Evolution of influenza A virus in intensive and free-range swine farms in Spain

Abstract

Swine harbor genetically diverse influenza A viruses (IAVs) with the capacity to host-switch to humans, causing global pandemics. Spain is the largest swine producer in Europe and has a mixed production system that includes 'white coat' pigs raised intensively in modern buildings and free-range Iberian pigs that interface differently with humans, wildlife, and other swine. Through active longitudinal IAV surveillance in nine Spanish provinces during 2015-9, we generated forty-seven complete or near-complete genome sequences from IAVs collected from swine in both systems. Genetically diverse IAVs were identified in intensively raised white pigs and free-range Iberian pigs, including new H3N1 reassortants. Both systems are dynamic environments for IAV evolution, but driven by different processes. IAVs in white pigs were genetically related to viruses found in swine raised intensively in other European countries, reflecting high rates of viral introduction following European trade routes. In contrast, IAVs in Iberian pigs have a genetic makeup shaped by frequent introductions of human IAVs, reflecting rearing practices with high rates of human contact. Transmission between white and Iberian pigs also occurred. In conclusion, Iberian swine with high rates of human contact harbor genetically diverse IAVs and potentially serve as intermediary hosts between white pigs and humans, presenting an understudied zoonotic risk that requires further investigation.

Keywords: ecology; evolution; genomic reassortment; influenza A virus; pandemic; reverse zoonosis.

Figure 1.

Map of Spanish swine and sample collection. Distribution and density of (A) all domestic pigs and (B) Iberian pigs in Spain in 2020. Each colored rectangle represents the number of pigs per municipality. The locations (Global Positioning System coordinate) of the farms for (C) white pigs and (D) Iberian pigs where respiratory specimens (squares) and serum samples (triangles) were collected for this study including positive (light color) and negative (dark color) farms. Samples were collected from white pigs in seven Spanish provinces (NA, Navarra; SA, Salamanca; SG, Segovia, TO, Toledo; VAL, Valencia; VA, Valladolid; ZA, Zaragoza) and from Iberian pigs in six provinces (BA, Badajoz; CC, Cáceres; CO, Córdoba; HU, Huelva; SA, Salamanca; TO, Toledo).

Figure 2.

Flowchart of sampling protocol in Iberian farms during 2015–9.

Figure 3.

IAV genotypes in Iberian pigs. The farm (#1–#5, see map) and genotype is listed for the eleven viruses sequenced for this study from Iberian pigs. Each circle represents a segment of the IAV genome, shaded by IAV lineages. HA and NA surface proteins are listed first, followed by the six internal gene segments.

Figure 4.

Twelve genotypes in Spanish swine. Each box represents a segment of the IAV genome, shaded by lineage, similar to Fig. 3. The Spanish provinces where each genotype was identified are listed for Iberian pigs and white pigs.

Figure 5.

H3N1 viruses in Spanish white pigs. A time-scale MCC tree of the H3 segment reconstructs the evolutionary relationships of 5 H3N1 viruses collected in Spanish white pigs, 12 closely related H3N2 viruses from swine in Denmark and the Netherlands, 40 human seasonal H3N2 viruses collected during 1997–2019 in Europe, and 3 human-like H3N2 viruses collected from swine in Russia. Posterior probabilities and estimated tMRCAs are provided for key nodes. Cartoons and pink boxes indicate the three introductions of human seasonal H3N2 viruses to swine in Europe since 1997. A white star indicates amino acid substitutions observed along the branch leading to the H3N1 Spanish clade (H3 numbering). A map shows the three locations where H3N1 viruses were identified in Spanish white pigs.

Figure 6.

Human seasonal 1990s-like N2 lineage identified in Iberian pigs. A ML tree reconstructs the evolutionary relationships of the N2 segment from H3N2 and H1N2 viruses collected in European swine and humans. Each virus is represented by a circle, shaded by host and location. Viruses sequenced for this study are highlighted in yellow. Bootstrap values are provided for key nodes. The genotype (Fig. 3) is listed for all viruses from Iberian pigs.

Figure 7.

Three mechanisms of IAV evolution in Spanish swine. A ML tree of viruses collected in European swine from 2009 to 2019 (PB2 segment). Red triangles indicate clades of viruses collected from Spanish swine for this study, with cartoons indicating host (Iberian or white pig). Three clades of interest have been expanded (A–C). Complete trees for all segments, including pdmH1N1 trees that include human and swine viruses, are available in the GitHub repository (https://github.com/mostmarmot/Iberian).

Figure 8.

Transmission of pdmH1N1 viruses from humans to swine in Europe, 2009–19. (A) Each pig represents a discrete introduction of a pdm-H1 virus from humans into swine, shaded by European country. Instances of onward transmission to swine in another European country are indicated by black lines. Viruses identified in Spanish swine in this study are highlighted by beige circles. Introductions are ordered chronologically by the seven winter epidemics in humans from 2009–10 to 2018–9 in which reverse zoonosis was observed. The total number of introductions from humans into European swine per winter season is provided. (B) The number of pdm-H1 viruses collected in humans in Spain during 2009–19. Data available from WHO FluNet.

Figure 9.

Sources of pdmH1N1 reassortants in Spanish swine. Arrows indicate introductions of two reassortant viruses with pdmH1N1 segments into Spanish white pigs from swine in other European countries and three pdmH1N1 viruses from humans into Iberian pigs, followed by reassortment with other swine viruses. Genotypes G4 and G7 (red font) have only been observed in Spain.