Claire Mary Smith

High pathogenicity avian influenza viruses (HPAIVs) remain a major threat to animal and human health. The clade 2.3.4.4b H5Nx panzootic has been marked by increasing cross-species adaptation, including outbreaks in dairy cattle. The surge in mammalian infections in both wild and captive populations heighten concerns about zoonotic and pandemic potential. Here, we analyse haemagglutinin (HA) evolution in clade 2.3.4.4b H5Nx viruses from 2020 to 2025, focusing on key residues within the receptor-binding domain. We identify HA E185K (mature H5 HA numbering; E189K in H3 numbering) as a novel immune escape mutation that confers increased binding to avian- and human-type sialic acid receptors. Strikingly, this mutation is increasingly prevalent in cattle-derived H5N1 viruses and enhances binding to human-like receptors. However, ferret studies demonstrated that whilst pathogenicity of the E185K virus was comparable to the cattle derived isolate, the E185K mutation did not confer transmission to direct or indirect contact ferrets. Our results reveal a mechanism linking immune escape with expanded receptor specificity, highlighting a potential pathway for mammalian adaptation. These findings underscore the need for continued surveillance of evolving H5Nx viruses to mitigate emerging zoonotic threats.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants, yet therapeutics are lacking. The aim of this study was to develop a pre‑clinical model that recapitulates key clinical outcomes in infants with RSV bronchiolitis, such as neutrophil activation and migration into the airways. Peripheral blood neutrophils from infants with severe RSV disease admitted to the Paediatric Intensive Care Unit showed elevated myeloperoxidase (MPO) in children with RSV, compared to age-matched controls. To mechanistically model this response, we established an air-liquid interface (ALI) system incorporating paediatric airway epithelial cells, endothelial cells and neutrophils from adults, to recapitulate the blood-airway barrier. Following RSV infection, with and without treatment with antivirals remdesivir or RSV604, neutrophil migration and activation were assessed using flow cytometry. While both drugs reduced viral load, only RSV604 attenuated MPO expression. This model suggests that MPO could be useful as a readout of therapeutic efficacy. Targeting neutrophil-driven inflammatory pathways may be critical for reducing pathology in infant RSV infection.

The outcome of virus spillover ranges from dead-end infections to pandemics and is underpinned by host-pathogen interactions as well as evolutionary and epidemiological processes. The emergence of novel influenza A viruses (IAVs) has been associated with reassortment events involving multiple species, highlighting the importance of reservoir and intermediate hosts in viral emergence. Highly pathogenic H5N1 IAVs of the 2.3.4.4b genotype have caused a panzootic affecting a broad range of mammals. The role of dogs -arguably the most popular companion animal and a natural host of IAVs- in the ecology of IAVs under this new zooepidemiological scenario is unknown. To address this, we characterised the glycome of the dog respiratory epithelium, infected canine tracheal explants with multiple IAVs (including canine H3N2 and H3N8, equine H3N8, avian H3N8 and H5N1, swine H1N1, human H1N1 and H3N2, and bovine H5N1 viruses), and determined their cellular tropism. We show that the respiratory tract of dogs presents abundant sialylated glycans known to act as IAV receptors. Further, most IAVs (including 2.3.4.4b viruses) infected and replicated in dog tracheas, targeting mainly ciliated cells. Serological testing showed evidence of influenza spillover infections in dogs from the UK. Overall, our results show that the canine respiratory tract can provide a suitable environment for the generation of new IAVs. Given the multi-host contact networks of dogs in nature, they could act as recipients, bridging hosts, and/or mixing vessels for multiple IAV lineages, playing a central role in the ecology of influenza emergence.

