Biology subjects

Digard, P.

Publications and source records attributed to Digard, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Investigating the evolutionary plasticity of influenza virus splicing using influenza A virus segment 7 as a model system

Influenza viruses A, C and D (IAV, ICV, IDV respectively) utilise mRNA splicing to produce two proteins from their matrix gene segment; the matrix (M1) protein and an ion channel (M2). In IAV, the M1 and M2 open reading frames (ORFs) partially overlap and splice donor (SD) and splice acceptor (SA) sites in the M1 gene are used to produce an M2 polypeptide with an N-terminal sequence shared with M1. In ICV and IDV, the M1 and M2 ORFs are concatenated and splice sites downstream of the M1 coding region are used to produce a matrix protein from the spliced mRNA. To probe how these different splicing strategies might have evolved, we used reverse genetics to test the evolutionary flexibility of IAV SD and SA sites. We found that relatively small numbers of mutations were sufficient to activate a variety of alternative SD and SA sites in a mini-replicon system, several of which led to the production of ion channel isoforms with variant ectodomains, as well as one that produced an M1 variant. Furthermore, several of the viruses with altered splicing patterns were viable, including five with both SD and SA sites changed. Three of these 'rewired' viruses had only modest fitness penalties, despite up to 3 nonsynonymous changes in M1 and a 34 amino acid insertion in M2. These results suggest that IAV segment 7 splicing strategies are evolutionarily flexible and have implications for how IAV, ICV and IDV matrix segments might have diversified from a common ancestor.

microbiology↗

bioRxiv2026

Bovine H5N1 influenza viruses have adapted to more efficiently use receptors abundant in cattle

Sustained mammal-to-mammal transmission of high pathogenicity H5N1 avian 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 in early 2024. Genomic analyses 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 identified alternative haemagglutinin substitutions that confer NeuGc usage and represent surveillance markers for emerging cattle H5 lineages.

microbiology↗

bioRxiv2025

Identification of cis-Acting Elements that Regulate Influenza A Virus Segment 7 Differential mRNA Splicing

Influenza A virus segment 7 encodes the M1 matrix protein and M2 ion channel from an unspliced primary transcript mRNA1 and spliced mRNA2, respectively. Both proteins are essential for efficient virus replication, but the intrinsic sequences that determine segment 7 splicing balance are not fully defined. Here, we systematically investigated how conserved cis-acting splicing elements regulate 7 splicing. We found that while the splice acceptor sequence is optimal, both potential upstream polypyrimidine tracts regulate splicing, the 5 region acting positively and the 3 region negatively to maintain M1 expression. We could not unambiguously identify the mRNA2 branch point, but two closely spaced motifs can act redundantly. Saturation mutagenesis of the mRNA2 splice donor sequence showed that it is nearly optimal but that nucleotides 51 and 55 modestly downregulate splicing efficiency. Most introduced mutations that change splicing balance are nonsynonymous in M1. Nevertheless, mutations in the polypyrimidine tract and branch points that significantly perturbed M1:M2 balance resulted in viable viruses. In contrast, the splice donor sequence was intolerant of mutation. We conclude that M1:M2 balance is maintained via suboptimal splice donor and polypyrimidine tract splicing signals, with the latter allowing more evolutionary flexibility to vary the ratio and/or M1 sequence.

microbiology↗

bioRxiv2025

Identification of key host genes for influenza A virus in avian cells using a genome-wide CRISPR-Cas9 screen

