US feral swine were exposed to both avian and swine influenza A viruses

39 Influenza A viruses (IAVs) in swine can cause sporadic infections and pandemic outbreaks among 40 humans, but how avian IAV emerges in swine is still unclear. Unlike domestic swine, feral swine are 41 free ranging and have many opportunities for IAV exposure through contacts with various habitats and 42 animals, including migratory waterfowl, a natural reservoir for IAVs. During 2010–2013, 8,239 serum 43 samples were collected from feral swine across 35 US states and tested against 45 contemporary 44 antigenic variants of avian, swine, and human IAVs; of these, 406 (4.9%) samples were IAV-antibody 45 positive. Among 294 serum samples selected for antigenic characterization, 271 cross-reacted with ≥1 46 testing virus whereas the other 23 did not cross-react with any testing virus. Of the 271 IAV-positive 47 samples, 236 cross-reacted with swine IAVs, 1 with avian IAVs, and 16 with avian and swine IAVs, 48 indicating that feral swine were exposed to both swine and avian IAVs but predominantly to swine 49 IAVs. Our findings suggest that feral swine could potentially be infected with both avian and swine 50 IAVs, generating novel IAVs by hosting and reassorting IAVs from wild birds and domestic swine and 51 facilitating adaptation of avian IAVs to other hosts, including humans, before their spillover. Continued 52 surveillance to monitor the distribution and antigenic diversities of IAVs in feral swine is necessary to 53 increase our understanding of the natural history of IAVs. 54 individual pens (2 per pen), were intranasally inoculated with a 10 6 50% tissue culture infectious dose (TCID 50 ) of A/mallard/Wisconsin/A00661712/2009 (H3N2). Nasal wash samples were collected from all eight feral swine daily from 1-10 days post infection (DPI) and titrated in SPF embryotic eggs; and serum was collected on 0, 7, 14, and 21 DPI to determine seroconversion. To detect pathogenesis of virus to swine, two infected and one control swine were necropsied at 5 and 7 DPI, respectively; turbinate, trachea and lung of each feral swine were collected and virus titer in these tissues were detected in SPF eggs. For H6N2 virus, four feral swine were intranasally inoculated with a 10 6 TCID 50 of influenza A/mallard/Ohio/648/2002 (H6N2) in a 1-mL volume. Serum was collected on 0, 5, 7, and 21 DPI to determine seroconversion. The animals used as the negative control were inoculcated with 186 sterile PBS. Swine were monitored daily for subjective signs of influenza infection (e.g., lethargy, nasal 187 discharge, coughing, and dyspnea) and objective signs (e.g., body temperature) until 14 DPI. Data analyses. To understand the risk factors (i.e. seasonality and host factors) affecting IAV seroprevalence in feral swine, Chi-square tests were used to assess the differences of IAV seroprevalence between sex, age group, and month, year, and state of sample collection. We developed a multilevel multivariable logistic regression model to test potential risk factors associated with IAV seroprevalence in feral swine, focusing on the population sizes of domestic swine and poultry. The logistic regression model was developed using generalized estimation equations with binomial distribution and logit link function and accounted for clustering of pigs samples on the same date and location. Variables were manually selected if they contributed significance to the likelihood ratio statistic for Type 3