Isolation, identification and whole genome sequence analysis of influenza B virus from Shanghai during 2023–2024

Abstract

Influenza B virus, a member of the Orthomyxoviridae family, is a significant contributor to global respiratory infections alongside influenza A virus. This study investigated the prevalence and genomic characteristics of influenza B virus during the 2023–2024 season in Shanghai, China. Among 174 clinical samples collected from the Nanxiang Branch of Ruijin Hospital, the positivity rate for influenza B virus was 62.36%. Genomic analysis revealed that all characterized isolates belonged to the B/Victoria lineage and were classified within subclade C.5.7, corresponding to the legacy designation V1A.3a.2. No B/Yamagata lineage strains were detected. Comparative genomic analysis further revealed close genetic similarity between the Shanghai isolates and strains from Shanxi Province, suggesting a potential geographical or evolutionary relationship. These findings highlight the substantial activity of influenza B virus in Shanghai during the study period and underscore the importance of continued genomic surveillance to monitor viral evolution and inform public health strategies.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1007/s00705-026-06754-7.

Highlights

The Influenza B positivity rate was 62.36% during the 2023–2024 season in Shanghai, indicating significant activity of the virus in this region.

Seven Influenza B strains were successfully isolated from 174 positive samples analyzed.

All positive samples belonged to the B/Victoria lineage, while no B/Yamagata lineage strains were detected.

Whole genome sequencing revealed that the isolated strains belong to the B/Victoria. The hemagglutinin (HA) genes of these strains were classified within clade C.5.7 of the B/Victoria lineage, corresponding to the legacy designation V1A.3a.2.

Comparative analysis showed close genetic similarity with sequences from Shanxi, suggesting potential geographical or evolutionary connection.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1007/s00705-026-06754-7.

Introduction

Influenza viruses, belonging to the Orthomyxoviridae family, are composed of eight gene segments: hemagglutinin (HA), neuraminidase (NA), nonstructural (NS), nucleoprotein (NP), matrix (M), polymerase acidic (PA), polymerase basic 1 (PB1), and polymerase basic 2 (PB2) [1]. These viruses are significant human pathogens responsible for widespread respiratory illnesses. According to the World Health Organization (WHO), influenza viruses are associated with an estimated 290,000 to 650,000 deaths annually [2]. There are four types of influenza viruses: A, B, C, and D. Among these, influenza C and D are less prevalent and typically do not cause major epidemics or pandemics. Influenza A is particularly notable for its involvement in global pandemics and co-circulates with influenza B. While the H3N2 subtype of influenza A is more commonly observed, influenza B plays a crucial role in seasonal epidemics, sometimes surpassing the prevalence of influenza A (H1N1). Despite its public health importance, the global evolution, genomic, and epidemiological characteristics of the influenza B virus remain incompletely understood.

Influenza B is classified into two distinct lineages: the Yamagata lineage (B/Yamagata/16/88) and the Victoria lineage (B/Victoria/2/87) [3]. Prior to the COVID-19 pandemic, both lineages were widely prevalent. However, since the onset of the pandemic, there has been a notable decline in reported cases of the Yamagata lineage. Although a few isolated cases have been observed, their genomic sequences are not publicly available in databases, such as GISAID, complicating efforts to track the lineage. Influenza infections can manifest with symptoms ranging from mild symptoms such as sore throat, fever, and chills to more severe complications including pneumonia and potentially death [4]. Vaccination remains the most effective preventive strategy against influenza. However, the virus frequently undergoes antigenic drift, allowing it to mutate and evade immune responses. Notably, influenza B exhibits slower antigenic changes compare to influenza A. Nowadays, neuraminidase (NA) and polymerase acidic (PA) inhibitors are the primary antiviral treatments [5]. However, widespread use of these antiviral drugs has led to the emergence of drug-resistant strains, limiting their overall effectiveness.

In China, influenza exhibits the highest prevalence during the winter and spring months, specifically from October to March. As an international metropolis and a major domestic transportation hub, Shanghai has established a comprehensive influenza surveillance system. During the COVID-19 pandemic, strict non-pharmaceutical interventions (NPIs) and standard operating procedures (SOPs) were implemented in Shanghai, which may have affected the transmission dynamics of the influenza virus. Given Shanghai’s large population, the risk of influenza outbreaks remains a significant concern.