Influenza A virus (IAV) causes major economic losses to the poultry industry and poses a zoonotic threat to human health. Potential pandemic outbreaks are underpinned by the ability of the virus to jump from one species to another. Host-virus interactions can dictate the success of such events and while systematic studies have successfully mapped host virus interactions in human cells, few studies have been performed in relevant animal host cell lines. Here, we conducted two independent genome-wide CRISPR/Cas9 knockout screens in chicken lung epithelial cells infected with either the human-adapted PR8 vaccine strain or the avian UDL 3:5 reassortant virus encoding PR8 HA, NA and M segments. Rather than selecting solely for cell survival, we used anti-M2 antibody staining and fluorescence-activated cell sorting to capture host factors influencing multiple stages of the IAV life cycle. Across both screens, we identified 104 genes required for efficient replication in chicken cells, including 16 with strong effects (log2 fold change > 2). Comparative analysis with published human screens revealed 17 conserved host factors, 19 human-specific factors, and 42 chicken-specific factors, highlighting potential species-specific interactions. Top hits included genes involved in sialic acid biosynthesis and N-linked glycosylation--SLC35A1, SLC35A2, and the avian-specific influenza polymerase cofactor ANP32A. Functional validation demonstrated that MOGS, MGAT1, DENR, DMXL1, ENO1, IPO9, KLF6, PTAR1, and TSG101 contribute to multiple stages of the IAV life cycle. In particular, MOGS and MGAT1 were essential for N-glycan processing and modulated cell-surface sialic acid abundance, with strain- and species-specific effects. These findings define a genetic landscape of IAV dependency factors in chicken cells and suggest shared and species-specific host requirements that could impact cross-species transmission.

cell biology↗

bioRxiv2025

Characterisation of the Tilapia Lake Virus proteome and identification of an 11th protein, S9-F3

Tilapia Lake Virus (TiLV) is an emerging negative-sense, single-stranded RNA virus that poses a significant threat to global tilapia aquaculture. Of the proteins encoded by its ten genomic segments, the first four, which encode a viral polymerase (segments 1-3) and a nucleoprotein (segment 4) have been characterised. However, the functions of the polypeptides encoded by the remaining segments remain largely unknown. Here, we systematically investigated the expression and subcellular localisation of all ten predicted TiLV-encoded proteins using in vitro translation and expression in mammalian and fish cells as well as mass spectrometry of virus infected cells. These approaches confirmed the synthesis of major polypeptides from each segment and identified an additional 11th protein, S9-F3, translated from an alternative reading frame of segment 9. Microscopy of individually expressed green fluorescent protein (GFP)-tagged constructs revealed that S2 and S10 polypeptides were predominantly nuclear, while S1, S3, S5, S8 and S9-F3 were mostly cytoplasmic with S5 and S6 displaying perinuclear foci. Bioinformatic analysis suggested a potential nuclear export signal in S9-F3 and use of the inhibitor leptomycin B confirmed its CRM1-dependent nuclear export. Evolutionary analyses indicated that both S9 and S9-F3 are under selective pressure, as well as the presence of an S9-F3 homologue in a TiLV-like guppy virus. These findings uncover an alternative translation product and a regulated nuclear export mechanism in TiLV, providing new insights into the molecular biology of this virus and its interaction with host cellular pathways.

microbiology↗

bioRxiv2025

The cow udder is a potential mixing vessel for influenza A viruses

The incursion of high pathogenicity avian influenza A virus (IAV) into US dairy cows is unprecedented in the era of molecular diagnosis and pathogen sequencing. This raises questions over the likelihood of further outbreaks and whether dairy cattle could be a "mixing vessel" for novel strains of IAV. Using a panel of BSL2-safe reassortant viruses representing clade 2.3.4.4b H5 epizootic lineages circulating since 2020, we found that a cow B3.13 isolate displayed enhanced replication in cow mammary gland cells, along with increased viral polymerase activity and stronger interferon antagonism in cow cells compared to an earlier EA-2020-C genotype virus. However, multiple avian and mammalian IAV strains, including other clade 2.3.4.4b high pathogenicity genotypes, were replication competent in bovine cells, particularly those of the mammary gland, suggesting that there is a diverse circulating IAV pool with the potential to infect cows. Moreover, we show that cow mammary cells co-express -2,3 and -2,6 - linked sialic acids, and are susceptible to co-infection with human and avian IAVs. We conclude that the US cow influenza outbreak does not simply reflect a unique adaptation of the B3.13 genotype virus; rather, the bovine udder represents a permissive niche for IAV and a plausible site for reassortment, underscoring its potential role in generating novel influenza viruses with pandemic risk.

microbiology↗

bioRxiv2025

Genetic reassortment and diversification of host specificity have driven evolutionary trajectories of lineages of panzootic H5N1 influenza

