Icosahedral viruses organize and compact their genomes within volumetrically constrained capsids. Double-stranded (ds) DNA viral genomes form ordered spool-like structures when packaged into preformed capsids. By contrast, single-stranded (ss) RNA viral coat proteins recognize genomic sequences or structures, nucleating assembly around folded genomes. A similar mechanism may occur in some ssDNA systems; however, in parvo- and microviruses, the ss genome is concurrently synthesized and packaged into a preformed shell. In the øX174 X-ray virion structure, only ~12% of the genome is ordered. Consequently, the mechanism by which the genome is accommodated within the constrained capsid remains obscure. Sequence motifs within the øX174 genome produce a periodic segmentation pattern consistent with T = 1 icosahedral symmetry. To determine the function of these motifs, a subset within the first five packaged segments was altered. No regulatory elements or encoded amino acids were changed. The resulting mutant, øXDO5, displayed a phenotype consistent with those of previously characterized packaging mutants. To further understand this phenomenon, the DNA replication-packaging pathway was characterized in cells co-infected with wild-type and øXDO5. Each genome could both be separately tracked and distinguished throughout the pathway. Early, pre-packaging øXDO5 genome replication was comparable to the wild-type. However, øXDO5 genomes were severely diminished within virions, suggesting a defect in the transition from packaging intermediates to mature virions.IMPORTANCEConcurrent genome biosynthesis and packaging are specific to some families of single-stranded (ss) DNA icosahedral viruses. This evolutionary strategy combines elements found in both dsDNA and ssRNA systems. Like dsDNA viruses, the genome is packaged into a preformed capsid. Like ssRNA viruses, there are numerous capsid-genome associations. However, in microviruses, such as øX174, these interactions do not facilitate capsid assembly around the genome. They occur after the ss genome enters the preformed procapsid. Sequence motifs within the øX174 genome produce a periodic segmentation pattern consistent with T = 1 icosahedral symmetry. The data provided herein demonstrate that altering these periodic motifs can lead to packaging defects, suggesting an additional level of selective pressure acting on ssDNA genomes.

In case of emerging or re-emerging infections, vaccine platform technologies are needed to rapidly develop effective vaccines to aid public healthcare in pandemics. Besides mRNA vaccines, also viral platform technologies, that is, the adenovirus-derived vaccines Vaxzevria and JCOVDEN, have proven to be of immense value during the COVID-19 pandemic. For future pandemics, it is crucial to understand the factors in vector design that modulate immunogenicity. This knowledge allows the tailoring of vaccine vectors to fit specific target product profiles, for example, to build vectors which trigger an accentuated T cell or, alternatively, antibody response against an antigen of interest. Our study using the live-attenuated measles vaccine backbone as a promising example is therefore crucial in demonstrating that very minor differences in the vaccine backbone can alter the antigen expression profile of the vector-antigen system and impact the relative induction of T-cell or antibody responses against the added target antigen.

Extrachromosomal DNA (ecDNA) drives extensive oncogene amplification and intrinsic tumor heterogeneity in human cancers. Recent studies have uncovered an unexpected convergence between viral oncogenesis and ecDNA biology: integration of human papillomavirus (HPV) can trigger the formation of hybrid viral-human ecDNA structures in approximately 30% of HPV-associated oropharyngeal cancers (HPVOPC), with analogous structures reported in cervical cancer models. These chimeric circular elements fuse viral oncogenes with captured human genomic sequences, co-amplifying viral and human genes, and generating de novo enhancer complexes absent from either parental structure. Hybrid ecDNA exhibits canonical ecDNA features, including disproportionately high gene expression, accessible chromatin landscape, and the capacity to act as mobile trans-activating elements that potentiate oncogenic programs. Emerging evidence further suggests that hybrid ecDNA operates through distinct regulatory mechanisms and may create specific therapeutic vulnerabilities that can be leveraged for personalized cancer therapy. Elucidation of the mechanisms governing hybrid ecDNA biogenesis, maintenance, and function may therefore uncover exploitable dependencies in hybrid ecDNA-associated cancers. In this review, we synthesize current insights into hybrid ecDNA formation, regulatory mechanisms, and therapeutic opportunities and discuss how these advances may guide the development of precision cancer therapies.

