Giant viruses are the size of small bacteria and have double-stranded DNA genomes that are as long as the genomes of some small bacteria. Tupanvirus deep ocean (TVdo), a giant virus discovered in deep sea sediment, has a >1.4 Mbp genome that contains many genes associated with translation, a function that all viruses need their host to perform. Electron microscopic analyses of the virus extracted from infected Acanthamoeba castellanii show an icosahedral head with an approximate diameter of 400 nm and a wide (~300 nm diameter) cylindrical tail, both covered by thin fibrils radiating from the surface. Proteomic analysis of purified virions identified >220 viral proteins, including double jelly-roll capsid proteins similar to the adenovirus hexon, several proteins associated with transcription, and many uncharacterized proteins. These results suggest that TVdo enters its amoeba host ready to transcribe its genes. We identified two candidate fibril proteins from the large list of uncharacterized structural proteins based on their predicted structures from AlphaFold3. Infectivity assays, the insertion of a GFP transgene into the TVdo genome, and fluorescence microscopy will allow for future characterization of the viral infection cycle.

Hepatitis E virus (HEV) remains an important but largely understudied zoonotic pathogen. A universally available vaccine is lacking, and current treatments are limited by severe side effects, teratogenicity, and frequent therapeutic failures. Drug repurposing may therefore accelerate the discovery of effective anti-HEV therapies. We screened a library of 496 nucleoside and nucleotide analogs and derivatives to identify potential antiviral candidates. Ten compounds inhibited HEV replication by more than 60% while maintaining cell viability above 75%, including four that showed clear dose–response relationships. Among these, 4′-fluorouridine (4′-FIU) exhibited potent antiviral activity in both replication (EC50 ~ 5 µM) and infection (EC50 ~ 10 µM) assays against HEV-Kernow-C1/p6 and HEV-1 Sar55 strains. Its efficacy was further confirmed in extrahepatic cell lines (placental JEG-3, intestinal Caco-2, and renal HEK293T) as well as in primary human hepatocytes. Combination treatment with ribavirin produced an additive antiviral effect, while exogenous uridine reversed the inhibitory activity of 4′-FIU on HEV-3 replicon replication, indicating that the in vitro activity of 4′-FIU depends on depletion of intracellular UTP pools. To validate the in vivo efficacy of 4′-FIU, experiments in athymic nude rats as well as HEV-1 infection experiments in mice are currently ongoing. Altogether, these findings highlight 4′-FIU as a promising candidate for HEV therapy, addressing a critical unmet need in antiviral treatment.

Lassa virus (LASV), a member of the Arenaviridae family, poses a significant global health threat, particularly in West Africa, where outbreaks continue to occur, including as recently as October 2025¹. Despite its impact, there are currently no approved medical countermeasures for LASV, and the genetic diversity of circulating clades further complicates therapeutic development. Addressing this challenge, we employed a human monoclonal antibody (mAb) discovery platform developed by USAMRIID and collaborators to identify cross-clade neutralizing mAbs using plasma, serum, and peripheral blood mononuclear cells (PBMCs) from LASV survivors in Sierra Leone.

Through a systematic in vitro down-selection process, including MAGPIX multiplex assays, qualitative and quantitative ELISAs, and microneutralization assays, we identified promising convalescent LASV samples for further interrogation. LASV-GPC antigen specific B cells were sorted, and the resulting cloned IgG antibodies were evaluated for affinity and kinetics by biolayer interferometry, epitope specificity, and neutralization activity against six diverse clades (I, II, III, IV, and VII) prevalent in West Africa. Notably, candidate mAb0074 demonstrated cross-neutralizing and binding activity against clades II, IV, and VII. Additionally, antibody-dependent cellular phagocytosis (ADCP) and binding assays revealed that non-neutralizing antibodies mAb0090 and mAb0095 exhibited potential as components of a cross-protective antibody cocktail.

This study highlights the potential of monoclonal antibody platforms to address the genetic diversity of LASV and provides a foundation for the development of cross-clade therapeutics. We will discuss the implications of these findings and outline future directions for advancing lead mAb candidates toward clinical evaluation.

Autophagy is a key cellular recycling process necessary for maintaining homeostasis. The protein ATG16L1 serves as a critical regulator and structural component of the autophagy machinery. One of its major roles is to facilitate the formation of the phagophore, a membrane structure that engulfs cytoplasmic material and directs it to lysosomes for degradation.

During SARS-CoV-2 infection, the virus triggers the production of double-membrane vesicles (DMVs), which are essential for viral replication. These vesicles share structural similarities with autophagosomes, and both arise from specialized domains of the endoplasmic reticulum (ER), suggesting that related molecular mechanisms may underlie their biogenesis. Yet, the precise molecular connection between autophagy and viral DMV formation has remained unclear.

Our study demonstrates that ATG16L1 is indispensable for DMV formation in SARS-CoV-2-infected cells. Notably, ATG16L1 variants that fail to support autophagosome formation are similarly defective in promoting DMV assembly. By combining functional analysis, biochemical approaches, and electron microscopy, we delineated the molecular basis of DMV formation.