Since 2021, subclade 2.3.4.4b A(H5N1) high pathogenicity avian influenza (HPAI) viruses have undergone changes in ecology and epidemiology, causing a panzootic of unprecedented scale in wild and domestic birds with spill-over infections and perceptible transmission in a range of mammalian species, raising concern over zoonotic potential. HPAI viruses readily exchange gene segments with low pathogenicity avian influenza viruses via reassortment, a mechanism that facilitates pronounced phenotypic change. Observations suggest changes in the seasonality and host range of panzootic viruses, however, data on the role of reassortment in determining such features are limited. Using phylodynamic approaches, we describe the emergence of the panzootic lineage and using a novel global genotype classification system we describe the subsequent emergence and global structuring of genotypes generated by reassortment. Focusing on evolutionary dynamics in Europe, we show reassortment has produced high fitness genotypes with enhanced capacity for transmission and further we show such advantages can be host-dependent, contrasting successful generalist genotypes with a specialist lineage (EA-2022-BB) adapted to birds of the order Charadriiformes. Experimental investigation of NS1-mediated shutoff indicates this Charadriiformes-specialist does not inhibit host cellular gene expression and hamper the defences of more typical hosts such as water- and land-fowl. We attribute this primarily to variation at position 127 of the NS1 protein. Our results emphasise that reassortment has driven phenotypic change, affected viral fitness, and caused diversification of host specificity and seasonality. Such factors should be considered in studies that seek to identify drivers of HPAI spread and map spillover risk. Additionally, relaxation of host specialisation, ecological diversification, and potential endemicity in atypical host populations present new reassortment opportunities that could result in further novel phenotypes.

microbiology↗

bioRxiv2025

The Winners Take It All? Evolutionary Success ofH5Nx Reassortants in the 2020-2024 Panzootic

Avian influenza viruses undergo frequent genetic reassortment, which can coincide with phenotypic changes in transmission, pathogenicity, and host species niche. Since 2020, clade 2.3.4.4b H5 high pathogenicity avian influenza viruses (HPAIVs) have driven a global panzootic, causing mass mortality in wild birds, poultry, and, for the first time, repeated spillover infections in a variety of mammalian species. This worldwide resurgence of H5 HPAIV has coincided with a dramatic increase in the number of circulating reassortant strains; however, the scale, impact and drivers of these reassortants remain unclear. Here, we combined statistical and phylodynamic modelling to reconstruct the global evolutionary dynamics of H5Nx viruses across four epizootic seasons (2020-2024). We identified 209 genetically distinct reassortants, stratified into three transmission categories based on their phylogenetic and epidemiological profiles. Accounting for sampling depth and HPAIV incidence, we estimated that reassortants emerged most frequently from Asia, but major reassortants associated with increased host range, inter-seasonal persistence, and long-range dissemination, more frequently emerged from Europe. Altogether, reassortant emergence followed an episodic pattern in which most reassortants were transient, but 2% seeded large clusters of secondary reassortants soon after their own emergence. Statistical modelling revealed that reassortant success was strongly shaped by ecological factors, including sustained circulation in specific wild bird orders and detection across a wider range of host niches. Collectively, our findings uncover global reassortment dynamics in H5 HPAIVs and identify key virological and ecological drivers underpinning the emergence and spread of successful reassortants. These insights support the importance of enhanced surveillance to track evolution of H5 HPAIV and identify traits relevant for consideration in pandemic risk assessment.

evolutionary biology↗

bioRxiv2025

Synergy between HA cleavage site sequence and NA-mediated plasminogen recruitment as a virulence mechanism for low pathogenic avian influenza

