Unlike most RNA and DNA viruses that broadly stimulate mRNA decay and interfere with host gene expression, human cytomegalovirus (HCMV) extensively remodels the host translatome without producing an mRNA decay enzyme. By performing a targeted loss‐of‐function screen in primary human fibroblasts, we here identify the host CCR4‐NOT deadenylase complex members CNOT1 and CNOT3 as unexpected pro‐viral host factors that selectively regulate HCMV reproduction. We find that the scaffold subunit CNOT1 is specifically required for late viral gene expression and genome‐wide host responses in CCR4‐NOT‐disrupted cells. By profiling poly(A)‐tail lengths of individual HCMV and host mRNAs using nanopore direct RNA sequencing, we reveal poly(A)‐tails of viral messages to be markedly longer than those of cellular mRNAs and significantly less sensitive to CCR4‐NOT disruption. Our data establish that mRNA deadenylation by host CCR4‐NOT is critical for productive HCMV replication and define a new mechanism whereby herpesvirus infection subverts cellular mRNA metabolism to remodel the gene expression landscape of the infected cell. Moreover, we expose an unanticipated host factor with potential to become a therapeutic anti‐HCMV target.

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 chickens. Following direct inoculation, pigeons did not develop clinical signs, and only those inoculated with the highest dose shed viral RNA (vRNA) or seroconverted to H5N1-AB, revealing a 50% minimum infectious dose (MID) of 10⁵ 50% egg infectious dose. Even in the high-dose group, only low-level shedding and environmental contamination were observed, and low-level viral RNAs 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.

Porcine reproductive and respiratory syndrome viruses (PRRSV-1 and -2) are the causative agents of one of the most important infectious diseases affecting the global pig industry. Previous studies, largely focused on PRRSV-2, have shown that non-structural protein-1α (NSP1α) and NSP1β modulate host cell responses; however, the underlying molecular mechanisms remain to be fully elucidated. Therefore, we aimed to identify novel PRRSV-1 NSP1–host protein interactions to improve our knowledge of NSP1-mediated immunomodulation. NSP1α and NSP1β from a representative western European PRRSV-1 subtype 1 field strain (215-06) were used to screen a cDNA library generated from porcine alveolar macrophages (PAMs), the primary target cell of PRRSV, using the yeast-2-hybrid system. This identified 60 putative binding partners for NSP1α and 115 putative binding partners for NSP1β. Of those taken forward for further investigation, 3 interactions with NSP1α and 27 with NSP1β were confirmed. These proteins are involved in the immune response, ubiquitination, nuclear transport, or protein expression. Increasing the stringency of the system revealed NSP1α interacts more strongly with PIAS1 than PIAS2, whereas NSP1β interacts more weakly with TAB3 and CPSF4. Our study has increased our knowledge of the PRRSV-1 NSP1α and NSP1β interactomes, further investigation of which could provide detailed insight into PRRSV immunomodulation and aid vaccine development.

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 MID ₅₀ of 10 ⁵ EID ₅₀ . 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.

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 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 protein was antigenically similar across clade 2.3.4.4b H5 HPAIVs, but neuraminidase 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.

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. Importance: Clade 2.3.4.4b H5N1 high pathogenicity avian influenza viruses (HPAIVs) have caused widespread outbreaks in birds and increasingly frequent spill-over into wild and domestic mammals, including recent infections in cattle. Greater circulation in mammalian hosts raises concern that viruses may acquire mutations associated with human adaptation. We assessed the risk of such “pre-adapted” viruses, already possessing mammalian-adaptive markers, re-entering and persisting in avian populations. We show that the key adaptation PB2-627K does not compromise viral fitness in birds and remains genetically stable following transmission in chickens and ducks. These findings indicate that mammalian-adaptive mutations acquired during spill-over can be maintained in avian reservoirs, potentially lowering the barrier to zoonotic infection. This work highlights the importance of genomic surveillance in both mammalian and avian populations to support any alterations to zoonotic risk.