Influenza A virus continues to circulate among children in Linyi, northern China, after the relaxation of COVID-19 control measures

doi:10.21203/rs.3.rs-4898499/v1

Following the relaxation of control measures for COVID-19 in China in December 2022, there was a resurgence of influenza A among children in Linyi. This research aims to analyze the epidemiology and genetic characteristics of influenza A viruses circulating among children in Linyi from April 2022 to March 2024. Throat swab specimens were collected from children with influenza-like illness (ILI), and the types of influenza were identified. The hemagglutinin (HA) genes of influenza A viruses were amplified using reverse transcription-polymerase chain reaction (RT-PCR) and then sequenced. Subsequently, the gene characteristics and phylogenetics of these sequences were analyzed. A total of 6258 cases of influenza A were tested from 54926 children with ILI over two years, showing three epidemic waves: one in summer 2022 and two in spring and winter 2023. The epidemic waves in summer 2022 and winter 2023 were caused by influenza A(H3N2) viruses. In spring 2023, influenza A(H1N1)pdm09 viruses accounted for 77.1% and A(H3N2) viruses accounted for 22.9%, respectively. The influenza A(H1N1)pdm09 viruses belonged to clade 6B.1A.5a.2a, while the influenza A(H3N2) viruses evolved from clade 3C.2a1b.2a.1a to 3C.2a1b.2a.2a.3a.1. Compared with the vaccine strain of the Northern Hemisphere of the current year, multiple amino acid substitutions and glycosylation sites changes were observed in the HA protein that most likely resulted in antigenic drift. The influenza A viruses have been circulating and evolving persistently in Linyi from 2022 to 2024. Continuous surveillance is essential for comprehending the dynamics of

influenza in the post-COVID-19 era.

Influenza A virus

Children

Epidemiology

Genetic characteristics

Influenza is an acute respiratory infectious disease caused by influenza viruses. Seasonal influenza leads to approximately one billion cases annually, with 3-5 million cases of severe illness and an estimated annual death toll from respiratory complications ranging from 290000 to 650000 [1].Children are highly susceptible to contracting influenza. In 2018, an estimated 109.5 million global cases of influenza were reported among children under five years old [2]. In China, the annual average number of influenza cases in children was approximately 3.6 million from 2010 to 2020 [3]. Children are also at an increased risk of experiencing severe illness and requiring hospitalization following the influenza infection. The annual hospitalization rate for influenza and related illnesses among children under five years old was 1000 per 100000 individuals [4]. Similarly, the burden and demand for medical care related to influenza are higher in children than in adults. Influenza outbreaks can lead to a significant number of school-age children being absent from school and their parents being absent from work, resulting in a substantial social and economic burden [5].

Influenza viruses are classified into four types: A, B, C, and D. At present, the predominant types of influenza viruses prevalent in humans are A and B. Influenza viruses are subdivided into different subtypes based on viral hemagglutinin (HA) and neuraminidase (NA). Each subtype of influenza virus can be further divided into different genetic clades based on HA gene sequences [6]. Up to now, eighteen HA (H1-18) and eleven NA (N1-11) subtypes have been identified. Currently, the predominant circulating subtypes of influenza A virus in humans are A(H1N1)pdm09 and A(H3N2) [7]. The influenza A(H3N2) virus often prevails in most influenza seasons due to its faster antigenic evolution, whereas the influenza A(H1N1)pdm09 virus exhibits slower antigenic evolution and may prevail in some influenza seasons [8]. HA is the major target of the host immune response to infection with influenza virus. Previous studies have identified five major antigenic determinant regions for H1 (Sa, Sb, Ca1, Ca2, and Cb), as well as for H3 (A-E). HA plays an important role in the viral invasion of host cells. The influenza virus can escape antibody neutralisation through amino acid substitutions in HA, which leads to a localized high-intensity epidemic [9]. Vaccination is the major method for reducing the prevalence of Influenza. However, the effectiveness of influenza vaccination can be reduced by amino acid substitutions in HA [10].