This study aims to isolate and identify influenza B virus strains circulating in Shanghai from 2023 to 2024, with a focus on their genomic characteristics. Our primary objective was to assess the currently circulating strains and their unique features to inform and enhance strategies for outbreak prevention. The workflow of our investigation is illustrated in Supplementary Fig. 1.

Materials and methods

Specimen collection and preliminary screening

Surveillance was conducted on children aged 2 to 12 years who presented with influenza-like illness (ILI) at the Pediatric Department of Nanxiang Branch, Ruijin Hospital, Shanghai, China, between 2023 and 2024. Nasopharyngeal swabs were collected from each patient and placed in virus transport media. The samples were then transported to the Pathogen Discovery and Preservation Platform at the Shanghai Institute of Immunity and Infection for further processing.

Initial RNA extraction was performed using the Roche MagNA Pure LC 2.0 system, followed by quantitative polymerase chain reaction (qPCR) for influenza B virus detection. A total of 279 clinical specimens were screened using qPCR, of which 174 were confirmed positive for influenza B virus.

Isolation

Influenza B virus-positive samples were cultured in Madin-Darby Canine Kidney (MDCK) cell lines for virus isolation. The cells were maintained in Eagle’s Minimum Essential Medium (EMEM) supplemented with 1% penicillin-streptomycin (PS) and 10% fetal bovine serum (FBS). A total of 174 positive samples were inoculated and passaged from P1 to P4 using MDCK cells with virus growth medium (VGM), consisting of EMEM, 1% penicillin-streptomycin (PS), and 2 µg/mL TPCK treated trypsin. The samples were incubated for 1 h before infection, followed by a post-infection incubation period of 5 to 7 days at 33 °C. Samples were monitored throughout the incubation, and RT-qPCR was performed on the 3rd, 5th, and 7th days to assess viral load.

Whole genome sequencing

Whole genome sequencing was performed using Illumina sequencing technology. Nucleic acid extraction was carried out to maximize viral material purity and concentration while minimizing host contamination. Extracted RNA was fragmented using magnesium ions (Mg²⁺). The fragmented RNA underwent reverse transcription to synthesize double-stranded complementary DNA (cDNA), followed by library construction using the TruSeq™ DNA Sample Prep Kit. The libraries were purified through magnetic bead selection to eliminate fragments outside the 200–500 bp range.

Bridge PCR was used to amplify these fragments by immobilizing them on a flow cell surface to form DNA clusters. Sequencing was performed using engineered DNA polymerase and fluorescently labeled nucleotides (dNTPs) on the Illumina NovaSeq6000 PE150 platform. Each cycle incorporated a single base, and nucleotide identification was achieved by laser detection and fluorescence signal capture. The sequence data were processed and stored in FASTQ format, providing essential base sequence and quality information for bioinformatics analysis. This included contamination removal, quality control, and genome assembly using SPAdes or SOAPdenovo software. Sequence alignment was performed against viral databases to ensure accurate strain identification and genomic completeness for downstream analysis.

Genome assembly and mutation analysis

The whole genome sequence of the seven influenza B isolates compared with the vaccine strain B/Austria/1,359,417/2021, as well as the 2023–2024 influenza B virus whole genome sequence in China from the GISAID database. The evolutionary tree of the eight gene segments of the influenza virus was constructed using the Neighbor-Joining method in MEGA 10.2.0 software. Amino acid sequence alignment was performed using the alignment tool in MEGA to analyze amino acid differences. The data presented in this study have been deposited in the NCBI GenBank database. The clinical sequences were aligned using MAFFT Version 7.520, and multiple sequence alignments were visualized using Jalview Version 2.11.5.1.

Results

Viral isolation and amplification in MDCK cells

A total of 174 influenza B virus-positive clinical samples were processed for viral isolation using MDCK cell culture. After the first passage (P1), qPCR analysis on day 7 revealed that 49 samples showed no amplification and were transferred to the second passage (P2). At P2, 47 additional samples did not exhibit amplification and were excluded from further analysis.

