Estimating reassortment rates in co-circulating Eurasian swine influenza viruses

Abstract

Swine have often been considered as a mixing vessel for different influenza strains. In order to assess their role in more detail, we undertook a retrospective sequencing study to detect and characterize the reassortants present in European swine and to estimate the rate of reassortment between H1N1, H1N2 and H3N2 subtypes with Eurasian (avian-like) internal protein-coding segments. We analysed 69 newly obtained whole genome sequences of subtypes H1N1-H3N2 from swine influenza viruses sampled between 1982 and 2008, using Illumina and 454 platforms. Analyses of these genomes, together with previously published genomes, revealed a large monophyletic clade of Eurasian swine-lineage polymerase segments containing H1N1, H1N2 and H3N2 subtypes. We subsequently examined reassortments between the haemagglutinin and neuraminidase segments and estimated the reassortment rates between lineages using a recently developed evolutionary analysis method. High rates of reassortment between H1N2 and H1N1 Eurasian swine lineages were detected in European strains, with an average of one reassortment every 2-3 years. This rapid reassortment results from co-circulating lineages in swine, and in consequence we should expect further reassortments between currently circulating swine strains and the recent swine-origin H1N1v pandemic strain.

Fig. 1.

Maximum-likelihood tree for PB2 sequences. The branches of the tree have been coloured according to clade: human seasonal, orange; classical swine, pink; avian Eurasian, pale blue; Eurasian swine, purple; avian North America, dark blue; triple-reassortant swine, green; H1N1v pandemic, turquoise. Swine sequences generated as part of this study are coloured red, and the bootstrap support for selected nodes is shown. Bar, 0.05 nucleotide substitutions per site.

Fig. 2.

Time-resolved trees for HA-H1, HA-H3, NA-N1 and NA-N2. Marked isolates are all from swine and have Eurasian swine clade segment 1 sequences. (a) Segment 4 – H1; (b) segment 6 – N1; (c) segment 4 – H3; (d) segment 6 – N2. The detailed subtype abbreviations are: SwEa, Eurasian swine; SwCl, classical swine; HuS, human seasonal-origin. The marked clades represent: A, H1N2+H3N2 introduction; B, H3N2 introduction; C, classical swine introduction; D, classical swine introduction; E, Eurasian swine clade.

Fig. 3.

Estimated TMRCA for the Eurasian swine clade per segment, and for the subsequent H1, H3 and N2 introductions to Eurasia and Europe. The letter designation for the subsequent introductions (e.g. A Eurasia) corresponds to the marked clades in Fig. 2. There are only classical swine H1 introductions into Asia (none into Europe) in this dataset. The black bars represent the node age estimates from the beast trees and the grey bars indicate the 95 % HPD range.

Fig. 4.

Significant rates for reassortments (expressed as exchanges year⁻¹) onto the PB2 backbone, calculated using beast discrete traits. HA only (left), NA only (top right) and HA-NA joint (bottom right) traits results are shown. The detailed subtype abbreviations are as for Fig. 2. Only those edges with a probability of existing in the trees sample ≥0.5 and Bayes factor ≥4 are shown, and the colour reflects the support for the edge ranging from red (strongest) to yellow (just significant). The edge values are median transition rates.