Multipartite viruses, with genomes split across multiple segments each encapsidated in separate particles, deviate sharply from classical viral architectures. In these systems, full genetic information is recovered only at the population level. Theoretical models predict that such viruses—especially those with more than three or four segments—should be nonviable due to high loss risk during transmission. Yet, they comprise ~17% of all known viral species. Using a nanovirus with eight genomic segments, we have experimentally challenged prevailing assumptions in virology. We showed that spatial co-localization of all segments in single host cells is not essential: distinct genes act cooperatively at a supracellular scale. Similarly, segments’ transfer between hosts by aphid vectors does not require co-transmission. They can be separately acquired from different infected plants by one or multiple vectors and reassembled into a complete genome. Furthermore, we uncovered that segment abundance varies with the host plant, suggesting a mutation-free mechanism for tuning gene expression through gene dosing. This flexibility may confer a benefit in fluctuating environments. These findings prompt a broader question: are such collective, flexible strategies unique to multipartite viruses, or do they reveal a more general, overlooked mode of viral organization?

In September 2024, circulating vaccine-derived poliovirus type 2 (cVDPV2) was detected in wastewater from Barcelona. The detected strain was genetically linked to the Nigerian NIE-ZAS cVDPV2.

We describe the mutational and phenotypic landscape of the VP1 genome swarm present in Barcelona sewage, representing the ensemble of viral variants shed by the infected population. In total, 1.030 haplotypes were identified: one dominant haplotype accounting for 66% of all reads, 57 haplotypes representing 17%, and the remaining 972 haplotypes accounting for the final 17%. The dominant haplotype harboured 48 mutations, eight of which were nonsynonymous, whereas most other haplotypes contained 49–52 mutations, with 9–10 nonsynonymous changes. Most of these mutations mapped to antigenic sites that overlap with the receptor-binding domain. Using dynamic computational models of antibody and receptor interactions, we found that most haplotypes exhibited a significant reduction in antibody binding, while only a small fraction showed increased receptor binding. Although observed in only 0.5% of sequences, the highly concerning combination of antibody escape and enhanced receptor binding underscores the need for continued and intensive surveillance.

Human adenoviruses (AdV) are highly contagious and account for ~2-15% of the respiratory tract infections worldwide, depending on age and country (1, 2). They are transmitted by respiratory droplets, person-to-person contacts or contaminated surfaces. AdVs infect epithelial cells and immune cells. The former give rise to high levels of progeny, and viremic dissemination through lytic or nonlytic pathways (3, 4), affected by cell state (5), and cell cycle (6, 7). Infections of immune cell, in contrast, yield low amounts of progeny suggesting strong anti-viral defense (8, 9). Here, we discuss how inducible-pluripotent stem cell (iPSC)-derived human macrophages restrict infection by AdV-C5, a virus-type known to persist in immune cells of the gastro-intestinal tract of children, and cause life-threatening conditions upon immuno-suppression. I also discuss how AdV transactivation turns macrophage infections from a restricted to a productive state, with potential implications for pathogenesis. Beyond transcriptional control of AdV-C5 progeny formation by interferon-mediated repression of the E1A enhancer/promoter inducing a persistence state in normal human dermal fibroblasts (10), we present cell biological observations suggesting a mechanism for switching between lytic and nonlytic AdV egress in epithelial cells. The results have implications for oncolytic viral therapies, and modulating the inflammatory response triggered by AdV infections.

References

1. Radin JM, et al. 2014. PLoS One 9: e114871
2. Arnold A, MacMahon E. 2021. Medicine 49: 790-3
3. Georgi F, et al. 2020. Antimicrob Agents Chemother 64(9):e01002-20
4. Andriasyan V, et al. 2021. iScience 24: 102543
5. Petkidis A, et al. 2024. mSphere: e0045424
6. Suomalainen M, et al. 2020. J Cell Sci 134: jcs.252544
7. Snaider S, et al. 2022. J Virol 96: e0044222
8. Garnett CT, et al. 2009. J Virol 83: 2417-28
9. Sequeira DP, et al. 2025. J Virol: e0182524
10. Zheng Y, et al. 2016. PLoS Pathog 12: e1005415