Together, our findings demonstrate how ATG16L1 facilitates DMV biogenesis during SARS-CoV-2 infection, providing insights into how the virus repurposes host autophagy machinery for its replication. This work highlights potential molecular targets for therapeutic intervention aimed at disrupting viral propagation.

The RNA genomes of coronaviruses comprise many conserved structures and distal RNA-RNA interactions that play essential roles in the viral replication cycle. In the 3’ untranslated region of betacoronaviruses, a pseudoknot RNA structure involving stem–loop 3SL2 has been proposed to form a binding site for the viral RNA polymerase, enabling the initiation of negative-sense RNA synthesis during the processes of virus replication and discontinuous transcription. On the basis of bioinformatic analyses and a mutagenesis approach combined with electrophoretic experiments and viral replication assays, we report that, in addition to the local pseudoknot in the 3’UTR, 3SL2 can establish a distal contact with an exposed sequence in the coding region of the SARS-CoV-2 genome¹. The bases involved in this contact are highly conserved in all SARS-like coronaviruses and sequencing results indicate that this motif may form an alternative polymerase binding site, enabling the synthesis of non-canonical subgenomic RNA. These findings contribute information about the molecular mechanisms governing the processes of replication, discontinuous transcription and recombination in these viruses, and may expand the scope of therapeutic strategies against current or future infections by sarbecoviruses.

(1) López-Núñez, S., Cantero-Camacho, A., Simba-Lahuasi, A., Ye, C., Mena, I., García-Sastre, A., Martínez-Sobrido, L., Gallego, J. A potential RNA-RNA distal interaction involving a functional pseudoknot in the 3'-untranslated region of SARS-like coronaviruses. Submitted (2025).

HIV-1 remains one of the leading contributors to the global burden of disease. The complex between several monomers of the HIV-1 Rev protein and the Rev Recognition Element (RRE) in the viral RNA allows nuclear export of unspliced or singly spliced transcripts, an essential step in the virus cycle that is not targeted by any of the currently marketed antiretroviral treatments. The 234-nucleotide RNA component of this complex adopts a multi-domain structure whose three-dimensional details are currently unknown at atomic resolution. To shed light on RRE structure and on the mechanism of RRE-Rev complex assembly, we have set up a method based on fluorescence resonance energy transfer (FRET) that yields information on RRE interdomain distance. By combining this information with all-atom ensemble modelling and electrophoretic assays assessing Rev association, we have generated an atomic model of RRE structure consistent with the available evidence¹. We have also applied the FRET assay to discover small-molecule compounds capable of altering the spatial organization of RRE domains and disrupting the RRE-Rev interaction in vitro and in cellulo². Some of these compounds exhibited antiretroviral activity with favourable selectivity indexes and may serve as valuable leads for the development of novel HIV therapeutics based on specific RRE-Rev inhibition.

(1) Szewczyk, M. P.; Loharch, S.; López-Núñez, S.; Gallego, J. Insights about the structure of the Rev Response Element of HIV-1 revealed by FRET-monitored mutagenesis and modelling. J Mol Biol 2025, 437 (24), 169451. DOI: 10.1016/j.jmb.2025.169451.

(2) Szewczyk, M. P.; Loharch, S.; Izquierdo-Pujol, J.; Beltrán, M.; Puertas, M. C.; Morón-López, S.; Chumillas, S.; Marchán, V.; Alcamí, J.; Martínez-Picado, J.; Bedoya, L. M.; Gallego J. Conformational inhibitors of the HIV-1 Rev Response Element identified through a FRET-based screening approach. Submitted 2025.

Antimicrobial (AM) abuse is a major issue in underdeveloped nations, including Ghana, where it poses serious risks to public health. One effective and efficient way to combat AMR is to implement functional antimicrobial stewardship (AMS) programs at health facilities. However, research has shown that, due to several constraints, implementation and compliance in developing countries remain very low. Most studies focus on poor prescription practices and knowledge on AMR, with scant studies on determinants of compliance and its theoretical implications. The purpose of this study is to examine the compliance of healthcare professionals towards stewardship programs and analyze their challenges using the theory of planned behavior. This study employed both quantitative and qualitative methods. This study employed a purposive sampling approach to sample 300 healthcare professionals across 10 hospitals in Ghana. Descriptive statistics and partial least squares structural equation modeling were used for analysis. The results revealed that the majority (71.2) were nurses, while 28.8% were Doctors. Also, most (98%) respondents had training on AMS programs, while only 2% did not. The findings showed that healthcare workers' intention to follow AMS standards was significantly influenced by attitudes (β=0.41, p<0.001), subjective norms (β=0.31, p<0.001), and perceived behavioral control (β=0.38, p<0.001). This study also discovered that behavioral intention and perceived behavioral control both strongly predicted rational prescribing and actual compliance. The qualitative investigation showed that a heavy workload, incorrect diagnosis, inadequate information technology, and inconsistent enforcement of AMS policies all impede compliance. Therefore, it is recommended that hospitals implement interventions that enhance institutional resources and equipment, improve organizational culture, and boost funding of AMS programs. Additionally, this study recommends that the Government, in collaboration with the Ministry of Health, should strengthen AMS programs by providing funds and capacity building at the national and local levels in the country.

