Swine influenza A viruses (swIAVs) with a truncated NS1del126 protein were strongly attenuated in previous laboratory-based safety studies and therefore approved for use as LAIVs for swine in the United States. In the field, however, the LAIV strains were detected in diagnostic samples and could regain a wild-type NS1 via reassortment with endemic swIAVs.

Surveillance of swine influenza A viruses (swIAV) allows timely detection and identification of new variants with potential zoonotic risks. In this study, we aimed to identify swIAV subtypes that circulated in pigs in Belgium and the Netherlands between 2014 and 2019, and characterize their genetic and antigenic evolution. We subtyped all isolates and analyzed hemagglutinin sequences and hemagglutination inhibition assay data for H1 swIAV, which were the dominant HA subtype. We also analyzed whole genome sequences (WGS) of selected isolates. Out of 200 samples, 89 tested positive for swIAV. swIAV of H1N1, H1N2 and H3N2 subtypes were detected. Analysis of WGS of 18 H1 swIAV isolates revealed three newly emerged genotypes. The European avian-like H1 swIAV (lineage 1C) were predominant and accounted for 47.2% of the total isolates. They were shown to evolve faster than the European human-like H1 (1B lineage) swIAV, which represented 27% of the isolates. The 2009 pandemic H1 swIAV (lineage 1A) accounted for only 5.6% of the isolates and showed divergence from their precursor virus. These results point to the increasing divergence of swIAV and stress the need for continuous surveillance of swIAV.

In 2019 a low pathogenic H3N1 avian influenza virus (AIV) caused an outbreak in Belgian poultry farms, characterized by an unusually high mortality in chickens. Influenza A viruses of the H1 and H3 subtype can infect pigs and become established in swine populations. Therefore, the H3N1 epizootic raised concern about AIV transmission to pigs and from pigs to humans. Here, we assessed the replication efficiency of this virus in explants of the porcine respiratory tract and in pigs, using virus titration and/or RT-qPCR. We also examined transmission from directly, intranasally inoculated pigs to contact pigs. The H3N1 AIV replicated to moderate titers in explants of the bronchioles and lungs, but not in the nasal mucosa or trachea. In the pig infection study, infectious virus was only detected in a few lung samples collected between 1 and 3 days post-inoculation. Virus titers were between 1.7 and 4.8 log10 TCID50. In line with the ex vivo experiment, no virus was isolated from the upper respiratory tract of pigs. In the transmission experiment, we could not detect virus transmission from directly inoculated to contact pigs. An increase in serum antibody titers was observed only in the inoculated pigs. We conclude that the porcine respiratory tract tissue explants can be a useful tool to assess the replication efficiency of AIVs in pigs. The H3N1 AIV examined here is unlikely to pose a risk to swine populations. However, continuous risk assessment studies of emerging AIVs in pigs are necessary, since different virus strains will have different genotypic and phenotypic traits.

I nfluenza A viruses (IAVs) of the H3 subtype are endemic to humans, swine, and wild birds; they also cause outbreaks in horses and are often detected in domestic birds. An H3 IAV that crosses the species barrier from animals to humans can result in a pandemic if the virus carries a hemagglutinin (HA) against which humans lack protective antibodies and the virus readily replicates in and spreads among humans. For example, in 1968, transmission of an IAV with an avian-origin H3 HA to humans caused the influenza A(H3N2) pandemic (1).The natural IAV reservoir is considered to be wild waterfowl, but transmission to domestic poultry is frequent. Avian H3 IAVs are classified as Eurasian and North American lineages, although the HA of these viruses is antigenically closely related (2,3). In contrast, after being introduced to humans in 1968, the HA of human H3 IAVs quickly drifted away from that of the avian precursor IAV. Consequently, contemporary human H3 IAVs are antigenically divergent from those in birds (2). Similarly, avian H3 IAVs were introduced into horses in the 1960s, after which their HA antigenically drifted. That evolution was, however, different and slower than for human H3 IAVs (4). Equine H3 IAVs of Florida clade 1 (FC1) are currently predominant (5). All swine H3 IAVs derived their HA from human IAVs.H3 IAVs from swine in Europe originated from a human IAV that circulated in the late 1970s. Of the 2 major lineages cocirculating in North America, cluster IV-A was derived from human IAVs from the late 1990s and novel human-like swine H3 IAVs from human IAVs from the early 2010s (6). H3 IAVs undergo slower antigenic drift in swine than in humans. Consequently, persons born after the swine viruses' human ancestor IAV had circulated are unlikely to have cross-reactive antibodies against the swine H3 IAVs. Therefore, with time, human population immunity against swine H3 IAVs decreases, increasing the pandemic risk (7-10).The infectious potential of swine H3 IAVs for humans is evident from >400 recorded zoonotic infections in the United States caused by North American cluster IV-A or novel human-like H3 swine IAVs. Four zoonotic infections with H3 IAVs from swine in Europe have also been reported (6,(11)(12)(13). H3 IAVs from equids can infect humans under experimental conditions, but there are no confirmed cases of natural transmission (14). Animal H3 IAVs might, however, become more adapted to humans by accumulating mutations in their viral proteins, reassortment of gene segments with IAVs of different species, or both (6,15). Avian H3 IAVs can infect humans directly or via an intermediate host, such as poultry or swine (2,15). In 2019, an H3N1 IAV that