Since the global COVID-19 pandemic in 2019, the activity and epidemiological patterns of influenza have been altered. The implementation of control measures such as wearing masks and maintaining social distancing in response to COVID-19 outbreaks has led to a reduction in influenza activity during 2019 and 2020 [11,12]. However, there has been a recovery trend in various types of influenza since 2021 [13]. It is worth attention that outbreaks of influenza occurred in various areas of China after the relaxation of control measures for COVID-19 in December 2022 [14,15]. Similarly, two pandemics of influenza A broke out among children in Linyi in 2023. Nevertheless, it remains unclear how influenza activity will be modified in the post-COVID-19 era. Therefore, continuous monitoring of the etiology of the influenza virus is necessary. In this research, we revealed the epidemiology and genetic characteristics of influenza A virus in children in Linyi from 2022 to 2024. This information will be helpful for molecular surveillance of influenza and the selection of vaccine strains.

Case definitions and specimens collection

Throat swabs were collected from pediatric patients under 14 years old with influenza-like illness (ILI) at Women and Children's Health Care Hospital of Linyi between April 2022 and March 2024. ILI was defined according to the National Influenza Surveillance Program as fever (≥38 ℃) accompanied by cough or sore throat [16]. The specimens were transported at 4℃ and stored at -80℃.

Nucleic acid extraction and infuenza virus detection

Nucleic acid was extracted using the viral RNA extraction kit (Code No. Z-ME-0025, ZJ Bio-Tech Co., Ltd., Shanghai, China). The combined detection kit for influenza A and B (Code No. Z-RR-0097-50, ZJ Bio-Tech Co., Ltd., Shanghai, China) was used to detect the types of influenza viruses. The experimental procedures and reaction conditions were carried out in accordance with the manufacturer's instructions. All experiments were conducted using the Lightcycler 96 Real-Time PCR System (Roche, Switzerland).

Amplification and sequencing of HA gene

According to previously research [17], the primers were designed for amplifying the nucleotide sequence of the HA gene of influenza A virus. The HA gene was amplified using the one-step RT-PCR kit (Code No. RR057A, Takara, Japan). The RT-PCR was performed under the following conditions: 50℃ for 30 min; 94℃ for 2 min; 35 cycles of denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, and elongation at 72℃ for 60 s. The products were confirmed by running on a 1.5% agarose gel electrophoresis, and the positive products were sent to Sangon-Tech Biotech Company (Shanghai, China) for sequencing. Finally, the sequences were validated through BLAST analysis in National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/BLAST/).

Phylogenetic analysis

Three epidemic waves of influenza A occurred in Linyi from April 2022 to March 2024. During each epidemic wave, ten strains of each type of influenza A virus were selected, resulting in a total of forty sequences submitted to Genbank (https://www.ncbi.nlm.nih.gov/genbank/) with accession numbers PP479467-PP479476 (H1N1) and PP478127-PP478156 (H3N2). WHO recommended vaccine strain sequences and HA clade reference sequences were obtained from the Global Initiative of Sharing All Influenza Data (GISAID, https://gisaid.org/). Phylogenetic analysis was performed using the neighbor-joining method with MEGA software (version7.0, http://www.megasoftware.net/). The reliability of the maximum-likelihood tree was evaluated through bootstrap analysis consisting of 1000 replications [18].

Analysis of homology of HA gene and antigenic variant sites

The homology of nucleotide sequence and the antigenic variant sites of HA between the strains isolated from Linyi and the vaccine strain of current year were analyzed using DNAstar software (version7.1, DNASTAR, Inc., Madison, WI, USA). The 3D model of representative H1 (template PDB ID: 4M5Z) and H3 (template PDB ID: 4W28.1) proteins were built using SWISS MODEL protein homology modeling online server (https://swissmodel.expasy.org). The amino acid substitutions in the antigenic sites of HA proteins were illustrated using PyMOL software. (https://www.pymol.org).

N-glycosylation sites prediction

The NetNGlyc-1.0 server (https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/) was used to predict the N-glycosylation sites. The amino acid sequences of HA were submitted to the server, and when the potential threshold higher than 0.5, asparagine in the Asn-XSer/Thr sequence (X can represent any amino acid except proline) was predicted to be N-glycosylated.