The lack of animal models that reliably recapitulate the neurological manifestations of Coxsackievirus A16 infection has constrained progress in understanding CVA16 neuropathogenesis. Here, we describe a neonatal mouse model based on a mouse-adapted CVA16 strain (CVA16-P5) that consistently induces neurological disease and multisystem pathology. This model enables the analysis of tissue-specific immune responses, viral dissemination, and genetic determinants of neurovirulence, including a VP1 mutation associated with enhanced pathogenicity. By providing a reproducible and physiologically relevant system for studying severe CVA16 infection, the CVA16-P5-adapted strain infection model setup in this study supports mechanistic studies of CVA16 pathogenesis and facilitates the preclinical evaluation of vaccines and antiviral drugs against neurotropic enteroviruses.

Diseases caused by zoonotic agents are major public health concerns. Orthohantaviruses are typical examples of zoonotic viruses transmitted from wild rodent hosts. Despite extensive laboratory investigations, the mechanisms of viral persistence and transmission in natural hosts remain poorly understood. Our study demonstrated that Hokkaido virus (HOKV), a genotype of Orthohantavirus puumalaense, seems to establish both acute and persistent infections in wild grey red-backed voles (Myodes rufocanus bedfordiae) without inducing major pathological changes. Detection of infectious virus in saliva, urine, and feces revealed multiple virus-shedding routes. Saliva likely serves as the predominant source of transmission, given that infectious virus was recovered from oral swabs of all infected rodents captured in 2024. These findings expand understanding of orthohantavirus ecology, persistence, and maintenance in reservoir populations. This information is crucial for evaluating spillover risks and enhancing public health preparedness.

Herpesviruses employ sophisticated immune evasion strategies to establish lifelong infections, subverting type I interferon (IFN-I) responses critical for antiviral defense. However, their adaptive mechanisms across species remain poorly characterized. Using duck plague virus (DPV)-an avian alphaherpesvirus model-we identify a cooperative immune evasion axis wherein ICP27 orchestrates UL55-mediated immunosuppression through dual regulatory mechanisms: its RNA-binding domain (RGG) facilitates UL55 mRNA nuclear export, while its C-terminal domain (CTD) stabilizes UL55 protein via direct interaction. This partnership enables synergistic suppression of IFN-I signaling-co-expression of ICP27 and UL55 inhibits Poly(I:C)-induced immune genes (IFN-β, Mx, OASL, IL-6) more potently than either protein alone. UL55 functions as a precision-targeted IFN-I antagonist, selectively degrading RIG-I and IRF7 through proteasomal pathways-confirmed by proteasome inhibitor rescue (MG132), structural modeling (AlphaFold), and binding assays (Co-IP). Evolutionarily, UL55 homologs (DPV, Herpes simplex virus type 1 [HSV-1], Varicella zoster virus [VZV]) conserve RIG-I targeting but diverge in IRF3/IRF7 regulation-adaptations shaped by UL55 sequence divergence (38.68% identity) and host biology (e.g., waterfowl IRF3 deficiency). This work establishes ICP27-UL55 as a key regulatory axis in herpesviral immune evasion and redefines UL55 as a conserved yet adaptable immunosuppressor in Alphaherpesvirinae.IMPORTANCEThis study fundamentally advances herpesvirology by defining a novel immune evasion paradigm in duck plague virus. We reveal ICP27 as a master regulator that coordinates UL55 immunosuppression through a two-tiered mechanism: RGG domain-mediated mRNA nuclear export and CTD-dependent protein stabilization-an unreported strategy in herpesviruses. UL55 selectively degrades RIG-I and IRF7 via proteasomal pathways, enabling precise IFN-I suppression with minimal immune activation. Crucially, ICP27-UL55 synergy inhibits Poly(I:C)-induced immune genes (IFN-β, Mx, OASL, IL-6) more effectively than individual proteins. Evolutionary analyses demonstrate conserved targeting of RIG-I across alphaherpesvirus UL55 homologs (DPV, HSV-1, VZV) but host-adapted divergence in IRF3/IRF7 regulation, shaped by UL55 sequence variation (38.68% identity) and host biology (e.g., avian IRF3 deficiency). These findings provide the first evidence of effector coordination through integrated transcriptional/post-translational regulation in herpesviruses. Disrupting ICP27-UL55 interaction offers new antiviral targets, while UL55-deficient strains serve as vaccine candidates for poultry disease control.

