
==== Front
Emerg Microbes Infect
Emerg Microbes Infect
Emerging Microbes & Infections
2222-1751
Taylor & Francis

39193626
2396867
10.1080/22221751.2024.2396867
Version of Record
Emerging Seasonal and Pandemic Influenza Infections
Research Article
Whole-genome analysis of circulating influenza A virus (H3N2) strains in Shanghai, China from 2005 to 2023
EMERGING MICROBES & INFECTIONS
X. ZHAO ET AL.
https://orcid.org/0000-0003-1818-9457
Zhao Xue a*
Gu Yijing a*
Tang Xiaode c*
Jiang Chenyan a
Fang Fanghao a
Chu Wei d
Tao Lixin e
Zhang Xi a
Chen Min a
Wu Huanyu a
Xie Youhua b
https://orcid.org/0000-0002-9728-8893
Liu Jing b*
https://orcid.org/0000-0002-9901-7376
Teng Zheng a
a Virus Testing Laboratory, Pathogen Testing Center, Shanghai Municipal Center for Disease Control and Prevention, Shanghai, People’s Republic of China
b Key Laboratory of Medical Molecular Virology (MOE/MOH/CAMS) and Shanghai Key Laboratory of Medical Epigenetics, Department of Microbiology and Parasitology and Institutes of Biomedical Sciences, School of Basic Medical Sciences and Shanghai Institute of Infectious Diseases and Biosecurity, Shanghai Medical College, Fudan University, Shanghai, People’s Republic of China
c Microbiological Testing Department, Shanghai Baoshan District Center for Disease Prevention and Control, Shanghai, People’s Republic of China
d Microbiological Laboratory, Shanghai Huangpu District Center for Disease Prevention and Control, Shanghai, People’s Republic of China
e Microbiological Laboratory, Shanghai Fengxian District Center for Disease Prevention and Control, Shanghai, People’s Republic of China
CONTACT Jing Liu liujing212@fudan.edu.cn Key Laboratory of Medical Molecular Virology (MOE/MOH/CAMS) and Key Laboratory of Medical Epigenetics and Metabolism (Shanghai), Shanghai Institute of Infectious Diseases and Biosecurity and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, 138 Yixue Yuan Road, Xuhui District, Shanghai, People’s Republic of China
Zheng Teng tengzheng@scdc.sh.cn Virus Testing Laboratory, Pathogen Testing Center, Shanghai Municipal Center for Disease Control and Prevention, No. 1380 Zhongshan West Road, Changning District, Shanghai, People’s Republic of China
* X.Z., Y.G., X.T., and J.L. contributed equally to this article.

Supplemental data for this article can be accessed online at https://doi.org/10.1080/22221751.2024.2396867.

28 8 2024
2024
28 8 2024
13 1 239686711 6 2024
27 7 2024
22 8 2024
Nova techset28 8 2024
Converted to JATS 1.2 by Nova Techset28 8 2024
© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., Ltd
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

Seasonal influenza A virus subtype H3N2 (A/H3N2) circulates globally and has been linked to higher hospitalization rates and summer outbreaks in temperate regions. Here, A/H3N2 circulation in Shanghai, China was systematically studied using data and materials generated by the Shanghai influenza surveillance network from 2005 to 2023. Time-series analysis of incidence and subtyping data showed that A/H3N2 co-circulated with other (sub)types and dominated in multiple seasonal influenza peaks, preferentially in summer. Whole genomes of 528 representative strains were sequenced, and spatiotemporal phylodynamic analysis using these and GISAID-archived sequences demonstrated that in the years before the COVID-19 pandemic, phylogenetically similar strains were circulating locally and elsewhere. However, clade 1a.1 (within 3C.2a.1b.2a), circulated in and only in Shanghai and domestically in 2022, while the sibling clade 2 predominated in other regions. Interestingly, clade 1a.1 was swiftly and completely replaced by clade 2, mostly 2a.3a.1, at the start of 2023. In hemagglutination inhibition and neutralization assays, sera from healthy donors collected in 2022 displayed higher or similar reactivity against 2a.3a.1 compared to 1a.1. By contrast, transcription and replication competence of 2a.3a.1 in MDCK cells was higher than 1a.1. These results indicated that instead of antigenicity differences enabling evasion of pre-existing immunity, higher replicative capability more likely contributed to 2a.3a.1 viruses achieving dominance in China. In addition to summarizing patterns of A/H3N2 local circulation in Shanghai, this work revealed an unusual episode in A/H3N2 global circulation and evolution dynamics in connection to the COVID-19 pandemic and explored possible mechanistic explanations.

KEYWORDS

Phylodynamic analysis
virus evolution
hemagglutination inhibition
neutralization
CPE
the key research project of Three-Year Initiative Plan for Strengthening Public Health System Construction in Shanghai This work was supported by Three-Year Initiative Plan for Strengthening Public Health System Construction in Shanghai (2023-2025) (GWVI-3, GWVI-11.1-09), and National Key Scientific Instrument and Equipment Development Projects of China, NSFC (22127806).
==== Body
pmcIntroduction

Influenza viruses are enveloped viruses with segmented, negative-sense RNA genomes, and among the four known types (A-D), influenza A virus (IAV) and influenza B virus (IBV) cause influenza syndrome after infecting humans [1]. Both IAV and IBV circulate in human populations globally and display different seasonality patterns depending on geographic location. In regions with typical temperate climates, IAV/IBV mostly causes seasonal outbreaks or epidemics in winter [2,3]. Two subtypes of IAV, H1N1 (2009pdm) and H3N2, as well as two lineages of IBV, Victoria and Yamagata, have been the major circulating influenza viruses [4]. World Health Organization estimated that approximately 5–15% of the global population is infected with seasonal influenza viruses each year, including 3–5 million severe cases and approximately 250,000–650,000 deaths [5].

Each of the 8 segments of the IAV/IBV genome encodes at least one viral protein and is named accordingly. The two major viral envelope proteins, haemagglutinin (HA) and neuraminidase (NA), are encoded by the two corresponding genome segments and mediate attachment-entry and receptor degradation, respectively [6]. HA and NA are also the main targets of host humoral immune responses against IAV/IBV, and HA antibodies could show neutralizing or protective effects in natural infections [7]. Antigenic changes in these envelope proteins, through mutation and/or reassortment, may enable the viruses to escape pre-existing immunity in a population and cause epidemics or pandemics. Consequently, circulating IAV and IBV variants are under continuous surveillance globally, and the results are used to guide regular updates to seasonal influenza vaccines [8].

