Favipiravir is a nucleoside analogue that inhibits the replication and transcription of a broad spectrum of RNA viruses, including pathogenic arenaviruses. In this study, we isolated a favipiravir-resistant mutant of Junin virus (JUNV), which is the causative agent of Argentine hemorrhagic fever, and analyzed the antiviral mechanism of favipiravir against JUNV. Two amino acid substitutions, N462D in the RNA-dependent RNA polymerase (RdRp) and A168T in the glycoprotein precursor GPC, were identified in the mutant. GPC-A168T substitution enhanced the efficiency of JUNV internalization, which explains the robust replication kinetics of the mutant in the virus growth analysis. Although RdRp-N462D substitution did not affect polymerase activity levels in a minigenome system, comparisons of RdRp error frequencies showed that the virus with RdRp-D462 possessed a significantly higher fidelity. Our next generation sequence (NGS) analysis showed a gradual accumulation of both mutations as we passaged the virus in presence of favipiravir. We also provided experimental evidence for the first time that favipiravir inhibited JUNV through the accumulation of transition mutations, confirming its role as a purine analogue against arenaviruses. Moreover, we showed that treatment with a combination of favipiravir and either ribavirin or remdesivir inhibited JUNV replication in a synergistic manner, blocking the generation of the drug-resistant mutant. Our findings provide new insights for the clinical management and treatment of Argentine hemorrhagic fever.

Globally, influenza A virus (IAV) and respiratory syncytial virus (RSV) infection remain very high. There is also a high burden of IAV and RSV co-infection in developing countries. To develop universally protective vaccines against these infections, it is imperative that viral genes and immune correlates of pathology are elucidated. As such, we profiled virus genes expressions, histopathology and immunological responses of BALB/c mice infected with RSV and/or IAV in this study.RSV A2 and/or influenza A/H3N2/Perth/16/09 (Pr/H3N2) were induced over a seven-day period in BALB/c mice. Anaesthetized BALB/c mice (12–14 g) were divided into six groups (15–20 mice per group), inoculated with 32 μl each of 3LD50 Pr/H3N2 and/or 100 TCID50 RSV. Two groups (R or I) received RSV or Pr/H3N2 intranasally. Prior infection with either RSV or Pr/H3N2 was followed with a second challenge of the other virus 24 hours post inoculation in RI and IR groups. Another set was exposed to the two viruses simultaneously (I + R group) while the last group served as healthy controls. Five to seven mice per group were euthanized at days 2, 4 and 7. Lung and spleen organs were harvested for virus genes quantitation and immune cells phenotyping respectively.I + R group showed progressive downregulation of RSV F, G, NS1 and NS2 genes. IAV PB2 and M genes had high fold increase on day 2 and 4 post infections. However, by day 7 post infection, M and PB2 fold increase was lower. Also, increased proportions of NKT and T cell subsets were observed throughout the period in I + R group. Conversely, I group was characterized by reduced NKT cell counts and enhanced CD8 T cells levels while R group only showed an increased proportion of CD8 T cells towards the peak of infection.This study shows that RSV and IAV co-infection lead to reduced virulence and pathology compared to single infections. This information is very useful in combinatorial RSV/IAV vaccine design and development.

Although many arenaviruses cause severe diseases with high fatality rates each year, treatment options are limited to off-label use of ribavirin, and a Food and Drug Administration (FDA)-approved vaccine is not available. To identify novel therapeutic candidates against arenaviral diseases, an RNA polymerase I-driven minigenome (MG) expression system for Lassa virus (LASV) was developed and optimized for high-throughput screening (HTS). Using this system, we screened 2595 FDA-approved compounds for inhibitors of LASV genome replication and identified multiple compounds including pixantrone maleate, a topoisomerase II inhibitor, as hits. Other tested topoisomerase II inhibitors also suppressed LASV MG activity. These topoisomerase II inhibitors also inhibited Junin virus (JUNV) MG activity and effectively limited infection by the JUNV Candid #1 strain, and siRNA knockdown of both topoisomerases (IIα and IIβ) restricted JUNV replication. These results suggest that topoisomerases II regulate arenavirus replication and can serve as molecular targets for panarenaviral replication inhibitors.

Favipiravir is a nucleoside analogue that inhibits the replication and transcription of a broad spectrum of RNA viruses, including pathogenic arenaviruses. In this study, we isolated a favipiravir-resistant mutant of Junin virus (JUNV), which is the causative agent of Argentine hemorrhagic fever, and analyzed the antiviral mechanism of favipiravir against JUNV. Two amino acid substitutions, N462D in the RNA-dependent RNA polymerase (RdRp) and A168T in the glycoprotein precursor GPC, were identified in the mutant. GPC-A168T substitution enhanced the efficiency of JUNV internalization, which explains the robust replication kinetics of the mutant in the virus growth analysis. Although RdRp-N462D substitution did not affect polymerase activity levels in a minigenome system, comparisons of RdRp error frequencies showed that the virus with RdRp-D462 possessed a significantly higher fidelity. We also provided experimental evidence for the first time that favipiravir inhibited JUNV through the accumulation of transition mutations, confirming its role as a purine analogue against arenaviruses. Moreover, we showed that treatment with a combination of favipiravir and either ribavirin or remdesivir inhibited JUNV replication in a synergistic manner, blocking the generation of the drug-resistant mutant. Our findings provide new insights for the clinical management and treatment of Argentine hemorrhagic fever.