Despite the huge global health burden presented by respiratory viruses, effective broad-spectrum antiviral therapeutic options remain limited. Here we evaluated the antiviral activity of four RNA-dependent RNA polymerase (RdRp) inhibitors, remdesivir, ribavirin, favipiravir, and molnupiravir, as monotherapy or dual-drug combinations against respiratory syncytial virus (subtype A, RSVA) and human parainfluenza (serotype 3, hPIV3) using epithelial cell lines and primary human airway culture models. Remdesivir showed the greatest potency across both viruses, while ribavirin and favipiravir also demonstrated inhibition. Molnupiravir was active against RSVA but not hPIV3. Several dual-drug combinations, including remdesivir–favipiravir, remdesivir–molnupiravir and favipiravir–molnupiravir, produced marked synergy against RSVA, and more limited synergy for hPIV3. Antiviral efficacy was validated in primary airway epithelial cultures, where effective concentrations preserved epithelial integrity and attenuated viral disruption of ciliary function. Across both viruses, increasing antiviral exposure was associated with dose-dependent signature mutagenesis. Antivirals induced significantly higher RSVA mutation burden in the primary airway model. These findings highlight the therapeutic potential of RdRp inhibitor combinations for RSVA and hPIV3, provide mechanistic insight through antiviral-related mutational signatures, and demonstrate advantages of the primary human airway culture model for development of effective multi-drug regimens and broad-spectrum antiviral preparedness.

Sustained mammal-to-mammal transmission of high pathogenicity H5N1avian influenza viruses is reshaping the host range of these pathogens. One of the longest-running mammalian transmission chains involves the B3.13 genotype circulating in U.S. dairy cattle which was detected early in 2024. Genomic analysis revealed selection and rapid fixation of haemagglutinin mutations D104G and V147M. We demonstate, via glycomic profiling, that bovine tissues, including the mammary gland, are enriched in N- and O-linked glycans capped with N-glycolylneuraminic acid (NeuGc), a sialic acid absent in humans and birds, which instead express only N-acetylneuraminic acid (NeuAc). Early cattle H5 viruses poorly recognized NeuGc, but D104G and V147M enabled efficient engagement of both NeuAc- and NeuGc-containing receptors. These mutations enhanced replication in bovine mammary tissue without major attenuation of replication in human lung and primary nasal epithelial cells. NeuGc-driven receptor adaptation therefore promotes viral fitness in cattle while potentially limiting immediate zoonotic risk. Deep mutational scanning further identifies alternative haemagglutinin substitutions that confer NeuGc usage and represent surveillance markers for emerging cattle H5 lineages.

Background: Respiratory RNA viral infections significantly impact quality of life and productivity, in terms of both pandemics ( e.g. severe acute respiratory syndrome coronavirus 2, influenza A viruses) and seasonal infections, such as respiratory syncytial virus (RSV). Globally, there are an estimated 33.8 million cases of RSV annually in children aged <5 years, leading to 2.8–4.3 million hospital admissions and up to 199 000 deaths. Human-relevant drug screening models are key to addressing the urgent need for effective antiviral treatments for RSV, particularly in vulnerable patient groups. Methods: Here, we present a donor-derived differentiated primary human nasal epithelial cell, high-throughput screening (HTS) Transwell-96 air–liquid interface (ALI) model designed to investigate the effects of combination antiviral therapies on RSV infection in primary human ciliated airway epithelium. In addition, we describe novel analytical tools using R (ciliR) to screen drug combinations by concurrently measuring efficacy and ciliary beat frequency, which we used as a sensitive marker of cell toxicity. Results: Our results demonstrate that the higher-throughput HTS Transwell-96 ALI cultures retain epithelial composition and ciliary function consistent within donor replicates. These cultures are permissible to infection with an RSV-green fluorescent protein (GFP) reporter virus, enabling quantitative comparison and combination treatment across multiple epithelial cultures from the same donor. Conclusion: We anticipate that our disease-relevant system will serve as a foundation for larger-scale experiments aimed at optimising combination therapy for RSV and other respiratory viruses.

In 2024, an unprecedented outbreak of H5N1 high pathogenicity avian influenza was detected in dairy cattle in the USA resulting in spillbacks into poultry, wild birds and other mammals including humans. Here, we present molecular and virological evidence that the cattle B3.13 genotype H5N1 viruses rapidly accumulated adaptations in polymerase genes that enabled better replication in bovine cells and tissues, as well as cells of other mammals including humans. We find evidence of several mammalian adaptations in cattle including PB2 M631L, which is found in all cattle sequences, and PA K497R, which is found in the majority. Structurally, PB2 M631L maps to the polymerase-ANP32 interface, an essential host factor for viral genome replication. We show that this mutation adapts the polymerase to better interact with bovine ANP32 proteins, particularly ANP32A, and thereby enhances virus replication in bovine mammary systems and primary human airway cultures. We show that ongoing evolution in the PB2 gene, including E627K and a convergently arising D740N substitution, further increase polymerase activity and virus replication in a range of mammalian cells. Thus, circulation of H5N1 in dairy cattle allows virus adaption improving replicative ability in cattle and poses a continued risk of zoonotic spillover.