At the third passage (P3), qPCR analysis performed on day 7 indicated that 5 samples failed to amplify, whereas 53 samples had Cq values below 30. These 53 samples were subsequently transferred to the fourth passage (P4). After P4, seven samples showed Cq values below 25 on day 7 (Supplementary Table 1), and these seven isolates were selected for further characterization and whole-genome sequencing (Supplementary Table 2; Supplementary Figs. 2–4).

Positive rate and nucleotide similarity

From 2023 to 2024, a total of 279 specimens from patients with influenza-like illness (ILI) were tested, of which 174 (62.36%) were positive for influenza B virus. Seven of these samples were successfully isolated and subjected to whole-genome sequencing.

The nucleotide sequences of all eight genomic segments (PB2, PB1, PA, HA, NP, NA, M, and NS) were compared with the vaccine strain B/Austria/1,359,417/2021. PB1 showed 99.986% nucleotide similarity, PB2 99.9913%, PA 99.9949%, HA 99.9879%, NP 99.99471%, NA 99.9961%, M 99.9958%, and NS 99.9960% similarity to the vaccine strain.

Phylogenetics analysis

Phylogenetic relationships among the seven Shanghai B/Victoria isolates were evaluated using complete sequences of all eight genomic segments, with 1,000 bootstrap replicates used to assess branch support. The seven isolates clustered within the B/Victoria lineage. The HA sequences were classified within clade C.5.7 of the B/Victoria lineage, corresponding to the legacy designation V1A.3a.2. The seven isolates clustered closely with B/Austria/1,359,417/2021 in the phylogenetic analyses.

Pairwise nucleotide distances between the seven isolates and the vaccine strain were low across the analyzed genomic segments, consistent with the high nucleotide similarities reported in Sect.  3.2. The phylogenetic tree for the HA and NA segments is shown in Fig. 1, while phylogenetic trees for the remaining segments are provided in Supplementary Fig. 5.

Fig. 1.

Phylogenetic tree of HA and NA segments

Amino acid variation

Amino acid variation sites of the B/Victoria influenza viruses analyzed in this study are summarized in Table 1. In PB2, T110N and I396M were shared among all seven isolates. A301T was detected in five isolates (B623122410, B623122704, B623122801, B623112801, and B623120303), whereas K392E was detected only in B623122410. In PB1, D51N, M65I, and I462V were shared among all seven isolates. Y38H was detected only in B623120303. In PA, S228N, I376M, and S530T were shared among all seven isolates. R312K was detected in B623122410 and B623122801, whereas V709I was detected only in B623122501. In HA, E143G, E195K, and D209E were shared among all seven isolates. N141D was detected only in B623122501, whereas K90R and G268R were detected only in B623122704. A28T in NP and V105I in BM2 were shared among all seven isolates. In NA, A47V, G378E, and I459V were shared among all seven isolates. S15L was detected only in B623112801, whereas R295S was detected only in B623121503. In M1, R105K was shared among all seven isolates. R101K was detected only in B623122410, whereas I55M was detected only in B623121503. In NS1, Y104H was shared among all seven isolates. P123L was detected only in B623122410, H63Q only in B623122704, and A96V only in B623121503. In NS2, I11L and E12Q were shared among all seven isolates. The NA and PA amino acid sequences were also examined for substitutions listed by the WHO as associated with reduced susceptibility to neuraminidase inhibitors or baloxavir, respectively. None of the NA substitutions identified in the seven isolates S15L, A47V, R295S, G378E, and I459V corresponded to the currently listed NA inhibitor resistance-associated substitutions, and none of the PA substitutions identified in the seven isolates S228N, R312K, I376M, S530T, and V709I corresponded to the currently recognized baloxavir reduced-susceptibility substitutions. Therefore, no known NA inhibitor- or baloxavir resistance-associated substitution was identified in the seven isolates based on the WHO reference lists. However, the absence of known resistance-associated substitutions in sequence data does not by itself establish phenotypic antiviral susceptibility [6]. Overall, the amino acid substitutions identified in the seven isolates were distributed across PB2, PB1, PA, HA, NA, M1, M2, NS1, and NS2, whereas the NP sequence showed only the shared A28T substitution.

