Species-specific emergence of H7 highly pathogenic avian influenza virus is driven by intrahost selection differences between chickens and ducks

Highly pathogenic avian influenza viruses (HPAIVs) cause severe hemorrhagic disease in terrestrial poultry and are a threat to the poultry industry, wild life, and human health. HPAIVs arise from low pathogenic avian influenza viruses (LPAIVs), which circulate in wild aquatic birds. HPAIV emergence is thought to occur in poultry and not wild aquatic birds, but the reason for this species-restriction is not known. We hypothesized that, due to species-specific tropism and replication, intrahost HPAIV selection is favored in poultry and disfavored in wild aquatic birds. We tested this hypothesis by co-inoculating chickens, representative of poultry, and ducks, representative of wild aquatic birds, with a mixture of H7N7 HPAIV and LPAIV, mimicking HPAIV emergence in an experimental setting. Virus selection was monitored in swabs and tissues by RT-qPCR and immunostaining of differential N-terminal epitope tags that were added to the hemagglutinin protein. HPAIV was selected in four of six co-inoculated chickens, whereas LPAIV remained the major population in co-inoculated ducks on the long-term, despite detection of infectious HPAIV in tissues at early time points. Collectively, our data support the hypothesis that HPAIVs are more likely to be selected at the intrahost level in poultry than in wild aquatic birds and point towards species-specific differences in HPAIV and LPAIV tropism and replication levels as possible explanations. Highly pathogenic avian influenza viruses (HPAIVs) cause severe disease in poultry with mortality rates reaching 100% and, therefore, pose a large burden on the poultry industry. Additionally, some HPAIVs have spilled back from poultry into wild bird populations, increasing their geographic spread. HPAIVs arise from low pathogenic avian influenza viruses (LPAIVs), which circulate in wild aquatic birds and occasionally spillover into poultry. LPAIV to HPAIV conversion is associated with terrestrial poultry species, but the reasons underlying this species-restriction are unknown. The second step of HPAIV emergence, following HPAIV genesis, constitutes of the intrahost selection of the HPAIV from the large pool of replicating LPAIVs. Here, we investigated whether the intrahost selection efficiency differs between chickens and ducks, models for poultry and wild aquatic birds respectively, by co-inoculating them with HPAIV and LPAIV. Tagged viruses were utilized to monitor LPAIV and HPAIV frequencies at both the RNA and protein level. The HPAIV was selected in a majority of the chickens, demonstrated by the development of canonical HPAI disease and infectious HPAIV shedding, whereas all ducks solely shed infectious LPAIV. These results confirm that intrahost selection of HPAIVs is species-specific, which likely contributes to the restriction of HPAIV-emergence to poultry populations.