividing mammalian cells. We demonstrated such mutagenic potential in a simple mammalian cell detection scheme. Molnupiravir has considerable potential as an orally bioavailable direct acting antiviral against SARS-CoV2 early in infection, especially in high risk patients. However, clinical use should be carefully considered in light of its potential mutagenic effects on the host.