Reassortment with dominant chicken H9N2 influenza virus contributed to the fifth H7N9 virus human epidemic

Abstract

H9N2 Avian influenza virus (AIV) is regarded as a principal donor of viral genes through reassortment to co-circulating influenza viruses that can result in zoonotic reassortants. Whether H9N2 virus can maintain sustained evolutionary impact on such reassortants is unclear. Since 2013, avian H7N9 virus had caused five sequential human epidemics in China; the fifth wave in 2016-2017 was by far the largest but the mechanistic explanation behind the scale of infection is not clear. Here, we found that, just prior to the fifth H7N9 virus epidemic, H9N2 viruses had phylogenetically mutated into new sub-clades, changed antigenicity and increased its prevalence in chickens vaccinated with existing H9N2 vaccines. In turn, the new H9N2 virus sub-clades of PB2 and PA genes, housing mammalian adaptive mutations, were reassorted into co-circulating H7N9 virus to create a novel dominant H7N9 virus genotype that was responsible for the fifth H7N9 virus epidemic. H9N2-derived PB2 and PA genes in H7N9 virus conferred enhanced polymerase activity in human cells at 33°C and 37°C, and increased viral replication in the upper and lower respiratory tracts of infected mice which could account for the sharp increase in human cases of H7N9 virus infection in the 2016-2017 epidemic. The role of H9N2 virus in the continual mutation of H7N9 virus highlights the public health significance of H9N2 virus in the generation of variant reassortants of increasing zoonotic potential.IMPORTANCEAvian H9N2 influenza virus, although primarily restricted to chicken populations, is a major threat to human public health by acting as a donor of variant viral genes through reassortment to co-circulating influenza viruses. We established that the high prevalence of evolving H9N2 virus in vaccinated flocks played a key role, as donor of new sub-clade PB2 and PA genes in the generation of a dominant H7N9 virus genotype (G72) with enhanced infectivity in humans during the 2016-2017 N7N9 virus epidemic. Our findings emphasize that the ongoing evolution of prevalent H9N2 virus in chickens is an important source, via reassortment, of mammalian adaptive genes for other influenza virus subtypes. Thus, close monitoring of prevalence and variants of H9N2 virus in chicken flocks is necessary in the detection of zoonotic mutations.

FIG 1

Phylogenetic analysis of H9N2 influenza viruses isolated from chickens. (A) HA phylogenetic tree of H9N2 influenza viruses from 2010 to 2017 denoted by different colors. Viruses labeled with a red dot were selected for HI assays. (B) PB2 phylogenetic tree of H9N2 influenza viruses. (C) Abundance of the subgenotypes of H9N2 influenza virus during the period from 2014 to 2017. The top graph depicts the percentages of strains of the five most dominant subgenotypes in each year; the bottom graph depicts the total numbers of subgenotypes in each year from 2014 to 2017. The total numbers of H9N2 viruses in each year are 72, 101, 29, and 41.

FIG 2

Antigenic cartography representation of the HI data generated by a panel of chicken antisera. The map was drawn by performing multidimensional scaling (MDS) with downscaling of the HI data to three-dimensional space after log₂ transformation, using K-means clustering. Circles and triangles represent the locations of strains classified as belonging to the F and G antigen groups, respectively. Different colors represent different years of isolation. Virus names are indicated by using their abbreviations, and details of the HI data are shown in Table S2 in the supplemental material.

FIG 3

Rate of isolation (percent) of H9N2 influenza viruses in chicken flocks reporting illness and number of human H7N9 cases in the five epidemic waves. Red horizontal lines (with connecting dots) indicate mean annual isolation rates, and gray horizontal dashed lines indicate 95% confidence intervals (CIs). The 2013 H9N2 isolation rate data obtained during the first epidemic wave are from our previous study (8). The total numbers of human cases from wave 1 to wave 5 were 135, 320, 226, 119, and 758, respectively.

FIG 4

Genetic evolution of H7N9 influenza viruses from epidemic wave 1 through wave 5. (A) Abundance of different genotypes of H7N9 influenza virus. The top graph shows the rate of prevalence of the five most abundant genotypes, and the bottom graph illustrates the total number of genotypes in each wave. (B) Number of G13 and G72 H7N9 strains in each month from 2013 to 2017 in humans. The first isolation of G72 H7N9 virus was in January 2015 during the third wave. The total numbers of G72 viruses isolated from humans in the third, fourth, and fifth waves were 3, 15, and 286, respectively. (C) Genomic constitution of G13 and G72 H7N9 viruses. Virus particles are represented by ovals. The eight gene segments are horizontal bars (from the top, PB2, PB1, PA, HA, NP, NA, MP, and NS). Scarlet bars represent the internal segments in the G72 genotype that are different from the G13 H7N9 viruses.

FIG 5

Genetic relatedness of H7N9 and H9N2 influenza viruses in PB2 and PA genes. (A) PB2 phylogenetic tree of H7N9 and H9N2 influenza viruses. On the right side of the tree, PB2 genes of G13 H7N9, G72 H7N9, and 6.2 subclade H9N2 viruses are denoted by different-colored bars. (B) PA phylogenetic tree of H7N9 and H9N2 influenza viruses. On the right side of the tree, PA genes of G13 H7N9, G72 H7N9, and 3.1 subclade H9N2 viruses are denoted by different-colored bars. (C) Prevalence of PB2-6.2 and PA-3.1 in H7N9 and H9N2 viruses over time. (D) Prevalence of critical amino acid residues encoded by the PB2 and PA genes of the indicated H7N9 and H9N2 viruses. Red indicates high-prevalence (up to 100%) substitutions, and blue indicates no mutation or no virus isolated in the given year.

FIG 6

Contribution of H9N2-derived PB2 and PA genes to polymerase activity in H7N9 virus. Viral polymerase activities in 293T cells (A) were determined by minigenome replication assays at 33°C and 37°C, and those in DF-1 cells (B) were determined at 37°C and 39°C (expressed as mean percentages ± standard deviations, with the activity of the corresponding wild-type G13 H7N9 virus being set to 100%, from three independent experiments). Test RNP complexes were variants of a G13 H7N9 virus with the indicated substitutions of PB2 and PA genes. rG13:PB2/PA-H9N2 refers to the RNP complex with the PB2 and PA genes from an H9N2 virus; rG13:PB2/PA-G72-1 and rG13:PB2/PA-G72-2 refer to RNP complexes with the PB2 and PA genes from different G72 H7N9 viruses. All substituted genes were from the H9N2-derived PB2-6.2 and PA-3.1 subclades. Details of segment and viral information for these reassortants are shown in Table S5 in the supplemental material. Statistical significance was based on one-way ANOVA (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). EV, empty vector.

FIG 7

Contribution of PB2 and PA genes of H9N2 virus origin to infectivity of H7N9 virus in mice. (A) Weight loss and percent survival of mice (n = 5) inoculated with 10⁶ TCID₅₀ of each virus. Mice that lost >25% of their baseline weight were euthanized. (B) Virus production from nasal turbinates and lungs. Five mice from each group were euthanized at 3 and 5 dpi to determine viral titers in nasal turbinates and lung tissues. Statistical significance was based on one-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (C) Representative histopathological changes in lung sections at 3 dpi by hematoxylin and eosin (H&E) staining (left) and immunodetection of influenza viral NP antigen (right). Mice infected with rG13:PB2/PA-H9N2, rG13:PB2/PA-H7N9-1, and rG13:PB2/PA-H7N9-2 viruses presented with more severe histopathology and a greater abundance of viral antigen-positive cells in the lung fields.