High-throughput screening (HTS) is a powerful tool for identifying modulators of viral replication, but its success depends heavily on the robustness, scalability, and comparability of the underlying assay formats. In the context of vesicular stomatitis virus (VSV) production in HEK293F cells, we aimed to establish a reliable and scalable analytical platform to support compound screening for host-directed viral titer enhancement.

To this end, we developed a tiered analytical funnel comprising three complementary assays using a reporter VSV: a fluorescence-based readout measuring mean fluorescence intensity (MFI) in producer cells for primary screening, flow cytometry to quantify infected cells at single-cell resolution for secondary validation, and an already established method, kinetic infectious titer assay (KITA), to directly assess infectious virus output.

A key focus of this work was the systematic comparison of assay performance across 96- and 384-well formats. Miniaturization to the 384-well format enabled increased throughput and accelerated workflows, both critical for large-scale screening. Comparative analysis revealed consistent signal dynamics across formats, with the 384-well setup maintaining sufficient sensitivity to detect ≥5-fold changes in viral output. Importantly, strong correlations between MFI-based fluorescence readouts and infectious titers confirmed the reliability of the miniaturized assays.

This study highlights the importance of scale compatibility and analytical consistency in HTS assay development. Our optimized platform provides a scalable and validated framework for screening host-targeting compounds aimed at enhancing viral yields in biotechnological applications.

A family of antiviral molecules against human coronaviruses was identified using a phenotypic cell-based screening approach. Optimized preclinical candidates with optimal therapeutic windows in different cell culture models were characterized in cell culture, revealing interference with the formation of functional replication organelles (ROs). Coronavirus ROs are characterized by a series of cytoplasmic membranous structures, among which double-membrane vesicles (DMVs) are the main site of RNA replication.

To further characterize the lead compounds, we produced recombinant DMVs through overexpression of its minimal components (nsp3/nsp4) in a replication-independent manner to test the ability of the compounds to interfere with their formation. Viral protein overexpression is sufficient to observe numerous DMVs using transmission electron microscopy (TEM), together with less characterized structures such as multiple membrane vesicles (MMVs) or zippered membranes (ZM) in the cell cytoplasm. Nsp3/nsp4 polyprotein overexpression and processing was not affected by the presence of the lead antiviral compounds. However, DMV frequency per explored surface was significantly reduced, suggesting that the compounds indeed interfere with DMV assembly. No significant changes in the diameter of the DMVs were observed.

These results are supported by the study of resistance-associated mutations, which preferentially map in the ECTO-domain of nsp4, a region of nsp4 involved in the interaction with nsp3, interaction that is essential for DMV formation. Resistance-associated mutations from three different lineages were mapped into an atomic 3D model obtained via cryo-electron tomography of the DMV pore, a key structure for the functionality of the DMVs. All nsp4 mutations are located in the luminal side of the pore, close the ECTO-domain of nsp3.

Overall, these results support a model in which nsp3/nsp4 interaction or nsp4 oligomerization are molecular targets for this family of pan-coronavirus antivirals, constituting a first-in-class antiviral family, optimal for combination therapies.

The hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide. HEV infection can become chronic in immunocompromised individuals, in whom virus–host recombinant variants (VHRVs) can be detected. These variants often harbor host-derived insertions in the polyproline rich region (PPR) and most display enhanced replication in vitro. However, the mechanisms underlying this replicative advantage remain unclear. It is likely that genes of the infected cells are differentially expressed according to the replicative capacity of the strain. The host factors involved in the improvement of the replicative capacity of these VHRVs are yet to be identified.

In this study, we analyzed the host transcriptional response to 7 VHRVs in HepG2/C3A cells using bulk RNA sequencing at 48 h and 168 h post-infection. Five VHRVs (RNF19A, ZNF787, KIF1B, RPS17, and EEF1A1) previously associated with a high replication rate induced more significant distinct transcriptomic changes than low-replicative variants (RNA18 and RPL6), particularly at 168 h post-infection. A shared set of 25 genes, especially interferon-stimulated genes (ISGs), was upregulated in cells infected with high-replicating variants. Interestingly, ISG induction was limited at 48 h post-infection despite high viral RNA concentrations, suggesting a delayed antiviral response. At 168 h post-infection, high ISG expression coincided with high viral loads, indicating that VHRVs may evade or exploit immune defenses. Our findings reveal candidate ISGs such as IFIT1 and ISG15 that may influence HEV persistence and immune escape. These results offer new insights into the interplay between VHRV replication and host immunity.