Oropouche virus (OROV), a member of the Peribunyaviridae family endemic to South America, is a current public health threat. The recent OROV outbreak driven by a novel reassortant strain has caused a dramatic increase in cases in 2024 (13,785 in Brazil, versus only 261 from 2015 to 2022) with sustained levels of transmission in 2025. Previously underreported outcomes have been recognized, including miscarriage, microcephaly, encephalitis, and death. OROV lethality in humans has been attributed to severe coagulopathy with liver involvement, and epidemiological data suggest that acute hepatitis occurs in mild cases of Oropouche fever, highlighting the underrecognized role of the liver in OROV pathogenesis. We present two discrete mouse models of OROV hepatic disease-a lethal model that is similar to the severe coagulative liver necrosis seen in fatal human cases and a model of self-resolving acute hepatitis, which is similar to mild human disease. In both models, OROV causes focal hepatic necrosis in mice, which progresses to massive necrosis and death when the Type I interferon receptor is antagonized. Additionally, we found that a contemporary OROV isolate is less pathogenic in mice than a historic prototypical strain. These studies enhance our understanding of OROV pathogenesis and provide additional models for potential therapeutic development and evaluation.IMPORTANCEThe disease burden of Oropouche fever has been underrecognized and underreported, as highlighted by the increased testing seen in an ongoing outbreak in South America. Specifically, the role of the liver in Oropouche virus pathogenesis has been understudied. Given the recent increase in cases and severe disease manifestations, there is a present need to understand Oropouche virus pathogenesis and provide models to test potential therapeutics. The mouse models of Oropouche-induced hepatitis presented here provide a means to understand how Oropouche virus causes liver damage in both a lethal and sublethal context. These models will be useful for the preclinical evaluation of vaccines and therapeutic treatments. Additionally, we compare the pathogenicity of a historical Oropouche virus isolate to a contemporary human isolate in a lethal mouse model.

Secondary bacterial pneumonia is a major complication of influenza A virus (IAV) infection, frequently caused by Streptococcus pneumoniae. Pneumococci asymptomatically colonize the upper respiratory tract by forming biofilms. Viral infections, such as IAV, disrupt this stable colonization, leading to bacterial migration to the lower respiratory tract, and the development of severe pneumonia. However, the specific organs and tissues where IAV infection enhances the replication capacity of pneumococci in the biofilm state remain unclear. In this study, we engineered a mutant S. pneumoniae strain carrying the near-infrared luciferase gene, Akaluc, which is selectively expressed during the log phase. Using this strain, we established a mouse model that enables in vivo imaging of pneumococcal activation following IAV infection. In this model, IAV infection resulted in a marked increase in luminescence signals in the larynx, accompanied by increased bacterial proliferation in the lower respiratory tract. These results suggest that the larynx serves as a key site for pneumococcal activation and transition from biofilm to planktonic state during secondary bacterial pneumonia. Our imaging approach offers a powerful tool for elucidating host-pathogen interactions in vivo and may contribute to the development of preventive strategies against secondary bacterial pneumonia following IAV infection.IMPORTANCESeasonal influenza virus infection increases the risk of severe bacterial pneumonia, but the underlying mechanisms remain unclear. In this study, we used a mouse model and a pneumococcal mutant strain expressing a near-infrared luminescent protein in the log phase, enabling in vivo monitoring of the enhanced replication of pneumococci in deep tissues. We discovered that following influenza virus infection, bacterial replication is predominantly activated in the larynx, indicating that the larynx serves as a key site for activation of commensal pneumococci. This insight may inform the development of better strategies to prevent secondary bacterial pneumonia.

