Evolutionary interactions between haemagglutinin and neuraminidase in avian influenza

Abstract

Background: Reassortment between the RNA segments encoding haemagglutinin (HA) and neuraminidase (NA), the major antigenic influenza proteins, produces viruses with novel HA and NA subtype combinations and has preceded the emergence of pandemic strains. It has been suggested that productive viral infection requires a balance in the level of functional activity of HA and NA, arising from their closely interacting roles in the viral life cycle, and that this functional balance could be mediated by genetic changes in the HA and NA. Here, we investigate how the selective pressure varies for H7 avian influenza HA on different NA subtype backgrounds.

Results: By extending Bayesian stochastic mutational mapping methods to calculate the ratio of the rate of non-synonymous change to the rate of synonymous change (d(N)/d(S)), we found the average d(N)/d(S) across the avian influenza H7 HA1 region to be significantly greater on an N2 NA subtype background than on an N1, N3 or N7 background. Observed differences in evolutionary rates of H7 HA on different NA subtype backgrounds could not be attributed to underlying differences between avian host species or virus pathogenicity. Examination of d(N)/d(S) values for each subtype on a site-by-site basis indicated that the elevated d(N)/d(S) on the N2 NA background was a result of increased selection, rather than a relaxation of selective constraint.

Conclusions: Our results are consistent with the hypothesis that reassortment exposes influenza HA to significant changes in selective pressure through genetic interactions with NA. Such epistatic effects might be explicitly accounted for in future models of influenza evolution.

Figure 1

H7 HA1 phylogeny. The tree was inferred using the PhyML software under the GTR + Γ model of DNA substitution, with 6 rate categories. 1000 bootstrap replicates were performed. Major geographical lineages are labelled in red and bootstrap support values (proportion of bootstrap replicates) for major clades are labelled in blue. An H15 sequence was used as an outgroup, but was removed in this figure for the purpose of presentation. Lineages are coloured by the background NA subtype of the virus at the tips of the tree, and clades of sequences of the same subtype have been collapsed for the purpose of presentation (numbers of sequences in collapsed clades are given in brackets). Note: FPV = 'fowl plague virus’, a term used to describe H7 avian influenza viruses isolated in the 1920s-1940s.

Figure 2

90% HPD plots for H7 HA1 evolutionary rates, split by viral NA subtype. The boxes show the limits of the narrowest interval containing 90% of the estimates. The horizontal lines inside the boxes indicate the location of the mean for each subtype. Individual points shown outside the boxes are values which lie below the lower limit, or above the upper limit, of the 90% HPD interval. For each subtype, values for d_S are the number of synonymous changes per synonymous site, scaled by the total branch lengths in the tree sample for lineages corresponding to that subtype. Similarly, d_N is given in terms of the number of non-synonymous changes per non-synonymous site, scaled by the total branch lengths in the tree sample for lineages corresponding to that subtype.

Figure 3

90% HPD plots for H7 HA1 evolutionary rates, split by virus pathogenicity. The coloured boxes show the limits of the narrowest interval containing 90% of the posterior estimates. The horizontal lines inside the boxes indicate the location of the mean for highly pathogenic (HP) or low pathogenic (LP) viruses. The similarity in evolutionary rates for HP and LP viruses can be observed from the overlap in the distributions and the location of the means of the distribution for HP viruses within the 90% HPD limits of the corresponding LP distribution and vice versa.

Figure 4

90% HPD plots for H7 HA1 evolutionary rates, split by avian host order. The means and HPD limits for d_N/d_S and rates of synonymous and non-synonymous substitution were similar for anseriform (Ans.), galliform (Gal.) and other avian hosts. This indicated that the taxonomic order of the avian host from which influenza viruses were isolated did not have a significant effect on evolutionary rates or selective pressure experienced by the virus.

Figure 5

Distribution of d_N/d_S values across avian influenza H7 HA1 sites, on different NA subtype backgrounds. The d_N value for each site was divided by the average d_S across all sites for that subtype to obtain a d_N/d_S value for each site on each background NA subtype. Sites with d_N/d_S > 1, i.e. under putative positive selection, are highlighted in red. Sites under putative positive selection were distributed across the HA1 for all background NA subtypes. Although there is some variation between NA backgrounds in terms of the sites under putative positive selection, there is also some commonality between the subtypes (see Additional file 1: Table S1). A coloured key is provided, which indicates the HA1 domain: fusion (pink), vestigial esterase (green) or receptor binding (blue). The signal peptide region is indicated in yellow.

Figure 6

Log(d_N/d_S) values across avian influenza H7 HA1 sites, on different NA subtype backgrounds. The natural logarithm of the d_N/d_S values from was taken, so that sites with log(d_N/d_S) > 0 corresponded to d_N/d_S > 1, and sites with log(d_N/d_S) < 0 corresponded to d_N/d_S < 1 (the value log(d_N/d_S) = 0, i.e. d_N/d_S = 1, is shown as a dotted red line). The d_N/d_S values for each site are colour coded according to the background NA subtype. Codon sites correspond to the H3 numbering.