Influenza A(H1N1)pdm09 virus infection in swine : virus characterization and NK cell responses to infection

Influenza viruses balance on the interface between animals and humans, and swine represents a possible intermediate host for human viruses. It is therefore of great importance for animal welfare and to the public health to monitor the viral reservoir in swine. To better understand the mechanisms deciding clinical outcome, one also needs to study the host immune responses. Natural killer (NK) cells in general and the NKp46 receptor in particular, have been assigned a special role in influenza A virus infections. Little is known about NK cells during influenza A virus infections in swine. From the first detection of influenza A virus in Norwegian swine in 2009 until 2013, viral hemagglutinin (HA) genes were sequenced and compared to HA sequences from human viruses for the same period (paper 1). The formation of a distinct phylogenetic group was detected in late 2011, showing that the influenza A(H1N1)pdm09 virus (pH1N1) lineage had evolved in Norwegian swine, indicating that these viruses have established themselves in the Norwegian swine population. Two pH1N1 virus infection experiments were performed in swine. Lymphocytes were isolated from blood and lung and analyzed by flow cytometry and lung tissue sections were stained by immunofluorescence markers. Decreased numbers of NKp46+ NK cells were found in the blood of infected animals, while increased numbers were found in the lung (paper 2). Furthermore, flow cytometric analysis showed the same pattern for CD3+NKp46+ cells (paper 3), indicating a recruitment of cells expressing the NKp46 receptor to the lungs during pH1N1 virus infections in swine. Increased amounts of mRNA encoding tumor necrosis factor (TNF) were found in isolated lung lymphocytes by real-time RT-PCR (paper 2). To further study porcine NK cell responses to pH1N1 viruses, NKp46+ cells were isolated from blood and spleen and kept in proliferative cultures for 10 days. Subsequently, cells were stimulated with pH1N1 virus in vitro and analyzed by flow cytometry. Cultured NK cells degranulated and started the production of TNF upon exposure to the virus (paper 4). Taken together, these results indicate that NK cells migrate from blood into the lungs following pH1N1 virus infection, and that they participate in viral clearance by killing virus infected cells and by producing TNF