Sadeyen, J.-R. — Page 2 | bioRxiv Science

bioRxiv2020

PA-X is an avian virulence factor in H9N2 avian influenza virus

Influenza A viruses encode several accessory proteins that have host- and strain-specific effects on virulence and replication. The accessory protein PA-X is expressed due to a ribosomal frameshift during translation of the PA gene. Depending on the particular combination of virus strain and host species, PA-X has been described as either acting to either reduce or increase virulence and/or virus replication. In this study, we set out to investigate the role PA-X plays in H9N2 avian influenza viruses, focussing particularly on the natural avian host, chickens. We found H9N2 PA-X induced robust host shutoff in both mammalian and avian cells and increased replication in mammalian, but not avian cells. We further showed that PA-X affected embryonic lethality in ovo and led to more rapid viral shedding and widespread organ dissemination in vivo in chickens. Overall, we conclude PA-X may act as a virulence factor for H9N2 viruses in chickens, allowing faster replication and wider organ tropism.

microbiology↗

bioRxiv2018

The molecular basis of antigenic variation among A(H9N2) avian influenza viruses

Avian influenza A(H9N2) viruses are an increasing threat to global poultry production and, through zoonotic infection, to human health where they are considered viruses with pandemic potential. Vaccination of poultry is a key element of disease control in endemic countries, but vaccine effectiveness is persistently challenged by the emergence of antigenic variants. Here we employed a combination of techniques to investigate the genetic basis of H9N2 antigenic variability and evaluate the role of different molecular mechanisms of immune escape. We systematically tested the influence of published H9N2 monoclonal antibody escape mutants on chicken antisera binding, determining that many have no significant effect. Substitutions introducing additional glycosylation sites were a notable exception, though these are relatively rare among circulating viruses. To identify substitutions responsible for antigenic variation in circulating viruses, we performed an integrated meta-analysis of all published H9 haemagglutinin sequences and antigenic data. We validated this statistical analysis experimentally and allocated several new residues to H9N2 antigenic sites providing molecular markers that will help explain vaccine breakdown in the field and inform vaccine selection decisions. We find evidence for the importance of alternative mechanisms of immune escape, beyond simple modulation of epitope structure, with substitutions increasing glycosylation or receptor-binding avidity exhibiting the largest impacts on chicken antisera binding. Of these, meta-analysis indicates avidity regulation to be more relevant to the evolution of circulating viruses, suggesting that a specific focus on avidity regulation is required to fully understand the molecular basis of immune escape by influenza, and potentially other viruses.

microbiology↗