Influenza A viruses of divergent pathogenicity elicit distinct host immune responses, yet the underlying cellular and spatial dynamics remain poorly defined. Here, we integrate single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics to systematically compare the immune landscapes in murine lungs following infection with seasonal H1N1 or a highly pathogenic avian influenza H5N1 virus belonging to clade 2.3.2.1. Analysis of 84,162 immune cells identifies 12 distinct cell types, suggesting that H1N1 infection is associated with robust interferon and inflammatory responses, characterized by alveolar macrophage depletion and extensive infiltration of monocytes and neutrophils. In contrast, H5N1 clade 2.3.2.1 infection is characterized by attenuated interferon signaling, delayed monocyte activation, dysregulated neutrophil maturation, and impaired intercellular communication-features that may be distinct from previously characterized H5N1 strains. Spatial mapping indicates distinct distribution and interaction patterns of immune cells between the two infections. Functional validation through myeloid cell depletion supports the critical role of the monocyte-macrophage axis in disease progression. These findings provide a high-resolution atlas of pulmonary immune responses to influenza viruses of differing pathogenicity and highlight cell-type-specific mechanisms of immune modulation.IMPORTANCESeasonal H1N1 influenza virus causes annual epidemics, while highly pathogenic avian H5N1 virus has a high mortality rate and pandemic potential. Understanding why H5N1 causes more severe disease is critical for developing better treatments. In this study, we used advanced single-cell and spatial technologies to create a detailed map of the immune response in the lungs of mice infected with either H1N1 or a clade 2.3.2.1 H5N1 virus. We discovered that H1N1 triggers a strong, well-organized immune response that controls the virus. In contrast, H5N1 infection leads to a disorganized and weakened response where key immune cells fail to activate and communicate properly. These findings suggest that the H5N1 virus may evade and suppress the host's immune system. Our study provides a high-resolution immune atlas and identifies potential targets for new therapies against severe influenza.

Hepatitis C virus (HCV) exploits lipid droplets (LDs) during the production of infectious particles. Although several host factors, including stress granule (SG) proteins, have been reported to localize to LDs in HCV-infected cells, the mechanism by which these proteins are recruited to LDs has remained unclear. In this study, we show that Ras-GAP SH3 domain-binding protein 1 (G3BP1), an essential SG component, is recruited to LDs with a time lag of several hours or more after the HCV Core protein accumulates on LDs following infection, and that G3BP1 retains its ability to assemble SGs even when localized to LDs during the late phase of infection. Treatment of HCV-infected cells with compounds that disrupt liquid-liquid phase separation (LLPS) abolished the LD localization of G3BP1, as well as another SG protein, T-cell intracellular antigen-1, whereas the LD localization of the viral proteins Core and NS5A was not affected. Finally, we demonstrate that ectopic expression of HCV Core alone is insufficient to recruit G3BP1 to LDs; however, co-expression of Core together with subgenomic HCV RNA, even a replication-defective replicon RNA, leads to the accumulation of G3BP1 around LDs. Collectively, these findings suggest that during the HCV life cycle, the initial localization of Core to LDs, followed by the recruitment of newly synthesized viral RNA to the LD surface, drives LLPS-mediated assembly of biomolecular condensates containing G3BP1 and other host SG proteins around LDs.IMPORTANCEDuring the late stages of hepatitis C virus (HCV) infection, various viral and host proteins are known to accumulate around lipid droplets (LDs), where virion assembly is thought to occur. However, it has remained unclear how the expression of a limited subset of viral factors can induce such an extensive cellular reorganization. Here, we identify that HCV infection induces the relocalization of the stress granule (SG)-associated proteins G3BP1 and TIA-1 onto LDs through liquid-liquid phase separation (LLPS). We further demonstrate that the minimal requirement for G3BP1 recruitment to LDs is the coexistence of the HCV Core and the viral RNA. Because G3BP1 plays a central role in the interaction network underlying SG formation and is capable of mobilizing additional SG components, our findings suggest that LLPS-dependent assembly of biomolecular condensates occurs around LDs during HCV infection. This work provides mechanistic insight into HCV particle formation, including viral genome packaging, and offers a conceptual basis for developing future antiviral strategies.