The currently circulating H3N2 subtype of IAV (A/H3N2) entered the human population in 1968 as a reassortant between previously circulating seasonal A/H2N2 virus and an avian A/H3 virus [9,10]. The novel and highly transmissible virus caused a global pandemic with widespread illness and mortality, and its descendants have since been circulating as a seasonal virus [11]. Clinically, seasonal A/H3N2 viruses have been linked to higher attack rates and hospitalizations compared to other (sub)types [12–14]. In addition, A/H3N2 has been associated with unusual influenza outbreaks or epidemics that occur in summer in temperate regions [15–19].

Shanghai is a population centre and domestic/international transport hub with a typical temperate climate. As part of the China Influenza Surveillance Network System [20], the city has been conducting systematic influenza surveillance since 2005 through the Shanghai influenza surveillance network. Similar to other parts of mainland China, strict COVID-19-related non-pharmaceutical intervention (NPI) policies [21,22] were implemented in Shanghai during the pandemic up until the end of 2022. This could have significantly modified local influenza circulation patterns due to the shared transmission routes between COVID-19 and influenza. On the other hand, restrictions on international travel as well as stringent screening and quarantine of international travellers would have also limited the international spread of influenza virus variants, due to shared early symptoms such as fever.

In this work, we first summarized and reviewed influenza surveillance data obtained through the Shanghai network from 2005 to 2023, focusing on A/H3N2 viruses and epidemic seasons during and around the COVID-19 pandemic. To reveal characteristics of A/H3N2 variants circulating in Shanghai, we performed whole-genome sequencing on more than 500 A/H3N2 clinical strains isolated by the Shanghai network between 2005 and 2023 and subjected the sequence data to spatiotemporal phylodynamic analysis along with A/H3N2 sequences in GISAID archive. Among other observations, we identified an A/H3N2 subclade, 1a.1 within 3C.2a.1b.2a, that exclusively circulated in Shanghai during 2022, but was swiftly and completely replaced by an internationally circulating sibling subclade 2 at the start of 2023. To probe factors underlying this unique event, hemagglutination and neutralization assays were performed using sera collected from healthy donors in 2022 and 2023 to assess possible differences in reactivity to the two subclades. Finally, the two subclades in question were compared with regard to CPE development, viral transcription and replication in infected cells.

Materials and methods

Ethics statement

This study was approved by the Ethics Review Committee of the Shanghai Municipal Center for Disease Control and Prevention (No.2022-29), and did not involve experimentation on humans.

Clinical respiratory specimens and isolation of influenza viruses

This study was conducted within the framework of the Shanghai influenza surveillance network following the guidelines of the National Influenza Surveillance Program of China (Editions 2000, 2010, and 2017). Influenza-like illness (ILI) was defined as fever (body temperature ≥ 38.0°C) accompanied by either cough or sore throat. Nasopharyngeal swab specimens were collected from ILI patients admitted to sentinel hospitals enrolled in the network and transported to corresponding network laboratories for multiplex quantitative real-time PCR testing of seasonal and pandemic influenza viruses (JC10202NW, Bioperfectus, China) within 2 working days. Influenza-positive samples were then subjected to virus isolation using Madin-Darby canine kidney (MDCK) cells and/or embryonated SPF chicken eggs (Beijing Boehringer Ingelheim Vital Biotechnology, China) within 24 hours. Virus culture was subjected to hemagglutination (HA) and hemagglutination inhibition (HAI) tests using reference viruses and antibodies for subtype and titre determination. Isolates with a titre greater than 8 HA units were transported to the laboratory of the Shanghai Municipal Centre for Disease Control and Prevention (SMCDCP) for confirmation and preservation. A total of 3028 A/H3N2 isolates from 16 district-level network laboratories were collected between 1 September 2005 and 31 July 2023. For maximal spatio-temporal coverage of the circulation A/H3N2 viruses, the isolates were first grouped by sample collection year and month. A subset of isolates was then selected from each group while ensuring that every district lab in the group contributed at least one isolate, and labs with markedly more than average isolates in the group proportionally contributed two or more isolates. A total of 530 isolates were thus selected, and subjected to high-throughput whole-genome sequencing.

Serum samples

Two panels of serum samples collected within the framework of the Shanghai Influenza Surveillance Program (2016 Edition) were used for testing reactivity against reference and circulating A/H3N2 strains. The Program mandates that at least 150 serum samples from healthy donors covering all age groups, regardless of influenza vaccination status, be collected between June and November every year and tested for HI titres against A/H1N1(pdm09), A/H3N2, B/Victoria and B/Yamagata by district branches of SMCDCP. All samples were collected with written informed consent from donors or their guardians and stored at −80°C until testing. A panel consisting of 150 serum samples collected from the Baoshan district between 19 August and 24 September 2022 and another panel of 150 samples from the Fengxian district collected between 14 June and 24 June 2023 were used in this work.

Whole-genome sequencing of A/H3N2 isolates

Total nucleic acids were extracted from virus culture media using MagNA Pure 96 System (Roche, USA) and subjected to amplification using One Step RT–PCR Kit (12574-026, Invitrogen, USA). Uni/Inf primer set for amplifying all 8 segments of influenza virus genome [23] was used for amplification in 50 uL reactions containing 14 μL nuclease-free water, 25 μL 2× Reaction buffer, 0.4 μL Uni12/Inf1 (10 μmol/L), 0.6 μL Uni12/Inf3 (10 μmol/L), 1 μL Uni13/Inf1 (10 μmol/L), 1 μL RT/High Fidelity enzyme. The following thermal profile was used: 45°C for 60 min; 94°C for 2 min; 5 cycles of 30 s at 94°C, 30 s at 44°C, 3 min at 68°C; 31 cycles of 30 s at 94°C, 30 s at 57°C, 3 min at 68°C with a final extension of 7 min at 68°C. Amplicons were quantified using Quant-iT dsDNA High Sensitivity Assay (Q33231, Invitrogen, China) and diluted to 2 ng/uL. Indexed paired-end libraries were prepared using Nextera XT Sample Preparation Kit (Illumina, China) following the manufacturer’s protocol. Prior to sequencing, the amplified libraries were checked for quantity and quality using Qubit 4.0 Green double-stranded DNA assay and a bioanalyzer (Agilent 2100, USA). The libraries were pooled in equimolar ratios and sequenced using the Illumina Novaseq 6000 system (Illumina, USA) according to the manufacturer’s instructions. In total, whole genome sequences of 528 H3N2 isolates were successfully obtained and deposited in GenBank (Accession Nos. OR717627-OR721850).

