Novel influenza A viruses in pigs with zoonotic potential, Chile

Abstract

Novel H1N2 and H3N2 swine influenza A viruses (IAVs) have recently been identified in Chile. The objective of this study was to evaluate their zoonotic potential. We perform phylogenetic analyses to determine the genetic origin and evolution of these viruses, and a serological analysis to determine the level of cross-protective antibodies in the human population. Eight genotypes were identified, all with pandemic H1N1 2009-like internal genes. H1N1 and H1N2 were the subtypes more commonly detected. Swine H1N2 and H3N2 IAVs had hemagglutinin and neuraminidase lineages genetically divergent from IAVs reported worldwide, including human vaccine strains. These genes originated from human seasonal viruses were introduced into the swine population since the mid-1980s. Serological data indicate that the general population is susceptible to the H3N2 virus and that elderly and young children also lack protective antibodies against the H1N2 strains, suggesting that these viruses could be potential zoonotic threats. Continuous IAV surveillance and monitoring of the swine and human populations is strongly recommended.IMPORTANCEIn the global context, where swine serve as crucial intermediate hosts for influenza A viruses (IAVs), this study addresses the pressing concern of the zoonotic potential of novel reassortant strains. Conducted on a large scale in Chile, it presents a comprehensive account of swine influenza A virus diversity, covering 93.8% of the country's industrialized swine farms. The findings reveal eight distinct swine IAV genotypes, all carrying a complete internal gene cassette of pandemic H1N1 2009 origin, emphasizing potential increased replication and transmission fitness. Genetic divergence of H1N2 and H3N2 IAVs from globally reported strains raises alarms, with evidence suggesting introductions from human seasonal viruses since the mid-1980s. A detailed serological analysis underscores the zoonotic threat, indicating susceptibility in the general population to swine H3N2 and a lack of protective antibodies in vulnerable demographics. These data highlight the importance of continuous surveillance, providing crucial insights for global health organizations.

Keywords: influenza A virus; pigs; zoonosis.

Fig 1

Novel H1 and H3 swine influenza viruses identified in Chile. Maximum clade credibility trees depicting TMRCA estimates, reconstructed using HA gene sequences of human and swine IAVs reported globally. (A) Tree reconstructed using H1 subtype sequences. Branches of the tree containing the sequences from novel Chilean H1 swine IAVs are highlighted in red, whereas branches with sequences from the Chilean swine A(H1N1)pdm09-like IAVs are colored in brown. (B) Tree reconstructed using H3 subtype sequences. Branches with novel Chilean H3 swine IAVs are highlighted in yellow.

Fig 2

Two divergent H1N2 swine IAVs derived from old human seasonal viruses co-circulate with A(H1N1)pdm09-like strains. Maximum clade credibility tree constructed depicting TMRCA estimates of HA gene sequences of the H1 subtype. (A) Novel Chilean H1 swine IAVs are grouped into two phylogenetic clusters (ChH1A and ChH1B), highlighted in red. The closest reference sequences, from the human seasonal and swine delta clusters, were used. Chilean human IAV sequences are highlighted in blue. (B) H1 maximum clade credibility tree constructed using only sequences belonging to the A(H1N1)pdm09 IAV lineage. Chilean swine sequences are highlighted in brown and are interleaved with human reference sequences, including Chilean human sequences, suggesting at least seven different introductions from human to swine (●).

Fig 3

All N1 segments belong to the A(H1N1)pdm09-like lineage, and most of the N2 genes belong to a novel N2 cluster named Chilean N2. Maximum clade credibility trees depicting TMRCA estimates of NA gene segment of human and swine IAVs. (A) Four N1 lineages are shown in the tree: human seasonal, North American classic swine, Eurasian avian like, and N1 A(H1N1)pdm09 like. All Chilean swine N1 sequences belong to the A(H1N1)pdm09-like lineage and are colored according to the original HA clustering (Fig. 1 and 2). (B) N2 sequences are colored according to the original HA clustering (Fig. 1 and 2). Most of the N2 sequences are grouped in a major monophyletic cluster, named Chilean, and are associated to H1 genes from the Chilean H1 clusters (ChH1A and ChH1B).

Fig 4

The M gene segment of Chilean swine IAVs is derived from early A(H1N1)pdm09 human strains. Maximum clade credibility tree depicting TMRCA estimates of M gene segment of human and swine IAVs. All Chilean swine M sequences belong to the A(H1N1)pdm09-like lineage. Most Chilean swine viruses were grouped within the same cluster and had a most recent common ancestor with A(H1N1)pdm09 human strains from 2009. The M gene segment from three swine A(H1N1)pdm09-like strains and from swine H3 strains was grouped with other A(H1N1)pdm09-like viruses.

Fig 5

The NS A(H1N1)pdm09 gene has been introduced multiple times in the swine population. Maximum clade credibility tree depicting TMRCA estimates of NS gene segment of human and swine IAVs. All Chilean swine NS sequences belong to the A(H1N1)pdm09-like lineage. Multiple introductions of the NS pandemic gene have been introduced into the swine population since the 2009 H1N1 pandemic.

Fig 6

Serological protection of humans of different ages against the Chilean swine IAVs. (A) Influenza A strains that circulated in human populations since 1918. H1N1: 1918–1957 and 1977–2009; H2N2: 1957–1968; H3N2: 1968–to date: A(H1N1)pdm09: 2009–to date. (B) 237 human sera collected from 2009 to 2015 were evaluated against the Chilean swine viruses by hemagglutination inhibition assay. Serum titers against the A(H1N1)pdm09, H3N2, ChH1N2A, and ChH1N2B swine viruses are grouped and shown according to individual’s date of birth.

Fig 7

Model of the introduction of human influenza viruses into swine in Chile leading to the generation of novel swine IAVs. Human H1N1 and H3N2 seasonal viruses were introduced into swine as early as the mid to late 1980s. The H1N1 were introduced at two time points during this decade, whereas the H3N2 subtype was introduced in the early 1990s and then again sometime after 2005. This likely generated double reassortment events that gave rise to the ChH1N2A and ChH1N2B lineages and to the rare H3N2 viruses found in Chile. Upon the emergence of the 2009 H1N1 human pandemic, this strain was rapidly introduced into the Chilean swine population early during the pandemic and thereafter in multiple occasions generating multiple additional reassortment events that gave rise to the increased diversity of genotypes found in the country. In all cases, all the viruses have six internal genes belonging to the A(H1N1)pdm09 strain, which replaced the previously exiting internal genes (currently of unknown origin).