An outbreak of H3N1 low pathogenic avian influenza virus (LPAIV) in Belgium in 2019 caused unexpected levels of mortality and morbidity in poultry. These viruses possess an NA polymorphism associated with plasminogen binding, as well as an unusual sequence around the HA cleavage site; accordingly, HA cleavage mediated by NA-driven plasminogen recruitment has been proposed to underly their systemic spread and pathogenicity. To test this, we established a reverse genetics system for A/chicken/Belgium/460/2019 and created single mutations in HA (K345R), and NA (S122N) that restored the viruses to normal consensus, as well as an HA/NA double mutant. Confirming previous work, trypsin-independent spread and HA cleavage of wild type Ck/Belgium was observed in the presence of fetal bovine serum containing plasminogen in vitro. Dose-dependent HA cleavage and trypsin-independent spread was also observed in the presence of purified chicken plasminogen. Compared to wild type virus, both HA cleavage and virus spread in vitro were reduced by the HA K345R mutation and further blocked by NA mutation S122N. Plasminogen-mediated HA cleavage was seen in a variety of avian cell lines and chicken organoids, excluding cell type-dependent effects. Furthermore, in ovo tests showed that mutant viruses unable to recruit plasminogen were less able to replicate systemically in chicken embryos. Bioinformatics analyses revealed other viruses which could potentially recruit plasminogen, including two independent outbreaks of H6N1 viruses, one of which we confirmed PLG-driven spread in vitro. We conclude that PLG-recruitment by NA is a general virulence mechanism of N1 LPAIVs Author summaryAvian influenza viruses (AIV) are divided into two broad categories - high or low pathogenicity - based on the sequence of their haemagglutinin (HA) and their lethality in chickens. The majority of AIV strains circulating in the wild are low pathogenicity both in waterfowl and when they spill over into domestic poultry. However, some low pathogenicity strains can cause severe disease in poultry despite not being classified as H5 or H7 high pathogenic AIVs with an HA polybasic protease cleavage site. A severe 2019 outbreak of an H3N1 strain has been suggested to result from the neuraminidase (NA) of the virus recruiting cellular plasminogen to proteolytically activate HA. Here, we tested this hypothesis by using reverse genetics to mutate the virus in a way predicted to block this. We found that indeed, the sequence of the NA at position 122 is the primary determinant of plasminogen-driven HA cleavage but that the unusual sequence at the HA cleavage site of the outbreak virus also contributes to pathogenicity. Furthermore, we show that N1 NA sequence can be used to identify other unexpectedly virulent strains of AIV. This work therefore adds to our ability to risk assess AIV strains from sequence-based surveillance.

microbiology↗

bioRxiv2025

Functional redundancy in Oxa1-family insertases supports robust membrane insertion of influenza A virus M2 viroporin

The biogenesis of thousands of highly diverse membrane proteins in humans is facilitated by an array of ER-resident membrane protein translocases. While some membrane proteins have a strict requirement for a specific insertion machinery, membrane proteins with short translocated domains may be able to access multiple pathways. Here, we quantify the functional importance of redundancy in membrane protein translocation during influenza A virus (IAV) infection by examining the biogenesis of the viroporin M2. Given the wide host and cellular tropism of IAV, the virus likely evolved mechanisms to leverage host translocation pathways efficiently. We demonstrate that although M2 utilizes the ER membrane protein complex (EMC), driven by signals encoded in its transmembrane and C-terminal domains, M2 maintains an approximately 50% membrane insertion rate in the absence of the EMC. This influences viral cell-to-cell transmission across different IAV strains, with a greater impact on those expressing lower levels of M2. We identify alternative translocation of M2 via Oxa1-family translocons independent of canonical targeting chaperones. These findings reveal how the exploitation of multiple redundant pathways can ensure robust IAV infection. SIGNIFICANCE STATEMENTIAV must rapidly replicate in diverse mammalian hosts, which requires efficient integration of viral proteins into host cell membranes. This study uncovers how the viral proton channel M2 utilizes multiple redundant protein insertion pathways, accessing EMC and alternative Oxa1-family translocases. Revealing these redundant strategies clarifies how cells triage membrane proteins, offering insights into both viral adaptation and host cell robustness.

cell biology↗

bioRxiv2025

Polymerase mutations underlie adaptation of H5N1 influenza virus to dairy cattle and other mammals.

In early 2024, an unprecedented outbreak of H5N1 high pathogenicity avian influenza was detected in dairy cattle in the USA1. As of mid-2025 the epidemic is ongoing, resulting in spillbacks into poultry, wild birds and other mammals including humans2. 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 mammalian species including humans and pigs. We find evidence of several mammalian adaptations gained early in the evolution of these viruses 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. Importantly, we show that ongoing evolution during 2024 and 2025 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, the continued circulation of H5N1 in dairy cattle not only allows virus adaption improving replicative ability in cattle, but also increases the risk of zoonotic spillover.

microbiology↗

bioRxiv2024

An emerging PB2-627 polymorphism increases the pandemic potential of avian influenza virus by breaking through ANP32 host restriction in mammalian and avian hosts

