Reassortment patterns of avian influenza virus internal segments among different subtypes

Background The segmented RNA genome of avian Influenza viruses (AIV) allows genetic reassortment between co-infecting viruses, providing an evolutionary pathway to generate genetic innovation. The genetic diversity (16 haemagglutinin and 9 neuraminidase subtypes) of AIV indicates an extensive reservoir of influenza viruses exists in bird populations, but how frequently subtypes reassort with each other is still unknown. Here we quantify the reassortment patterns among subtypes in the Eurasian avian viral pool by reconstructing the ancestral states of the subtypes as discrete states on time-scaled phylogenies with respect to the internal protein coding segments. We further analyzed how host species, the inferred evolutionary rates and the dN/dS ratio varied among segments and between discrete subtypes, and whether these factors may be associated with inter-subtype reassortment rate. Results The general patterns of reassortment are similar among five internal segments with the exception of segment 8, encoding the Non-Structural genes, which has a more divergent phylogeny. However, significant variation in rates between subtypes was observed. In particular, hemagglutinin-encoding segments of subtypes H5 to H9 reassort at a lower rate compared to those of H1 to H4, and Neuraminidase-encoding segments of subtypes N1 and N2 reassort less frequently than N3 to N9. Both host species and dN/dS ratio were significantly associated with reassortment rate, while evolutionary rate was not associated. The dN/dS ratio was negatively correlated with reassortment rate, as was the number of negatively selected sites for all segments. Conclusions These results indicate that overall selective constraint and host species are both associated with reassortment rate. These results together identify the wild bird population as the major source of new reassortants, rather than domestic poultry. The lower reassortment rates observed for H5N1 and H9N2 may be explained by the large proportion of strains derived from domestic poultry populations. In contrast, the higher rates observed in the H1N1, H3N8 and H4N6 subtypes could be due to their primary origin as infections of wild birds with multiple low pathogenicity strains in the large avian reservoir.


Figure S2
Bayesian maximum clade credibility phylogenies for the five internal segments (A: HA subtype, B: NA subtype, C: Combined HA-NA subtype) of Eurasian Avian influenza viruses. Branches were coloured according to the different subtypes of their descendent nodes. The colour markers are the same as Figure 2.

Figure S3
Distribution of variance of reassortment rate using BSSVS and Bayes Traits. PB2 segment of HA subtype (A) and NA subtype (B). Variance of estimated reassortment rate between pairs of subtypes using BayesTraits (blue); Variance of estimated reassortment rate between pairs of subtypes using BSSVS (red).

Figure S4
Robustness of reassortment rate (HA, NA and HA-NA combined subtype) estimates using BSSVS among 3 random sampling subgroups. As for clarity, only one reassortment rate per HA,NA and HA-NA combined subtype for PB2 segment was shown respectively, with mean reassortment rates and 95% Highest probability density (HPD) intervals. Blue: group1; Red: group 2; Green: group 3.The complete rate of each transition were shown in Table S2.

Figure S6
Distribution of reassortment rates in the PB2 heat maps for H, N, and H-N subtypes in rows (blue) and columns (red) ( Fig. 2 and Table S2). The variance of the reassortment rate in the rows (blue) is lower than that in the columns (red); indicating the asymmetry of the reassortment rates.
Reassortment rates from H and N subtypes as donors (rows) to other subtypes are less variable than rates from all subtypes to H and N subtypes as recipients (columns). Correlation between proportion of AIV in wild birds and reassortment rate of different subtypes. The scatterplot of reassortment rate of HA-NA combined subtype against proportion of wild birds per subtype were represented for 6 internal segments by different colors: PB2 (red); PB1 (green); PA (blue); NP (orange); M1 (purple); NS1 (black).

Figure S9
Correlation between proportion of AIV in anseriformes and reassortment rate of different subtypes.

Figure S10
Correlation between the proportion of AIV in wild anseriformes and the reassortment rate of different subtypes. The scatterplot of reassortment rate of HA-NA combined subtype against proportion of wild anseriformes per subtype were represented for 6 internal segments by different colors: PB2 (red); PB1 (green); PA (blue); NP (orange); M1 (purple); NS1 (black).

Figure S11
Correlation between proportion of AIV in domestic anseriformes and reassortment rate of different subtypes. The scatterplot of reassortment rate of HA-NA combined subtype against proportion of domestic anseriformes per subtype were represented for 6 internal segments by different colors: PB2 (red); PB1 (green); PA (blue); NP (orange); M1 (purple); NS1 (black).

Figure S12
Correlation between proportion of AIV in domestic galliformes and reassortment rate per subtype.