Biology subjects

Warren, C. J.

Publications and source records attributed to Warren, C. J..

13 recordsLinked to original sources

bioRxiv2026

The mammalian-adaptive PB2-E627K substitution preserves viral fitness of clade 2.3.4.4b H5N1 HPAIV in birds.

Since emerging in 2020, clade 2.3.4.4b H5N1 high pathogenicity avian influenza virus (HPAIV) has disseminated globally, causing substantial infection and mortality in wild birds and poultry. Additionally, numerous spill-over events into mammals have occurred, including mass mortalities and sporadic human infections. Such events can drive the acquisition of mammalian-adaptive mutations, which further increase the risk to humans; the most well characterised mutation is the glutamate to lysine change at amino acid position 627 in the polymerase basic 2 protein (PB2-627K). Across avian influenza viruses, PB2-627K enhances polymerase activity and replication in mammalian cells, increasing zoonotic and pandemic potential. However, bioinformatic analyses indicate that PB2-627K remains rare among clade 2.3.4.4b H5N1 viruses circulating in avian populations. Here, we investigated the impact of PB2-627K on viral fitness in avian hosts to assess the potential for mammalian-adapted viruses to re-establish in birds, mimicking infection of avian species following scavenging on mammalian species where PB2-627K has already emerged. Using a representative H5N1 virus (European genotype AB), PB2-627K increased polymerase activity and replication in human cells without compromising replication in avian cells. A mammalian-derived PB2-627K H5N1 isolate from a fox exhibited comparable replication kinetics and transmission efficiency in chickens and ducks to a closely related PB2-627E virus. Notably, PB2-627K remained genetically stable at consensus level following infection and transmission in both avian hosts. These findings demonstrate that mammalian-adaptive mutations acquired during spill-over can be maintained in avian populations, potentially elevating zoonotic risk and underscoring the importance of surveillance for such mutations in birds.

microbiology↗

bioRxiv2026

Bovine-derived H5N1 influenza virus efficiently infects lactating swine via the mammary gland

Since 2024, highly pathogenic influenza A(H5N1) viruses have spread extensively among U.S. dairy cattle, where they replicate efficiently in the mammary gland and are shed at high titers in milk. To directly assess susceptibility of commercial swine populations to bovine-derived H5N1 virus, lactating sows with prior influenza virus vaccination histories representative of U.S. commercial swine production systems were inoculated via the intramammary route and co-housed with their 1-week-old piglets to evaluate disease outcomes, viral replication, and potential for vertical transmission. Intramammary inoculation of lactating sows resulted in sustained viral RNA shedding in milk, while piglets exhibited sporadic oral viral RNA positivity that mirrored viral kinetics in milk. Lesions in mammary tissue and viral antigen staining, as well as development of neutralizing antibody responses and changes in milk color and consistency, further confirmed infection in the sows. Despite these molecular findings, none of the animals developed overt clinical disease, and respiratory involvement was not noted during the study period. Collectively, we demonstrate that intramammary exposure results in productive influenza A(H5N1) virus infection in lactating sows despite their vaccination histories, indicating the potential threat of viral spillover into commercial swine populations. The clinically inapparent nature of infection presents a risk of subclinical spread and underscores the importance of expanding viral surveillance to swine.

microbiology↗

bioRxiv2026

A precision-cut lung slice platform for evaluating respiratory virus replication dynamics

Understanding respiratory virus replication, tropism, and disease mechanisms requires experimental systems that balance physiological relevance with scalability. Although immortalized cell lines remain widely used, they fail to capture the cellular complexity of the lung, while animal models--though informative--are often costly, low-throughput, and limited in their ability to model human disease. Here, we describe a rapid and scalable precision-cut lung slice (PCLS) platform that overcomes many of these limitations. Our workflow generates infection-ready lung tissue slices within 24 hours and maintains tissue viability over extended culture periods. Using influenza A virus as a model pathogen, we demonstrate that PCLS support robust viral replication, recapitulate characteristic infection-associated pathology, and elicit localized immune cell responses within the respiratory epithelium. Together, these features establish PCLS as a versatile and physiologically relevant platform for studying the biology of influenza virus and other respiratory pathogens.