Alterations in the PB2-627 domain could substantially increase the risk of an avian influenza virus (AIV) pandemic. So far, a well-known mammalian mutation PB2-E627K has not been maintained in AIV in poultry, which limits the spread of AIVs from avian to humans. Here, we discovered a variant, PB2-627V, which combines the properties of avian-like PB2-627E and human-like PB2-627K, overcoming host restrictions and posing a risk for human pandemics. Specifically, by screening the global PB2 sequences, we discovered a new independent cluster with PB2-627V emerged in the 2010s, which is prevalent in various avian, mammalian, and human isolates of AIVs, including H9N2, H7N9, H3N8, 2.3.4.4b H5N1, and other subtypes. And, the increasing prevalence of PB2-627V in poultry is accompanied by a rise in human infection cases with this variant. Then we systematically assessed its host adaptation, fitness, and transmissibility across three subtypes of AIVs (H9N2, H7N9, and H3N8) in different host models, including avian and human cells, chickens, mice, and ferrets where infections naturally occur. We found that PB2-627V facilitates AIVs to efficiently infect and replicate in chickens and mice by utilizing both avian- and human-origin ANP32A proteins. Importantly, and like PB2-627K, PB2-627V promotes efficient transmission between ferrets through respiratory droplets. Deep sequencing in passaged chicken samples and transmitted ferret samples indicates that PB2-627V remains stable across the two distinct hosts and has a high potential for long-term prevalence in avian species. Therefore, the mutation has the ability to continue spreading among poultry and can also overcome the barrier between birds and humans, greatly enhancing the likelihood of AIVs infecting humans. Given the escalating global spread of AIVs, it is crucial to closely monitor influenza viruses carrying PB2-627V to prevent a pandemic.

microbiology↗

bioRxiv2023

Understanding off-target growth defects introduced to influenza A virus by synonymous recoding

CpG dinucleotides are under-represented in the genomes of most RNA viruses. Synonymously increasing CpG content of a range of RNA viruses reliably causes replication defects due to the recognition of CpG motifs in RNA by cellular Zinc-finger Antiviral Protein (ZAP). Prior to the discovery of ZAP as a CpG sensor, we described an engineered influenza A virus (IAV) enriched for CpGs in segment 5 that displays the expected replication defects. However, we report here that this CpG-high ( CpGH) mutant is not attenuated by ZAP. To understand this, we sought to uncover the alternative attenuation mechanism(s). IAV segment 5 encodes NP, a component of the viral RNA replication complex. Unexpectedly, while CpG enrichment resulted in depleted segment 5 transcript and NP protein abundance, this did not impair viral polymerase activity. A pair of nucleotide changes, introduced as compensatory changes to maintain base frequencies, were instead found to be responsible for the replication defect. These mutations resulted in the encoding of a stretch of eight consecutive adenosines (8A), a phenomenon not seen in natural IAV isolates. Sequencing experiments revealed evidence of viral polymerase slippage at this site, resulting in the production of aberrant peptides and type I interferon induction. When the nucleotides in either of these two positions were restored to wildtype sequence, no viral attenuation was seen, despite the 86 extra CpGs encoded by this virus. Conversely, when these two adenosines were introduced into wildtype virus (thereby introducing the 8A tract), viral attenuation, polymerase slippage, aberrant peptide production and type I interferon induction were apparent. That a single nucleotide change can offset the growth defects in a virus designed to have a formidable barrier to wild-type reversion highlights the importance of understanding the processes underlying viral attenuation. The lessons from this study will inform improved recoding designs in the future.

microbiology↗

bioRxiv2023

Novel technology to develop non-transmissible live influenza virus vaccines through alterations in M2/M42 ion channel expression