Sequence alignment and phylogenetic analysis

Multiple sequence alignment was performed using the Multiple Alignment Program for Amino Acid or Nucleotide Sequences (MAFFT, version 7.427) and alignments were minimally edited in Molecular Evolutionary Genetic Analysis (MEGA, version 7.0). Sequences and metadata associated with A/H3N2 strains available at GISAID [24] up to 27 August 2023 were retrieved and de-duplicated based on strain name. After discarding sequences derived from 19 strains sequenced in this work that were previously deposited in GISAID, sequences of a total of 81989 strains were used for phylodynamic analysis using Nextstrain (build-20231009T203029Z) [25] workflow for seasonal influenza A/H3N2 viruses (https://github.com/nextstrain/seasonal-flu/, revision 16b66fbd3029d6c9511a6751b90aee6f7f0fcd8d). All 528 isolates sequenced in this work, and ∼2000 isolates archived by GISAID that were isolated from mainland China, Hong Kong/Macau/Taiwan (H/M/T) regions of China or other country/regions of the world were used to generate maximum likelihood timetrees in Nextstrain. The GISAID isolates were randomly selected to maximally represent all geographic locations (by country or, in the case of China, by division) and isolation time (by month) combinations by Nextstrain. Generated timetrees were viewed using the built-in auspice function of Nextstrain [26] and provided as raw data along with viewing instructions in Supplementary Data 1. For the exhaustive representation of specific subclades, Nucleotide-Nucleotide BLAST [27] (version 2.14.1+) was used to search for highly similar GISAID sequences, which were then added to Nextstrain analysis to build new timetrees.

Hemagglutination inhibition (HI) and microneutralization (NT) assays

HI assay was performed using reference strain A/Darwin/9/2021(H3N2) and two strains isolated and sequenced in this work, A/Shanghai-FX/1804/2022(H3N2) and A/Shanghai-JD/1390/2023(H3N2). The inactivated reference virus and corresponding antiserum were kindly provided by the Chinese National Influenza Center. HI tests were performed as previously described [28]. Briefly, virus preparations were first titrated and diluted to 4 HA units/25 uL. Serum samples were treated with receptor-destroying enzyme (RDE, 340122, Denka Seiken, Japan) at 37°C for 17–24 hours and heat-inactivated at 56°C for 30 minutes. RDE-treated sera were then subjected to two-fold serial dilution to prepare 1:10 through 1:1280 diluted sera, and mixed with equal volume (25 uL: 25 uL) of viruses and incubated at room temperature for 30 minutes. Hemagglutination was performed by adding 50 uL of 1% guinea pig RBC to each well and incubating at room temperature. HI titer was defined as the highest dilution displaying full inhibition of agglutination, or 1:5 if no inhibition was observed with any of the diluted serum.

Microneutralization assay was carried out by mixing serially diluted sera with 100 TCID50 of viruses, followed by incubation for 1 hour at 37°C and adding to an MDCK cell monolayer. Progeny virus production was measured at 4 days after inoculation using quantitative real-time PCR as described above. NT titer was defined as the highest dilution displaying full inhibition of progeny virus production, or 1:5 if no inhibition was observed with any of the diluted serum.

Viral replication analysis

MDCK cells were infected with A/Shanghai-FX/1804/2022 and A/Shanghai-JD/1390/2023 viruses at a multiplicity of infection (MOI) of 0.02. After 1 h of virus adsorption at room temperature, cells were washed twice with PBS and overlaid with DMEM containing 0.25% BSA, 2% NaHCO3, 25 mM HEPES, 100 IU/ml penicillin and 2 μg/ml TPCK treated trypsin, and cultured at 35°C. At indicated time points, cell morphology was observed, and culture supernatant and cell samples were collected. Progeny virus in the supernatant was measured using quantitative real-time PCR as described above. TCID50 was determined as described previously [29], and expressed as log10 TCID50/ml. Total RNA was extracted from harvested cells and reverse transcribed using the Hot-Start protocol as previously described [30]. Unique tags consisting of an additional 18–20 nucleotides unrelated to the influenza virus were added to distinguish the three types of influenza RNAs: negative-sense viral RNA (vRNA), replication intermediate positive-sense complementary RNA (cRNA), and capped positive-sense viral mRNA [30]. The obtained cDNA was then measured using quantitative PCR on Roche LightCycler 480 using SYBR Green Super Mix (Vazyme Biotech, China).

Data processing and statistical analysis

Reciprocal HI and NT titers were divided by 5 (normalized to titer 1:5), and geometric mean titers (GMT) were calculated before statistical analysis using SPSS (version 25.0).

Results

Circulation pattern of influenza viruses in Shanghai from 2005 to 2023

The Shanghai influenza surveillance network was established circa 2004–2005 as part of the National Influenza Surveillance Network of China. We first performed a time series analysis of influenza-like illness (ILI) incidence data reported by sentinel hospitals in the network between September 2005 and July 2023 (Figure 1(A)). Prior to the onset of the COVID-19 pandemic at the start of 2020, ILI case ratios displayed a seasonal pattern typical for temperate zones: a low and rather stable baseline throughout the year with annual peaks in the winter, usually between December and February (Figure 1(A)). In some years (2007, 2008, 2009, 2010, 2012, 2014, 2015, 2017), an additional summer peak, usually between July and October, was also observed. Such a pattern was disrupted after the 2019–2020 winter peak was over when the COVID-19 pandemic started. No ILI peak was observed in the winter of 2020–2021, and ILI case ratios became especially erratic during early 2022 and the winter of 2022–2023, which coincided with drastic COVID-19 NPI policy changes in Shanghai (Figure 1(A)). Figure 1. Time series analysis of influenza incidence in Shanghai between 2005 and 2023. (A) The average percentage of influenza-like illness (ILI) among out-patients received by sentinel hospitals in the Shanghai influenza surveillance network. (B) Monthly numbers of ILI specimens tested in-network labs and percentages of flu-positive samples. (C) Percentages of listed influenza types and subtypes among flu-positive samples. Blue arrows, winter influenza peaks; red arrows, summer influenza peaks; black arrows, non-influenza ILI peaks.

