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

To investigate the epidemiological status of SIVs, from 2011 to 2018, we performed active surveillance and collected a total of 29,918 nasal swab samples from normal pigs in slaughterhouses in 10 provinces with high-density pig populations (SI Appendix, Fig. S1). We isolated 136 influenza viruses from these samples, with an isolation rate of 0.45% (SI Appendix, Table S1). In the same period, 1,016 nasal swabs or lung samples were collected from pigs showing respiratory symptoms in our school’s veterinary teaching hospital, of which 43 were positive for influenza virus, at an isolation rate of 4.23% (SI Appendix, Table S1). Based on sequence analysis of the hemagglutinin (HA) and neuraminidase (NA) genes of the combined 179 SIVs, they were identified as EA H1N1 (n = 165), pdm/09 H1N1 (n = 7), CS H1N1 (n = 1), H3N2 (n = 4), and H9N2 (n = 2) viruses (SI Appendix, Table S1), indicating that EA H1N1 is the predominant subtype virus circulating in pig populations in China. Among them, only EA H1N1 virus was isolated every year, while other SIVs such as CS H1N1 and H3N2 were only found in certain years. Seven pdm/09 H1N1 viruses were only found in 2011, indicating that pdm/09 H1N1 virus was not maintained in pigs even though it was generated from pigs (2). All 43 viruses isolated from diseased pigs were of EA H1N1 subtype. It is noted that the mean virus isolation rates from diseased pigs increased annually from 1.40% in 2011 to 8.21% in 2018, with a sharp increase from 2014 (SI Appendix, Fig. S2), indicating that EA H1N1 viruses are a growing problem in pig farms.

To understand the phylogenetic evolution of the prevailing EA H1N1 viruses, a total of 77 viruses were selected for full genome sequencing on the basis of isolation time and location, with at least one strain sequenced per province (SI Appendix, Table S2). The whole genomes of 77 viruses were analyzed along with all available EA H1N1 sequences from both swine and human viruses in mainland China from 2011 to 2018. Based on the unified swine H1 HA nomenclature system (24), the HA genes of all of the EA H1N1 viruses isolated in the study belonged to clade 1C.2.3 (SI Appendix, Fig. S3). Viruses isolated during 2011–2013 had relative short evolutionary branches. However, long branches leading to several lineages were found in viruses isolated after 2013 (Fig. 1A). NA genes had a similar genetic evolution pattern (SI Appendix, Fig. S3). Notably, also after 2013, the six internal genes exhibited distinct diversity, with multiple origins from original EA, pdm/09, Avian, and TR lineages (SI Appendix, Fig. S3). These results suggest that the genomes of EA H1N1 SIVs have undergone increased diversity since 2013.

To show viral evolution, we conducted molecular clock phylogenic analysis and genotype characterization (Fig. 2A and SI Appendix, Fig. S4). Based on lineage classification, six genotypes of G1–G6 were found in EA H1N1 viruses from 2011 to 2018 (Fig. 2B). The virus with all eight genes from the “pure” EA H1N1 lineage was designated as G1. G1 viruses were predominately circulating in both southern and northern China from 2011 to 2013 (SI Appendix, Fig. S5). However, prototypical EA H1N1 viruses have largely disappeared since 2014 (Fig. 2B and SI Appendix, Fig. S5). G2, G3, and G6 reassortant EA viruses appeared transiently during 2011–2015. In 2013, two reassortant G4 and G5 viruses emerged in southern China (SI Appendix, Fig. S5). G5 virus possesses the HA, NA, and matrix (M) genes from the original EA H1N1 lineage, the viral ribonucleoprotein (vRNP) genes from the pdm/09 lineage, and the nonstructural (NS) gene from the TR lineage. G5 virus was detected continuously from 2013 to 2017, but it has declined since 2015 and was not found in 2018 (Fig. 2B). Similar to G5, G4 was also a triple reassortant, except its M gene was derived from pdm/09 lineage. G4 virus has shown a sharp increase since 2016, and is the predominant genotype in circulation in pigs detected across at least 10 provinces (Fig. 2B and SI Appendix, Fig. S5).

To assess the zoonotic potential of the G4 reassortant EA viruses, four representative G4 viruses (A/swine/Shandong/1207/2016 [SW/SD/1207/16], A/swine/Hebei/0116/2017 [SW/HB/0116/17], A/swine/Henan/SN13/2018 [SW/HN/SN13/18], and A/swine/Jiangsu/J004/2018 [SW/JS/J004/18]) were selected for further biological characterization. Two G1 strains (A/swine/Henan/08/2011 [SW/HN/08/11] and A/swine/Hebei/T37/2013 [SW/HB/T37/13]) and a pdm/09 H1N1 virus, A/California/04/09 (CA04), were also selected for comparison.