SARS-CoV-2 has undergone rapid genetic diversification since its emergence in late 2019, giving rise to multiple lineages and variants of concern (VOCs). While many mutations in the viral Spike (S) protein affect antibody recognition and receptor binding, the contribution of non-S mutations to viral replication is less well understood. To address this, we generated a panel of recombinant variants of concern (rVOCs) expressing the same S:D614G gene. rAlpha, rBeta, rDelta, rGamma, rOmicron(BA.1) and rOmicron(BA.5) S:D614G replicated comparably to parental rSARS-CoV-2 S:D614G in VeroE6-TMPRSS2 (VTN) cells. In contrast, rBeta and rOmicron BA.5 S:D614G showed significantly attenuated replication in Calu-3 cells. Alpha, Beta and BA.5 S:D614G viruses formed significantly smaller plaques in VTN cells, suggesting that non-S lineage defining mutations alter cell-to-cell spread. Competition assays in Calu-3 cells demonstrated reduced fitness of rBeta and rOmicron BA.5 S:D614G, both of which were outcompeted by rSARS-CoV-2 S:D614G after a single passage. Attenuated replication of rBeta S:D614G was rescued by blocking JAK-STAT signalling with ruxolitinib. Transcriptomic analysis of Calu-3 cells infected with rBeta S:D614G +/- ruxolitinib revealed comparable induction of host innate antiviral response genes to rSARS-CoV-2 S:D614G at significantly lower RNA levels. Therefore, the interferon response is a major, but not the sole, barrier for replication of rBeta S:D614G in Calu-3 cells. The Beta, BA.1 and BA.5 S:D614G viruses replicated to significantly lower titres in the lung, nasal tissue and brain of K18-hACE2 mice than rSARS-CoV-2 S:D614G and did not cause significant weight loss. Transcriptomic analysis of murine lung tissue is currently in progress to establish if comparable transcription patterns are observed in vitro and in vivo. Together, these data highlight the importance of monitoring non-S lineage-defining mutations as viral variants emerge, as they can significantly alter viral replication phenotypes.

Nucleoside triphosphate analogues in which the γ-phosphate has two different non-cleavable lipophilic alkyl residues were observed to potently inhibit HIV-1 replication in vitro. In the present work, a series of γ-phosphonate monoalkyl nucleotides were found to inhibit the RNA-dependent RNA polymerase (RdRp) function of the SARS-CoV-2 nsp12 protein. These nucleotide analogues were synthesized with multiple nucleobase derivatives, sharing one alkyl group of two different lengths on the γ-phosphonate. The enzymatic PAGE-based assay of SARS-CoV-2 nsp12 in the presence of cofactors nsp7/8 revealed IC₅₀ values in the low micromolar range, whose potency appeared to be dependent on the length of the alkyl group. Notably, the assay displayed an unexpected mechanism of action, with no incorporation of the analogues and complete loss of enzymatic function. Competition assay revealed that the tested nucleotide analogues do not compete with the natural triphosphate nucleotides independently of sequence complementarity, hence acting as non-nucleoside inhibitors. Using multiple techniques, we were able to identify the mechanism of action in the compound-induced dissociation of the nsp7 cofactor from the nsp12 subunit. Molecular docking confirmed the binding site, identifying putative amino acid residues relevant for compound interaction. Different prodrugs of the most potent nucleotide analogues were synthesized and showed inhibitory activity against SARS-CoV-2 replication in cell culture.

We developed a universal influenza (UFlu) DNA vaccine to protect from annual influenza and future influenza variants with pandemic potential encoding HA stem or long alpha helix (LAH), the M2e consensus sequences from human, swine and avian strains to elicit broad protective antibody and nucleoprotein to induce protective T cell responses. We co-delivered genetic adjuvants encoding IL-12 to enhance T cell responses and the heat labile enterotoxin from E. Coli (LT) that we previously showed increases DNA vaccine induction of mucosal immune responses when delivered by gene gun into the epidermis of the skin and induces protective levels of immune responses in large animals and humans with very low doses of DNA (< 20 micrograms/dose). In mice, UFlu induced broad antibody and T cell responses, protection from representative influenza viruses, and superior protection vs. a strain-matched inactivated vaccine. In macaques, GG delivery of UFlu on weeks 0, 6, 19, and 43 induced robust HA and M2e antibody. mucosal and systemic NP-specific T cell responses in blood and lungs and protection from clinical disease after challenge with an H3N2 strain (A/Texas/71/2017) that is moderately virulent in macaques and after re-challenge with the highly virulent pandemic H1N1 strain A/California/04/2009. Peak viral loads in bronchoalveolar lavages at 3 days post-challenge were 2-3 Logs lower than in control macaques challenged with the same viruses. UFlu is being advanced to phase I human trials using a clinical gene gun (MACH-1^TM) engineered by Orlance, Inc with innovations that are user friendly and increase penetration and distribution of the DNA/gold particles. When compared to legacy gene guns, MACH-1 improves local gene/antigen expression and immunogenicity and can deliver either DNA or RNA vaccines. Together, these studies support clinical development of a MACH-1 GG-delivered universal influenza DNA vaccine.

