Mutation of Influenza A Virus PA-X Decreases Pathogenicity in Chicken Embryos and Can Increase the Yield of Reassortant Candidate Vaccine Viruses.

Saira Hussain,Philippa M Beard,Othmar G Engelhardt,Brett W Jagger,Paul Digard,Lonneke Vervelde,Matthew L Turnbull,Helen M Wise,Jeffery K Taubenberger,Kristina Kovacikova

doi:10.1128/jvi.01551-18

Abstract

The PA-X protein of influenza A virus has roles in host cell shutoff and viral pathogenesis. While most strains are predicted to encode PA-X, strain-dependent variations in activity have been noted. We found that PA-X protein from the A/PR/8/34 (PR8) strain had significantly lower repressive activity against cellular gene expression than PA-X proteins from the avian strains A/turkey/England/50-92/91 (H5N1) (T/E) and A/chicken/Rostock/34 (H7N1). Loss of normal PA-X expression, either by mutation of the frameshift site or by truncating the X open reading frame (ORF), had little effect on the infectious virus titer of PR8 or PR8 7:1 reassortants with T/E segment 3 grown in embryonated hens' eggs. However, in both virus backgrounds, mutation of PA-X led to decreased embryo mortality and lower overall pathology, effects that were more pronounced in the PR8 strain than in the T/E reassortant, despite the low shutoff activity of the PR8 PA-X. Purified PA-X mutant virus particles displayed an increased ratio of hemagglutinin (HA) to nucleoprotein (NP) and M1 compared to values for their wild-type (WT) counterparts, suggesting altered virion composition. When the PA-X gene was mutated in the background of poorly growing PR8 6:2 vaccine reassortant analogues containing the HA and neuraminidase (NA) segments from H1N1 2009 pandemic viruses or from an avian H7N3 strain, HA yield increased up to 2-fold. This suggests that the PR8 PA-X protein may harbor a function unrelated to host cell shutoff and that disruption of the PA-X gene has the potential to improve the HA yield of vaccine viruses.IMPORTANCE Influenza A virus is a widespread pathogen that affects both humans and a variety of animal species, causing regular epidemics and sporadic pandemics, with major public health and economic consequences. A better understanding of virus biology is therefore important. The primary control measure is vaccination, which for humans mostly relies on antigens produced in eggs from PR8-based viruses bearing the glycoprotein genes of interest. However, not all reassortants replicate well enough to supply sufficient virus antigen for demand. The significance of our research lies in identifying that mutation of the PA-X gene in the PR8 strain of virus can improve antigen yield, potentially by decreasing the pathogenicity of the virus in embryonated eggs.

Highlights

IMPORTANCE Influenza A virus is a widespread pathogen that affects both humans and a variety of animal species, causing regular epidemics and sporadic pandemics, with major public health and economic consequences
Recombinant influenza viruses are made by reverse genetics (RG) [7,8,9], which relies on the transfection of cells with plasmids engineered to express both viral genomic RNA and proteins from each of the eight segments and, to initiate virus production; the resultant virus is subsequently amplified in eggs
Here, we show that ablating expression of PA-X resulted in reduced pathogenicity in the chicken embryo model despite the PR8 PA-X protein having relatively low host cell shutoff activity compared to that of PA-X from other Influenza A viruses (IAV) strains

Summary

Introduction

IMPORTANCE Influenza A virus is a widespread pathogen that affects both humans and a variety of animal species, causing regular epidemics and sporadic pandemics, with major public health and economic consequences. Seasonal CVVs are widely produced by classical reassortment This process involves coinfecting embryonated hens’ eggs with the vaccine virus along with a high-yielding donor virus adapted to growth in eggs (most commonly the A/Puerto Rico/8/34 strain, or PR8). Recombinant influenza viruses are made by reverse genetics (RG) [7,8,9], which relies on the transfection of cells with plasmids engineered to express both viral genomic RNA and proteins from each of the eight segments and, to initiate virus production; the resultant virus is subsequently amplified in eggs. RG is the only viable method for production of CVVs for potentially pandemic highly pathogenic avian influenza viruses since it allows removal of genetic determinants of high pathogenicity in the virus genome as vaccines are manufactured in biosafety level 2 laboratories. There is a clear need for new reagents and methods for IAV production, for a response to a pandemic

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