ILI specimens collected by sentinel hospitals were tested for influenza viruses including influenza A/H1N1, A/H1N1pdm (after 2009), A/H3N2, B/Yamagata and B/Victoria. The results showed that influenza viruses played significant roles in nearly all observed ILI peaks between 2005 and 2023, both in winter and in summer, contributing as high as 60–70% of ILI cases in certain peaks (Figure 1(B)). The few exceptions were two ILI peaks in early 2020 and at the end of 2022, which were largely caused by SARS-CoV-2, and a peak in early 2023, which was mostly attributable to a hike in other respiratory pathogen(s) infections (data not shown).

Influenza virus (sub)typing results demonstrated alternating dominance by different types/subtypes over the epidemic seasons (Figure 1(C)). Notably, over the summer and winter of 2009, the pandemic H1N1(pdm09) lineage replaced the previously circulating seasonal H1N1 lineage and drove the latter out of circulation. On the other hand, the B/Yamagata lineage, which was mainly or partially responsible for influenza peaks in the summer of 2007 as well as in the winters of 2014–2015 and 2017-2018, disappeared from circulation after 2019. Both of these events were simultaneously observed elsewhere [31,32], showing that the local circulation of influenza viruses in Shanghai constitutes an integral part of their global circulation.

Among the influenza (sub)types, A/H3N2 circulated in Shanghai throughout the surveillance period, intermittently dominating a subset of seasonal influenza peaks (Figure 1(B, C)). Notably, with the exception of winters of 2007–2008 and 2016–2017, A/H3N2 dominance during an influenza peak was mostly observed in summer, with A/H3N2 accounting for all summer influenza peaks after 2010, including two peaks in 2022 and 2023.

Phylodynamic analysis of A/H3N2 viruses isolated in Shanghai from 2005 to 2023

A total of 3028 A/H3N2 viruses were isolated from ILI specimens collected at sentinel hospitals during the surveillance period. From these, 530 isolates covering all winter and summer epidemic seasons and all districts of Shanghai were selected for whole genome sequencing, and 528 A/H3N2 genomes were successfully sequenced (GenBank Acc. OR717627-OR721850). No indication of co-infection or contamination with different isolates/subtypes were encountered while processing the sequencing data. These strains were then subjected to phylodynamic analysis using Nextstrain, along with Nextstrain built-in reference strains including vaccine strains, and GISAID-archived strains isolated in China and other countries/regions prior to Aug. 2023.

As shown in Figure 2, in a maximum likelihood timetree built using HA segment sequences, the majority of A/H3N2 strains sequenced in this work are closely grouped with strains isolated around the same time from other domestic and international geographic locations. This is corroborated by similar analysis using sequences of NA and the other 6 segments (Supplementary Figures 1–7), and reiterated the close association of A/H3N2 circulation in Shanghai with other parts of the world. Figure 2. Maximum-likelihood phylodynamic analysis of HA sequences of A/H3N2 viruses isolated in Shanghai between 2005 and 2023. HA segment sequences of A/H3N2 isolates sequenced in this work (*) and about 2000 GISAID isolates isolated in mainland China, Hong Kong/Macau/Taiwan (H/M/T) regions of China or other countries/regions of the world were used to generate maximum likelihood timetree in Nextstrain. Red arrow, 1a.1 clade that exclusively circulated in mainland China before the end of 2022. ×, vaccine strains.

Subclade 1a.1 A/H3N2 viruses exclusively circulated in Shanghai and mainland China before swift and complete replacement by subclade 2a.3a.1

A notable exception to the general observation of similar A/H3N2 viruses simultaneously circulating in Shanghai and elsewhere, however, was A/H3N2 variants isolated in Shanghai between July 2022 and Jan. 2023. In the HA timetree, all 149 of these strains cluster with multiple strains isolated elsewhere in China between Sep. 2021 and Jan. 2023 as monophyletic subclade 1a.1 within clade 3C.2a1b.2a (Figure 2). By contrast, strains isolated outside of mainland China during this period are grouped into multiple branches within the sibling subclade 2 of 3C.2a1b.2a, except only a couple of isolates that belong to the more distantly related 3C.2a1b.1a clade. On the other hand, all but one A/H3N2 strain isolated in Shanghai after Jan. 2023 are grouped with strains isolated in other Chinese and international locations around the same time as multiple branches within subclade 2, predominantly in subclade 2a.3a.1. The only exception was a strain isolated in Mar. 2023, which was one of the last seen members of 1a.1 (Figure 2).

Phylodynamic analysis by Nextstrain involved subsampling (random selection) of GISAID-archived viruses to reduce the number of phylogenetically analyzed strains, and this step might have left out isolates that are inconsistent with the above observation. To rule out this possibility, we used HA nucleotide sequences of subclades 1 and 2 of 3C.2a1b.2a as queries and ran blast searches against GISAID sequences to identify all potential subclade 1 viruses isolated outside of mainland China, and all potential subclade 2 viruses isolated in mainland China before 2023. Addition of the obtained blast results to Nextstrain analysis then identified only one subclade 1a.1 virus (A/Washington/47/2022, isolated in August), and only one subclade 2 virus (A/Beijing-Huairou/1417/2022, subclade 2a.1, isolated in March) that meet the criteria (Figure 3). Furthermore, similar blast searches against all influenza A virus sequences in GenBank using these criteria failed to find any additional isolate (data not shown). These two isolates most likely represent international export events that failed to lead to subsequent local circulation and confirmed the exclusive circulation of subclade 1a.1 in China and its absence elsewhere. Figure 3. Maximum-likelihood phylodynamic analysis of HA sequences of A/H3N2 clades 1 and 2 within 3C.2a1b.2a. Maximum likelihood timetree was generated using the same strains as used in Figure 2, plus all clade 1 strains isolated outside of mainland China and clade 2 viruses isolated in mainland China before 2023 identified in GISAID archive using blast. Only clade 3C.2a1b.2a from the tree is shown here, and the full tree as well as corresponding timetrees of other segments are provided in Supplementary Data 1. Red arrow, 1a.1 clade that exclusively circulated in mainland China before the end of 2022. ×, vaccine strains.