Distribution of infuenza

A total of 54926 children with ILI were enrolled in this study, and 9615 (17.5%) cases were positive for influenza. Among all influenza cases, 6258 cases (65.1%) were influenza A and 3357 cases (34.9%) were influenza B. Three epidemic waves of influenza A appeared in Linyi from April 2022 to March 2024. The first epidemic wave of influenza A occurred in summer 2022, from July to August. The numbers of ILI and positive cases of influenza were 1366 and 273 (20.0%) during this period, and all the strains were influenza A(H3N2). The second epidemic wave of influenza A occurred in Spring 2023, from February to May, with 6529 cases of ILI and 864 positive cases of influenza A (13.2%). The proportions of A(H1N1)pdm09 strains and A(H3N2) strains were 77.1% and 22.9% during this period. The third epidemic wave of influenza A occurred in winter 2023, from November 2023 to February 2024, with the numbers of ILI and positive cases of influenza A being 35157 and 5121 (14.6%) respectively, and all strains were influenza A(H3N2). During this period, we also detected 3357 (9.5%) cases of influenza B (Fig. 1).

Phylogenetic analysis

The results of the HA gene-based phylogenetic analysis showed that the influenza A(H1N1)pdm09 strains isolated from Linyi in spring 2023 fell into clade 6B.1A.5a.2a. The strains isolated from the USA and Japan in 2022, as well as the strains isolated from other provinces of China and other countries such as the USA, Canada, Italy, Peru, Japan, Australia, and Korea in 2023, all belong to this clade. The 2022-2023 vaccine strain of influenza A(H1N1)pdm09 (A/Victoria/2570/2019) belongs to clade 6B.1A.5a.2 (Fig. 2). The influenza A(H3N2) strains isolated from Linyi in summer 2022 fell into clade 3C.2a1b.2a.1a. These strains are the same clade as the 2021-2022 vaccine strain of influenza A(H3N2) (A/Cambodia/e0826360/2020). In spring 2023, three influenza A(H3N2) strains belonged to the same clade as the strains isolated in summer 2022. Additionally, seven strains fell into clade 3C.2a1b.2a.2a.3a.1, and this was also the clade circulated in winter 2023. These strains were closely related to the strains that circulated in Canada and Germany in 2022, as well as the strains isolated from the other areas of northern China such as Beijing, Tianjin, Liaoning province in 2023. The 2024-2025 vaccine strain of influenza A(H3N2) (A/Thailand/8/2022) also belonged to this clade. (Fig. 3).

Nucleotide homology analysis

The nucleotide homology of the HA gene of influenza A(H1N1)pdm09 viruses isolated in spring 2023 was 99.2-100%. Compared to the 2022-2023 vaccine strain of influenza A(H1N1)pdm09 (A/Victoria/2570/2019), the nucleotide homology of influenza A(H1N1)pdm09 viruses was 98.4-98.8%. The nucleotide homology of the HA gene of influenza A(H3N2) viruses was 99.3–100% in summer 2022, 96.7-99.9% in spring 2023, and 99.7-100% in winter 2023, respectively. The nucleotide homogeneity between the H3N2 strains isolated in summer 2022 and the 2021-2022 vaccine strain of influenza A(H3N2) (A/Cambodia/e0826360/2020) was 98.6-99.1%. Compared to the 2022-2023 vaccine strain of influenza A(H3N2) (A/Darwin/9/2021), the nucleotide homology of the influenza A(H3N2) viruses isolated in spring and winter 2023 were 97.5-98.3% and 98.0-98.2%, respectively.

Analysis of amino acid sequence variations

Amino acid substitutions were detected in all the sequences (Table 1). Antigenic determinant regions Sa, Sb, Ca 1, Ca2, and Cb were displayed on a representative 3D homology model of H1 protein (template PDB ID: 4M5Z) (Fig 4a). Compared to the 2022-2023 vaccine strain of influenza A(H1N1)pdm09 (A/Victoria/2570/2019), mutations A186T and Q189E were observed in all influenza A(H1N1)pdm09 strains isolated from Linyi , located in the Sb antigenic determinant region. Additionally, two strains exhibited the mutation A139D in the Ca2 antigenic determinant region, and one strain exhibited the mutation P159Q in the Sa2 antigenic determinant region (Fig 4b). Antigenic determinant regions A, B, C, D, and E of H3 protein were displayed on a representative 3D homology model of H3 protein (template PDB ID: 4WE8.1) (Fig. 4c). Compared to the 2021-2022vaccine strain of influenza A(H3N2) (A/Cambodia/e0826360/2020), all the influenza A(H3N2) strains isolated in summer 2022 showed mutations K160T, R186S, and I48T. One strain of each had mutation S146G or L194R. All mutations are located in the A and B antigen determinant regions (Fig 4d). During spring 2023, compared to the 2022-2023 vaccine strain of influenza A(H3N2) (A/Darwin/9/2021), seven influenza A(H3N2) strains showed multiple mutations including E50K, D53N, N96S, N122D, I140K, N186D, and I192F. Three strains showed mutations I48T, S156H, N159Y, I160T, Q164L, N190D, S198P, and K171N. Additionally, one strain of each had mutation G62E or V102A (Fig 4e) . In winter 2023, all influenza A(H3N2) strains exhibited mutations E50K, D53N, N96S, N122D, I140K, N186D, I192F, and K276E compared to the 2022-2023 vaccine strain of influenza A(H3N2) (A/Darwin/9/2021). One strain of each had mutation E83G or N159T (Fig 4f).

