Aberrant RNA replication of highly pathogenic avian influenza viruses and it's impact in the mammalian-associated cytokine storm

Highly pathogenic avian influenza viruses (HPAIVs), can sporadically cross the species barrier from their natural host (aquatic waterfowl) into mammalian species. Such zoonotic infections in humans are associated with high fatality rates and coupled with their potential to adapt for airborne transmissibility, pose a serious public health threat. Four factors have been identified in mammalian HPAIV pathogenesis; high viral replication, virus dissemination, diverse tissue tropism and a dysregulated host innate immune response. The latter is characterised by the release of type I interferons (IFN) and elevated levels of pro-inflammatory cytokines resulting in extensive tissue damage and is referred to as a "cytokine storm" or hypercytokinemia.

How HPAIV infections can cause hypercytokinemia in mammalian hosts are still undefined. Innate sensing of influenza A virus (IAV) is performed primarily by the helicase RIG-I, which binds to double stranded RNA with 5'-triphosphate extremities; a pattern found in the IAV viral genome. Once RIG-I is bound, signalling cascades are activated which regulate transcription factors necessary to produce type I IFN and pro-inflammatory cytokines. Errors made by the viral polymerase during RNA replication can generate defective viral genomes (DVGs) and these are potent RIG-I ligands. Recently a class of DVGs termed mini viral RNAs (mvRNAs) were shown to be made by HPAIV polymerases and certain avian specific mutations within the polymerase genes conferred a higher propensity for their formation within mammalian cells. HPAIVs are also able to replicate within myeloid immune cells. These cells play a key role in orchestrating the immune response and it has been hypothesised that high viral replication within them could contribute to hypercytokinemia by triggering inappropriate levels of type I IFN and pro-inflammatory cytokines.


In this study we aim to investigate the role that aberrant replication of HPAIVs has in the mammalian-associated cytokine storm. Thus far we have detected immunostimulatory DVGs generated de novo by an HPAIV H5N1 polymerase in vitro by utilising minigenome assays. We have also detected DVGs in lung homogenates from mice infected with the virus containing the internal genes (including the polymerase genes) from the H5N1 subtype, and plan to establish whether DVGs are correlated to pathogenicity and high pro-inflammatory cytokine levels in vivo. As mammalian- adapted IAVs do not cause hypercytokinemia, we have begun to create a panel of mutant polymerases and corresponding recombinant viruses, which incorporate mammalian adaptive substitutions within the polymerase genes of the H5N1 subtype; the aim is to identify key residues that drive DVG formation and/or hypercytokinemia.

We are currently in the process of establishing the use of ex vivo murine bone marrow derived immune cells for analysis of replication levels by viruses containing the H5N1, mammalian-adapted or mutated (recombinant) internal genes. Cytokine and IFN levels will be analysed to correlate replication levels to pathogenicity. DVG levels will also be investigated to clarify whether this correlates with cytokine levels within these cells. Ultimately this study wishes to enhance our understanding of the molecular mechanisms driving hypercytokinemia within mammalian hosts following infection with HPAIV.