The binding preference of HA to host SAα2,6Gal receptor is a critical determinant for cross-species transmission of IAVs to humans (25, 26). We determined the binding affinity of EA H1N1 viruses to SAα2,3Gal and SAα2,6Gal sialylglycopolymers. Like pdm/09 virus CA04, all four of the G4 EA H1N1 viruses as well as the two G1 viruses bound SAα2,6Gal receptors with high affinity but bound poorly to SAα2,3Gal receptors (SI Appendix, Fig. S6A). Furthermore, all of the EA H1N1 viruses were found to bind to human tracheal epithelial lining to an extent similar to CA04 pdm/09 H1N1 virus, but control avian H5N1 virus showed no binding (SI Appendix, Fig. S6B). Thus, these results demonstrate that G4 EA H1N1 viruses preferentially bind human-like SAα2,6Gal receptor, a key prerequisite for infecting human cells.

Next, we assessed the replication of G4 viruses in normal human bronchial epithelial (NHBE) cells and alveolar epithelial (A549) cells, the major target cells in human influenza virus infection. In NHBE cells, G4 and pdm/09 viruses replicated to similar levels at each time point, and both of them produced more viable progeny viruses during 36 h to 60 h postinfection (pi) than did G1 viruses (P < 0.05 or P < 0.01, ANOVA) (SI Appendix, Fig. S7A).

Infection of human A549 cells with G4 viruses gave similar progeny results. G4 and pdm/09 viruses produced more infectious virus than did G1 viruses from 24 h to 60 h pi (P < 0.05 or P < 0.01, ANOVA), reaching highest titers of 10^7.75 median tissue culture infective dose (TCID₅₀)/mL and 10^7.5 TCID₅₀/mL, respectively. In contrast, G1 viruses showed peak titers of 10^6.5 TCID₅₀/mL (SI Appendix, Fig. S7B). Collectively, G4 EA reassortant viruses replicate efficiently in human airway epithelial cells, similarly to replication of pdm/09 H1N1 virus.

Ferrets have been widely used as an experimental model to study human infection and transmission of influenza virus (27). Here, three ferrets were infected intranasally (i.n.) with each virus at a dose of 10⁶ TCID₅₀ in a 1.0-mL volume. We found that G1 EA or pdm/09 viruses caused only mild clinical signs (SI Appendix, Table S4). Infection with G4 EA viruses, on the other hand, resulted in more severe clinical symptoms such as pyrexia, sneezing, wheezing, and coughing, with higher mean maximum weight loss ranging from 7.3 to 9.8% (SI Appendix, Table S4). Postmortem and histopathology revealed that G4 virus-infected lungs had more-severe lesions than G1 or pdm/09 virus-infected lungs, with pronounced multifocal areas of consolidation, hemorrhage, and edema, and exhibited more severe peribronchiolitis and bronchopneumonia (SI Appendix, Fig. S8A). All four G4 viruses replicated to higher titers in the upper respiratory tract (nasal turbinate and trachea) of ferrets, which were similar to pdm/09 viruses and significantly higher than the two G1 viruses (P < 0.05 or P < 0.01, ANOVA), while no infectious virus was recovered from extrapulmonary tissues (SI Appendix, Fig. S8B). Overall, current G4 reassortant EA H1N1 viruses showed increased replication and pathogenicity in ferrets, indicating that G4 viruses are likely to cause more severe infection than G1 EA H1N1 viruses in humans.

Efficient human-to-human transmission is a critical feature of pandemic influenza viruses. To assess the transmissibility of G4 viruses, we performed direct contact (DC) and respiratory droplet (RD) virus transmission experiments on ferrets. The results showed that CA04 pdm/09 H1N1 virus efficiently transmitted to all ferrets by DC and RD (Fig. 3). All four G4 viruses were transmitted to all DC animals. Importantly, three of four G4 viruses, SW/SD/1207/16, SW/HN/SN13/18, and SW/JS/J004/18, were transmitted to all three RD ferrets. The remaining G4 virus, SW/HB/0116/17, was transmitted to one of three RD ferrets (Fig. 3). By contrast, with G1 viruses, neither virus transmission in DC or RD groups (Fig. 3) nor seroconversion at 14 day pi was detected in all recipient ferrets (SI Appendix, Table S4). Thus, there is compelling evidence to show that current predominant G4 reassortant EA H1N1 viruses are highly transmissible by DC and RD among ferrets, suggesting their capacity to readily infect humans.

Preexisting immunity can protect humans from related influenza viruses, but antigenic drift can decrease such protection in a population. Antigenic change is mainly due to variation of the HA gene. In this study, we found that the HA gene of EA H1N1 viruses isolated after 2013, including G4 viruses, formed an independent phylogenetic group. To determine the extent of antigenic drift of G4 viruses, 14 representative EA H1N1 viruses (10 G4 and 4 G1 viruses) were selected, based on their HA phylogenic topology, for antigenicity test.