Table 1 The list of amino acid substitutions in the HA protein of influenza A(H1N1)pdm09 and A(H3N2) strains isolated from Linyi compared with vaccine Strains

Strain	List of mutations
●A/Linyi/215/2023 (H1N1)	A186T, Q189E
●A/Linyi/216/2023 (H1N1)	A186T, Q189E
●A/Linyi/305/2023 (H1N1)	A186T, Q189E
●A/Linyi/310/2023 (H1N1)	A186T, Q189E
●A/Linyi/313/2023 (H1N1)	A139D, A186T, Q189E
●A/Linyi/314/2023 (H1N1)	A139D, A186T, Q189E
●A/Linyi/323/2023 (H1N1)	A186T, Q189E
●A/Linyi/327/2023 (H1N1)	P159Q, A186T, Q189E
●A/Linyi/331/2023 (H1N1)	A186T, Q189E
●A/Linyi/336/2023 (H1N1)	A186T, Q189E
■A/Linyi/5/2022 (H3N2)	I48T, S146G,K160T, R186S
■A/Linyi/9/2022 (H3N2)	I48T, K160T, R186S
■A/Linyi/12/2022 (H3N2)	I48T, K160T, R186S, L194R
■A/Linyi/16/2022 (H3N2)	I48T, K160T, R186S
■A/Linyi/27/2022 (H3N2)	I48T, K160T, R186S
■A/Linyi/28/2022 (H3N2)	I48T, K160T, R186S
■A/Linyi/36/2022 (H3N2)	I48T, K160T, R186S
■A/Linyi/40/2022 (H3N2)	I48T, K160T, R186S
■A/Linyi/42/2022 (H3N2)	I48T, K160T, R186S
■A/Linyi/44/2022 (H3N2)	I48T, K160T, R186S
▲A/Linyi/201/2023 (H3N2)	I48T, S156H, N159Y, I160T, Q164L, K171N, N190D, S198P
▲A/Linyi/205/2023 (H3N2)	I48T, V102A, S156H, N159Y, I160T, Q164L, K171N, N190D, S198P
▲A/Linyi/210/2023 (H3N2)	I48T, S156H, N159Y, I160T, Q164L, K171N, N190D, S198P
▲A/Linyi/218/2023 (H3N2)	E50K, D53N, G62E, N96S, N122D, I140K, N186D, I192F
▲A/Linyi/220/2023 (H3N2)	E50K, D53N, N96S, N122D, I140K, N186D, I192F
▲A/Linyi/221/2023 (H3N2)	E50K, D53N, N96S, N122D, I140K, N186D, I192F
▲A/Linyi/315/2023 (H3N2)	E50K, D53N, N96S, N122D, I140K, N186D, I192F
▲A/Linyi/319/2023 (H3N2)	E50K, D53N, N96S, N122D, I140K, N186D, I192F
▲A/Linyi/320/2023 (H3N2)	E50K, D53N, N96S, N122D, I140K, N186D, I192F
▲A/Linyi/328/2023 (H3N2)	E50K, D53N, N96S, N122D, I140K, N186D, I192F
▲A/Linyi/1202/2023 (H3N2)	E50K, D53N, N96S, N122D, I140K, N186D, I192F, K276E
▲A/Linyi/1206/2023 (H3N2)	E50K, D53N, N96S, N122D, I140K, N186D, I192F, K276E
▲A/Linyi/1208/2023 (H3N2)	E50K, D53N, N96S, N122D, I140K, N186D, I192F, K276E
▲A/Linyi/1210/2023 (H3N2)	E50K, D53N, N96S, N122D, I140K, N186D, I192F, K276E
▲A/Linyi/1218/2023 (H3N2)	E50K, D53N, N96S, N122D, I140K, N186D, I192F, K276E
▲A/Linyi/1220/2023 (H3N2)	E50K, D53N, N96S, N122D, I140K, N186D, I192F, K276E
▲A/Linyi/1221/2023 (H3N2)	E50K, D53N, N96S, N122D, I140K, N186D, I192F, K276E
▲A/Linyi/1225/2023 (H3N2)	E50K, D53N, E83G, N96S, N122D, I140K, N186D, I192F, K276E
▲A/Linyi/1227/2023 (H3N2)	E50K, D53N, N96S, N122D, I140K, N186D, I192F, K276E
▲A/Linyi/1230/2023 (H3N2)	E50K, D53N, N96S, N122D, I140K, N159T, N186D, I192F, K276E

