Aberrant RNA replication products of highly pathogenic avian influenza viruses and its impact in the mammalian associated cytokine storm

Highly pathogenic avian influenza viruses (HPAIVs), can sporadically cross the species barrier and cause zoonotic infections in mammalian hosts (including humans), with often fatal consequences. Severe disease has been associated with an overexuberant host innate immune response known as hypercytokinema (cytokine storm) where excessive levels of pro-inflammatory cytokines are produced. Previous work in our laboratory shows that high levels of viral replication by HPAIV in murine myeloid immune cells correlated with high cytokine levels and mapped this phenotype to the polymerase genes. Innate sensing of IAV is performed by the cytoplasmic helicase RIG-I, which recognises blunt ended double stranded RNA with 5’-triphosphate extremities, a pattern present in the viral genome. Defective viral genomes (DVGs) can be produced by aberrant RNA replication and these are also recognised by RIG-I and could play a role in hypercytokinemia. Our studies aim to probe for the presence of DVGs in influenza infected cells in vitro, and in lung samples from infected mice in vivo as well as in murine immune cells ex vivo, using RT-PCR and sequencing. We will establish whether DVGs are correlated to pathogenicity and high pro-inflammatory cytokine levels. Preliminary data utilising minigenome assays show that the polymerase genes from an HPAIV H5N1 strain do generate DVGs, whereas DVGs could not be detected from a H3N2 seasonal polymerase. Ultimately, we aim to identify mutations within the polymerase genes that contribute to virulence and by using reverse genetics, create mutant viruses that test the hypothesis that aberrant polymerase activity drives hypercytokinemia.