Genetic and antigenic characterization of H1 influenza viruses from United States swine from 2008

Abstract

Prior to the introduction of the 2009 pandemic H1N1 virus from humans into pigs, four phylogenetic clusters (α-, β-, γ- and δ) of the haemagglutinin (HA) gene from H1 influenza viruses could be found in US swine. Information regarding the antigenic relatedness of the H1 viruses was lacking due to the dynamic and variable nature of swine lineage H1. We characterized 12 H1 isolates from 2008 by using 454 genome-sequencing technology and phylogenetic analysis of all eight gene segments and by serological cross-reactivity in the haemagglutination inhibition (HI) assay. Genetic diversity was demonstrated in all gene segments, but most notably in the HA gene. The gene segments from the 2009 pandemic H1N1 formed clusters separate from North American swine lineage viruses, suggesting progenitors of the pandemic virus were not present in US pigs immediately prior to 2009. Serological cross-reactivity paired with antigenic cartography demonstrated that the viruses in the different phylogenetic clusters are also antigenically divergent.

Fig. 1.

HA1 protein sequence alignment reveals critical amino acid changes between α-, β- γ- and δ-clusters. The 12 2008 H1 predicted HA1 proteins were aligned and compared to that of 2009 pandemic H1N1 (A/MX/4108/2009, GenBank accession no. GQ162170) and A/swine/Minnesota/07002083/2007 (δ2 cluster, GenBank accession no. FJ611898) and numbered using the mature HA1. Two human seasonal influenza viruses HA sequences were included: A/Michigan/2/2003 (GenBank accession no. CY016324) and A/Wisconsin/04/2009 (GenBank accession no. GQ475852). Amino acid differences are visible in the antigenic sites Cb (red), Ca2 (green), Sa (light blue), Sb (violet) and Ca1 (yellow) between the α-, β-, γ-cluster viruses and the human-like δ-cluster viruses. The residues forming the receptor-binding pocket are indicated by a diamond (⋄). Viruses belonging to the δ-cluster preserve remnants of the human lineage. A/swine/Iowa/02039/2008 contains a G (blue) at position 68 and N (violet in the Sb antigenic site) at position 187. The 187N human virus-derived residue is present in A/swine/Texas/01976/2008 as well.

Fig. 2.

(a, b) Full phylogenetic analysis of the 2008 H1 North American isolates. Neighbour-joining trees inferred from multiple nucleotide sequence alignment of (a), PB2 (2280 nt), PB1 (2274 nt), PA (2151 nt), NP (1497 nt), M (982 nt), NS (844 nt) and (b), HA (1701 nt) and NA (1410 nt) gene segments. HA and NA gene sequences of 24 swine and human pandemic H1N1 (including the five isolates included in the antigenic mapping), five classical H1N1 lineage and 15 δ1 isolates were included, but condensed in the HA and NA phylogenetic analyses. Fig. 2(b) HA analysis shows five clusters of related viruses, H1α, H1β, H1γ, H1δ and δ2 as indicated by the bars on the right of the tree. Bars indicate the estimated numbers of nucleotide substitutions per site. The TRIG constellation is indicated by the bar to the right and other lineages are indicated by group labels of compressed branches. The swine HA-cluster specificity of each 2008 virus is indicated in each tree: open diamond, α cluster; closed triangle, β cluster; closed diamond, γ cluster; closed circle, δ cluster; open circle, human seasonal H1N1/N2 influenza viruses; open triangle, 2009 pandemic H1N1. Hu, Human; sw, swine; du, duck; ch, chicken.

Fig. 2.

(a, b) Full phylogenetic analysis of the 2008 H1 North American isolates. Neighbour-joining trees inferred from multiple nucleotide sequence alignment of (a), PB2 (2280 nt), PB1 (2274 nt), PA (2151 nt), NP (1497 nt), M (982 nt), NS (844 nt) and (b), HA (1701 nt) and NA (1410 nt) gene segments. HA and NA gene sequences of 24 swine and human pandemic H1N1 (including the five isolates included in the antigenic mapping), five classical H1N1 lineage and 15 δ1 isolates were included, but condensed in the HA and NA phylogenetic analyses. Fig. 2(b) HA analysis shows five clusters of related viruses, H1α, H1β, H1γ, H1δ and δ2 as indicated by the bars on the right of the tree. Bars indicate the estimated numbers of nucleotide substitutions per site. The TRIG constellation is indicated by the bar to the right and other lineages are indicated by group labels of compressed branches. The swine HA-cluster specificity of each 2008 virus is indicated in each tree: open diamond, α cluster; closed triangle, β cluster; closed diamond, γ cluster; closed circle, δ cluster; open circle, human seasonal H1N1/N2 influenza viruses; open triangle, 2009 pandemic H1N1. Hu, Human; sw, swine; du, duck; ch, chicken.

Fig. 3.

Antigenic map of North American swine influenza viruses subtype H1. The relative positions of strains (coloured dots) and swine hyperimmune antisera (open squares) were computed such that the distances between strains and antisera in the map with the least error represent the corresponding HI measurements (Smith et al., 2004). Strain colour indicates α-cluster (cyan), β-cluster (pink), γ-cluster (blue), δ-cluster (gold) and 2009 H1N1 (red). Each grid square represents 1 U of antigenic distance, corresponding to a twofold difference in the HI assay.