● Comparison with the 2022-2023 vaccine strain of influenza A(H1N1)pdm09 (A/Victoria/2570/2019);

■ Comparison with the 2021-2022 vaccine strain of influenza A(H3N2) (A/Cambodia/e0826360/2020);

▲ Comparison with the 2022-2023 vaccine strain of influenza A(H3N2) (A/Darwin/9/2021)

The prediction of N-glycosylation sites

All the influenza A(H1N1)pdm09 strains isolated in spring 2023 shared the same seven glycosylation sites (11NST, 23NVT, 87NGT, 162NQT, 287NTS, 481NGT, and 540NGS) with the 2022-2023 vaccine strain of influenza A(H1N1)pdm09 (A/Victoria/2570/2019). Ten common glycosylation sites (8NST, 22NGT, 38NAT, 45NSS, 63NCT, 126NWT, 133NGT, 165NVT, 246NST, and 285NGS) were identified in the HA protein of the 2021-2022 vaccine strain (A/Cambodia/e0826360/2020) and 2022-2023 vaccine strain (A/Darwin/9/2021) of influenza A(H3N2). Compared to the vaccine strain, all the influenza A(H3N2) strains isolated in summer 2022 and three strains isolated in spring 2023 had a new glycosylation site 158NYT and lacked site 45NSS. The other seven influenza A(H3N2) strains isolated in spring 2023 and all the strains isolated in winter 2023 had a new glycosylation site 94NSS and lacked site 126NWT (Table 2).

Table 2 The glycosylation sites in the HA protein of the vaccine strains and the infuenza A strains isolated from Linyi

