Highly pathogenic avian influenza viruses (HPAIVs) such as the H5N1 subtype, can sporadically cross the species barrier from their natural host (aquatic waterfowl) into mammalian species including humans with often fatal outcomes. Mammalian HPAIV pathogenesis is associated with an overzealous innate immune response characterised by elevated levels of pro-inflammatory cytokines, referred to as a “cytokine storm.” Aberrant RNAs including defective viral genomes (DVGs) and mini viral RNAs (mvRNAs) are made by the viral polymerase in error during replication and cytokine induction is associated with their emergence in vivo. High viral replication of HPAIVs within myeloid immune cells could also trigger inappropriate levels of type I interferon (IFN) and pro-inflammatory cytokines.
Here we investigated the role that aberrant replication of HPAIVs has in the mammalian-associated cytokine storm. Firstly, we demonstrated that viruses containing the internal genes of H5N1 did not generate higher levels of vRNA in macrophages than those containing internal genes from an H1N1pdm09 virus. Next, by manipulating the levels of DVGs in H5N1 recombinant viruses, high DVG stocks displayed reduced viral replication but increased type I IFN and pro-inflammatory cytokines in various cell types. In BALB/c mice, DVG levels in the initial virus inoculum as well as their amplification kinetics during the infection, impacted pro-inflammatory cytokine levels, viral load, and pathogenesis. Furthermore, we showed that HPAIV polymerases generated aberrant RNAs de novo and limiting NP increased mvRNA synthesis. Finally, we showed that the introduction of human adapting amino acid residues into the H5N1 PB2 protein led to increased type I IFN in vitro but did not impact severity in mice. Overall, our results suggest that the timing and levels of aberrant RNAs generated during infection contribute to H5N1 pathogenesis. This knowledge could help guide better treatments and highlights the need to consider aberrant RNA replication products in future research.