Despite the huge global health burden presented by respiratory viruses, effective broad-spectrum antiviral therapeutic options remain limited. Here we evaluated the antiviral activity of four RNA-dependent RNA polymerase (RdRp) inhibitors, remdesivir, ribavirin, favipiravir, and molnupiravir, against respiratory syncytial virus (RSV) and human parainfluenza (hPIV) using monotherapy or dual-drug combinations with epithelial cell lines and primary human airway culture models. Remdesivir showed the greatest potency across both viruses, while ribavirin and favipiravir also demonstrated inhibition. Molnupiravir was active against RSVA but not hPIV3. Several dual-drug combinations, including remdesivir–favipiravir, remdesivir–molnupiravir and favipiravir–molnupiravir, produced marked synergy against RSVA, and more limited synergy for hPIV3. Antiviral efficacy was validated in primary airway epithelial cultures, where effective concentrations preserved epithelial integrity and attenuated viral disruption of ciliary function. Across both viruses, increasing antiviral exposure was associated with dose-dependent signature mutagenesis. Importantly, antivirals induced significantly higher RSVA mutation burden in the primary airway model. These findings highlight the therapeutic potential of RdRp inhibitor combinations for RSVA and hPIV3, provide mechanistic insight through antiviral-related mutational signatures and demonstrate advantages of the primary human airway culture model for development of effective multi-drug regimens and broad-spectrum antiviral preparedness. Graphical

High pathogenicity avian influenza virus (HPAIV) has significantly impacted upon avian and mammalian populations across the Antarctic region. All viruses detected have been genotype B3.2 with phylogenetic analyses indicating multiple independent incursions from continental South America to, and transmission between, sub-Antarctic islands. From a zoonotic perspective, several isolates contained markers of mammalian adaptation in PB2 with functional characterisation of mutants demonstrating efficient replication in primary human airway epithelial cell cultures, demonstrating that these PB2-mutations alone contributed to enhanced polymerase activity in human cell lines. No mammalian-adaptive mutations were detected in the haemagglutinin or neuraminidase genes, with viruses retaining avian receptor binding preferences. Antigenic characterisation demonstrated cross-reactivity with existing pre-pandemic candidate vaccine strains and all viruses remained susceptible to licensed frontline antiviral therapeutics. We demonstrate a complex evolving viral ecology in the sub-Antarctic region involving both avian and marine mammal hosts, with significant implications for regional wildlife populations and zoonotic risk.

This work was conducted during the COVID-19 pandemic prior to the licensing of any vaccines against COVID-19. Although several COVID-19 vaccines are now commercially available, this research on the development of a combination Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV), SARS-coronavirus 2 (SARS-CoV-2), and Middle Eastern Respiratory Syndrome (MERS) is still relevant and shows how a combination vaccine can be designed, produced and rapidly tested in the laboratory. We present the development of a combination vaccine designed to provide immunity against Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV), SARS-coronavirus 2 (SARS-CoV-2), and Middle Eastern Respiratory Syndrome (MERS). The primary objective of this vaccine design is twofold: to mitigate the burden of coronavirus and to address the specific vulnerability of regions prone to recurrent MERS outbreaks. Our combination vaccine incorporates antigenic components from zoonotic sources, specifically SARS-CoV, SARS-CoV-2, and MERS. We assess the impact of combining different Spike protein’s S1 subunit antigens, due to its recognised immunogenic potential, and adjuvants on serum antibody titres, virus neutralizing capabilities, and inter-antigen immune responses. We report a robust and broad antibody response against SARS-CoV-2 and related coronaviruses, which was amplified by different adjuvant formulations, including alum, MPLA, CpG, and Squalene-in-Water Emulsion.