Conserved Expression and Functionality of Furin between Chickens and Ducks as an Activating Protease of Highly Pathogenic Avian Influenza Virus Hemagglutinins

Highly pathogenic avian influenza viruses (HPAIVs) typically emerge from low-pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes upon spillover from wild aquatic birds into poultry. The conversion from LPAIV to HPAIV is characterized by the acquisition of a multibasic cleavage site (MBCS) at the proteolytic cleavage site in the viral binding and fusion protein, hemagglutinin (HA), resulting in cleavage and activation of HA by ubiquitously expressed furin-like proteases. The ensuing HPAIVs disseminate systemically in gallinaceous poultry, are endotheliotropic, and cause hemorrhagic disease with high mortality. HPAIV infections in wild aquatic birds are generally milder, often asymptomatic, and generally not associated with systemic dissemination nor endotheliotropic. As MBCS cleavage by host proteases is the main virulence determinant of HPAIVs in poultry, we set out to determine whether cleavage of HPAIV HA by host proteases might influence the observed species-specific pathogenesis and tropism. Here, we sequenced, cloned, and characterized the expression and functionality of duck furin. The furin sequence was strongly conserved between chickens and ducks, and duck furin cleaved HPAIV and tetrabasic HA in an overexpression system, confirming its functionality. Furin was expressed ubiquitously and to similar extents in duck and chicken tissues, including in primary duck endothelial cells, which sustained multicycle replication of H5N1 HPAIV but not LPAIVs. In conclusion, differences in furin-like protease biology between wild aquatic birds and gallinaceous poultry are unlikely to largely determine the stark differences observed in species-specific pathogenesis of HPAIVs. IMPORTANCE HPAIV outbreaks are a global concern due to the health risks for poultry, wildlife, and humans and their major economic impact. The number of LPAIV-to-HPAIV conversions, which is associated with spillover from wild birds to poultry, has been increasing over recent decades. Furthermore, H5 HPAIVs from the A/goose/Guangdong/1/96 lineage have been circulating in migratory birds, causing increasingly frequent epizootics in poultry and wild birds. Milder symptoms in migratory birds allow for dispersion of HPAIVs over long distances, justifying the importance of understanding the pathogenesis of HPAIVs in wild birds. Here, we examined whether host proteases are a likely candidate to explain some differences in the degree of HPAIV systemic dissemination between avian species. This is the first report to show that furin function and expression is comparable between chickens and ducks, which renders the hypothesis unlikely that furin-like protease differences influence the HPAIV species-specific pathogenesis and tropism.