microbiology↗

bioRxiv2026

Efficient replication of influenza D virus in the human airway underscores zoonotic potential

Influenza D virus (IDV), primarily found in livestock species, has demonstrated cross-species transmission potential, yet its threat to humans remains poorly understood. Here, we curated a panel of IDV isolates collected during field surveillance from 2011 to 2020 from swine and cattle to assess their ability to infect human airway cells as a proxy for zoonotic threat assessment. Using lung epithelial cell lines, primary well-differentiated airway epithelial cultures, and precision-cut lung slices, we demonstrated that IDV efficiently propagates in cells and tissues from the human respiratory tract, reaching titers comparable to human influenza A virus (IAV). Infection kinetics in primary porcine airway cultures and respiratory tissues mirrored those from human, suggesting similar infectivity across species. To define host responses to IDV infection, we evaluated innate immune sensing and downstream interferon signaling in human respiratory cells. IDV infection resulted in markedly reduced activation of interferon regulatory factor (IRF) signaling and diminished induction of interferon lambda 1 and interferon-stimulated genes compared to IAV, indicating inefficient activation of innate immune sensing pathways. However, IDV replication was potently restricted in interferon-pretreated cells, demonstrating sensitivity to interferon-mediated antiviral effector mechanisms once an antiviral state was established. Together, these findings show that IDV can efficiently infect the human airway while limiting innate immune sensing, a feature that may facilitate zoonotic spillover. Our study highlights the need for enhanced surveillance of IDV at the animal-human interface and provides a foundation for further investigation into its biology and potential for causing human infection and disease. SIGNIFICANCE STATEMENTInfluenza D virus (IDV) is a poorly understood virus type in the Orthomyxoviridae family. Although initially considered incapable of infecting humans, high seropositivity rates among cattle and swine workers suggest that zoonotic infections may already be occurring. However, the extent of human compatibility--and the potential for spillover--remains poorly understood. Our study demonstrates that IDV replicates efficiently in multiple human respiratory models while largely evading innate immune defenses, raising concern that only minimal evolutionary changes may be required for sustained human transmission. These findings underscore the need for further investigation into IDV biology and zoonotic risk. Such studies are critical for identifying viruses with the potential to adapt to humans before they become public health threats.

microbiology↗

bioRxiv2026

Natural H5N1 immunity in dairy cows is durable and cross-protective but non-sterilizing

Ongoing transmission of influenza A virus (H5N1) in U.S. dairy cattle threatens both animal and human health, underscoring the need to understand the durability of host immunity against reinfection with evolving genotypes. We challenged naive and convalescent cows, infected one year prior with H5N1 genotype B3.13, with either homologous B3.13 or heterologous D1.1 genotype virus. Homologous rechallenge resulted in complete clinical protection with no infectious viral shedding. Conversely, heterologous rechallenge led to transient clinical disease and limited infectious viral shedding. Convalescent cows experienced significantly milder disease than naive cows, which developed severe illness with high viral shedding and required early euthanasia, regardless of the strain. These findings indicate that naturally acquired immunity offers strong protection against severe illness but may allow silent transmission of divergent strains. Therefore, natural herd immunity alone is unlikely to eliminate the virus; controlling H5N1 in cattle will likely require vaccination strategies that address viral evolution.

immunology↗

bioRxiv2025

Pigeons exhibit low susceptibility and poor transmission capacity for H5N1 clade 2.3.4.4b high pathogenicity avian influenza virus

