Molecular evidence for interspecies transmission of H3N2pM/H3N2v influenza A viruses at an Ohio agricultural fair, July 2012

Abstract

Evidence accumulating in 2011-2012 indicates that there is significant intra- and inter-species transmission of influenza A viruses at agricultural fairs, which has renewed interest in this unique human/swine interface. Six human cases of influenza A (H3N2) variant (H3N2v) virus infections were epidemiologically linked to swine exposure at fairs in the United States in 2011. In 2012, the number of H3N2v cases in the Midwest had exceeded 300 from early July to September, 2012. Prospective influenza A virus surveillance among pigs at Ohio fairs resulted in the detection of H3N2pM (H3N2 influenza A viruses containing the matrix (M) gene from the influenza A (H1N1) pdm09 virus). These H3N2pM viruses were temporally and spatially linked to several human H3N2v cases. Complete genomic analyses of these H3N2pM isolates demonstrated >99% nucleotide similarity to the H3N2v isolates recovered from human cases. Actions to mitigate the bidirectional interspecies transmission of influenza A virus between people and animals at agricultural fairs may be warranted.

Keywords: H3N2pM; fair; human H3N2v; influenza; swine.

Figure 1

Phylogeny of HA gene segment from swine lineages of H3N2 viruses. The percentage of replicate trees in which the associated HA gene segments clustered together in the bootstrap test using the Maximum Parsimony method are shown next to the branches. The analysis involved 45 nucleotide sequences and there were a total of 863 positions in the final dataset. The cluster-based classification is derived from previously established nomenclature.^, The four major clusters of H3 that have been circulating in the swine populations are shown. All H3N2v/H3N2pM HA segments (identified by a dot alongside the isolate) clustered together as a sublineage of previously described Cluster IV viruses.

Figure 2

Maximum Parsimony analysis of NA gene from classical swine H3N2 and H3N2pM/H3N2v viruses. The NA genes of all the isolates (identified by a dot alongside the isolates) clustered together in a single lineage suggesting a clonal pattern. NA, neuraminidase.

Figure 3

Phylogenies of the polymerase complex (PA, PB1, PB2) genes. All sequences of the respective genes from the H3N2v/H3N2pM viruses clustered together. PA, polymerase A; PB1, polymerase B1; PB2, polymerase B2.

Figure 4

Molecular phylogenetic analysis for NP gene. Shown is the evolutionary history of NP segment from swine-origin H3N2pM and human-origin H3N2v isolates inferred by using the Maximum Likelihood method based on the Hasegawa–Kishino–Yano model. All NP sequences from the H3N2pM/H3N2v (Ohio and other geographical locations) share high levels of identity. Initial tree(s) for the heuristic search were obtained automatically as follows. When the number of common sites was <100 or less than one-fourth of the total number of sites, the maximum parsimony method was used; otherwise BIONJ method with MCL distance matrix was used. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories (+G, parameter=0.3714)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 22 nucleotide sequences. There were a total of 1497 positions in the final dataset. BIONJ, BIO neighbor joining; MCL, Markov cluster; NP, nucleocapsid protein.

Figure 5

Phylogenetic analysis of the NS gene. The bootstrap consensus tree inferred using the Maximum Parsimony method is taken to represent the evolutionary history of the NS segments analyzed. The analysis involved 16 nucleotide sequences with a total of 838 positions in the final dataset. All NS genes of H3N2v/H3N2pM influenza A viruses clustered in a single clade suggesting clonality. NS, non-structural protein.

Figure 6

Phylogenetic analysis of gene coding for the MP from the (H1N1) pdm09, H3N2v/H3N2pM, North American swine-origin H3N2 and human-origin seasonal H3N2 influenza A viruses. Shown is a bootstrap consensus tree inferred using the Maximum Parsimony method. The analysis involved 22 nucleotide sequences of matrix genes from human, swine and recent pandemic influenza A viruses. All positions containing gaps and missing data were eliminated. There were a total of 978 positions in the final dataset. Matrix of all H3N2v/H3N2pM isolates clustered with the pandemic H1N1 segment sequence (boxed sequences in the tree). There is an indication of minor microscale evolution away from influenza A (H1N1) pdm09 virus within this segment with specific polymorphisms that favor human infection being maintained. MP, matrix protein.