Respiratory syncytial virus (RSV) is the leading cause of severe respiratory infection in infants and an increasingly recognized pathogen in the elderly. Conventional in vitro culture models for RSV rely on poorly differentiated cell lines that lack key airway epithelial features such as polarization, tight junctions and cilia, despite ciliated cells being the main RSV target. Primary airway epithelial cultures better reflect in vivo conditions, but they are difficult to access. We evaluated the human bronchial epithelial cell line BCi-NS1.1 differentiated at an air-liquid interface (ALI) as an alternative model. Three weeks after ALI, BCi-NS1.1 cultures exhibited advanced differentiation, including mature tight junctions, IL-13-induced MUC5AC production, a transepithelial potential difference of around −20 mV and 19% ciliated cells. These cultures expressed CX3CR1 receptor in cilia, which may serve as an entry point for RSV. These cultures were readily infected by RSV from the apical side, with infected cell numbers increasing from day 1 to day 5 post-infection. Apical viral titres reached ~2×10 5 PFU ml −1 between day 2 and day 4. Infection led to the formation of typical viral factories with inclusion-body-associated granules, apical release of new infectious virions and controlled shedding of infected cells. Because shedding limits subcellular analysis, we generated an RSV lacking NS2, a protein promoting cell rounding and shedding. Cells infected with RSV-Delta NS2 exhibited slower shedding and maintained their shape longer, enabling visualization of ribonucleoprotein migration towards and across the apical membrane. Overall, ALI-differentiated BCi-NS1.1 cells provide a suitable tool for studying epithelial responses to RSV infection.
The human bronchial/tracheal epithelial air–liquid interface (HBTEC-ALI) culture system is widely used for isolating and propagating respiratory viruses; however, variability in differentiation status complicates quality control and may influence viral replication efficiency. Practical criteria for selecting optimally differentiated cultures remain limited. In this study, we classified HBTEC-ALI cultures into three morphological categories – Thick, Intermediate and Thin – based on cell size, ciliary activity and mucus production. These categories were associated with epithelial thickness and expression of the basal cell marker KRT5 . Thin-type cultures exhibited significantly reduced transepithelial electrical resistance and diminished apical glycan staining, suggesting compromised barrier integrity and altered apical glycan composition. We next evaluated replication of multiple respiratory viruses. Most viruses tended to replicate more efficiently in Thick- and Intermediate-type cultures, whereas adenovirus replication was enhanced in Thin-type cultures. Bocavirus replication did not differ between types. Our findings suggest that morphological assessment of HBTEC-ALI cultures is associated with functional barrier status and virus-specific replication efficiency. In addition, the association between KRT5 expression and morphological phenotypes suggests that KRT5 expression may reflect differences in epithelial characteristics among HBTEC-ALI cultures. These results may provide a practical framework for selecting HBTEC-ALI cultures and may help improve the reproducibility and efficiency of respiratory virus isolation and propagation.
This narrative review explores the complex landscape of coinfections involving Human Pegivirus Type 1 (HPgV), a member of the Hepaciviridae family previously known as GB virus type C or hepatitis G virus. Structurally similar to hepatitis C virus (HCV), HPgV is lymphotropic and while viraemia in healthy donors is asymptomatic, it has been associated with the onset of several non-Hodgkin lymphoma subtypes. We begin with an overview of HPgV’s genomic features, classification, transmission routes and natural history, including spontaneous clearance and post-infection anti-E2 antibody development. As of 03 February 2026, a PubMed search yielded 346 relevant studies from which data were synthesized, reporting HPgV coinfection across human immunodeficiency virus (HIV), HCV, hepatitis B virus and other viral diseases – highlighting genotype distributions, geographic variability and population-specific patterns. In summary, HPgV shows a reproducible signal in HIV coinfections, suggesting potential therapeutic benefits, while its impact on chronic hepatitis, acute infections and lymphoma risk remains preliminary and requires further investigation. This review highlights the need for rigorous prospective studies with standardized protocols to clarify HPgV’s true impact on human health and its potential for therapeutic application.
Mouse-adapted dengue virus type 2 (DENV2), designated the AG-P10 strain, was established through serial blind passages between AG129 mice and mosquito C6/36 cells. Compared with the parental strain INDIA/09-74, the AG-P10 strain exhibited increased virulence in AG129 mice, leading to rapid body weight loss and earlier mortality. In vitro characterization of the AG-P10 strain demonstrated significantly enhanced viral replication and larger focus formation in mammalian Vero and A549 cells but not in mosquito-derived C6/36 cells, suggesting host-specific adaptation. Whole-genome sequencing of the AG-P10 strain identified two nonsynonymous mutations, A119T in NS4B and E802Q in NS5. Through an analysis of recombinant viruses, we determined that the A119T mutation alone is sufficient to confer enhanced pathogenicity in AG129 mice and increased viral replication in Vero and A549 cells, suggesting its potential role in viral adaptation and pathogenesis. The DENV2 with the A119T mutation, as well as the AG-P10 strain, thus provides a robust animal model for developing antiviral therapeutics and vaccines against DENV and for investigating the molecular mechanisms governing DENV2 replication and virulence.
Mpox remains a significant global public health threat, highlighting the need for new antiviral therapies. Ridinilazole is a symmetric bisbenzimide developed for the treatment of Clostridium difficile infection that has recently been shown to inhibit human cytomegalovirus. Here, we investigated its antiviral activity against orthopoxviruses (OPXV). Ridinilazole potently inhibited vaccinia virus replication in primary human foreskin fibroblasts, with an IC 50 of 21 nM and minimal cytotoxicity. Importantly, ridinilazole also reduced infectious monkeypox virus (the causative agent of mpox) yields in primary fibroblasts by more than 150-fold. Mechanistic studies demonstrated that ridinilazole did not affect early viral gene expression or viral DNA replication, but significantly reduced the expression of late viral genes at both transcript and protein levels. These findings identify ridinilazole as an effective inhibitor of OPXV replication and support its potential repurposing as a novel therapeutic for mpox.
