Elucidation of virulence determinants of highly pathogenic non-H5, non-H7 avian influenza viruses

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The 16 hemagglutinin (HA) subtypes of avian influenza viruses (AIV) in birds are classified into low pathogenic (LP) or highly pathogenic (HP). Up to now, HP avian influenza (fowl plague) was only caused by viruses specifying subtypes H5 and H7 with a polybasic cleavage site (CS) within the HA protein. Interestingly, there are few non-H5/H7 strains complying with the official definition of HPAIV. Virulence markers of those viruses are not known to date but can be investigated using reverse genetics systems for AIV that have been established in our laboratory. A unique H4N2 virus with a polybasic CS at the HA was isolated from quails in 2012. However, the virus displayed low virulence in chickens and did not grow in cell culture without the addition of exogenous trypsin. The potential of H4N2 to shift to an HP phenotype was investigated (1) after serial passages in embryonated chicken eggs or chickens, (2) by increasing the number of basic amino acids at the HA CS, (3) removal of high conserved glycosylation sites close to the CS and (4) by reassortment with HPAI H5/H7 viruses. Moreover, mortalities in harbor seals in Europe in 2014 was attributed to H10N7 virus, which was closely related to AIV in wild birds. Several genetic markers were observed in the seal-H10N7 virus compared to H10Nx from birds. In this proposal, we will study the impact of these mutations on virulence, pathogenesis and adaptation in vivo and in vitro. In summary, the low virulent, recent H4N2 virus is the first naturally occurring non-H5/H7 AIV with polybasic HA CS. Mutations and gene segments conferred high virulence of this virus in chickens resembling H5/H7 HPAIV were determined, however, their biological functions remain to be studied. Likewise, the impact of mutations peculiar to seal-H10N7 virus on adaptation in mammals will be investigated.