Restriction of influenza A virus replication via serine protease targeting in chicken cells and organ cultures

Influenza A viruses represent a significant threat to human health and animal welfare. Viral hemagglutinin cleavage by host proteases is essential for viral infectivity. The transmembrane serine protease 2 ( TMPRSS2 ) is responsible for the posttranslational modification of the viral surface protein of low pathogenic avian Influenza viruses (LPAIVs), representing a promising target for the restriction of viral replication. This study used gene targeting and chemical inhibition of TMPRSS2 -related proteases to evaluate the suitability of chicken cells and organ cultures for investigating host proteases. We generated TMPRSS2 knockout (KO) chicken DF-1 cells and tested their susceptibility to H1N1. We implemented tracheal organ cultures (TOCs) and lung cultures (LCs) to assess the efficacy of Camostat mesylate (CAM) during infection with H9N2 and H6N8. Both methods, genetic modification and chemical inhibition, led to significantly lower virus replication compared to the control groups. Furthermore, chemical inhibition of host proteases prolonged the ciliary activity in TOCs and the survival of LCs upon virus infection. Our data demonstrate the importance of TMPRSS2 -related proteases as a promising target for limiting avian influenza infections and reveal the usefulness of TOCs and LCs for evaluating therapeutics. • CRISPR/Cas9 targeting of the chicken TMPRSS2 in DF-1 cells limited the replication of influenza A viruses. • Treatment with Camostat mesylate efficiently limited the replication of avian influenza viruses in organ cultures. • Tracheal and lung cultures are suitable for evaluating the efficacy of protease inhibitors in birds.