New Threats from H7N9 Influenza Virus: Spread and Evolution of High- and Low-Pathogenicity Variants with High Genomic Diversity in Wave Five

Abstract

H7N9 virus has caused five infection waves since it emerged in 2013. The highest number of human cases was seen in wave 5; however, the underlying reasons have not been thoroughly elucidated. In this study, the geographical distribution, phylogeny, and genetic evolution of 240 H7N9 viruses in wave 5, including 35 new isolates from patients and poultry in nine provinces, were comprehensively analyzed together with strains from first four waves. Geographical distribution analysis indicated that the newly emerging highly pathogenic (HP) and low-pathogenicity (LP) H7N9 viruses were cocirculating, causing human and poultry infections across China. Genetic analysis indicated that dynamic reassortment of the internal genes among LP-H7N9/H9N2/H6Ny and HP-H7N9, as well as of the surface genes, between the Yangtze and Pearl River Delta lineages resulted in at least 36 genotypes, with three major genotypes (G1 [A/chicken/Jiangsu/SC537/2013-like], G3 [A/Chicken/Zhongshan/ZS/2017-like], and G11 [A/Anhui/40094/2015-like]). The HP-H7N9 genotype likely evolved from G1 LP-H7N9 by the insertion of a KRTA motif at the cleavage site (CS) and then evolved into 15 genotypes with four different CS motifs, including PKGKRTAR/G, PKGKRIAR/G, PKRKRAAR/G, and PKRKRTAR/G. Approximately 46% (28/61) of HP strains belonged to G3. Importantly, neuraminidase (NA) inhibitor (NAI) resistance (R292K in NA) and mammalian adaptation (e.g., E627K and A588V in PB2) mutations were found in a few non-human-derived HP-H7N9 strains. In summary, the enhanced prevalence and diverse genetic characteristics that occurred with mammalian-adapted and NAI-resistant mutations may have contributed to increased numbers of human infections in wave 5.IMPORTANCE The highest numbers of human H7N9 infections were observed during wave 5 from October 2016 to September 2017. Our results showed that HP-H7N9 and LP-H7N9 had spread virtually throughout China and underwent dynamic reassortment with different subtypes (H7N9/H9N2 and H6Ny) and lineages (Yangtze and Pearl River Delta lineages), resulting in totals of 36 and 3 major genotypes, respectively. Notably, the NAI drug-resistant (R292K in NA) and mammalian-adapted (e.g., E627K in PB2) mutations were found in HP-H7N9 not only from human isolates but also from poultry and environmental isolates, indicating increased risks for human infections. The broad dissemination of LP- and HP-H7N9 with high levels of genetic diversity and host adaptation and drug-resistant mutations likely accounted for the sharp increases in the number of human infections during wave 5. Therefore, more strategies are needed against the further spread and damage of H7N9 in the world.

Keywords: H7N9; HPAIV; avian influenza virus; dynamic reassortment; evolution; genetic diversity; origin; wave five.

FIG 1

The temporal and spatial distributions of H7N9 human infections during wave 5. (A) The numbers of human H7N9 cases in each wave are listed by month. The fatality rates in January of wave 2 to 5 are shown. (B) Three provinces (QH, NX, and HaiN) without human or poultry H7N9 infections during wave 5 are colored in dark gray. The other provinces reporting human or poultry H7N9 infections are colored in red or faint yellow. Red indicates that HP-H7N9 was found in these provinces. The blue border indicates the new H7N9 viruses isolated from poultry and humans by our group. The numbers of HP- and LP-H7N9 strains analyzed in the present study are listed within the brackets as indicated in the provinces. Abbreviations in the map are as follows: AH, Anhui; BJ, Beijing; CQ, Chongqing; FJ, Fujian; GD, Guangdong; GS, Gansu; GX, Guangxi; GZ, Guizhou; HaiN, Hainan; HB, Hebei; HK, Hong Kong; HLJ, Heilongjiang; HN, Henan; HuB, Hubei; HuN, Hunan; IM, Inner Mongolia; JL, Jilin; JS, Jiangsu; JX, Jiangxi; LN, Liaoning; MC, Macao; NX, Ningxia; QH, Qinghai; SaX, Shaanxi; SC, Sichuan; SD, Shandong; SH, Shanghai; SX, Shanxi; TB, Tibet; TJ, Tianjin; TW, Taiwan; XJ, Xinjiang; YN, Yunnan; ZJ, Zhejiang.

FIG 2

Distinctive amino acid variations in HA and NA of HP-H7N9 compared to LP-H7N9. The amino acid sequences of HA and NA were aligned between HP-H7N9 and the consensus sequences of LP-H7N9. The specific residues in HA and NA are listed. “X” of A/GX/18902/2017 at site 235 indicates an H or L residue, and “X” of A/GD/17SF064/2017 at site 289 indicates a K or R residue. The numbers in brackets represent the numbers of HP-H7N9 strains. CK, chicken; DK, duck; EN, environment; GD, Guangdong; GX, Guangxi; HuN, Hunan; HY, Heyuan; HZ, Huizhou; SZ, Shenzhen.

FIG 3

Phylogenies of HA and NA genes of H7N9 viruses from different waves. All of the H7N9 viruses from waves 1 to 5, including our 35 new isolates, were used to perform the phylogenetic analysis. Panel A shows the HA gene tree, and the HP-H7N9 viruses in panel A were magnified as shown in panel B. Panel C shows the NA gene tree. The HP strains with different NA lineages are marked as stars with different colors in panels B and C; stars with the same color represent the same strain. H7N9 viruses from waves 1, 2, 3, 4, and 5 are colored blue, red, orange, light green, and lime green, respectively.

FIG 4

Schematic representation of the evolutionary pathway of the H7N9 viruses during wave 5. (A) The schematic evolutionary pathway of H7N9 in wave 5 on the basis of the timeline of the first and last detection for each genotype during wave 5. Three dominant genotypes (G1, G3, and G11) were expressed as bigger “virions.” The eight gene segments (shown as horizontal bars starting from top to bottom of the “virion”) are PB2, PB1, PA, HA, NP, NA, M, and NS. Different colors of gene segments represent different virus lineages. (B) The dominant genotypes, the genotypes that included more than eight strains, and the transient genotypes are listed along with their isolation times. Symbols represent the corresponding genotypes of the H7N9 viruses.