A panel of ferret sera were used for hemagglutination inhibition (HI) assays, including sera against pdm/09 H1N1 virus A/Michigan/45/2015 from the current H1N1 human influenza vaccine lineage, G1 EA H1N1 viruses SW/HN/08/11 and SW/HB/T37/13, and G4 EA H1N1 viruses SW/SD/1207/16 and SW/HN/SN13/18. On the basis of reactivity levels in HI assays, EA viruses could be classified into antigenic groups A and B (Fig. 1B and SI Appendix, Table S5). The original G1 EA viruses were in antigenic group A, while G4 viruses belonged to antigenic group B. The cross-reactive titers between the two antigenic groups were 8- to 64-fold lower than those of homologous reactions. Antisera against pdm/09 H1N1 viruses (A/Michigan/45/2015) cross-reacted with antigenic group A viruses (titers 1:160 to 320) but reacted poorly with antigenic group B strains (titers ≤ 40) (SI Appendix, Table S5). Further analysis showed several amino acid differences in the HA antigenic sites among G1 and G4 EA H1N1 viruses, including 135 (H1 numbering) and 222 in Ca2, and 185 in Sb (SI Appendix, Table S6). However, which amino acid contributes to the observed antigenic change needs to be determined in future. Thus, predominant G4 reassortant EA H1N1 viruses are antigenically distinct from the earlier G1 EA and pdm/09 H1N1 viruses.

To assess cross-protection of human seasonal influenza vaccine against G4 EA viruses, HI assays were performed with 20 serum samples collected from 4-y-old children vaccinated with trivalent vaccines (pdm/09 H1N1+ H3N2 + B/Victoria). All serum samples were reactive (titers ≥ 1:40) to pdm/09 H1N1 and H3N2 viruses (Fig. 1C). However, none of the serum samples cross-reacted with the G4 or even the G1 EA H1N1 virus (Fig. 1C). Collectively, predominant G4 reassortant EA viruses are antigenically distinct from current human influenza vaccine strains, indicating that preexisting immunity derived from the present human seasonal influenza vaccines cannot provide protection against G4 viruses.

To determine whether the G4 reassortant EA H1N1 virus can infect across species from swine to humans, serological surveillance was conducted to detect prevalence of virus exposure in swine production workers. From 2016 to 2018, a total of 338 serum samples were collected from swine workers in 15 farms. Serum samples (n = 230) from ordinary households were also collected as a population comparison group. G4 EA virus SW/SD/1207/16, which belonged to antigenic group B, was used as virus antigen in HI assays. To control for interference of H1N1 antibody against pdm/09 and earlier G1 EA viruses, pdm/09 virus A/Michigan/45/2015 and G1 EA virus SW/HN/08/11 were included as viral antigens. Disconcertingly, 10.4% (35/338) of swine workers and 4.4% (10/230) of general population were positive (titers ≥ 1:40) for G4 virus SW/SD/1207/16. In the multivariable analysis, after adjusting for confounders, swine workers had an increased odds ratio (aOR = 2.60, 95% CI [1.24 to 5.45], P = 0.012) compared with the general population group. After controlling for possible cross-reactivity with pdm/09 virus, the odds ratio remained elevated (aOR = 2.25, 95% CI [1.05 to 4.83], P = 0.038) (Table 1). It is noted that swine workers in 4 of 15 farms were more than 15% seropositive against G4 virus SW/SD/1207/16 (SI Appendix, Table S7). In contrast, 6.5% (22/338) of swine workers and 2.2% (5/230) of the general population were positive for G1 virus SW/HN/08/11, with no statistically significant difference (P = 0.068) between the two groups after controlling for possible cross-reactivity with pdm/09 virus (Table 1). In addition, the swine workers group and general population were 38.8% (131/338) and 31.7% (73/230) seropositive, respectively, for pdm/09 H1N1 virus A/Michigan/45/2015 (P = 0.082). These results demonstrate that the newly prevalent G4 reassortant EA H1N1 viruses in pigs are more infectious to humans than their predecessors of G1 viruses.

We further investigated the association of sera collection year, age, or gender with seroprevalence of G4 reassortant EA H1N1 virus. In the swine workers group, the seropositive rates of G4 EA H1N1 virus were 6.7%, 11.7%, and 11.7% from 2016, 2017, and 2018, respectively (SI Appendix, Table S8). It is noteworthy that participants 18 y to 35 y of age had a 20.5% (9/44) seropositive rate against G4 EA H1N1 virus SW/SD/1207/16, which had an increased odds ratio (OR = 3.2, 95% CI [1.3 to 7.7], P < 0.01) compared with other age groups (SI Appendix, Table S8). For gender factor, no statistically significant difference in the seroprevalence of any virus tested according to sex was observed (P > 0.05). These results indicate that young adult swine workers carry a higher risk of infection with G4 reassortant EA H1N1 virus.

The sequences generated in this study have been deposited in the GenBank database (accession nos. are listed in SI Appendix, Table S3).