Subclades 1 and 2 within 3C.2a1b.2a are estimated to have diverged from each other in mid-2019, before the onset of the COVID-19 pandemic, and non-1a.1 clade 1 viruses were circulating in other countries up until mid-2021 (Figures 2 and 3). It therefore appears that circulation of A/H3N2 in Shanghai and other parts of mainland China was in effect separated from other regions during the pandemic: subclade 1a.1 exclusively circulated in, and only in, China before the end of 2022, while subclade 2 predominated elsewhere (Figure 2). When NPI policies in mainland China changed at the end of 2022, subclade 2, especially subclade 2a.3a.1, was introduced in early 2023, and within less than 2 months, completely replaced 1a.1 as the dominant A/H3N2 subclade both in Shanghai and elsewhere in mainland China (Figures 2 and 3). No 1a.1 virus was isolated after Mar. 2023.

Although relevant samples in GISAID archive were comparatively few, a similar but earlier transition from clade 1a.1 to clade 2 could be observed for Hong Kong SAR: multiple strains isolated in July 2022 all belong to clade 1a.1, whereas only clade 2a viruses were isolated after Sep. 2022. This apparently coincided with the COVID-19 NPI policy change regarding international travellers entering Hong Kong.

Comparative analysis of HA antigenicity of 1a.1 and 2a.3a.1 A/H3N2 viruses using sera from healthy donors

To probe possible factors underlying the swift and complete replacement of 1a.1 clade by 2a.3a.1 in Shanghai, A/Shanghai FX/1804/2022 isolated in October and A/Shanghai JD/1390/2023 isolated in May were selected to represent 1a.1 and 2a.3a.1 clades, respectively, and hereafter referred as such for brevity. Multiple amino acid sequence differences exist between the two clades, especially in HA and NA genes (Supplementary Table 1). We first tested their reactivity in HI assay with reference antiserum raised against vaccine strain A/Darwin/9/2021 (clade 2a), which was selected for the vaccine in Sep. 2021 and in use for 2022–2023 winter season in China. As shown in Table 1, the reference antiserum displayed the highest HI reactivity (1:1280) against the cognate vaccine virus, while reactivity against the 1a.1 and 2a.3a.1 viruses was 4 and 2 folds lower, respectively. This result is consistent with the closer phylogenetic relationship between clades 2a and 2a.3a.1 but also suggested a potential mismatch between vaccine and circulating strains for the winter of 2022–2023 (Figures 2 and 3). Table 1. Reactivity of reference A/Darwin/9/2021 antiserum in HI assay.

Virus	Clade (within 3C.2a1b.2a)	Reciprocal HI titer	
A/Darwin/9/2021	2a	1280	
A/Shanghai-FX/1804/2022	1a.1	320	
A/Shanghai-JD/1390/2023	2a.3a.1	640	

Next, the HA antigenicity of the two clinical isolates was compared in HI and NT assays using two serum panels, each collected from 150 healthy donors covering all age groups. These were collected in Aug.-Sep. 2022 and June 2023, respectively, roughly around the isolation time of the selected 1a.1 and 2a.3a.1 viruses.

In HI assay, both serum panels displayed the lowest reactivity (geometric mean titer, GMT) against 2a vaccine virus, slightly to moderately higher reactivity against 1a.1 virus, and highest reactivity against 2a.3a.1 virus (∼2 fold higher compared to 2a) (Figure 4(A, B)). Similarly, in both panels, the percentage of donors with putative protection, defined as having an HI titer ≥ 1:40 against the virus in question, was lowest for 2a, higher for 1a.1, and highest for 2a.3a.1 (Figure 4(C)). For any of the three tested viruses, the 2023 panel displayed higher HI titers and a higher percentage of protected donors compared to the 2022 panel (Figure 4(B,C)). Interestingly, in both panels, a majority of donors with protective HI titers against 1a.1 also displayed protective HI titers against 2a.3a.1 (Figure 4(C)). Figure 4. Hemagglutination inhibition titres of sera from healthy donors against clade 2a, 1a.1 and 2a.3a.1 A/H3N2 viruses. Two serum panels each containing 150 samples collected in Aug.-Sep. 2022 (2022 sera) and June 2023 (2023 sera) were tested in HI assay against A/H3N2 vaccine strain for 2022–2023 season in China A/Darwin/9/2021 (clade 2a), A/Shanghai-FX/1804/2022(H3/N2) (clade 1a.1) and A/Shanghai-JD/1390/2023 (clade 2a.3a.1). (A) HI titres grouped by donor’s age are shown as heatmaps and the distribution of titres within age groups are shown as violin plots at the bottom. (B) Geometric mean HI titres (GMT) including 95% CI of each panel against indicated viruses. (C) Percentages of serum with HI titre ≥ 1:40 against indicated viruses in each panel. Dashed line, putative protective HI titre threshold of 1:40.

The higher reactivity of the 2022 serum panel with 2a.3a.1 virus was unexpected, as this virus had not been introduced into Shanghai at the time of serum collection. NT assay was then performed to assess the effects of HA recognition by serum antibodies on infection. The 2022 panel displayed similar NI titers against 1a.1 and 2a.3a.1 viruses (Figure 5(A, B)), and percentages of donors with putative protection (NI titer ≥ 1:20) were also similar for both viruses (Figure 5(C)). This is in distinct contrast to HI assay results described above. On the other hand, sera collected in 2023 displayed higher NI titers against 2a.3a.1 virus compared to 1a.1 virus, although the percentages of donors with protection-level antibodies were not markedly different between the two viruses (Figure 5(B, C)). For both these viruses, the 2023 panel displayed higher NI titers and higher protection percentages compared to the 2022 panel. The NT assay data suggest that donors in 2022 would have been equally susceptible to infection by 1a.1 and 2a.3a.1 viruses, and consequently, evasion of pre-existing immunity due to antigenicity differences is not likely to have played a major role in the swift replacement of 1a.1 by 2a.3a.1. Figure 5. Neutralization titres of sera from healthy donors against clade 2a, 1a.1 and 2a.3a.1 A/H3N2 viruses. Two serum panels each containing 150 samples collected in Aug.-Sep. 2022 (2022 sera) and June 2023 (2023 sera) were tested in neutralization (NT) assay against A/H3N2 vaccine strain for 2022–2023 season in China A/Darwin/9/2021 (clade 2a), A/Shanghai-FX/1804/2022(H3/N2) (clade 1a.1) and A/Shanghai-JD/1390/2023 (clade 2a.3a.1). (A) NT titres grouped by donor’s age are shown as heatmaps and the distribution of titres within age groups are shown as violin plots at the bottom. (B) Geometric mean NT titres (GMT) including 95% CI of each panel against indicated viruses. Statistical significance was calculated using a two-tailed unpaired t test. *, P < 0.05. (C) Percentages of serum with NT titre ≥ 1:20 against indicated viruses in each panel. Dashed line, putative protective HI titre threshold of 1:20.