Continued global outbreaks of H5Nx highly pathogenic avian influenza viruses (HPAIV) have been reported in poultry since the emergence of the Asian Goose/Guangdong lineage of HPAIV H5N1 in 1996. Consequently, vaccines have been developed and employed to protect commercial and non-commercial poultry flocks. However, constant changes in virus immunogenetics makes vaccine candidates that prevent morbidity, mortality, reduce shedding, and prevent transmission a moving target. Here, we tested the effects of disrupting the mRNA splice sites and thus expression of either of the two isoforms of the viral ion channel M2 and M42 expressed by the H5N2 low pathogenic avian influenza virus (LPAIV) progenitor of the 1983 Pennsylvania HPAIV outbreak. Both G52C ({Delta}M2) and G145A ({Delta}M42) versions of the A/chicken/Pennsylvania/1/83 (Ck/Penn) virus replicated well in vitro and in ovo, but showed altered virion morphology, with the G145A virus exhibiting filamentous budding. The G52C and G145A viruses also infected chickens efficiently and stimulated robust immune responses; however, unlike the wild type virus, they did not transmit to naive-contact birds. In protection studies, vaccination of birds with G52C or G145A viruses protected 100% of birds from lethal challenge with homologous and distant heterologous H5 HPAIV strains from North American and Asian lineages and significantly reduced virus shedding compared to controls. Furthermore, the live virus vaccines decreased virus shedding 10,000-fold more than corresponding inactivated forms of the vaccine virus. Taken together, these studies demonstrate a strategy for developing a non-transmittable but highly protective live attenuated virus for AIV.

microbiology↗

bioRxiv2022

One for all - Human kidney Caki-1 cells are highly susceptible to infection with corona- and other respiratory viruses

In vitro investigations of host-pathogen interactions of viruses are reliant on suitable cell and tissue culture models. Results are only as good as the model they have been generated in. However, choosing cell models for in vitro work often depends on what is available in labs or what has previously been used. Despite the vast increase in coronavirus research activity over the past few years, researchers are still heavily reliant on: non-human cells, for example Vero E6, highly heterogeneous or not fully differentiated cells, such as Calu-3, or naturally unsusceptible cells requiring overexpression of receptors and other accessory factors, such as ACE2 and TMPRSS2. Complex cell models, such as primary cell-derived air-liquid interface epithelial models are highly representative of human tissues but are expensive and time-consuming to develop and maintain. They have limited suitability for large-scale and high-throughput analysis. Using tissue-specific expression pattern as a selection criteria, we identified human kidney cells as an ideal target for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and broader coronavirus infection. We show the use of the highly characterized human kidney cell line Caki-1 for infection with three human coronaviruses: Betacoronaviruses SARS-CoV-2 and Middle Eastern respiratory syndrome coronavirus (MERS-CoV) and Alphacoronavirus human coronavirus 229E (hCoV-229E). Caki-1 cells show equal or superior susceptibility to all three coronaviruses when compared to other commonly used cell lines for the cultivation of the respective virus. Antibody staining against SARS-CoV-2 N protein shows comparable replication rates. Using a panel of 21 antibodies in infected Caki-1 cells using immunocytochemistry shows the location of viral proteins during replication. In addition, Caki-1 cells were found to be susceptible to two other human respiratory viruses, influenza A virus and respiratory syncytial virus, making them an ideal model for cross-comparison of not only a broad range of coronaviruses but respiratory viruses in general. Author SummaryInvestigating how viruses interact with their host relies models used for laboratory research. The closer a model matches the host, the more conclusive results are. Complex cell systems based on primary epithelial or stem cells are the gold standard of in vitro research. However, they are expensive, time consuming, and laborious to establish. Therefore, cell lines remain the backbone of virus research. Despite vastly increased research into human coronaviruses following the COVID-19 pandemic, researchers continue to rely on suboptimal cell line models, for example ells of non-human origin like the VeroE6 African Green Monkey cell line. Using known expression patterns of the entry factors of the COVID-19 causative agent severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) we identified the Caki-1 cell line as a target for SARS-CoV-2. This cell line could be shown to be infectable with a wide range of coronaviruses including common cold virus hCoV-229E, epidemic virus MERS-CoV, and SARS-CoV-2 as well as other important respiratory viruses influenza A virus and respiratory syncytial virus. We could show that SARS-CoV-2 proteins can be stained for and localized in Caki-1 cells and the cells are competent of forming a cellular immune response. Together, this makes Caki-1 cells a unique tool for cross-virus comparison in one cell line.

