Rapid surge of reassortant A(H1N1) influenza viruses in Danish swine and their zoonotic potential

Abstract

In 2018, a single detection of a novel reassortant swine influenza A virus (swIAV) was made in Denmark. The hemagglutinin (HA) of the virus was from the H1N1 pandemic 2009 (H1N1pdm09) lineage and the neuraminidase (NA) from the H1N1 Eurasian avian-like swine lineage (H1N1av). By 2022, the novel reassortant virus (H1pdm09N1av) constituted 27% of swIAVs identified through the Danish passive swIAV surveillance program. Sequencing detected two H1pdm09N1av genotypes; Genotype 1 contained an entire internal gene cassette of H1N1pdm09 origin, Genotype 2 differed by carrying an NS gene segment of H1N1av origin. The internal gene cassette of Genotype 2 became increasingly dominant, not only in the H1pdm09N1av population, but also in other Danish enzootic swIAV subtypes. Phylogenetic analysis of the HA genes from H1pdm09N1av viruses revealed a monophyletic source, a higher substitution rate compared to other H1N1pdm09 viruses and genetic differences with human seasonal and other swine adapted H1N1pdm09 viruses. Correspondingly, H1pdm09N1av viruses were antigenically distinct from human H1N1pdm09 vaccine viruses. Both H1pdm09N1av genotypes transmitted between ferrets by direct contact, but only Genotype 1 was capable of efficient aerosol transmission. The rapid spread of H1pdm09N1av viruses in Danish swine herds is concerning for swine and human health. Their zoonotic threat is highlighted by the limited pre-existing immunity observed in the human population, aerosol transmission in ferrets and the finding that the internal gene cassette of Genotype 2 was present in the first two zoonotic influenza infections ever detected in Denmark.

Keywords: H1pdm09N1av; Influenza A virus; novel reassortant virus; swine influenza surveillance; zoonotic potential.

Figure 1.

Proportion of IAV positive/negative and H1pdm09 positive submissions (A) and the proportion of the different HA and NA lineage combinations (subtypes) (B) registered from 2019-2022. 1A) The percentages of submissions testing negative and positive for influenza A virus including the proportion of positive submissions containing a hemagglutinin (HA) of H1N1pmd09 origin. 1B) The percentage of the H1pdm09N1av, H1N1pdm09 and H1avN2sw are highlighted on the columns to illustrate the relation between the increase of H1pdm09N1av with the decrease of H1avN2sw, while the proportion of H1N1pdm09 remains stable.

Figure 2.

Genotypes of the Danish swIAV detected between 2019-2022. The origin of each gene segment “HA, NA, NP, NS, PA, PB1 and PB2” is indicated by the colour and a letter for the internal genes, where A = Eurasian avian-like H1N1 (H1N1av) origin and P = H1N1pdm09 origin. The column “genotype” refers to the genotype according to the terminology defined by two previous studies by Wastson et al., 2015 and Henritzi et al., 2020. The number of each genotype is presented for the years 2019–2022 and as a total in the last column.

Figure 3.

Bayesian phylogenetic tree of Danish H1pdm09Nx sequences and H1pdm09Nx reference sequences. A_California_07_2009 was used as an outgroup. Node labels represent posterior probabilities. Red and pink taxon = Danish H1pdm09N1av reassortant; red with full internal gene cassette of H1N1pdm09 origin and pink similar but with an Eurasian avian-like NS gene segment. Blue = Danish H1pdm09N2sw viruses. Black and brown = Danish H1N1pdm09 viruses; black with full internal cassette of H1N1pdm09 origin and brown similar but with an Eurasian avian-like NS gene segment. Grey = other European swine H1pdm09Nx viruses clustering with Danish H1pdm09Nx viruses. Purple taxon and red star = recent human zoonotic cases. Green = human H1N1pdm09 seasonal strains from 2016-2022. Light blue = swine H1N1pdm09 vaccine strain of Respiporc FluPan H1N1 (Ceva Animal Health).

Figure 4.

Strict molecular clock tree of the Danish H1pdm09 sequences derived from H1pdm09Nx viruses collected during the swIAV surveilance from 2013-2022. Node labels represent posterior probabilities. Black taxons indicate H1N1pdm09 viruses, blue taxons indicate H1pdm09N2sw viruses and red taxons indicate H1pdm09N1av viruses. The timeline from 2009–2022 is indicated below.

Figure 5.

Level of pre-existing immunity against H1pdm09N1av genotype 1 and 2 in the general public. The figure represents the proportion of HI positive sera (HI titer ≥40) of the 119 sera from six different age groups of the general public against the A/swine/Denmark/19922-5/2021 and A/swine/Denmark/15063-1/2020 strains, respectively.

Figure 6.

Viral titers of the nasal washes of the donors, direct – and aerosol contacts ferrets innoculated with the two different H1pdm09N1av strains; Genotype 1 (A) and Genotype 2 (B). It should be noted that “Donor 4092” have a similar decrease as “Donor 4093” on DPI 7. The exact titers of each ferret are included in Supplementary File 1.