Molecular characterization of H3N2 influenza A viruses isolated from Ontario swine in 2011 and 2012

Abstract

Background: Data about molecular diversity of commonly circulating type A influenza viruses in Ontario swine are scarce. Yet, this information is essential for surveillance of animal and public health, vaccine updates, and for understanding virus evolution and its large-scale spread.

Methods: The study population consisted of 21 swine herds with clinical problems due to respiratory disease. Nasal swabs from individual pigs were collected and tested by virus isolation in MDCK cells and by rtRT-PCR. All eight segments of 10 H3N2 viruses were sequenced using high-throughput sequencing and molecularly characterized.

Results: Within-herd prevalence ranged between 2 and 100%. Structurally, Ontario H3N2 viruses could be classified into three different groups. Group 1 was the most similar to the original trH3N2 virus from 2005. Group 2 was the most similar to the Ontario turkey H3N2 isolates with PB1 and NS genes originating from trH3N2 virus and M, PB2, PA and NP genes originating from the A(H1N1)pdm09 virus. All Group 3 internal genes were genetically related to A(H1N1)pdm09. Analysis of antigenic sites of HA1 showed that Group 1 had 8 aa changes within 4 antigenic sites, A(1), B(3), C(2) and E(2). The Group 2 viruses had 8 aa changes within 3 antigenic sites A(3), B(3) and C(2), while Group 3 viruses had 4 aa changes within 3 antigenic sites, B(1), D(1) and E(2), when compared to the cluster IV H3N2 virus [A/swine/Ontario/33853/2005/(H3N2)].

Conclusions: The characterization of the Ontario H3N2 viruses clearly indicates reassortment of gene segments between the North American swine trH3N2 from cluster IV and the A(H1N1)pdm09 virus.

Figure 1

Within-herd prevalence of positive samples for influenza virus during testing of individual samples by virus isolation in Madin-Darby canine kidney (MDCK) cells, and during testing of 3:1 pools of nasal swabs on real-time reverse transcription (rtRT)-PCR in Ontario swine herds during 2011–2012.

Figure 2

Phylogenetic tree of the HA gene nucleotide sequences of 10 Ontario H3N2 viruses.

Figure 3

Alignment of the 10 Ontario H3 HA1 amino acid sequences without signal peptide. Amino acids of the HA1 subunit of the 10 Ontario H3N2 isolates and prototype cluster IV trH3N2 virus [A/swine/ON/33853/2005]. Residues shown in boxes represent previously identified antigenic sites A, B, C, D and E; respectively. Potential glycosylation sites are underlined.

Figure 4

Alignment analysis of M2 sequences of 10 Ontario swH3N2 isolates showing amino acid substitution S31N and V27I within the transmembrane domain of M2 protein, and R77Q substitution.

Figure 5

Presentation of PB1-F2 variants of mammalian and avian influenza A viruses. Nine PB1 lineages from A to I have been described and some selected sequences within these lineages are presented. Lines at the top of the Figure represent amino acids of the predicted helical region (black) and the putative mitochondrial targeting sequence (dashed). Amino acids that are considered to enhance viral pathogenesis are marked in grey. The first stop codon has been shown by asterisk and following stop codons are indicated by subsequent asterisk.