The ongoing panzootic of H5N1 high pathogenicity avian influenza virus (HPAIV) has caused the deaths of over half a billion wild birds and poultry, and has led to spillover events in both wild and domestic mammals, alongside sporadic human infections. A key driver of this panzootic is the apparent high viral fitness across diverse avian species, which facilitates an increased interface between wild and domestic species. Columbiformes (pigeons and doves) are commonly found on poultry premises, yet little is known about their potential role in contemporary HPAIV disease ecology. Here we investigated the epidemiological role of pigeons (Columba livia) by determining their susceptibility using decreasing doses of clade 2.3.4.4b H5N1 HPAIV (genotype AB). We investigated infection outcomes and transmission potential between pigeons and to chickens. Following direct inoculation, pigeons did not develop clinical signs, and only those inoculated with the highest dose shed viral RNA or seroconverted to H5N1-AB, revealing a MID50 of 105 EID50. Even in the high-dose group, only low-level shedding and environmental contamination was observed, and low-level viral RNA were present in the tissues of directly inoculated pigeons, with no distinct pathological lesions. Pigeons did not transmit the virus to pigeons or chickens placed in direct contact. We observed distinct differences in sialic acid receptor distribution in the pigeon respiratory tract compared to chickens and ducks. Together, these findings suggest that pigeons have low susceptibility to clade 2.3.4.4b H5N1 HPAIV and are unlikely to contribute significantly to virus maintenance, transmission to poultry, or zoonotic infection.

microbiology↗

bioRxiv2025

Investigating Factors Driving Shifts in Subtype Dominance within H5Nx Clade 2.3.4.4b High-Pathogenicity Avian Influenza viruses

H5Nx clade 2.3.4.4b high-pathogenicity avian influenza viruses (HPAIVs) have decimated wild bird and poultry populations globally since the autumn of 2020. In the United Kingdom (UK) and in continental Europe, the H5N8 subtype predominated during the first epizootic wave of 2020/21, with few detections of H5N1. However, during the second (2021/22) and third (2022/23) epizootic waves, H5N1 was the dominant subtype. The rapid shift in dominance from H5N8 to H5N1 was likely driven by a combination of virological, immunological, and/or host-related factors. In this study, we compared viral fitness and immunological responses in ducks, a key reservoir species, using dominant genotypes of H5N1 (genotype AB) and H5N8 (genotype A) from the second wave. While viral shedding dynamics were similar for both viruses, H5N8 was more pathogenic. Antigenic analysis of post-infection duck sera revealed that the haemagglutinin (HA) protein was antigenically similar across clade 2.3.4.4b H5 HPAIVs, but neuraminidase (NA) proteins displayed different patterns of cross-reactivity. We also modelled a scenario where ducks were pre-exposed to H5N1 (genotype C) or H5N8 (genotype A) from the first wave and subsequently challenged with either homologous or heterologous subtypes from the second wave (genotype AB or A). Despite the absence of seroconversion, pre-exposure to different subtypes resulted in varying clinical outcomes following challenge. These findings indicate that both viral and immunological factors likely played significant roles in the emergence and spread of H5Nx HPAIVs in wild bird populations.

microbiology↗

bioRxiv2024

Rapid mortality in captive bush dogs (Speothos venaticus) caused by influenza A of avian origin (H5N1) at a wildlife collection in the United Kingdom

Europe has suffered unprecedented epizootics of high pathogenicity avian influenza (HPAI) clade 2.3.4.4b H5N1 since Autumn 2021. As well as impacting upon commercial and wild avian species, the virus has also infected mammalian species more than ever observed previously. Mammalian species involved in spill over events have primarily been scavenging terrestrial carnivores and farmed mammalian species although marine mammals have also been affected. Alongside reports of detections in mammalian species found dead through different surveillance schemes, several mass mortality events have been reported in farmed and wild animals. During November 2022, an unusual mortality event was reported in captive bush dogs (Speothos venaticus) with clade 2.3.4.4b H5N1 HPAIV of avian origin being the causative agent. The event involved an enclosure of fifteen bush dogs, ten of which succumbed during a nine-day period with some dogs exhibiting neurological disease. Ingestion of infected meat is proposed as the most likely infection route.

microbiology↗

bioRxiv2023

Transmission dynamics and pathogenesis differ between pheasants and partridges infected with clade 2.3.4.4b H5N8 and H5N1 high-pathogenicity avian influenza viruses

