Genetic and biological characterisation of an avian-like H1N2 swine influenza virus generated by reassortment of circulating avian-like H1N1 and H3N2 subtypes in Denmark

Background: The influenza A virus subtypes H1N1, H1N2 and H3N2 are the most prevalent subtypes in swine. In 2003, a reassorted H1N2 swine influenza virus (SIV) subtype appeared and became prevalent in Denmark. In the present study, the reassortant H1N2 subtype was characterised genetically and the infection dynamics compared to an avian-like H1N1 virus by an experimental infection study. Methods: Sequence analyses were performed of the H1N2 virus. Two groups of pigs were inoculated with the reassortant H1N2 virus and an avian-like H1N1 virus, respectively, followed by inoculation with the opposite subtype four weeks later. Measurements of HI antibodies and acute phase proteins were performed. Nasal virus excretion and virus load in lungs were determined by real-time RT-PCR. Results: The phylogenetic analysis revealed that the reassorted H1N2 virus contained a European avian-like H1-gene and a European swine-like N2-gene, thus being genetically distinct from most H1N2 viruses circulating in Europe, but similar to viruses reported in 2009/2010 in Sweden and Italy. Sequence analyses of the internal genes revealed that the reassortment probably arose between circulating Danish avian-like H1N1 and H3N2 SIVs. Infected pigs developed cross-reactive antibodies, and increased levels of acute phase proteins after inoculations. Pigs inoculated with H1N2 exhibited nasal virus excretion for seven days, peaking day 1 after inoculation two days earlier than H1N1 infected pigs and at a six times higher level. The difference, however, was not statistically significant. Pigs euthanized on day 4 after inoculation, had a high virus load in all lung lobes. After the second inoculation, the nasal virus excretion was minimal. There were no clinical sign except elevated body temperature under the experimental conditions. Conclusions: The avian-like H1N2 subtype, which has been established in the Danish pig population at least since 2003, is a reassortant between circulating swine avian-like H1N1 and H3N2. The Danish H1N2 has an avian-like H1 and differs from most other reported H1N2 viruses in Europe and North America/Asia, which have H1-genes of human or classical-swine origin, respectively. The variant seems, however, also to be circulating in countries like Sweden and Italy. The infection dynamics of the reassorted avian-like H1N2 is similar to the older avian-like H1N1 subtype.