Background: Although influenza A viruses (IAVs) are respiratory pathogens, infections may occur via nonrespiratory routes. However, the effects of different routes of exposure on the course of infection and disease are not well characterized.

Methods: This study assessed the pathogenicity and host responses in ferrets inoculated with the low pathogenic A/Anhui/1/2013 (H7N9) influenza A virus via different routes. Ferrets were inoculated through various routes, and viral replication in the respiratory tract was evaluated using nasal wash samples as well as respiratory and nonrespiratory tissues. Host immune responses were analyzed using peripheral blood collected from the virus-inoculated ferrets.

Results: Inoculation of ferrets with H7N9 via the intranasal (IN), intraocular (IO), or intraesophageal (IE) routes revealed that IN inoculation led to the greatest distribution of virus, whereas IO and IE inoculation led to more restricted viral spread. However, despite different routes, the respiratory tract remained the preferred site of IAV replication. IN- and IE-inoculation led to greater symptom severity compared to IO inoculation. Proinflammatory cytokine expression was highest in IN-inoculated ferrets and was not always associated with cumulative viral loads.

Conclusions: Overall, these results indicated that the route of inoculation can influence tissue distribution and disease outcomes, but the respiratory tract remains the primary site of viral replication.