Comparative analysis of growth properties of 1a.1 and 2a.3a.1 A/H3N2 viruses in cell culture

In addition to interactions with host antibodies through envelope proteins, intracellular steps of the viral life cycle also contribute towards the fitness of a virus. Amino acid sequence differences exist between 1a.1 and 2a.3a.1 clades in polymerase (PA/PB1/PB2) and nucleoprotein (NP) genes (Supplementary Table 1), which drive viral transcription and replication. To test possible differences in growth properties, MDCK cells were infected with 1a.1 and 2a.3a.1 viruses at the same MOI. CPE development was monitored daily and the results showed that CPE started to appear at 24 hours post infection (h.p.i.) for both viruses and progressed over time up to the end of observation (96 h.p.i.) (Figure 6). From 48 h.p.i. onward, however, CPE in 2a.3a.1-infected cells was more prominent compared to 1a.1-infected cells (Figure 6). Figure 6. CPE development in MDCK cells infected with clade 1a.1 and 2a.3a.1 A/H3N2 viruses. MDCK cells infected with A/Shanghai-FX/1804/2022(H3/N2) (clade 1a.1) or A/Shanghai-JD/1390/2023 (clade 2a.3a.1), or mock-infected, were monitored at indicated time points for CPE development.

Next, progeny virus production was compared between the two viruses by measuring supernatants of infected cells using TCID50 assay. As shown in Figure 7(A), infective progeny viruses were detectable at roughly similar levels at 6 h.p.i. for both viruses, yet from 24 h.p.i. onward, 2a.3a.1-infected cells produced 0.5–1 log more progeny viruses compared to 1a.1-infected cells, and this difference persisted up to the end of observation (96 h.p.i.). Similarly, when HA and NA segment genomic RNA in supernatants were quantified, the results were comparable for both viruses at 6 h.p.i., but from 24 h.p.i. onward, 2a.3a.1-infected cells produced 2–3 logs higher levels of viral RNA (Figure 7(B, C)). Figure 7. Replication of clade 1a.1 and 2a.3a.1 A/H3N2 viruses in MDCK cells. MDCK cells infected with A/Shanghai-FX/1804/2022(H3/N2) (clade 1a.1) or A/Shanghai-JD/1390/2023 (clade 2a.3a.1) were sampled at indicated time points for measurement of infective progeny viruses in the supernatants using TCID50 assay (A), and measurement of HA (B) and NA (C) segment RNA in the supernatants, and intracellular viral mRNA (D), cRNA(E) and vRNA (F), using quantitative real-time PCR.

As both CPE development and progeny virus production indicated faster propagation of 2a.3a.1 compared to 1a.1 viruses, we went on to test viral transcription and replication by measuring intracellular viral mRNA, anti-genomic RNA (cRNA) and genomic RNA (vRNA). As shown in Figure 7(D), viral transcription was ten-fold higher in 2a.3a.1-infected cells at 6 h.p.i., and the difference increased to nearly 3 logs from 24 to 96 h.p.i. In contrast, 1a.1 virus had a temporary and slight lead in viral cRNA production at 6 h.p.i., but 2a.3a.1 closed the gap and overtook by about 1 log from 24 to 96 h.p.i. (Figure 7(E)). Genomic vRNA levels were nearly the same at 6 h.p.i. for both viruses, but from 24 h.p.i. onward until 96 h.p.i., 2a.3a.1-infected cells produced 2 logs more vRNA compared to 1a.1-infected cells (Figure 7(F)).

Collectively, these data showed that at least for the two selected isolates and the virus culture condition tested here, 2a.3a.1 virus displayed quicker and higher transcription, higher replication, and higher production of progeny viruses, and caused more prominent CPE in infected cells, compared to 1a.1 virus. Such a growth advantage might have contributed towards the swift and complete replacement of 1a.1 clade by 2a.3a.1 clade in Shanghai and other parts of mainland China after the latter’s introduction in early 2023.

Discussion

Traditionally, seasonal influenza has been expected to cause increased transmission and diseases in temperate regions in winter. Due to improved surveillance infrastructure and techniques, however, influenza cases during summer in such regions have become increasingly recognized. A/H3N2 has been associated with summertime outbreaks or epidemics in various locations in both hemispheres [15–19]. This is also illustrated by data obtained through the Shanghai surveillance network, which show that A/H3N2 played dominant roles in influenza peaks in multiple summers between 2005 and 2023, and many of these summer peaks are comparable to neighbouring winter influenza peaks in intensity (Figure 1). Such over-representation of A/H3N2 in summertime influenza diseases is not yet fully explained but has been suggested to involve multiple factors relating to both the subtype itself, as well as its interaction with other (sub)types [33].

Prior to the COVID-19 pandemic, A/H3N2 had been circulating globally with constant exchange of variants between geographic locations, including Shanghai and other parts of China. This is clearly reflected in the phylodynamic timetrees generated using sequence data of local, domestic and international A/H3N2 isolates (Figure 2 and S1-S7). NPI measures to combat COVID-19 were also effective in reducing transmission of other respiratory tract pathogens, and no winter ILI or influenza peak was observed in Shanghai at the end of 2021 (Figure 1). More importantly, restrictions on international travel and travellers during the early part of the pandemic apparently interrupted A/H3N2 global circulation and created a relatively isolated niche in mainland China wherein a unique subclade (1a.1) spread and evolved independently from A/H3N2 variants circulating elsewhere (Figures 2 and 3). This offered a unique opportunity for studying A/H3N2 circulation and evolution, while strains isolated and sequenced in this work would provide valuable support for future work in this direction. In addition, it would be interesting to examine whether other globally circulating pathogens underwent similar geographical isolation during the same period.

