Payel Chatterjee

4′-fluorouridine (4′-FlU, also called EIDD-2749) is a nucleoside analog with broad-spectrum antiviral activity. To assess the potential for Lassa virus (LASV) to develop 4’-FlU resistance, we selected variants able to grow in the presence of this compound. We identified two coding changes in the polymerase gene, C1201S and N1315S, associated with growth in the presence of 4′-FlU and an approximately 9-fold increase in EC50. When introduced into the LASV minigenome, the C1201S substitution reduced susceptibility to 4′-FlU with a 3-fold increase in EC50 but was also associated with reduced minigenome replication. This loss in replicative fitness was reversed when C1201S was present in combination with the N1315S substitution. Both the C1201 and N1315 residues were found to be completely conserved across LASV clades and located proximal to the active site in the polymerase structure. These data contribute to the development of 4′-FlU and related polymerase inhibitors as potential antiviral therapies for LASV.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus that can cause severe human disease; no licensed antiviral is broadly available. Antiviral discovery is constrained by the need to study authentic CCHFV under biosafety level 4 containment, creating a need for lower-containment platforms that capture glycoprotein-dependent entry. Here, we developed replication-incompetent and competent vesicular stomatitis virus pseudotypes bearing a modified CCHFV M-segment glycoprotein precursor and used them as a quantitative biosafety level 2 platform for entry-focused screening. We screened 186 small molecules by replication-incompetent VSV and identified eltrombopag olamine and quercetin as inhibitors of CCHFV entry and limited cytotoxicity over the active concentration range. Mechanistic profiling showed that both compounds were most effective when present during virus exposure and early entry, and both reduced CCHFV glycoprotein-mediated cell-cell fusion. Docking analyses nominated candidate interaction sites at the GP38-Gn interface and within a Gc fusion-loop pocket, providing testable hypotheses for future binding and resistance studies. Both compounds inhibited authentic recombinant CCHFV expressing ZsGreen1 in fluorescence and focus-forming assays under biosafety level 4 conditions, validating their antiviral activity in an authentic-virus setting. These findings establish a practical CCHFV entry-screening workflow that links biosafety level 2 pseudotype discovery to authentic-virus validation and identified eltrombopag olamine and quercetin as early-entry inhibitors for further mechanistic follow-up and antiviral optimization.

Filovirus outbreaks caused by Ebola virus (EBOV) and Marburg virus (MARV), pose severe global health threats characterized by high rates of fatal hemorrhagic fever. While species-specific vaccines and therapeutic monoclonal antibodies are approved for Zaire ebolavirus, broadly-active therapeutics remain unavailable, leaving populations vulnerable to MARV and other pathogenic Ebola species, such as Bundibugyo (BDBV) and Sudan (SUDV) ebolaviruses. Here we report a computationally guided, infectious virus validated screening platform for the rapid discovery of broad-spectrum filovirus antivirals. By leveraging quantitative structure–activity relationship (QSAR) models, we screened 142,382 compounds in silico to prioritize 125 high-potential candidates. Subsequent dose–response and viability profiling identified 23 compounds exhibiting potent, low-micromolar pan-filovirus activity and favorable cytotoxicity profiles. Molecular docking indicates these compounds target conserved structural and functional domains—primarily the VP35 and L proteins—which may disrupt essential viral replication and immune antagonism. Furthermore, systematic combinatorial screening revealed three highly synergistic compound pairs, notably NCGC00113249-01 and NCGC00118008-01, demonstrating robust cross-species efficacy. By targeting conserved vulnerabilities across the filovirus family, this integrated in silico and in vitro pipeline provides a scalable framework to rapidly nominate and optimize synergistic therapeutic regimens against both endemic and emerging viral threats including BDBV. Graphical

Hantaviruses, including the Sin Nombre virus (SNV) and Andes virus (ANDV), are associated with severe global health risks, causing high mortality rates in hantavirus pulmonary syndrome (HPS) patients. Neutralizing antibodies are essential for virus clearance and survival, making neutralization assays critical for understanding immunity and evaluating therapeutic strategies. In this study, we developed a recombinant vesicular stomatitis virus (VSV)-based surrogate system expressing SNV and ANDV glycoproteins (GPCs), enabling neutralization studies under biosafety level 2 conditions. The neutralization titers obtained with the VSV-based system closely matched the findings from authentic hantavirus assays performed under biosafety level 3 conditions, confirming its potential as a useful tool for determining immune responses and advancing hantavirus research.

Development of broad-spectrum antiviral therapies is critical for outbreak and pandemic preparedness against emerging and reemerging viruses. Viruses inducing hemorrhagic fevers cause high morbidity and mortality in humans and are associated with several recent international outbreaks, but approved therapies for treating most of these pathogens are lacking. Here, we show that 4′-fluorouridine (4′-FlU; EIDD-2749), an orally available ribonucleoside analog, has antiviral activity against multiple hemorrhagic fever viruses in cell culture, including Nipah virus, Crimean-Congo hemorrhagic fever virus, orthohantaviruses, and arenaviruses. We performed preclinical in vivo evaluation of oral 4′-FlU against two arenaviruses, Old World Lassa virus (LASV) and New World Junín virus (JUNV), in guinea pig models of lethal disease. 4′-FlU demonstrated both advantageous pharmacokinetic characteristics and high efficacy in both of these lethal disease guinea pig models. Additional experiments supported protection of the infected animals even when 4′-FlU delivery was reduced to a low dose of 0.5 milligram per kilogram. To demonstrate clinical utility, 4′-FlU treatment was evaluated when initiated late in the course of infection (12 or 9 days after infection for LASV and JUNV, respectively). Delayed treatment resulted in rapid resolution of clinical signs, demonstrating an extended window for therapeutic intervention. These data support the use of 4′-FlU as a potent and efficacious treatment against highly pathogenic arenaviruses of public health concern with a virus inhibition profile suggesting broad-spectrum utility as an orally available antiviral drug against a wide variety of viral pathogens.