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Most RNA viruses utilize RNA-dependent RNA polymerases for viral genome replication and synthesis of mRNAs, making them prime targets for mutagenic compounds to be utilized as broad-spectrum antivirals. Primer ID with next-generation sequencing (NGS) allows for accurate and deep characterization of viral populations, which provides for the ideal tool for screening mutagenic compounds and their effects on RNA viruses. In this study, we used this approach to study three potential mutagenic compounds (N4-beta-hydrocytidine, Favipiravir, and Ribavirin) on a panel of RNA viruses, including MERS coronavirus (MERS-CoV), Zika virus (ZIKV), and La Crosse virus (LACV). We hypothesize that these compounds exhibit antiviral effects by inducing lethal mutations in the viral genomes. We used a cell culture model where we grew the viruses in cell culture in the presence and absence of drugs, determined viral titers using plaque assays, and sequenced viruses at different time points to compare sequence differences by using primer ID and NGS. Using this data from the in vitro experiments, we developed a model to predict the percentage of defective viral genomes after treatment with mutagenic compounds. We also used an in vivo mice-MERS coronavirus model to study the antiviral effects and short-term toxicity of NHC. To study the long term toxicity of all three compounds, we used an 8E5 cell model, harboring a single HIV provirus per cell, to monitor the effects of these compounds on DNA dependent RNA polymerase (transcription) and DNA polymerase (DNA replication). The viral titers in the cell culture model showed that 10 μM of NHC had significant inhibition of MERS-CoV and LACV and moderate inhibition of ZIKV, which correlated strongly with an increase of cytosine (C) to uridine (U) transversion mutations, suggesting its mechanism for its antiviral activity is lethal mutagenesis. Both Favipiravir and Ribavirin exhibited no antiviral effects on MERS-CoV or ZIKV but had some antiviral effects on LACV. The In vivo mouse model showed that NHC had great inhibition in MERS-CoV with no significant observed mutations in the mice mRNA. The 8E5 cell model showed that neither Ribavirin or Favipiravir increased the mutation rate, but found that NHC caused an increase in the C to U mutations, suggesting there may be some long term effects of the drug. With the broad-spectrum antiviral effects of these compounds, they could potentially be able to be used to treat a wide variety of viral infections in humans, including newly emerged viruses that lack other forms of treatment. |