Table 1.

Amino acid variation sites of B/Victoria influenza viruses isolated in Shanghai during 2023–2024 compared with the reference vaccine strain B/Austria/1359417/2021

Sample ID	Segments & Mutations
	PB2	PB1	PA	HA	NP	NA	M		NS
							M1	M2	NS1	NS2
B623122410	T110N, A301T, K392E, I396M	D51N, M65I, I462V	S228N, R312K, I376M, S530T	E143G, E195K, D209E	A28T	A47V, G378E, I459V	R101K, R105K	V105I	Y104H, P123L	I11L, E12Q
B623122501	T110N, I396M	D51N, M65I, I462V	S228N, I376M, S530T, V709I	N141D, E143G, E195K, D209E	A28T	A47V, G378E, I459V	R105K	V105I	Y104H	I11L, E12Q
B623122704	T110N, A301T, I396M	D51N, M65I, I462V	S228N, I376M, S530T	K90R, E143G, E195K, D209E, G268R	A28T	A47V, G378E, I459V	R105K	V105I	H63Q, Y104H	I11L E12Q
B623122801	T110N, A301T, I396M	D51N, M65I, I462V	S228N, R312K, I376M, S530T	E143G, E195K, D209E	A28T	A47V, G378E, I459V	R105K	V105I	Y104H	I11L, E12Q
B623112801	T110N, A301T, I396M	D51N, M65I, I462V	S228N, I376M, S530T	E143G, E195K, D209E	A28T	S15L, A47V, G378E, I459V	R105K	V105I	Y104H	I11L, E12Q
B623120303	T110N, A301T, I396M	Y38H, D51N, M65I, I462V	S228N, I376M, S530T	E143G, E195K, D209E	A28T	A47V, G378E, I459V	R105K	V105I	Y104H	I11L, E12Q
B623121503	T110N, I396M	D51N, M65I, I462V	S228N, I376M, S530T	E143G, E195K, D209E	A28T	A47V, R295S, G378E, I459V	I55M, R105K	V105I	A96V, Y104H	I11L, E12Q

Discussion

The seven B/Victoria influenza viruses isolated in Shanghai during 2023–2024 showed high nucleotide similarity to the vaccine strain B/Austria/1,359,417/2021 across all eight genomic segments, with similarities ranging from 99.986% to 99.9961%. Phylogenetic analysis further placed the HA sequences of all seven isolates within clade C.5.7 of the B/Victoria lineage, corresponding to V1A.3a.2. The amino acid analysis identified substitutions shared among all seven isolates as well as substitutions restricted to individual isolates or subsets of isolates. Overall, the sequence profile indicates that the analyzed viruses formed a closely related group of B/Victoria viruses with limited amino acid variation. The HA substitutions shared among all seven isolates included E143G, E195K, and D209E, whereas N141D, K90R, and G268R were restricted to individual isolates. Published surveillance data describe C.5.7 as a subclade within the V1A.3a.2 lineage and report its circulation in multiple geographic regions during the 2023–2024 period [7]. C.5.7 has been described in surveillance analyses as carrying additional HA substitutions, including E128G and E183K, in viruses assigned to this subclade [8]. These substitutions were not identified among the HA substitutions listed for the seven isolates in the present study. Therefore, the substitutions detected here should be interpreted as sequence differences observed in the analyzed viruses relative to the selected vaccine reference and should not be considered, individually, as defining markers of C.5.7.

