Human Infection with Eurasian Avian-Like Swine Influenza A(H1N1) Virus, the Netherlands, September 2019

We report a zoonotic infection of a pig farmer in the Netherlands with a Eurasian avian-like swine influenza A(H1N1) virus that was also detected in the farmed pigs. Both viruses were antigenically and genetically characterized. Continued surveillance of swine influenza A viruses is needed for risk assessment in humans and swine.

sequencing (7) of both isolates confirmed that all genome segments were closely related to those of Eurasian avian-like H1N1 swine IAVs. A BLAST homology search (http://www.fludb.org) with both whole genomes showed highest nucleotide identities (96%) for hemagglutinin (HA) and neuraminidase with clade 1C.2.2 Eurasian avian-like H1N1 swine IAVs isolated in Germany and the Netherlands during 2011-2012. These databases contain limited numbers of sequences of this swine IAV clade, which explains the lack of similar recent viruses. A phylogenetic tree of Eurasian avian-like H1N1 swine IAVs isolated in Europe and Asia was constructed by using MEGA7 software (https://www.megasoftware.net). Phylogenetic analysis confirmed the genetic relationship of the HA1 genes of both isolates with Eurasian   Figure). The human H1N1v and swine H1N1 isolates differed in several positions in the genes coding for the 3 polymerase proteins (polymerase basic [PB] 2, PB1, and polymerase acidic), the HA gene, and the nonstructural protein gene. The other 3 gene segments (neuraminidase, nucleoprotein, and matrix) were 100% identical. HA gene sequences of the swine H1N1 and the human H1N1v isolates showed amino acid substitutions K142N, N195S, and V215I (H1 numbering). Position 142 is located in antigenic site Ca2 142 and position 195 in antigenic site Sb (8). In human seasonal influenza A(H1N1) viruses, a substitution at position 142 was reported to cause antigenic change (9). This substitution might explain the loss of reactivity with pH1N1 antiserum for the human H1N1v isolate versus the swine H1N1 isolate (Table 1). In addition, in H5 IAVs this substitution decreased the pH at which the HA underwent fusion (10).
Because human-adapted viruses undergo fusion at a lower pH (5.0-5.5) than swine-adapted and avian-adapted viruses (pH 5.6-6.0), such mutations might contribute to human adaptation of zoonotic viruses. We found multiple substitutions in the polymerase genes of the human H1N1v isolate: R739Q in PB2, L108I and T652A in PB1, and D682N in polymerase acidic. Based on analyses in the FluSurver database (http://flusurver.bii.a-star.edu.sg/), the R739Q Figure. Phylogenetic tree based on amino acid sequences of the hemagglutinin 1 of Eurasian avian-like swine influenza A(H1N1) virus isolates from a pig farmer and his pigs (green circles), the Netherlands, and reference sequences (see Table 2). Red triangles indicate reference sequences from humans. Phylogenetic relationships were estimated by using the maximum-likelihood method in MEGA7 software (https://www.megasoftware.net) and the Jones-Taylor-Thornton substitution model with a gamma distribution of among-site rate. Branch length is proportional to genetic distance. Scale bar indicates amino acid substitutions per site. substitution in the PB2 gene might influence the binding of PB2 to host protein(s). We also found 2 substitutions in the nonstructural protein gene: V18I and G227R. Sequences were made publicly available in GenBank (accession nos. MT395362-77), and GI-SAID (https://www.gisaid.org; accession nos. EPI_ ISL_430866 [A/swine/Netherlands/Gent-193/2019] and EPI_ISL_0865 [A/Netherlands/Gent-193/2019]).

Conclusions
We report another zoonotic infection with a Eurasian avian-like H1N1 swine IAV in Europe since the emergence of the virus in 1979 (2)(3)(4). No further humanto-human transmission was reported, although it cannot be excluded that the farmer was infected by the animal caretaker. The nasal swab sample from the caretaker might have tested negative because it was collected as late as 8 days after he reported influenzalike symptoms.
The swine antiserum against the pH1N1 virus cross-reacted with the swine H1N1 isolate from this investigation ( Table 1) but had a 192-fold lower virus neutralization titer against the human H1N1v isolate. Therefore, it is unlikely that current human seasonal vaccines would provide cross-protection against the human H1N1v isolate. This finding is consistent with our recent investigations of human serum samples for antibodies against 8 H1 swine IAVs representing 7 predominant H1 clades of swine IAVs; only 55 (10%) of 549 human serum samples had hemagglutination inhibition titers >40 against a European avian-like H1N1 swine IAV of clade 1C.2.1, which is predominant in swine in Europe (11), compared with 24%-54% against 5 other clades (12). These data point toward a relatively greater zoonotic risk for avianlike H1N1 swine IAVs from Europe and are consistent with previous studies about Eurasian avian-like H1N1 swine IAVs from China (5,13). Our data further support the notion that Eurasian avian-like H1N1 swine IAVs need to be monitored closely.
We found several amino acid substitutions between the H1N1 swine isolate and the H1N1v human isolate, but their role remains obscure. The past 2 decades have seen an unprecedented increase of data for putative mammalian-adaptive mutations of avian influenza viruses. The known genetic markers are mainly based on studies with wholly avian viruses of various HA subtypes in mammalian cell culture or in ferrets. Knowledge of amino acid substitutions that might enable adaptation of swine-adapted influenza viruses to humans, in contrast, is almost nonexistent (14). This finding is true for Eurasian avian-like H1N1 swine IAVs, as well as for the pH1N1 virus, which is the only known swine-origin virus with the ability to spread efficiently between humans. Our study highlights the need for experimental research on this topic and for continued surveillance of swine IAVs because of the risk for human infection or zoonotic spread.