Prevalent Eurasian avian-like H1N1 swine influenza virus with 2009 pandemic viral genes facilitating human infection

Abstract

Pigs are considered as important hosts or "mixing vessels" for the generation of pandemic influenza viruses. Systematic surveillance of influenza viruses in pigs is essential for early warning and preparedness for the next potential pandemic. Here, we report on an influenza virus surveillance of pigs from 2011 to 2018 in China, and identify a recently emerged genotype 4 (G4) reassortant Eurasian avian-like (EA) H1N1 virus, which bears 2009 pandemic (pdm/09) and triple-reassortant (TR)-derived internal genes and has been predominant in swine populations since 2016. Similar to pdm/09 virus, G4 viruses bind to human-type receptors, produce much higher progeny virus in human airway epithelial cells, and show efficient infectivity and aerosol transmission in ferrets. Moreover, low antigenic cross-reactivity of human influenza vaccine strains with G4 reassortant EA H1N1 virus indicates that preexisting population immunity does not provide protection against G4 viruses. Further serological surveillance among occupational exposure population showed that 10.4% (35/338) of swine workers were positive for G4 EA H1N1 virus, especially for participants 18 y to 35 y old, who had 20.5% (9/44) seropositive rates, indicating that the predominant G4 EA H1N1 virus has acquired increased human infectivity. Such infectivity greatly enhances the opportunity for virus adaptation in humans and raises concerns for the possible generation of pandemic viruses.

Keywords: 2009 pandemic H1N1 virus; Eurasian avian-like H1N1 virus; pandemic potential; reassortant; swine influenza.

Fig. 1.

Phylogenetic relationship of HA gene and antigenic characterization of EA H1N1 SIVs in China from 2011 to 2018. (A) Phylogenetic tree of HA gene. Phylogenetic tree was estimated using genetic distances calculated by maximum likelihood under the GTRGAMMA + I model. SIVs isolated in this study are green; sequences of viruses with names in black were downloaded from databases. Node labels represent bootstrap values. Viruses labeled with a red triangle were selected for antiserum generation. A full detailed HA gene tree with the consistent topology is shown in SI Appendix, Fig. S3. (Scale bar is in units of nucleotide substitutions per site.) (B) Antigenic map based on the HI assay data. Open squares and filled circles represent the positions of antisera and viruses, respectively. Clusters were identified by a k-means clustering algorithm. Strains belonging to the same antigenic cluster are encircled in an oval. The vertical and horizontal axes both represent antigenic distance. The spacing between grid lines is 1 unit of antigenic distance, corresponding to a twofold dilution of antiserum in the HI assay. Details of the HI assay data are shown in SI Appendix, Table S5. (C) Antigenic analysis of EA H1N1 and human influenza vaccine strains. Twenty serum samples, collected from 4-y-old children vaccinated with trivalent vaccines (A/Michigan/45/2015 [pdm/09 H1N1] + A/Singapore/INFIMH-16-0019/2016 [H3N2] + B/Colorado/06/2017 [B/Victoria]), were subjected to HI assays. The pdm/09 H1N1 virus A/Michigan/45/2015, human H3N2 virus A/Singapore/INFIMH-16-0019/2016, G1 EA H1N1 virus SW/HN/08/11, and G4 EA H1N1 virus SW/SD/1207/16 were used as antigens. HI titers ≥ 40 were considered positive.

Fig. 2.

Phylogenetic analysis of EA H1N1 SIVs in China from 2011 to 2018. (A) Phylogeny and divergence time of the HA gene and genotype evolution of EA H1N1 SIVs. The phylogenetic tree of HA gene was generated by Bayesian Markov Chain Monte Carlo framework, using the GTR substitution model with gamma-distributed among site rate heterogeneity and a “strict molecular clock” model. Colored boxes show the lineage classification of each gene segment of EA H1N1 viruses. Purple node bars represent 95% credible intervals of lineage divergence times. A detailed phylogenetic tree including sequence names is shown in SI Appendix, Fig. S4. (B) Diversity of genotypes of EA viruses isolated from swine in China, 2011–2018.

Fig. 3.

Horizontal transmission of EA H1N1 viruses between ferrets. Groups of three ferrets were inoculated i.n. with 10⁶ TCID₅₀ of indicated viruses. The next day, infected animals were individually cohoused with an uninfected DC ferret; an uninfected RD contact animal was also housed in a wire frame cage adjacent to the infected ferret. Nasal washes for virus shedding detection were collected every other day from all animals from day 2 of the initial infection. Each color bar represents the virus titer of an individual animal. Dashed lines indicate the lower limit of virus detection.