The Ongoing Epidemics of Seasonal Influenza A(H3N2) in Hangzhou, China, and Its Viral Genetic Diversity

Abstract

This study examined the genetic and evolutionary features of influenza A/H3N2 viruses in Hangzhou (2010-2022) by analyzing 28,651 influenza-like illness samples from two sentinel hospitals. Influenza A/H3N2 coexisted with other subtypes, dominating seasonal peaks (notably summer). Whole-genome sequencing of 367 strains was performed on GridION platforms. Phylogenetic analysis showed they fell into 16 genetic groups, with multiple clades circulating simultaneously. Shannon entropy indicated HA, NA, and NS gene segments exhibited significantly higher variability than other genomic segments, with HA glycoprotein mutations concentrated in antigenic epitopes A-E. Antiviral resistance showed no inhibitor resistance mutations in PA, PB1, or PB2, but NA mutations were detected in some strains, and most strains harbored M2 mutations. A Bayesian molecular clock showed the HA segment exhibited the highest nucleotide substitution rate (3.96 × 10^-3 substitutions/site/year), followed by NA (3.77 × 10^-3) and NS (3.65 × 10^-3). Selective pressure showed A/H3N2 strains were predominantly under purifying selection, with only sporadic positive selection at specific sites. The Pepitope model demonstrated that antigenic epitope mismatches between circulating H3N2 variants and vaccine strains led to a significant decline in influenza vaccine effectiveness (VE), particularly in 2022. Overall, the study underscores the complex circulation patterns of influenza in Hangzhou and the global importance of timely vaccine strain updates.

Keywords: Bayesian molecular clock; Pepitope model; Shannon entropy; antiviral resistance; influenza A/H3N2; phylogenetic analysis; selective pressure; whole-genome sequencing.

Figure 1

The prevalence of influenza-like illness (ILI) cases at two sentinel hospitals in Hangzhou, China, during the 2010–2022 seasons. (A) Distribution of influenza-like illness cases sampled monthly during a 13-year surveillance period. (B) The positivity rate of the currently circulating subtypes of human seasonal influenza among influenza-like cases; sixteen wave influenza virus epidemics are seen (W1, 2010–2011 summer; W2, 2011–2012 winter; W3, 2012–2013 summer; W4, 2012–2013 winter; W5, 2013–2014 winter; W6, 2014–2015 summer; W7, 2015–2016 summer; W8, 2015–2016 winter; W9, 2016–2017 winter; W10, 2017–2018 summer; W11, 2017–2018 winter; W12, 2018–2019 winter; W13, 2019–2020 summer; W14, 2019–2020 winter; W15, 2022–2023 summer; W16, 2022–2023 winter). Blue font, winter influenza peaks; red font, summer influenza peaks. (C) The sequencing number of A(H3N2) virus-positive samples between 2010 and 2022.

Figure 2

The phylogenetic tree of the HA gene of influenza A(H3N2) virus from local circulating influenza A(H3N2) strains in Hangzhou between 2010 and 2022. The WHO-recommended vaccine strains of the northern hemisphere and reference clades from GISAID were included and annotated in the trees. The tree was constructed by the maximum likelihood method using MEGA v11.0.11 and visualized using the iTOL. Maximum likelihood phylogenies were estimated by the Hasegawa–Kishino–Yano model (HKY) and gamma distribution with bootstrap analysis (1000 replicates). The tree was rooted with A/Victoria/208/2009 as an outgroup.

Figure 3

Shannon entropy for each genome segment of influenza A/H3N2 strains in Hangzhou, 2010–2022. Each bar represents the Shannon entropy value of each amino acid position in each genome segment. The Shannon entropy value of positions in known antigenic epitope regions A (red; amino acid residues 122, 124, 126, 130–133, 135, 137, 138, 140, 142–146, 150, 152, 168), B (blue; residues 128, 129, 155–160, 163, 165, 186–190, 192–194, 196–198), C (yellow; residues 44–48, 50, 51, 53, 54, 273, 275, 276, 278–280, 294, 297, 299, 300, 304, 305, 307–312), D (green; residues 96, 102, 103, 117, 121, 167, 170–177, 179, 182, 201, 203, 207–209, 212–219, 226–230, 238, 240, 242, 244, 246–248), and E (pink; residues 57, 59, 62, 63, 67, 75, 78, 80–83, 86–88, 91, 92, 94, 109, 260–262, 265) of HA protein in the dominant circulating strains were labeled. The amino acid numbering is counted without the signal peptide.

Figure 4

P-distance and prediction of vaccine efficacy against influenza A/H3N2 strains in Hangzhou, 2010–2022. Annual vaccine candidates from corresponding epidemics are separated by a dotted line. (A) The P-distance values of the HA1 substitutions on epitope responsible for vaccine mismatch. (B) Prediction of potential vaccine efficacy (VE) against the circulating influenza A/H3N2 strains.