Dynamics of interactions between three major respiratory pathogens in reconstituted human epithelium

Respiratory viral coinfections pose a substantial global health burden, yet the underlying virus-virus interactions remain incompletely understood. Here, we systematically examined the interplay among influenza A virus (IAV), SARS-CoV-2, and respiratory syncytial virus (RSV) using a reconstituted human airway epithelium model. We monitored viral replication dynamics and host transcriptional responses under both simultaneous and sequential infection conditions. Our results reveal complex, asymmetric interactions strongly influenced by infection timing and sequence. IAV exerted a pronounced inhibitory effect on SARS-CoV-2, mediated mainly by a robust type III interferon response. However, transient early enhancement of SARS-CoV-2 by IAV and subsequent bidirectional inhibition were also observed. Transcriptomic profiling identified coinfection-specific gene expression signatures enriched for metabolic and cell death pathways. In IAV-RSV coinfections, IAV generally suppressed RSV replication; strikingly, prior RSV infection significantly enhanced subsequent IAV replication, potentially through RSV-induced cellular remodeling or syncytia formation. Overall, host responses to coinfection were integrated and non-additive, with distinct transcriptional programs suggesting activation of unique cellular pathways beyond canonical antiviral responses. Together, these findings highlight the pivotal role of innate immunity and the order of infection in determining the outcome of respiratory viral coinfections, providing mechanistic insights with implications for clinical management and epidemic modeling. Author SummaryRespiratory viruses often circulate simultaneously; however, their interactions within the same host remain poorly understood. We used a reconstituted human airway epithelium model to examine coinfections involving influenza A virus (IAV), SARS-CoV-2, and respiratory syncytial virus (RSV). Our findings reveal that these viruses do not simply compete or coexist: their interactions are dynamic, asymmetric, and highly dependent on the order in which infections occur. IAV generally suppresses SARS-CoV-2 replication through a strong type III interferon response, although brief early enhancement and later mutual inhibition can occur. Transcriptomic profiling reveals that coinfection triggers a complex array of host responses, including alterations in metabolic and cell-death pathways. In IAV/RSV coinfections, IAV consistently inhibits RSV; however, prior RSV infection markedly boosts subsequent IAV replication--likely due to RSV-induced cellular remodeling, such as syncytia formation. These transcriptional signatures point to altered cell adhesion and motility as key features of such interactions. Overall, our results demonstrate that innate immunity and the sequence of infection shape coinfection outcomes in non-additive ways, driving distinct cellular responses beyond classical antiviral pathways. Understanding these mechanisms is essential for improving clinical management and guiding the development of targeted antiviral strategies.