The genomic landscape of swine influenza A viruses in Southeast Asia

Abstract

Swine are a primary source for the emergence of pandemic influenza A viruses. The intensification of swine production, along with global trade, has amplified the transmission and zoonotic risk of swine influenza A virus (swIAV). Effective surveillance is essential to uncover emerging virus strains; however gaps remain in our understanding of the swIAV genomic landscape in Southeast Asia. More than 4,000 nasal swabs were collected from pigs in Cambodia, yielding 72 IAV-positive samples by RT-qPCR and 45 genomic sequences. We unmasked the cocirculation of multiple lineages of genetically diverse swIAV of pandemic concern. Genomic analyses revealed a novel European avian-like H1N2 swIAV reassortant variant with North American triple reassortant internal genes, that emerged approximately seven years before its first detection in pigs in 2021. Using phylogeographic reconstruction, we identified south central China as the dominant source of swine viruses disseminated to other regions in China and Southeast Asia. We also identified nine distinct swIAV lineages in Cambodia, which diverged from their closest ancestors between two and 15 B.P., indicating significant undetected diversity in the region, including reverse zoonoses of human H1N1/2009 pandemic and H3N2 viruses. A similar period of cryptic circulation of swIAVs occurred in the decades before the H1N1/2009 pandemic. The hidden diversity of swIAV observed here further emphasizes the complex underlying evolutionary processes present in this region, reinforcing the importance of genomic surveillance at the human-swine interface for early warning of disease emergence to avoid future pandemics.

Keywords: European avian-like virus; evolution; pandemic; zoonotic.

Fig. 1.

Geographical area of swine influenza surveillance study conducted in Cambodia, 2020 to 2022. Striped lines indicate the location by district (D1 to D9) of sampled pig slaughterhouses in Kampong Speu, Kandal, Phnom Penh, and Takeo provinces.

Fig. 2.

Diversity of swIAV strains. (A) Genomic constellations for each gene segment of swIAV strains from sampled pigs in Cambodia. Colored boxes represent the major swIAV lineages detected in this study. (B and C) Evolutionary relationships of the H1-HA (B) and N1-NA (C) genes of human and swine influenza viruses inferred by maximum likelihood method in RAxML. Human and swine influenza viruses are denoted by pink and white tip circles, respectively. Colored branches represent different IAV lineages; red branches and arrows indicate swIAV gene sequences generated from this study. Bootstrap support (≥50) are indicated at major nodes.

Fig. 3.

Time-scaled phylogenies of H1-HA genes of human and swine influenza viruses. (A) European avian-like H1N1 swine viruses, represented by green branches. (B) Human H1N1/pdm09 viruses, represented by blue branches. (C) Classical H1N1 swine viruses, represented by orange/yellow branches. Red branches represent swIAV sequences from this study. Persistent amino acid substitutions are indicated at the nodes. Bayesian PP values (≥0.95) are shown at major nodes.

Fig. 4.

Phylogenies of H3-HA and N2-NA gene segments. (A and B) Evolutionary relationships of the H3-HA (A) and N2-NA (B) genes of influenza viruses inferred by maximum likelihood method in RAxML. Human and swine influenza viruses are denoted by pink and white tip circles, respectively. Colored branches represent different IAV lineages. Bootstrap support (≥50) are indicated at major nodes. (C and D) Time-scaled phylogenies of H3-HA genes. (E) Seasonal H3 avian-like H1N1 swine viruses. Red branches and arrows represent swIAV sequences generated from this study. Bayesian PP values (≥0.95) are shown at major nodes. *The corresponding HA gene of A/swine/Cambodia/PFC26/2020 is H1N1/pdm09, whereas that of A/swine/Cambodia/PFC32/2020 contained both H1N1/pdm09 and H3.

Fig. 5.

Temporal phylogenetic trees highlighting the origin of EA H1N2 internal genes. Purple, blue, and yellow branches represent TRIG, H1N1/pdm09 and CS virus lineages, respectively. Red and green branches represent swIAV and EA H1N2 sequences generated from this study, respectively. Bayesian PP values (≥0.95) are shown at major nodes.

Fig. 6.

Discrete phylogeographic reconstruction of EA-like swine H1 diffusion dynamics. (A) Time-scaled maximum clade credibility tree of the Bayesian phylogeographic inference. Branch color corresponds to different geographic locations. The insert displays the circular migration plot based on log Bayes factor support between pairwise locations (see SI Appendix, Table S2 for support values). Abbreviations: EC, east China; EU, Europe; NC, north China; NEC, northeast China; SEA, Southeast Asia; SCC, south central China; WC, west China. Bayesian PP values (≥0.95) are shown at major nodes only. (B) Spatial migration pathways of EA swine viruses through time.