Strain	List of glycosylation sites
^✱A/Victoria/2570/2019	11NST, 23NVT, 87NGT, 162NQT, 287NTS, 481NGT, 540NGS
A/Linyi/215/2023 (H1N1)	11NST, 23NVT, 87NGT, 162NQT, 287NTS, 481NGT, 540NGS
A/Linyi/216/2023 (H1N1)	11NST, 23NVT, 87NGT, 162NQT, 287NTS, 481NGT, 540NGS
A/Linyi/305/2023 (H1N1)	11NST, 23NVT, 87NGT, 162NQT, 287NTS, 481NGT, 540NGS
A/Linyi/310/2023 (H1N1)	11NST, 23NVT, 87NGT, 162NQT, 287NTS, 481NGT, 540NGS
A/Linyi/313/2023 (H1N1)	11NST, 23NVT, 87NGT, 162NQT, 287NTS, 481NGT, 540NGS
A/Linyi/314/2023 (H1N1)	11NST, 23NVT, 87NGT, 162NQT, 287NTS, 481NGT, 540NGS
A/Linyi/323/2023 (H1N1)	11NST, 23NVT, 87NGT, 162NQT, 287NTS, 481NGT, 540NGS
A/Linyi/327/2023 (H1N1)	11NST, 23NVT, 87NGT, 162NQT, 287NTS, 481NGT, 540NGS
A/Linyi/331/2023 (H1N1)	11NST, 23NVT, 87NGT, 162NQT, 287NTS, 481NGT, 540NGS
A/Linyi/336/2023 (H1N1)	11NST, 23NVT, 87NGT, 162NQT, 287NTS, 481NGT, 540NGS
^✱A/Cambodia/e0826360/2020	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 126NWT, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/5/2022 (H3N2)	8NST, 22NGT, 38NAT, 63NCT, 126NWT, 133NGT, 158NYT, 165NVT, 246NST, 285NGS
A//inyi/9/2022 (H3N2)	8NST, 22NGT, 38NAT, 63NCT, 126NWT, 133NGT, 158NYT, 165NVT, 246NST, 285NGS
A//inyi/12/2022 (H3N2)	8NST, 22NGT, 38NAT, 63NCT, 126NWT, 133NGT, 158NYT, 165NVT, 246NST, 285NGS
A//inyi/16/2022 (H3N2)	8NST, 22NGT, 38NAT, 63NCT, 126NWT, 133NGT, 158NYT, 165NVT, 246NST, 285NGS
A//inyi/27/2022 (H3N2)	8NST, 22NGT, 38NAT, 63NCT, 126NWT, 133NGT, 158NYT, 165NVT, 246NST, 285NGS
A//inyi/28/2022 (H3N2)	8NST, 22NGT, 38NAT, 63NCT, 126NWT, 133NGT, 158NYT, 165NVT, 246NST, 285NGS
A//inyi/36/2022 (H3N2)	8NST, 22NGT, 38NAT, 63NCT, 126NWT, 133NGT, 158NYT, 165NVT, 246NST, 285NGS
A//inyi/40/2022 (H3N2)	8NST, 22NGT, 38NAT, 63NCT, 126NWT, 133NGT, 158NYT, 165NVT, 246NST, 285NGS
A//inyi/42/2022 (H3N2)	8NST, 22NGT, 38NAT, 63NCT, 126NWT, 133NGT, 158NYT, 165NVT, 246NST, 285NGS
A//inyi/44/2022 (H3N2)	8NST, 22NGT, 38NAT, 63NCT, 126NWT, 133NGT, 158NYT, 165NVT, 246NST, 285NGS
^✱A/Darwin/9/2021	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 126NWT, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/201/2023 (H3N2)	8NST, 22NGT, 38NAT, 63NCT, 126NWT, 133NGT, 158NYT, 165NVT, 246NST, 285NGS
A//inyi/205/2023 (H3N2)	8NST, 22NGT, 38NAT, 63NCT, 126NWT, 133NGT, 158NYT, 165NVT, 246NST, 285NGS
A//inyi/210/2023 (H3N2)	8NST, 22NGT, 38NAT, 63NCT, 126NWT, 133NGT, 158NYT, 165NVT, 246NST, 285NGS
A//inyi/218/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/220/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/221/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/315/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/319/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/320/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/328/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/1202/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/1206/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/1208/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/1210/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/1218/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/1220/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/1221/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/1225/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/1227/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS
A//inyi/1230/2023 (H3N2)	8NST, 22NGT, 38NAT, 45NSS, 63NCT, 94NSS, 133NGT, 165NVT, 246NST, 285NGS

^✱The vaccine strains of the current year

Influenza virus infection is extremely prevalent among children worldwide. Linyi is the largest and most populous city in Shandong Province, located in northern China. Historically, the epidemic of influenza in the Linyi area has occurred in spring and winter, from November to March of the following year [20]. However, an epidemic wave of influenza A was observed among children in Linyi in summer 2022, from July to September, which was a non-traditional influenza season for Linyi. This period also coincided with the implementation of the control measures for COVID-19 in China, so that only a low-intensity epidemic of influenza A occurred among children in Linyi. During this period, 20.0% of the children with ILI were identified as having influenza A(H3N2) virus infection, and the rate of influenza infection was generally consistent with those reported in children during the annual influenza seasons prior to the COVID-19 pandemic [21,22].The epidemic of influenza in a non-traditional season had not appeared in Linyi previously. Although the control measures for COVID-19 could prevented many respiratory pathogens such as influenza virus, and decreased exposure enabled a group of susceptible individuals to avoid respiratory infections. However, it also leads to a lack of pathogen-specific immunity, which could prevent future infections.This phenomenon is called immunity debt [23]. Since China adopted the control measures in response to COVID-19 outbreaks from 2019, there has been no epidemic of influenza A in Linyi untile summer 2022, and children are generally susceptible to influenza A due to the lack of specific antibodies against influenza A virus. It is important to investigate whether non-traditional seasonal epidemics may occur at irregular intervals after the relaxation of control measures for COVID-19. This issue requires continuous attention.

