Analysis of mucosal innate immune responses to human and swine adapted influenza A viruses using swab samples

Background: The mucosal surface of the respiratory tract function as the first line of defence against respiratory pathogens such as influenza A virus (IAV). Upon infection of respiratory epithelial cells, the local cytokine milieu change from a steady to an antiviral state within hours after infection. The innate antiviral immune response to IAV infection involves activation of the viral pattern recognition receptors (PRRs) and type I and III interferons (IFNs), further stimulating a wide range of interferon-stimulated genes (ISGs), that will protect the infected cell and neighbouring cells from viral infection and spread. Even though more than 90% of all infections are assumed to be recognized and controlled by the innate immune system, local viral recognition and immune response in the nasal mucosa is poorly understood.
Methods: Groups of 12 pigs were inoculated with 107 TCID50/ml of swine- (swH1N1pdm09) or human-adapted (huH1N1pdm09) influenza virus strains. Nasal mucosal swab samples were collected before viral infection and at days 1, 2, 3, 4, 7, 10, and 14 after inoculation. RNA was isolated from nasal swab samples, converted into cDNA, and analyzed by microfluidic qPCR (Biomark, Fludigm) targeting several PRRs, IFNs, ISGs, and mucins.
Results: Type I and type III IFNs, as well as several ISGs, such as OAS1, MX1, IFIT1, and ISG20, were highly upregulated after infection with fold changes ranging between 2 and 59. Peak expression varied between the swine- and human-adapted strains. Expression of seven different membrane-bound and secreted mucins were investigated. The excreted mucin MUC5AC was downregulated at several time points after infection. This regulation was most significant after infection with the swine-adapted strain. More innate immune factors found to be significantly regulated upon infection in swab samples were likewise regulated in nasal mucosal tissue of the same pigs at necropsy at day 3.
Conclusions: Non-invasive and cost-efficient swab sampling was successfully used to compare the innate mucosal immune response to a human-adapted and a swine-adapted strain of the influenza A H1N1pdm09 virus in pigs. Host factors centrally involved in early orchestration of the antiviral immune response were regulated in the nasal mucosa after infection. Expression levels and dynamics after infection differed between the two strains and might be linked to mechanisms important for host tropism and thereby zoonotic transmission.