The first member of clade 1a.1 was isolated in mainland China in Sep. 2021, and continued variation within the clade before 2023 resulted in multiple branches that include viruses isolated in multiple distantly separated domestic locations (Figures 2–3, Supplementary Data 1). In the summer of 2022, domestically circulating 1a.1 viruses caused a fairly severe outbreak in Shanghai and transmission continued well into winter (Figures 1–3). Since strict COVID-19 NPI measures in mainland China were not to change until Dec. 2022, this outbreak reiterated the high transmissibility of A/H3N2 in susceptible populations in hot temperatures. Theoretically, susceptibility in the population could be attributed to both lack of recent exposure due to reduced incidence in preceding years (Figure 1), and generally low seasonal influenza vaccination coverage in mainland China [34].

The swift and complete replacement of domestically circulating clade 1a.1 A/H3N2 by internationally circulating clade 2 (predominantly 2a.3a.1) in early 2023 was surprising (Figures 2 and 3). Although insufficient pre-existing immunity against 2a.3a.1 compared to 1a.1 seemed like a plausible explanation, sera collected from healthy donors in 2022 displayed higher, instead of lower, HI titres against 2a.3a.1 virus compared to 1a.1 virus (Figure 4), while NT titres were comparable against the two clades (Figure 5). The slight inconsistency between HI and NT comparisons was not unusual [35], and stressed the importance of multi-assay characterization in the assessment of anti-influenza immunity. Regardless, these results suggest that at least at the time of serum collection (summer of 2022), humoral immunity against the two clades was not markedly different in the local population. HA antigenicity changes caused by variations in amino acid sequences (Supplementary Table 1) were therefore unlikely to have been a major factor in determining clade dynamics.

Compared to 1a.1, clade 2a.3a.1 virus displayed higher replicative competence in the MDCK cell infection model with regard to all tested parameters (Figures 6 and 7). Such an advantage could have played an important role in helping 2a.3a.1 to drive 1a.1 out of circulation, but similar results must first be reproduced in more clinically relevant cell and animal models before any conclusion could be made. The underlying mechanisms of this difference also remain to be elucidated. Functional effects of clade-specific variations in the polymerase subunits (PA, PB1, PB2) and nucleoprotein (NP) should be tested, while contribution by variations in other viral proteins is also possible (Supplementary Table 1).

The interplay between influenza (sub)types and between influenza and other respiratory tract pathogens is another important factor affecting influenza virus circulation [36]. The replacement of clade 1a.1 A/H3N2 by clade 2 in Shanghai coincided with a COVID-19 peak after NPI policy changes and an ensuing influenza peak dominated by A/H1N1(pdm09) (Figure 1). Whether these events also influenced concurrent A/H3N2 clade dynamics is an interesting question that warrants further probing.

In summary, continuous and systematic surveillance of influenza circulation in Shanghai over the past 2 decades established local transmission patterns that are consistent with the temperate climate and identified A/H3N2 as the major subtype causing summer influenza peaks. Whole-genome sequencing of A/H3N2 isolates obtained through the Shanghai influenza surveillance network enabled spatiotemporal analysis of intra-subtype variations in the context of global circulation, which revealed unusual A/H3N2 circulation patterns during the COVID-19 pandemic. Complex evolution and circulation dynamics of A/H3N2 as illustrated by results presented in this work underpin the importance of surveillance in understanding and combating this important respiratory tract pathogen.

Supplementary Material

Supplementary Data 1.zip

Supplementary Table and Figures.pdf

Acknowledgements

The authors gratefully acknowledge all data contributors, i.e. the Authors and their Originating laboratories responsible for obtaining the specimens, and their Submitting laboratories for generating the genetic sequence and metadata and sharing via the GISAID Initiative, on which this research is partially based. We are also grateful to the medical and lab staff involved in collecting, testing and reporting the clinical samples used in this work. We are thankful for the support from the Chinese National Influenza Center throughout the monitoring period reported here.

Author contribution

Zheng Teng and Jing Liu designed the study, reviewed and approved the final version. Xi Zhang, Min Chen, Huanyu Wu, Fanghao Fang, and Youhua Xie provided administrative and technical support. Chenyan Jiang, Wei Chu and Lixin Tao collected the specimens and clinical data. Xue Zhao, Yijing Gu and Xiaode Tang performed the experiments. Jing Liu and Xue Zhao analyzed all the data, prepared the figures and tables, and wrote the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.
==== Refs
References