Chemical disinfection is a fundamental component of biosecurity strategies for controlling transboundary animal diseases (TADs). However, virucidal efficacy is often evaluated using simplified experimental designs that focus on single viruses or fixed exposure conditions, limiting comparative interpretation across biologically heterogeneous animal viruses. In this study, virus-specific virucidal response patterns were explored across four representative TAD viruses: avian influenza virus (AIV), African swine fever virus (ASFV), foot-and-mouth disease virus (FMDV), and lumpy skin disease virus (LSDV). Four disinfectant active ingredients representing distinct chemical classes—benzalkonium chloride, glutaraldehyde, potassium peroxymonosulfate, and citric acid—were evaluated using standardized quantitative suspension assays. Each disinfectant was tested across multiple concentration levels and contact times, and virucidal activity was assessed based on log₁₀ reduction values. At a fixed contact time, distinct virus-specific concentration–response patterns were observed. Enveloped viruses generally exhibited greater susceptibility to aldehyde- and oxidizing agent-based disinfectants, whereas marked limitations were observed for quaternary ammonium compounds against certain non-enveloped viruses. Organic acid-based disinfectants demonstrated strong virucidal activity under acidic conditions for selected viruses but showed variable performance depending on virus type. Extension of contact time reduced the concentration required for effective inactivation in some virus–ingredient combinations, while other combinations showed limited time-dependent enhancement, indicating that prolonged exposure does not uniformly compensate for lower intrinsic activity. These results highlight substantial heterogeneity in virucidal response patterns among TAD viruses under identical experimental conditions. Rather than emphasizing single-condition efficacy outcomes, this preliminary analysis underscores the importance of pattern-based interpretation across concentration and time dimensions when comparing disinfectant performance against diverse animal viruses.

The antigenic peptide transporter (TAP) plays a crucial role in the antigen presentation pathway by transporting antigenic peptides into the endoplasmic reticulum for loading onto MHC class I molecules. Certain alphaherpesviruses encode immunomodulatory proteins that inhibit TAP function, allowing viral evasion from host immune responses. Despite their significance, the structural basis of these TAP inhibitors remains poorly understood.

In our project, we analyze key structural elements of TAP inhibitors encoded by cowpox herpesvirus (CPXV), an infectious zoonotic virus belonging to the Poxviridae family. For experimental reasons, the sequence of the CPXV virus was divided into three peptides, each comprising various fragments of the whole virus. These peptides were analyzed with multidimensional NMR spectroscopy in DPC micelles, which were used as a model of the cell membrane. Based on the acquired NMR data, it was possible to reconstruct the 3D structure of CPVX in the cell membrane using Molecular Dynamics (MD) simulations. Based on available structural data, the CPXV freezes TAP in a specific, non-functional state. In particular, the bound CPVX stabilizes the antigen in its outward-facing conformation, which prevents TAP from rotating back to its inward-facing state to pick up peptides. As a result, the CPXV protein acts as a physical plug, completely obstructing the peptide channel.

Our results will provide insights into viral immune evasion strategies and be applicable for developing and designing novel antiviral treatments against CPXV infections.

Several members of the Schlafen (SLFN) protein family exhibit antiviral activity through distinct molecular mechanisms. SLFN11 and SLFN13 restrict HIV-1 infection by degrading specific subsets of tRNAs, thereby impairing translation of viral mRNAs enriched in rare codons. Notably, SLFN11, but not SLFN13, inhibits West Nile virus (WNV) replication. Cells lacking SLFN11 are more permissive to WNV infection, and the virions produced in these cells display increased infectivity. Because SLFN11 and SLFN13 are expressed in humans but absent in mice, in vivo studies of their antiviral roles have been limited. Two murine Schlafen proteins, SLFN8 and SLFN9, which originated through gene duplication, have been proposed as the functional counterparts of human SLFN11 and SLFN13. To investigate this relationship, we examined the ability of SLFN8 and SLFN9 to inhibit WNV replication. Human A172 cells, highly permissive to WNV infection, were engineered to lack SLFN11 and to stably express either murine SLFN8 or SLFN9. Following WNV infection, viral replication and virion production were quantified by plaque assay and RT-qPCR, respectively. Our results demonstrate that SLFN8, but not SLFN9, reduces WNV infection, indicating that SLFN8 functions as the murine ortholog of human SLFN11.

Respiratory Syncytial Virus (RSV) is an enveloped RNA virus that causes severe lower respiratory tract infections in all age groups. It is the main cause of hospitalization during infancy and in adults, RSV is less studied but started to be recognized as an important pathogen, especially among elderly living with comorbidities. While RSV imposes a huge disease burden on the public healthcare system, the existing effective treatment options are limited. The development of RSV specific antivirals is partially impeded by a lack of knowledge of virus-host interactions and molecular mechanisms underlaying virus multiplication and pathogenicity. Here we present preliminary data on an integrated multi-omics approach that includes a RSV-host interaction network and the cellular response to RSV infection. By using state-of-the-art-of proteomics and transcriptomics, we characterized the RSV interactome, analyzed the proteome of cells expressing the individual viral proteins (effectome), and assessed the influence of RSV infection on cellular mRNA expression, protein abundance, and phosphorylation. The impact of some top candidates on viral replication dynamics was assessed by using a CRISPR/Cas9-based loss-of-function approach and time-course viral replication analysis of a GFP expressing recombinant RSV. Overall, the proposed approach will thus reveal yet unstudied proteins and pathways determining RSV growth, viral host defense mechanisms or disease severity, which will shed light on unresolved questions of RSV biology and reveal hotspots amenable to therapeutic intervention.