During the UK 2020-2021 epizootic of H5Nx clade 2.3.4.4b high-pathogenicity avian influenza viruses (HPAIVs), high mortality occurred during incursions in commercially farmed common pheasants (Phasianus colchicus). Two pheasant farms, affected separately by H5N8 and H5N1 subtypes, included adjacently housed red-legged partridges (Alectoris rufa), which appeared to be unaffected. Despite extensive ongoing epizootics, H5Nx HPAIV partridge outbreaks were not reported during 2020-2021 and 2021-2022 in the UK, so it is postulated that partridges are more resistant to HPAIV infection than other gamebirds. To assess this, pathogenesis and both intra- and inter-species transmission of UK pheasant-origin H5N8-2021 and H5N1-2021 HPAIVs were investigated. Onward transmission to chickens was also assessed to better understand the risk of spread from gamebirds to other commercial poultry sectors. A lower infectious dose was required to infect pheasants with H5N8-2021 compared to H5N1-2021. However, HPAIV systemic dissemination to multiple organs within pheasants was more rapid following infection with H5N1-2021 than H5N8-2021, with the former attaining generally higher viral RNA levels in tissues. Intraspecies transmission to contact pheasants was successful for both viruses and associated with viral environmental contamination, while interspecies transmission to a first chicken-contact group was also efficient. However, further onward transmission to additional chicken contacts was only achieved with H5N1-2021. Intra-partridge transmission was only successful when high-dose H5N1-2021 was administered, while partridges inoculated with H5N8-2021 failed to shed and transmit, although extensive tissue tropism was observed for both viruses. Mortalities among infected partridges featured a longer incubation period compared to that in pheasants, for both viruses. Therefore, the susceptibility of different gamebird species and pathogenicity outcomes to the ongoing H5Nx clade 2.3.4.4b HPAIVs varies, but pheasants represent a greater likelihood of H5Nx HPAIV introduction into galliforme poultry settings. Consequently, viral maintenance within gamebird populations and risks to poultry species warrant enhanced investigation.

microbiology↗

bioRxiv2023

Regulation of interferon signaling by transposon exonization

SummaryInnate immune signaling is essential for clearing pathogens and damaged cells, and must be tightly regulated to avoid excessive inflammation or autoimmunity. Here, we found that the alternative splicing of exons derived from transposable elements is a key mechanism controlling immune signaling in human cells. By analyzing long-read transcriptome datasets, we identified numerous transposon exonization events predicted to generate functional protein variants of immune genes, including the type I interferon receptor IFNAR2. We demonstrated that the transposon-derived isoform of IFNAR2 is more highly expressed than the canonical isoform in almost all tissues, and functions as a decoy receptor that potently inhibits interferon signaling including in cells infected with SARS-CoV-2. Our findings uncover a primate-specific axis controlling interferon signaling and show how a transposon exonization event can be co-opted for immune regulation.

genomics↗

bioRxiv2023

The role of airborne particles in the epidemiology of clade 2.3.4.4b H5N1 high pathogenicity avian influenza virus in commercial poultry production units

Since October 2021, Europe has experienced the largest avian influenza virus (AIV) epizootic, caused by clade 2.3.4.4b H5N1 high pathogenicity AIV (HPAIV), with over 320 poultry and captive bird infected premises (IPs) and 2480 dead H5N1 positive wild birds detected in Great Britain alone. Many IPs have been detected as geographical clusters, raising questions around potential lateral spread between premises by airborne particles. Airborne transmission over short distances has been reported for some AIVs strains. However, the risk of airborne spread of this strain remains to be elucidated. We conducted extensive sampling from IPs where clade 2.3.4.4b H5N1 HPAIVs was confirmed during the 2022/23 epizootic, each representing a major poultry species (ducks, turkeys, and chickens). A range of environmental samples were collected inside and outside houses, including deposited dust, feathers, and other potential fomites. Viral RNA (vRNA) and infectious virus were detected in air samples collected from inside and outside, but in close proximity, of infected houses, with vRNA alone being detected greater distances (>10m) outside. Some dust samples collected outside of the affected houses contained infectious virus, while feathers from the affected houses, located up to 60m away, only contained vRNA. Together, these data suggest that airborne particles harbouring infectious HPAIV can be translocated short distances (<10m) through the air, while particles containing vRNA might travel further (<50m). Therefore, the potential for airborne transmission of clade 2.3.4.4b H5N1 HPAIV between premises is considered low. Other factors, including indirect contact with wild birds, fomite spread and the efficiency of biosecurity represent greater importance in disease incursion. IMPORTANCEUnderstanding the routes of avian influenza virus incursion into poultry premises is critical to ensure the implementation of effective preventative measures, reducing the risk of spread of the virus. Extensive environmental sampling across three infected premises, representing the major global commercial poultry species, detected viral RNA and infectious virus in air samples, dust, and other fomites inside of the houses containing infected poultry, as well as within the immediate proximity of infected houses. This suggests that particles harbouring infectious virus may travel short distances outside of affected houses, which may potentially contribute to house-to-house transmission on the same premises, but not at distances likely to contribute to the transmission to another poultry premises. Consequently, control efforts should be focused on traditional, robust biosecurity measures at key barriers on poultry houses. Human behaviour in and around poultry houses as factors contributing to biosecurity risks should be considered.

