Genetic and Antigenic Evolution of European Swine Influenza A Viruses of HA-1C (Avian-Like) and HA-1B (Human-Like) Lineages in France from 2000 to 2018

Abstract

This study evaluated the genetic and antigenic evolution of swine influenza A viruses (swIAV) of the two main enzootic H1 lineages, i.e., HA-1C (H1_av) and -1B (H1_hu), circulating in France between 2000 and 2018. SwIAV RNAs extracted from 1220 swine nasal swabs were hemagglutinin/neuraminidase (HA/NA) subtyped by RT-qPCRs, and 293 virus isolates were sequenced. In addition, 146 H1_avNy and 105 H1_huNy strains were submitted to hemagglutination inhibition tests. H1_avN1 (66.5%) and H1_huN2 (25.4%) subtypes were predominant. Most H1 strains belonged to HA-1C.2.1 or -1B.1.2.3 clades, but HA-1C.2, -1C.2.2, -1C.2.3, -1B.1.1, and -1B.1.2.1 clades were also detected sporadically. Within HA-1B.1.2.3 clade, a group of strains named "Δ146-147" harbored several amino acid mutations and a double deletion in HA, that led to a marked antigenic drift. Phylogenetic analyses revealed that internal segments belonged mainly to the "Eurasian avian-like lineage", with two distinct genogroups for the M segment. In total, 17 distinct genotypes were identified within the study period. Reassortments of H1_av/H1_hu strains with H1N1pdm virus were rarely evidenced until 2018. Analysis of amino acid sequences predicted a variability in length of PB1-F2 and PA-X proteins and identified the appearance of several mutations in PB1, PB1-F2, PA, NP and NS1 proteins that could be linked to virulence, while markers for antiviral resistance were identified in N1 and N2. Altogether, diversity and evolution of swIAV recall the importance of disrupting the spreading of swIAV within and between pig herds, as well as IAV inter-species transmissions.

Keywords: Eurasian avian-like lineage; H1N1; H1N2; antigenic drift; genetic diversity; genotype; matrix protein; surveillance; swine influenza; virus evolution.

Figure 1

Yearly distribution of swine influenza A viruses (swIAV) identified in France from 2000 to 2018. Data based on molecular subtyping using European swIAV hemagglutinin- (HA)- and neuraminidase- (NA)-specific RT-PCRs.

Figure 2

Bayesian inference tree of HA-1C (H1_avN_y) swIAV strains. Strains isolated in pigs in France are indicated in black type whereas those isolated in pigs in other countries are in grey type. Nodes supported by more than 50% of the sampled trees are indicated by a blue bar displaying the 95% credibility intervals of highest posterior density for the node heights. HA-1C clades are defined according to the classification of Anderson et al., 2016 [7]. The three boxes on the right grid are colored according to the genogroups defined on genomic segments 4 (HA), 6 (NA) and 7 (M) phylogenies, respectively. For segment 6, the boxes were colored in orange for N1 ‘Eurasian avian-like’ lineage, in blue for N2 ‘Scotland-like’ lineage, in yellow for N2 of ‘H3N2 human-like’ lineage, and in green for N2 of ‘Gent-like’ lineage (see Section 3.4.1). For segment 7, the boxes were colored in brown for ‘M ancestral Eurasian avian-like swine’ group, in red for ‘M recent Eurasian avian-like swine’ group, in green for ‘M European swine’ group, in yellow for ‘M Danish-like’ group, and in purple for M segment from the H1N1pdm lineage (see Section 3.4.2).

Figure 3

Geographical distribution of swIAVs of HA-1C/H1_av (A) and HA-1B/H1_hu (B) lineages identified from 2000 to 2018 in mainland France. The grey lines define the limits of each French administrative “département”. A “département” is colored according to the H1 clade when at least one strain of the clade was identified in this area. Stripes are used when several genogroups were identified in the same “département”.

Figure 4

Bayesian inference tree of HA-1B (H1_huNy) swIAV strains. Strains isolated in pigs in France are indicated in black type whereas those isolated in pigs in other countries are in grey type. Strains harboring nucleotide deletions leading to lack of one (*) or two (**) amino acid(s) in positions 147 or 146-147, respectively, are indicated by asterisks. Nodes supported by more than 50% of the sampled trees are indicated by a blue bar displaying the 95% credibility intervals of highest posterior density for the node heights. HA-1B clades are defined according to the classification of Anderson et al., 2016 [7]. The boxes on the right grid are colored according to the genogroups defined on genomic segment 4 (HA), 6 (NA) and 7 (M) phylogenies. For segment 6, the boxes were colored in orange for N1 ‘Eurasian avian-like’ lineage, in blue for N2 ‘Scotland-like’ lineage, in yellow for N2 of ‘H3N2 human-like’ lineage, and in green for N2 of ‘Gent-like’ lineage (see Section 3.4.1). For segment 7, the boxes were colored in brown for ‘M ancestral Eurasian avian-like swine’ group, in red for ‘M recent Eurasian avian-like swine’ group, in green for ‘M European swine’ group, in yellow for ‘M Danish-like’ group, and in purple for M segment from the H1N1pdm lineage (see Section 3.4.2).

Figure 5

Bayesian inference tree of NA-N1 segment of HxN1 swIAV strains. Strains isolated in pigs in France are indicated in black type, those isolated in pigs from other countries in grey type. Nodes supported by more than 50% of the sampled trees are indicated by a blue bar displaying the 95% credibility intervals of highest posterior density for the node heights. Vertical colored bars in right correspond to the color reported in cases of column #6 in the grids of HA trees (Figure 2 and Figure 4).

Figure 6

Bayesian inference tree of NA-N2 segment of HxN2 swIAV strains. Strains isolated in pigs in France are indicated in black type, those isolated in humans or pigs in other countries in grey type. Nodes supported by more than 50% of the sampled trees are indicated by blue bar displaying the 95% credibility intervals of highest posterior density for the node heights. Vertical colored bars at the right correspond to colors reported in cases of column #6 in the grids of HA trees (Figure 2 and Figure 4).

Figure 7

Bayesian inference tree of M-gene segment of H1Ny viruses. Strains isolated in pigs in France are indicated in black type, those isolated in pigs from other countries in grey type. Nodes supported by more than 50% of the sampled trees are indicated by blue bar displaying the 95% credibility intervals of highest posterior density for the node heights. Vertical colored bars at the right correspond to M genogroups with the colors reported in cases of column #7 in the grids of HA trees (Figure 2 and Figure 4).