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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

Ramona Trebbien1*, Karoline Bragstad2, Lars Erik Larsen1, Jens Nielsen3, Anette Bøtner3, Peter MH Heegaard1, Anders Fomsgaard2, Birgitte Viuff4 and Charlotte Kristiane Hjulsager1

Author Affiliations

1 National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, Frederiksberg C, DK-1870, Denmark

2 Statens Serum Institut, Artillerivej 5, Copenhagen S, DK-2300, Denmark

3 National Veterinary Institute, Technical University of Denmark, Lindholm Ø, Kalvehave, DK-4771, Denmark

4 Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Dyrlægevej 88, Frederiksberg C, 1870, Denmark

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Virology Journal 2013, 10:290  doi:10.1186/1743-422X-10-290

Published: 18 September 2013

Abstract

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.

Keywords:
Influenza virus; Swine; H1N2 subtype; Reassortment; Phylogeny; Infection