Genetic Characterization of Influenza A (A/H3N2) Viruses Reveals Antigenic Drift in Receptor Binding Domain and Possible Vaccine Mismatch in Strains Circulating in Riyadh, Saudi Arabia, 2024-2025

Genetic Characterization of Influenza A (A/H3N2) Viruses Reveals Antigenic Drift in Receptor Binding Domain and Possible Vaccine Mismatch in Strains Circulating in Riyadh, Saudi Arabia, 2024-2025 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genetic Characterization of Influenza A (A/H3N2) Viruses Reveals Antigenic Drift in Receptor Binding Domain and Possible Vaccine Mismatch in Strains Circulating in Riyadh, Saudi Arabia, 2024-2025 Shatha Ata Abdulgader, Ibrahim M. Aziz, Abdulhadi M. Abdulwahed, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8353173/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Feb, 2026 Read the published version in BMC Infectious Diseases → Version 1 posted 13 You are reading this latest preprint version Abstract Introduction: Influenza A/H3N2 viruses undergo continuous antigenic evolution, necessitating ongoing surveillance for informed vaccine strain selection. This study characterized the molecular epidemiology of H3N2 viruses circulating in Riyadh, Saudi Arabia, during the winter season of 2024-2025 and assessed their compatibility with current vaccine strains. Methods: Nasopharyngeal samples (NPAs) (n=363) were collected from patients presenting with influenza-like illness at King Khalid University Hospital in Riyadh. Influenza A/H3N2 detection and subtyping were performed using RT-PCR. Complete hemagglutinin (HA) and neuraminidase (NA) gene sequencing was conducted on confirmed A/H3N2 strains (n=7), followed by phylogenetic analysis, amino acid substitution mapping, and glycosylation site prediction. Results: Of 363 samples tested, 110 (30.3%) were positive for influenza A, with 42 (38.2%) identified as A/H3N2 and 68 (61.8%) as A/H1N1pdm09. Phylogenetic analysis revealed that all seven sequenced A/H3N2 strains belonged to clade 2a.3a.1, which is identical to the current vaccine strain clade. However, molecular analysis identified six amino acid substitutions in the HA gene and four in the NA gene that distinguished circulating strains from the A/H3N2 vaccine strain A/Thailand/8/2022. Notably, all study strains showed deletion of an N-glycosylation site (N122S) that is present in the vaccine strain. Conclusions: While phylogenetic clade compatibility suggests potential vaccine effectiveness, the observed amino acid differences and glycosylation site deletion highlight the importance of continued molecular surveillance to monitor antigenic drift and assess vaccine performance in the Saudi Arabian population. vaccine compatibility IAV phylogenetic analysis glycosylation sites molecular surveillance Figures Figure 1 Figure 2 Figure 3 1. Introduction Influenza viruses are highly contagious pathogens and the leading cause of acute febrile respiratory illness worldwide. They impose a major public-health and economic burden, triggering seasonal epidemics and, at times, devastating pandemics [ 1 – 3 ]. Several subtypes of influenza A virus (IAV) have caused global outbreaks in the past century, including the 1918 H1N1, 1957 H2N2, 1968 H3N2, and 2009 H1N1 pandemics [ 4 ]. The ability of IAV to cross species barriers and to evolve continuously through genetic change has enabled the emergence of novel antigenic variants such as H5N1, H7N9, H9N2, H5N8, and H7N7 [ 5 , 6 ]. Two main mechanisms drive the evolutionary of IAV: antigenic drift and antigenic shift. Antigenic drift arises from point mutations that accumulate gradually within the viral genome, particularly at the antigenic sites of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Because the viral RNA-dependent RNA polymerase lacks proofreading activity, these mutations occur frequently and can alter viral tropism, virulence, replication efficiency, and antigenicity [ 7 ]. In contrast, antigenic shift results from reassortment of the segmented viral RNA during co-infection, generating new genome arrangements with unique antigenic properties and the potential to cause pandemics [ 8 , 9 ]. Among IAV subtypes, A/H3N2 viruses have demonstrated remarkable antigenic drift since their first detection in 1968 [ 10 ]. The World Health Organization (WHO) has consequently updated the A/H3N2 vaccine component more than 28 times [ 11 ]. Despite these efforts, vaccine effectiveness often remains suboptimal; for example, efficacy during the 2016–2017 season was only 28–42% across age groups [ 12 ]. Such limited protection underscores the need for continuous genetic and antigenic monitoring to guide timely vaccine reformulation [ 13 ]. Saudi Arabia represents a unique setting for respiratory-virus transmission owing to its annual mass gatherings (Hajj and Umrah) and the movement of millions of foreign workers [ 14 ]. These dynamics can accelerate viral importation, circulation, and mutation. Although several local studies have described influenza A/H3N2 activity in previous years [ 15 , 16 ], information on its molecular epidemiology and genetic diversity remains limited—particularly for the 2024–2025 season. Our previous surveillance in Riyadh (2014–2020) showed that 48.8% of IAV isolates were A/H3N2 [ 17 ]. Also, our recently and a more recent surveillance in Riyadh (2020–2023) revealed that A/H3N2 subtype was found in 9.21% of IAV isolates, which overcame the number of A/H1N1 isolates 7.89% [ 18 ]. However, comprehensive molecular characterization of circulating strains during the recent 2024–2025 season remains absent, creating a critical knowledge gap for evidence-based vaccine policy. This study specifically aimed to (1) determine the prevalence of A/H3N2 viruses in Riyadh during 2024–2025; (2) characterize the complete HA and NA gene sequences of circulating strains; (3) assess phylogenetic relationships and clade classification; (4) identify amino-acid substitutions within key antigenic sites; (5) evaluate N-glycosylation patterns; and (6) determine vaccine-strain compatibility for the strains identified from Saudi population. Understanding these molecular patterns will provide critical insight for national vaccination policy, future vaccine-strain selection, and preparedness for potential influenza epidemics in Saudi Arabia. 