Impact of variation in equine influenza A virus pathogenicity determinants on vaccine effectiveness and interspecies transmission

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Influenza A viruses (IAV) are believed to have circulated in horses for centuries and to have crossed species barriers to infect humans in the past. In 2005, the first evidence for transmission of equine influenza A virus (eqIAV) to dogs was reported; the virus subsequently became endemic in the USA. We retrospectively demonstrated that eqIAV had also jumped from horses to foxhounds in the UK in 2002, but did not become established. Experimental infection of ponies with an eqIAV strain isolated in 2003 confirmed the more severe clinical signs seen (including prolonged coughing) than with earlier isolates. This was associated with greater induction of pro-inflammatory cytokines. Genome sequencing of the 2003 strain revealed a truncation in the viral NS1 protein, which modulates the host cell cytokine response. Using reverse genetics to switch the NS gene segment, we confirmed that the truncated NS1 induced a greater cytokine response in infected cells. This suggests that the species jump occurred due to emergence of an eqIAV strain with increased pathogenicity. Interestingly, the NS1 truncation was lost on adaptation to dogs. Vaccines against eqIAV have been available since the late 1960s and vaccination of UK racehorses has been compulsory since 1982. There is a process for updating vaccine strains but there continue to be periodic major epidemics of equine influenza - these have occurred in 1963, 1979, 1989, 2003 and 2019. These major epidemics are usually associated with some deaths among unvaccinated horses. Variation in the haemagglutinin surface glycoprotein as a cause of vaccine breakdown has been extensively studied. The aim of this project is to investigate the role of variation in viral proteins that determine the pathogenicity of IAV in vaccine breakdown and interspecies transmission. Reverse genetics is a technique where a recombinant influenza virus can be generated by transfecting eight plasmids each encoding one of the eight viral gene segments into mammalian cells in vitro. This allows specific changes between viruses to be studied individually, for example by swapping only the gene segment encoding NS1 on an 'isogenic' background. This technique will be complemented by bioinformatics analysis to compare the genes that determine the pathogenicity of IAV in epidemic eqIAV strains with inter-epidemic strains. The ability of viruses in which genes have been swapped or mutated to replicate and induce or resist pro-inflammatory cytokine responses will be tested in cell culture. The use of established techniques and involvement of collaborators with complementary skills should ensure any challenges can be addressed and the appropriate risk assessment for this work is already in place.Identification of patterns associated with the emergence of an epidemic strain could improve the ability of the World Organization for Animal Health (OIE) Expert Surveillance Panel to pre-empt the next equine influenza epidemic.