microbiology↗

bioRxiv2023

Clade 2.3.4.4b H5N1 high pathogenicity avian influenza virus (HPAIV) from the 2021/22 epizootic is highly duck adapted and poorly adapted to chickens

The 2021/2022 epizootic of high pathogenicity avian influenza (HPAIV) remains one of the largest ever in the UK, being caused by a clade 2.3.4.4b H5N1 HPAIV. This epizootic affected more than 145 poultry premises, most likely through independent incursion from infected wild birds, supported by more than 1700 individual detections of H5N1 from wild bird mortalities. Here an H5N1 HPAIV, representative of this epizootic (H5N1-21), was used to investigate its virulence, pathogenesis and transmission in layer chickens and pekin ducks, two species of epidemiological importance. We inoculated both avian species with decreasing H5N1-21 doses. The virus was highly infectious in ducks, with high infection levels and accompanying shedding of viral RNA, even in ducks inoculated with the lowest dose, reflecting the strong waterfowl adaptation of the clade 2.3.4.4 HPAIVs. Duck-to-duck transmission was very efficient, coupled with high environmental contamination. H5N1-21 was frequently detected in water sources, serving as likely sources of infection for ducks, but inhalable dust and aerosols represented low transmission risks. In contrast, chickens inoculated with the highest dose exhibited lower rates of infection compared to ducks. There was no evidence for experimental H5N1-21 transmission to any naive chickens, in two stocking density scenarios, coupled with minimal and infrequent contamination being detected in the chicken environment. Systemic viral dissemination to multiple organs reflected the pathogenesis and high mortalities in both species. In summary, the H5N1-21 virus is highly infectious and transmissible in anseriformes, yet comparatively poorly adapted to galliformes, supporting strong host preferences for wild waterfowl. Key environmental matrices were also identified as being important in the epidemiological spread of this virus during the continuing epizootic.

microbiology↗

bioRxiv2017

High-Risk Human Papillomavirus E7 Alters Host DNA Methylome And Represses HLA-E Expression In Human Keratinocytes

Human papillomavirus (HPV) infection distinctly alters methylation patterns in HPV-associated cancer. We have recently reported that HPV E7-dependent promoter hypermethylation leads to downregulation of the chemokine CXCL14 and suppression of antitumor immune responses. To investigate the extent of gene expression dysregulated by HPV E7-induced DNA methylation, we analyzed parallel global gene expression and DNA methylation using normal immortalized keratinocyte lines, NIKS, NIKS-16, NIKS-18, and NIKS-16{Delta}E7. We show that expression of the MHC class I genes is downregulated in HPV-positive keratinocytes in an E7-dependent manner. Methylome analysis revealed hypermethylation at a distal CpG island (CGI) near the HLA-E gene in NIKS-16 cells compared to either NIKS cells or NIKS-16{Delta}E7 cells, which lack E7 expression. The HLA-E CGI functions as an active promoter element which is dramatically repressed by DNA methylation. HLA-E protein expression on cell surface is downregulated by high-risk HPV16 and HPV18 E7 expression, but not by low-risk HPV6 and HPV11 E7 expression. Conversely, demethylation at the HLA-E CGI restores HLA-E protein expression in HPV-positive keratinocytes. Because HLA-E plays an important role in antiviral immunity by regulating natural killer and CD8+ T cells, epigenetic downregulation of HLA-E by high-risk HPV E7 may contribute to virus-induced immune evasion during HPV persistence.

immunology↗