Sharp, C. P. | Search bioRxiv

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↗

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↗