Viral lineage and mode of exposure modulate within-host spatial dynamics of influenza A viruses in a guinea pig model

The upper and lower respiratory tracts (URT and LRT) present distinct environments for influenza A virus (IAV) replication. Their differential features have major implications for viral evolutionary dynamics, transmission potential, and pathogenesis. To investigate the implications of differential viral replication in the URT and LRT, we assessed dispersal of IAVs throughout the guinea pig respiratory system. Guinea pigs were inoculated intranasally with a 300 μL volume to deliver inoculum to both the URT and LRT. Two strains were used to represent the circulating seasonal IAV lineages: influenza A/TX/50/2012 (H3N2) and influenza A/CA/07/2009 (H1N1). For the H1N1 virus, a genetically diverse barcode library enabled high-resolution tracking of viral dispersal. Infectious virus was consistently detected in the URT for both strains; however, only the H1N1 virus was detected in the LRT. To determine whether replication of the H1N1 virus in the LRT extends to other routes of infection, virus distribution was evaluated following infection via aerosol exposure or transmission. Infectious virus in lung homogenates was observed in both cases, confirming the LRT tropism of the H1N1 virus. Sequencing genetic barcodes revealed that diversity was largely maintained in nasal samples and trachea but was reduced upon dispersal to the lungs. This contraction of diversity was associated with increased distance and branching from the major airways, suggesting long-distance dispersal through the respiratory tract imposes within-host population bottlenecks. Together, these findings highlight how the distinct environments of the URT and LRT shape within-host IAV population dynamics.