Infection Dynamics of Swine Influenza Virus in a Danish Pig Herd Reveals Recurrent Infections with Different Variants of the H1N2 Swine Influenza A Virus Subtype

Abstract

Influenza A virus (IAV) in swine, so-called swine influenza A virus (swIAV), causes respiratory illness in pigs around the globe. In Danish pig herds, a H1N2 subtype named H1N2dk is one of the main circulating swIAV. In this cohort study, the infection dynamic of swIAV was evaluated in a Danish pig herd by sampling and PCR testing of pigs from two weeks of age until slaughter at 22 weeks of age. In addition, next generation sequencing (NGS) was used to identify and characterize the complete genome of swIAV circulating in the herd, and to examine the antigenic variability in the antigenic sites of the virus hemagglutinin (HA) and neuraminidase (NA) proteins. Overall, 76.6% of the pigs became PCR positive for swIAV during the study, with the highest prevalence at four weeks of age. Detailed analysis of the virus sequences obtained showed that the majority of mutations occurred at antigenic sites in the HA and NA proteins of the virus. At least two different H1N2 variants were found to be circulating in the herd; one H1N2 variant was circulating at the sow and nursery sites, while another H1N2 variant was circulating at the finisher site. Furthermore, it was demonstrated that individual pigs had recurrent swIAV infections with the two different H1N2 variants, but re-infection with the same H1N2 variant was also observed. Better understandings of the epidemiology, genetic and antigenic diversity of swIAV may help to design better health interventions for the prevention and control of swIAV infections in the herds.

Keywords: H1N2; antigenic diversity; phylogenetic analysis; pig herd; prevalence; swine influenza A virus (swIAV).

Figure 1

Phylogenetic analysis of the H1 gene segment. The nucleotide sequences were aligned and analysed using the maximum likelihood method in MEGA 7.0 [51], using the General Time Reversible (GTR+G+I) model [70] with a bootstrapping of 1000 replicates. The analysis included 34 H1 sequences of HA gene segments of different IAV H1Nx subtypes, of which 11 H1 gene sequences were obtained in this study (red taxon). The numbers at the nodes represent bootstrap values, and only bootstrap values at or above 60% are shown. Branch lengths are scaled according to the number of nucleotide substitutions per site.

Figure 2

Phylogenetic analysis of the N2 gene segment. The nucleotide sequences were aligned and analysed using the maximum likelihood method in MEGA 7.0 [51], using the General Time Reversible (GTR+G+I) model [70] with a bootstrapping of 1000 replicates. The analysis included 38 N2 sequences of which 11 N2 gene sequences were obtained in this study (red taxon). The numbers at the nodes represent bootstrap values, and only bootstrap values at or above 60% are shown. Branch lengths are scaled according to the number of nucleotide substitutions per site.

Figure 3

Phylogenetic analysis of the NEP-NS1 gene segment. The nucleotide sequences were aligned and analysed using the maximum likelihood method in MEGA 7.0 [51], using the Tamura three-parameter (T92+G) model [73] with a bootstrapping of 1000 replicates. The analysis included 30 sequences of the NEP-NS1 gene segment of IAV of which 11 NEP-NS1 gene sequences were obtained in this study (red taxon). The numbers at the nodes represent bootstrap values, and only bootstrap values at or above 60% are shown. Branch lengths are scaled according to the number of nucleotide substitutions per site.