The control measures for COVID-19 have been relaxed in China in winter 2022.

Winter and spring are also high incidence seasons for respiratory infections in China. After the relaxation of control measures for COVID-19, influenza outbreaks have occurred in various areas of China. In spring 2023, an epidemic wave of influenza was detected among children in Linyi. From February to May, influenza A(H1N1)pdm09 viruses and A(H3N2) viruses circulated together. After a period of silence, the numbers of children with ILI increased significantly in Linyi in winter 2023, from November 2023 to February 2024. During the same period, WHO has been monitoring data from the Chinese surveillance systems, which showed an increase in respiratory illness among children in northern China. Chinese authorities attributed this increase to the lifting of COVID-19 restrictions, the arrival of the cold season, and the circulation of known pathogens [24]. During this period, we also detected a large number of children with influenza B virus, adenovirus, and COVID-19 virus infection. The symptoms of these viral infections are essentially the same as those of influenza A virus [25]. It suggested that we should continue to monitor the spread of respiratory pathogens in children in the post-pandemic era of COVID-19.

The influenza A(H1N1)pdm09 strains isolated from Linyi belonged to clade 6B.1A.5a.2, which is also the main influenza A(H1N1)pdm09 epidemic clade in the world at present [26, 27]. However, the genetic relationship between the influenza A(H1N1)pdm09 strains isolated from Linyi and those from other regions was complicated. Ten influenza A(H1N1)pdm09 strains isolated from Linyi showed different genetic relationships with strains from other areas of China and other countries. Four strains were closely related to the strains isolated from Jilin and Guangdong province of China, as well as Korea. Four strains were closely related to the strains isolated from Shanghai, China, and Oregon, the USA. One strain was closely related to the strain isolated from Fujian province, China, One strain was closely related to the strain isolated from New York, the USA. This reflects that Linyi, as a logistics center in northern China, has increased the mobility of people after the relaxation of control measures for COVID-19, leading to the prevalence of influenza strains from different regions in Linyi in 2023, making it extremely susceptible to the cross-regional spread of influenza. Therefore, more attention should be paid to the trend of antigenicity and genetic properties variation of influenza virus.

The influenza A(H3N2) strains isolated from Linyi in the summer 2022 wave fell into clade 3C.2a1b.2a.1a. Since China was under strict control measures for the COVID-19 epidemic at that time, which reduced personnel exchanges with other countries, the Linyi strains were distantly related to the strains from foreign countries, but closely related to the strains from other areas of China, such as Guangdong, Zhejiang, Jiangsu and Hebei province. During this period, the circulating influenza A(H3N2) viruses belonged to clade 3C.2a1b.2a.2 in the USA [28]. In spring 2023, three influenza A(H3N2) strains isolated from Linyi belonged to the same clade as the strains of summer 2022. Seven strains belonged to clade 3C.2a1b.2a.2a.3a.1, and these strains were closely related to the strains that circulated in Canada and Germany in 2022，as well as the strains isolated from the other areas of northern China such as Beijing, Tianjin, Liaoning and Shanxi province in 2023. In winter 2023, the influenza A(H3N2) strains isolated from Linyi were also belong to clade 3C.2a1b.2a.2a.3a.1. Four strains were closely related to the strains isolated from Zhejiang province in 2023, and three strains were closely related to the strains isolated from HongKong in 2023. One strain in each was closely related to the epidemic strains in Jilin and Heilongjiang province, respectively. This indicates that the influenza A(H3N2) virus continued to evolve during its epidemic in Linyi, and the virus did not exist alone but co-circulated with other strains in China and foreign countries. The 2023-2024 Northern Hemisphere influenza vaccine strain recommended by WHO is A/Darwin/9/2021 and it belongs to the clade 3C.2a1b.2a.2a. However, during the last two influenza epidemic waves in Linyi, most of the influenza A(H3N2) strains belonged to clade 3C.2a1b.2a.2a.3a.1, which was consistent with the 2024-2025 vaccine strain recommended by WHO (A/Thailand/8/2022). These results indicate that HA gene of influenza A(H3N2) strains evolved rapidly. Therefore, it is essential to enhance vaccination with new influenza vaccines.