microbiology↗

bioRxiv2022

Zoonotic avian influenza viruses evade human BTN3A3 restriction

Cross-species transmission of avian influenza A viruses (IAVs) into humans could represent the first step of a future pandemic1. Multiple factors limiting the spillover and adaptation of avian IAVs in humans have been identified, but they are not sufficient to explain which virus lineages are more likely to cross the species barrier1,2. Here, we identified human BTN3A33 (butyrophilin subfamily 3 member A3) as a potent inhibitor of avian but not human IAVs. We determined that BTN3A3 is constitutively expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts at the early stages of virus replication by inhibiting avian IAV vRNA transcription. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F, or rarely 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, several serotypes of avian IAVs that spilled over into humans in recent decades evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure4. Importantly, we identified more than 150 avian IAV lineages with a BTN3A3-resistant genotype. In conclusion, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

microbiology↗

bioRxiv2022

CpG enrichment as a vaccine strategy for influenza A virus

Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. Problematically, recoding typically hinders virus growth, but this may be rectified using CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), and so in principle, removing ZAP sensing from a virus propagation system will reverse attenuation of a CpG-enriched virus, enabling high titre yield of a vaccine virus. We tested this using a vaccine strain of influenza A virus (IAV) engineered for increased CpG content in genome segment 1. Virus attenuation was mediated by the short isoform of ZAP, correlated with the number of CpGs added, and was enacted via turnover of viral transcripts. The CpG-enriched virus was strongly attenuated in mice, yet conveyed protection from a potentially lethal challenge dose of wildtype virus. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Unexpectedly, in both MDCK cells and embryonated hens eggs that are used to propagate live attenuated influenza vaccines, the ZAP-sensitive virus was fully replication competent. Thus, ZAP- sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform to augment existing live attenuated vaccines. AUTHOR SUMMARYCpG dinucleotides are under-represented in vertebrate genomes, wherein cytosines in the CpG conformation are methylated to regulate transcription. Methylated cytosines are prone to deamination, resulting in TpG dinucleotides replacing CpGs. The resultant CpG suppression has provided a route by which vertebrate cells can recognise RNA from invading pathogens, using cellular Zinc-finger Antiviral Protein (ZAP) as a CpG sensor. Vertebrate-infecting RNA viruses also genomically suppress CpGs, and it is believed that this is an evolved trait to evade detection by ZAP. Here, we engineered an influenza A virus (IAV) with elevated CpG content and characterised how this impacts viral replication. CpG addition resulted in viral attenuation, mediated by ZAP activity. CpG suppression is conserved in dog and chicken genomes (relevant for live attenuated IAV vaccine propagation), and it is logical to predict that ZAP-mediated CpG sensing would also be conserved in these species. However, when we propagated ZAP-sensitive IAV in cognate culture systems, we saw no replication defect. This unexpected result raises questions about why viruses infecting these species suppress CpG in their genomes, and importantly delivers a new, tractable approach to augment rational live attenuated IAV vaccine design.

microbiology↗

bioRxiv2022

Engineering a Vaccine Platform using Rotavirus A to Express SARS-CoV-2 Spike Epitopes

Human rotavirus (RV) vaccines used worldwide have been developed using live attenuated platforms. The recent development of a reverse genetics system for RVs has delivered the possibility of engineering chimeric viruses expressing heterologous peptides from other virus species to generate polyvalent vaccines. We tested the feasibility of this using two approaches. Firstly, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian SA11 RV strain viral protein (VP) 4. Secondly, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C-terminus of non-structural protein (NSP) 3 of the bovine RF strain RV, with or without an intervening T2A peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RF strain. Except for the RBD mutant, all NSP3 mutants delivered endpoint titres and replication kinetics comparable to that of the WT virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants but only RBM mutant showed cross reactivity with SARS-CoV-2 RBD antibody. The tolerability of large peptide insertions in the NSP3 segment highlights the potential for this approach in the development of vaccine vectors targeting multiple enteric pathogens simultaneously. IMPORTANCEWe explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an exemplar. Small SARS-CoV-2 peptide insertion (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titre and replication, thus limiting its use as a potential vaccine expression platform. To test RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids. With a T2A-separated 193 amino acid tag on NSP3, there was little effect on the viral rescue efficiency, titre and replication. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. This is the first report describing epitope tagging of VP4, and of a reverse genetics system for the RF strain. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously.

microbiology↗