Evolution of novel reassortant A/H3N2 influenza viruses in North American swine and humans, 2009-2011

Abstract

Novel H3N2 influenza viruses (H3N2v) containing seven genome segments from swine lineage triple-reassortant H3N2 viruses and a 2009 pandemic H1N1 (H1N1pdm09) matrix protein segment (pM) were isolated from 12 humans in the United States between August and December 2011. To understand the evolution of these novel H3N2 viruses in swine and humans, we undertook a phylogenetic analysis of 674 M sequences and 388 HA and NA sequences from influenza viruses isolated from North American swine during 2009-2011, as well as HA, NA, and M sequences from eight H3N2v viruses isolated from humans. We identified 34 swine influenza viruses (termed rH3N2p) with the same combination of H3, N2, and pM segments as the H3N2v viruses isolated from humans. Notably, these rH3N2p viruses were generated in swine via reassortment events between H3N2 viruses and the pM segment approximately 4 to 10 times since 2009. The pM segment has also reassorted with multiple distinct lineages of H1 virus, especially H1δ viruses. Importantly, the N2 segment of all H3N2v viruses isolated from humans is derived from a genetically distinct N2 lineage that has circulated in swine since being acquired by reassortment with seasonal human H3N2 viruses in 2001-2002, rather than from the N2 that is associated with the 1998 H3N2 swine lineage. The identification of this N2 variant may have implications for influenza vaccine design and the potential pandemic threat of H3N2v to human age groups with differing levels of prior exposure and immunity.

Fig 1

Proportion of influenza viruses collected in U.S. swine during 2005–2012 by subtype: H1N1, H1N2, H3N2, or mixed (note that mixed samples may come from more than one animal and that methods for detecting H1N2 viruses improved in 2010). Data are from a total of 6,043 influenza viruses subtyped by the University of Minnesota Veterinary Diagnostic Laboratory (UMVDL) during 2005–2012.

Fig 2

(a) Frequency of the trigM versus pM genome segments among 674 M segments collected from influenza viruses in North American swine, 2009–2011. (b) Frequency of the trigM versus pM segments among 129 M segments from influenza virus isolates that were subtyped as H3N2 by the NAHLN, 2009–2011. (c) Frequency of the pM segment among 259 H1δ, H1γ, and pH1 influenza viruses collected from North American swine, 2009–2011.

Fig 3

Phylogenetic relationships of pandemic matrix protein (pM) sequences from 246 influenza virus isolates collected in North American swine during 2009–2011 and 8 M sequences from H3N2v viruses collected from humans in 2011 (total data set = 256 M sequences). Each isolate is shaded by the corresponding HA lineage: pH1 isolates are blue, H1δ isolates are yellow, H3 isolates from swine are red, H1γ isolates are green, H1β isolates are light pink, and H3 segments isolated from human H3N2v cases are hot pink and labeled “hu” (associated with reassortment events 3, 4, and 7). Support for individual nodes defining reassortment events between H3N2 viruses and the pM segment is provided with approximate likelihood ratios (aLRT) of >70 and reassortment events are numbered 1 to 10, with reassortment events that are additionally supported by significant bootstrap values indicated by asterisks (percentage bootstrap support for reassortment event 1 = 99, 3 = 97, 5 = 95, and 6 = 84). The tree is midpoint rooted for clarity, and all branch lengths are drawn to scale. The identical phylogeny with tip labels included is available in Fig. S6 in the supplemental material.

Fig 4

Phylogenetic relationships of N2 sequences from 164 influenza virus isolates collected in North American swine during 2009–2011, for which information about the HA was available, and 8 N2 sequences from H3N2v viruses collected from humans in 2011 (total, 172 N2 sequences). The two major clades on N2 tree are labeled as those related to the original triple reassortant swine virus identified in 1998 and swine influenza viruses first identified in 2005 containing N2 segments that were most closely related to human H3N2 influenza viruses collected in 2001–2002 (2002/clade IV). Each isolate is colored by the combination of the HA lineage and the phylogenetic position of the M segment, as shown in the key. Support for individual nodes defining reassortment events between isolates containing the H3 and pM segments and the N2 segment is provided with approximate likelihood ratios (aLRT) of >70, and reassortment events are numbered 1 to 14, with bootstrap support of >70 indicated by asterisks (percentage bootstrap support for reassortment event 1 = 100, 2 = 100, 3 = 99, 4 = 83, 5 = 100, 6 = 85, 7 = 100, 8 = 100, 9 = 100, 10 = 80, 11 = 100, 12 = 94, and 14 = 83). The geographical locations (abbreviation for U.S. state or QB [Quebec, Canada]) of all rH3N2p (in swine) and H3N2v (in humans) isolates are labeled by N2 cluster. The tree is midpoint rooted for clarity, and all branch lengths are drawn to scale. The identical phylogeny with tip labels included is available in Fig. S7 in the supplemental material. The box encloses the seven of the eight human H3N2v isolates that belong to a new clade within the N2 2002/clade IV lineage that is separated by a long branch.