1 Uyeki TM, Hui DS, Zambon M, et al. Influenza. Lancet. 2022;400 (10353 ):693–706. doi:10.1016/S0140-6736(22)00982-5 36030813
2 Azziz Baumgartner E, Dao CN, Nasreen S, et al. Seasonality, timing, and climate drivers of influenza activity worldwide. J Infect Dis 2012;206 (6 ):838–846. doi:10.1093/infdis/jis467 22829641
3 Yu H, Alonso WJ, Feng L, et al. Characterization of regional influenza seasonality patterns in China and implications for vaccination strategies: spatio-temporal modeling of surveillance data. PLoS Med 2013;10 (11 ):e1001552. doi:10.1371/journal.pmed.1001552 24348203
4 Petrova VN, Russell CA. The evolution of seasonal influenza viruses. Nat Rev Microbiol. 2018;16 (1 ):47–60. doi:10.1038/nrmicro.2017.118 29081496
5 WHO. Influenza (Seasonal). World Health Organization Fact Sheet (3 October 2023) [cited 2024 June 1 2024]. https://www.who.int/news-room/fact-sheets/detail/influenza-(seasonal).
6 Gubareva L, Mohan T. Antivirals targeting the neuraminidase. Cold Spring Harbor Perspect Med. 2022 Jan 4;12(1):a038455. doi:10.1101/cshperspect.a038455
7 Wu NC, Wilson IA. Influenza hemagglutinin structures and antibody recognition. Cold Spring Harb Perspect Med 2020 Aug 3;10 (8 ):a038778.31871236
8 Tosh PK, Jacobson RM, Poland GA. Influenza vaccines: from surveillance through production to protection. Mayo Clin Proc 2010;85 (3 ):257–273. doi:10.4065/mcp.2009.0615 20118381
9 Parrish CR, Kawaoka Y. The origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza A viruses. Annu Rev Microbiol 2005;59 :553–586. doi:10.1146/annurev.micro.59.030804.121059 16153179
10 Cox NJ, Subbarao K. Global epidemiology of influenza: past and present. Annu Rev Med. 2000;51 :407–421. doi:10.1146/annurev.med.51.1.407 10774473
11 Jester BJ, Uyeki TM, Jernigan DB. Fifty years of influenza A(H3N2) following the pandemic of 1968. Am J Public Health. 2020;110 (5 ):669–676. doi:10.2105/AJPH.2019.305557 32267748
12 Zhang ZXZ, Kyaw M, Ho W, et al. Seasonal influenza-associated intensive care unit admission and death in tropical Singapore, 2011-2015. J Clin Virol. 2019;117 :73–79. doi:10.1016/j.jcv.2019.06.005 31238274
13 Sumner KM, Masalovich S, O'Halloran A, et al. Severity of influenza-associated hospitalisations by influenza virus type and subtype in the USA, 2010-19: a repeated cross-sectional study. Lancet Microbe. 2023;4 (11 ):e903–e912. doi:10.1016/S2666-5247(23)00187-8 37769676
14 Park JE, Ryu Y. Transmissibility and severity of influenza virus by subtype. Infect, Genet Evol: J Mol Epidemiol Evolut Genet Infect Dis. 2018;65 :288–292.
15 Kohn MA, Farley TA, Sundin D, et al. Three summertime outbreaks of influenza type A. J Infect Dis. 1995;172 (1 ):246–249. doi:10.1093/infdis/172.1.246 7797922
16 Centers for Disease C, Prevention. Update: outbreak of influenza A infection–Alaska and the Yukon Territory, July-August 1998. MMWR Morbidity and Mortality Weekly Report. 1998 Aug 28;47 (33 ):685–688.9733414
17 Young LC, Dwyer DE, Harris M, et al. Summer outbreak of respiratory disease in an Australian prison due to an influenza A/Fujian/411/2002(H3N2)-like virus. Epidemiol Infect. 2005;133 (1 ):107–112. doi:10.1017/S0950268804003243 15724717
18 Tsou TP, Su CP, Huang WT, et al. Influenza A(H3N2) virus variants and patient characteristics during a summer influenza epidemic in Taiwan, 2017. Euro Surveill. 2017 Dec;22(50):17–00767. doi:10.2807/1560-7917.ES.2017.22.50.17-00767
19 Wei D, Yu DM, Wang MJ, et al. Genome-wide characterization of the seasonal H3N2 virus in Shanghai reveals natural temperature-sensitive strains conferred by the I668 V mutation in the PA subunit. Emerg Microbes Infect. 2018 Oct 23;7 (1 ):171.30353004
20 Wang DY. Development and prospect of influenza surveillance network in China. Zhonghua Liu Xing Bing Xue Za Zhi. 2018 Aug 10;39 (8 ):1036–1040.30180424
21 Li H, Gao GF. Highly effective stratified hub-and-spoke non-pharmaceutical intervention provides new insight into the prevention of COVID-19 transmission. People’s Republic of China CDC Wkly. 2020;2 (51 ):985–986.
22 Bai W, Gu Y, Liu H, et al. Epidemiology features and effectiveness of vaccination and non-pharmaceutical interventions of delta and lambda SARS-CoV-2 variants. People’s Republic of China CDC Wkly. 2021 Nov 12;3 (46 ):977–982.
23 Zhou B, Wentworth DE. Influenza A virus molecular virology techniques. Methods Mol Biol. 2012;865 :175–192. doi:10.1007/978-1-61779-621-0_11 22528160
24 Shu Y, McCauley J. GISAID: global initiative on sharing all influenza data – from vision to reality. Euro Surveill. 2017 Mar 30;22(13):30494. doi:10.2807/1560-7917.ES.2017.22.13.30494
25 Hadfield J, Megill C, Bell SM, et al. Nextstrain: real-time tracking of pathogen evolution. Bioinformatics. 2018;34 (23 ):4121–4123. doi:10.1093/bioinformatics/bty407 29790939
26 Lee J, Hadfield J, Black A, et al. Joint visualization of seasonal influenza serology and phylogeny to inform vaccine composition. Front Bioinform. 2023;3 :1069487. doi:10.3389/fbinf.2023.1069487 37035035
27 Camacho C, Coulouris G, Avagyan V, et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009;10 :421. doi:10.1186/1471-2105-10-421 20003500
28 Truelove S, Zhu H, Lessler J, et al. A comparison of hemagglutination inhibition and neutralization assays for characterizing immunity to seasonal influenza A. Influenza Other Respir Viruses. 2016;10 (6 ):518–524. doi:10.1111/irv.12408 27406695
29 Karakus U, Crameri M, Lanz C, et al. Propagation and titration of influenza viruses. Methods Mol Biol. 2018;1836 :59–88. doi:10.1007/978-1-4939-8678-1_4 30151569
30 Kawakami E, Watanabe T, Fujii K, et al. Strand-specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA. J Virol Methods. 2011;173 (1 ):1–6. doi:10.1016/j.jviromet.2010.12.014 21185869
31 Cheng VC, To KK, Tse H, et al. Two years after pandemic influenza A/2009/H1N1: what have we learned? Clin Microbiol Rev 2012;25 (2 ):223–263. doi:10.1128/CMR.05012-11 22491771
32 Paget J, Caini S, Del Riccio M, et al. Has influenza B/Yamagata become extinct and what implications might this have for quadrivalent influenza vaccines? Euro Surveill 2022 Sep;27(39):2200753. doi:10.2807/1560-7917.ES.2022.27.39.2200753
33 Zhang B, Huang W, Pei S, et al. Mechanisms for the circulation of influenza A(H3N2) in China: A spatiotemporal modelling study. PLoS Pathog. 2022;18 (12 ):e1011046. doi:10.1371/journal.ppat.1011046 36525468
34 Zhao HT, Peng ZB, Ni ZL, et al. [Investigation on influenza vaccination policy and vaccination situation during the influenza seasons of 2020-2021 and 2021-2022 in China]. Zhonghua Yu Fang Yi Xue Za Zhi. 2022;56 (11 ):1560–1564.36372744
35 Sicca F, Martinuzzi D, Montomoli E, et al. Comparison of influenza-specific neutralizing antibody titers determined using different assay readouts and hemagglutination inhibition titers: good correlation but poor agreement. Vaccine. 2020;38 (11 ):2527–2541. doi:10.1016/j.vaccine.2020.01.088 32044163
36 Opatowski L, Baguelin M, Eggo RM. Influenza interaction with cocirculating pathogens and its impact on surveillance, pathogenesis, and epidemic profile: a key role for mathematical modelling. PLoS Pathog. 2018;14 (2 ):e1006770. doi:10.1371/journal.ppat.1006770 29447284
