Differential outcomes of viral co-infections with high pathogenicity avian influenza A(H5N6) and SARS-CoV-2 in mammalian in vitro systems.

Co-infection with two highly pathogenic viruses is often expected to worsen disease severity. However, increasing evidence suggests that viral interactions can be antagonistic, with one virus suppressing the other. In this study, we used various mammalian in vitro models to investigate the outcomes of co-infections with two highly pathogenic viruses: recombinant avian influenza A(H5N6) (A/chicken/Jiangxi/02.05 YGYXG023-P/2015) and an early isolate of SARS-CoV-2 (England/2/2020). Despite the use of identical viral strains and standardized experimental conditions, co-infection outcomes varied substantially across the models. In interferon-deficient Vero cells, SARS-CoV-2 (CoV) strongly outcompeted recombinant high pathogenicity avian influenza A(H5N6) virus (IV), leading to near-complete suppression of IV replication. This effect was reversed when IV was given a head start prior to secondary infection with CoV. In Calu-3 lung epithelial cells, both viruses could replicate simultaneously, with limited interference, however IV showed a dominant effect during both sequential co-infections (pre-infection and secondary infection), significantly limiting CoV replication. In contrast, human nasal airway epithelial cells (hNAECs) cultured at the air-liquid interface (ALI) supported efficient co-replication of both viruses with minimal interference. Bioimaging of co-infected hNAECs revealed that both viruses were spatially segregated, with no evidence of co-infection within the same cells. These findings demonstrate that the IV/CoV interactions are highly context-dependent and can shift significantly even with identical viral strains.