Antigen drift refers to the occurrence of point substitutions in the viral genome, which results in minor changes in crucial viral epitopes, and it is closely associated with the prevalence of influenza virus [29]. Specifically, the HA gene contains antigenic sites that are targeted by neutralizing antibodies. Substitutions in the HA gene can lead to immune evasion and a reduction in vaccine efficacy. Antigenic drift occurs infrequently in influenza A(H1N1)pdm09 virus and influenza B virus, while influenza A(H3N2) virus undergoes more frequent antigenic drift [30]. We have identified only one epidemic wave of influenza A(H1N1)pdm09 viruses in Linyi from 2022 to 2024. The influenza A(H1N1)pdm09 strains had only A186T and Q189E substitutions compared with the 2022-2023 vaccine strain (A/Victoria/2570/2019), and all substitutions were located in the Sb antigenic determinant region. Therefore, there was no obvious antigenic drift in these strains,, which may have contributing to the lack of continued circulation of influenza A(H1N1)pdm09 viruses in 2024. However, further attention is needed to determine whether the A(H1N1)pdm09 viruses will continue to mutate and spread in Linyi.

Compared with the 2021-2022 vaccine strain (A/Cambodia/e0826360/2020), the influenza A(H3N2) viruses isolated from Linyi in summer 2022 showed only three substitutions. There was no obvious antigenic drift in these influenza A(H3N2) strains. Meanwhile, due to the implementation of control measures for COVID-19 in Linyi, the epidemic intensity of influenza was low. During spring 2023, significant antigenic drift was observed in the influenza A(H3N2) viruses compared to the 2022-2023 vaccine strain (A/Darwin/9/2021). Concurrently, the control measures for COVID-19 were relaxed by the Chinese government. All these have led to the epidemic wave of influenza A in spring 2023. During this period, three influenza A(H3N2) strains were closely related to those from summer 2022, while seven strains were closely related to those from winter 2023. This indicates that the influenza A(H3N2) virus continued to circulate in Linyi. All influenza A(H3N2) viruses isolated from Linyi in 2023 exhibited noticeable antigenic drift compared with the 2022-2023 vaccine strain (A/Darwin/9/2021), resulting in reduced vaccine efficacy. This may contribute to why influenza A(H3N2) viruses continue to circulate in Linyi. The composition of the 2024-2025 vaccine strain has been updated to A/Thailand/8/2022 by WHO. The influenza A(H3N2) strains isolated in winter 2023 and the 2024-2025 vaccine strain (A/Thailand/8/2022) all belong to clade 3C.2a1b.2a.2a.3a. Therefore, it is imperative to update the vaccine strain and expand vaccination efforts promptly.

The glycosylation sites of HA protein play an important role in stabilizing the protein structure and escaping the host immunity. The increase or decrease in the number of glycosylation sites of HA protein will affect the infectivity, antigenicity and pathogenicity of viruses [31]. Compared with the vaccine strains of the current year, all influenza A(H3N2) strains isolated from Linyi have experienced loss of the original glycosylation sites and generation of new sites. This is likely to affect the biological characteristics of influenza A(H3N2), but further study is needed to determine the specific effects.

During summer 2022, despite the implementation of control measures by the Chinese government in response to COVID-19 outbreaks, there was still a low-intensity anti-seasonal epidemic of influenza A among children in Linyi. After the relaxation of public health and social measures for COVID-19, there was a resurgence of influenza A among children in Linyi. Antigenic drift and glycosylation sites changes occurred in the HA gene of influenza A(H3N2) viruses. Due to the rapid mutation in the HA gene of influenza A viruses, continuous molecular surveillance and timely updating of vaccine strains are essential.

Ethical Statement

This study was in compliance with the Helsinki Declaration and approved by the Ethics Committee of Women and Children's Health Care Hospital of Linyi.

Funding

This research is funded by Shandong Medical and Health Science and Technology Development Project (202206011119).

Author contributions

T. and P. designed the experiments; T. wrote the main manuscript text; J. performed the experiments; H. and X. collected data; N. prepared figures. C. and Z. checked the data. All authors reviewed the manuscript.

Statements and declarations

The authors have no competing interests to declare that are relevant to the content of this article.

Data availability

The data are obtainable from the corresponding authors upon reasonable request.

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No competing interests reported.

Influenza A virus continues to circulate among children in Linyi, northern China, after the relaxation of COVID-19 control measures

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Abstract

Figures

Introduction

Materials and methods

Results

Discussion

Conclusion

Declarations

References

Additional Declarations

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