The relationship between the circulating viruses and the vaccine strain is also consistent with recent WHO antigenic surveillance. WHO reports that B/Victoria viruses within the V1A.3a.2 lineage, including C.5.7 viruses, were generally well recognized by antisera raised against B/Austria/1,359,417/2021-like viruses [9]. In the WHO assessment for the 2025–2026 Northern Hemisphere season, post-infection ferret antisera recognized the majority of tested C.5.1, C.5.6, and C.5.7 viruses well [9]. These observations support a cautious interpretation of the present sequence findings: the identification of amino acid substitutions in HA does not, by itself, demonstrate antigenic escape or reduced vaccine effectiveness. The non-HA segments also showed a limited number of shared and isolate-specific substitutions. Several substitutions were shared among all seven isolates, including T110N and I396M in PB2; D51N, M65I, and I462V in PB1; S228N, I376M, and S530T in PA; A28T in NP; A47V, G378E, and I459V in NA; R105K in M1; V105I in BM2; Y104H in NS1; and I11L and E12Q in NS2. Other substitutions were restricted to individual isolates or subsets of isolates. Because the present study did not include reverse-genetics, replication, receptor-binding, antigenic, protein-stability, or host-interaction experiments, the functional consequences of these individual substitutions cannot be established from the sequence data alone.

Antiviral resistance analysis did not identify any of the substitutions present in the seven isolates among the currently recognized NA inhibitor resistance-associated substitutions or baloxavir reduced-susceptibility substitutions listed by WHO [10]. WHO identifies several NA substitutions associated with reduced inhibition by neuraminidase inhibitors and emphasizes that their interpretation requires consideration of phenotypic data. For baloxavir, PA I38T is considered clinically relevant, whereas other substitutions have been observed less frequently and their clinical significance may remain uncertain [11]. None of the PA substitutions identified in the present study corresponded to the currently recognized baloxavir-associated substitutions. The recent WHO GISRS global analysis also emphasizes surveillance of susceptibility to neuraminidase inhibitors and the cap-dependent endonuclease inhibitor baloxavir. In addition, global surveillance studies have demonstrated that antiviral resistance-associated substitutions may occur sporadically and that their interpretation should be based on standardized genotypic and phenotypic analyses [12]. Therefore, the absence of recognized resistance-associated substitutions in the present dataset should not be interpreted as definitive evidence of antiviral susceptibility. WHO recommends phenotypic testing where appropriate, particularly when unusual substitutions or reduced susceptibility are detected. Accordingly, the present findings indicate that no known genotypic markers of NA inhibitor or baloxavir reduced susceptibility were detected in the seven isolates, but phenotypic susceptibility testing would be required to directly assess antiviral susceptibility.

Overall, the seven B/Victoria viruses characterized in this study were genetically closely related to B/Austria/1,359,417/2021 and belonged to the C.5.7 clade within the V1A.3a.2 lineage. Although multiple amino acid substitutions were identified across the eight genomic segments, most were shared among the seven isolates or occurred in individual isolates, and sequence analysis alone does not establish their functional consequences. Continued genomic, antigenic, and antiviral susceptibility surveillance will therefore be important for monitoring the evolution of B/Victoria viruses and identifying substitutions that may warrant further phenotypic or functional investigation.

Limitations

This study has several limitations. First, the phylogenetic analysis was restricted to Chinese sequences, which limits global interpretation. Second, sequencing was performed on MDCK cell-adapted isolates due to low viral loads in most clinical specimens, introducing potential selection bias and reducing representation of the full circulating diversity. Third, antigenic effects of HA mutations could not be experimentally confirmed because HI assays were not feasible.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization and Experiments, M.A.A; writing-original draft preparation, M.A.A; writing-review and editing, M.A.A., L.Y., M.A.R., B.S. M.N.A., G.D., L.C., H.X., H.Y., R.H., Y.T., A.D., A.A.B and Y.H.; visualization, M.A.A.; supervision, Y.H.; project administration Y.H., H.Y., A.D.; funding acquisition, Y.H. All authors have read and agreed to the published version of the manuscript.

Data availability

Not Applicable.

Declarations

Ethics statement

This study was approved by the Ethics Committees of Shanghai Nanxiang Branch of Ruijin hospital, and complied with all relevant ethical regulations. The handling respiratory viruses was conducted in strict accordance with standard procedures for an advanced Biosafety Level 2 laboratory.

Institutional review board statement

Not applicable.

Informed consent

Not applicable.

Conflict of interest

The authors declare no conflict of interest.