2. Materials and Methods 2.1. Ethical approval The study was conducted in accordance with the Declaration of Helsinki and approved by the Research Ethics Committee at King Saud University, Riyadh, Saudi Arabia (Institutional Review Board Nos. E-24-9609 and E-25-9609, approved in November 2023 and May 2025, respectively). All NPAs that tested positive for IAV were handled in accordance with ethical standards, ensuring patient confidentiality and appropriate medical follow-up as per King Saud University regulations. 2.2. Acquisition of clinical samples A total of 363 NPAs were collected from individuals presenting with influenza-like illness (ILI) at King Khalid University Hospital, Riyadh, between September 2024 and February 2025. Patients exhibiting symptoms such as fever, cough, sore throat, runny nose, muscle or body aches, headache, or fatigue were enrolled after providing informed consent. Individuals who had been vaccinated with seasonal influenza vaccines were excluded from the study. Each specimen was mixed with 2 mL of virus minimum essential medium (MEM) transport medium (Gibco, Invitrogen, Grand Island, NY, USA) and transported under refrigerated conditions to the Virology Research Laboratory, College of Science, King Saud University. Samples were briefly vortexed for 15 s, centrifuged at 1,000× g for 10 min at 4 °C, aliquoted, and stored at −80 °C until further analysis. 2.3. Typing, detection and sequencing of IAV 2.3.1. Detection and typing Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Detection and subtyping of IAV were carried out using the One-Step Ahead RT-PCR Kit with Taq High-Fidelity DNA Polymerase (Qiagen, Hilden, Germany; Cat. No. 220213). The reaction was performed on a GeneAmp 9700 Thermal Cycler (Applied Biosystems, USA) under the following cycling conditions: reverse transcription at 50 °C for 30 min, initial denaturation at 95 °C for 15 min, followed by 35 cycles of denaturation at 94 °C for 15 s, annealing at 52 °C for 30 s, and extension at 72 °C for 2 min. A final extension was performed at 72 °C for 10 min. PCR products were visualized by electrophoresis on a 1% agarose gel stained with ethidium bromide and compared against a 100 bp DNA ladder (Qiagen, Germany). 2.3.2. Amplification of full-length HA and NA genes The HA and NA genes of A/H3N2 strains were amplified using the same kit and thermal protocol described above, with two sets of overlapping primers to obtain the complete gene sequences. Primer sequences and PCR conditions for HA and NA gene amplification are detailed in Table 1. All primers were designed based on conserved regions and validated using reference strains before clinical application. Seven representative A/H3N2 isolates (n = 7) were selected for sequencing based on sample quality and temporal distribution across the study period (2024 and 2025). Sequencing was performed by Macrogen Inc. (Seoul, South Korea). Raw sequence data were edited using BioEdit v7.0 (Ibis Biosciences, Carlsbad, CA, USA) and assembled using the EditSeq and MegAlign modules of Lasergene software v3.18 (DNAStar, Madison, WI, USA). All generated sequences were submitted to GenBank under accession numbers EPI_ISL_653577, EPI_ISL_653578, EPI_ISL_653579, EPI_ISL_653580, EPI_ISL_653581, EPI_ISL_653582, EPI_ISL_653583 for HA gene. EPI_ISL_653552, EPI_ISL_653553, EPI_ISL_653554, EPI_ISL_653555, EPI_ISL_653556, EPI_ISL_653557, EPI_ISL_653558. 2.4. Sequence and phylogenetic analysis Multiple sequence alignment of complete HA and NA genes were performed using the ClustalW algorithm implemented in the MegAlign program. The analysis compared the A/H3N2 Riyadh strains with 100 local and international reference and vaccine strains retrieved from GISAID and GenBank databases (see Supplementary Table S1). Sequence variations including; amino-acid substitutions, indels, nucleotide divergences were considered in the analysis. In addition, potential N- and O-glycosylation sites were identified using NetNGlyc 1.0 [ 20 ] and NetOGlyc 3.1 [ 21 ] . Phylogenetic trees were constructed by the neighbor-joining method using MEGA v11 (Pennsylvania State University, University Park, PA, USA), with 1,000 bootstrap replicates to assess branch reliability. 2.5. Statistical analysis: Statistical analysis was performed using IBM SPSS Statistics v26.0 (IBM Corp., Armonk, NY, USA). Categorical variables were analyzed using Fisher's exact test, and post-hoc comparisons were conducted using the Z-test with Bonferroni correction. A p-value < 0.05 was considered statistically significant. 3. Results 3.1. Detection and typing of influenza A/H3N2 Among the 363 NPAs analyzed, 110 (30.3%) were positive for IAV, and 68 (18.7%) were confirmed cases of influenza B (IBV). Although these IBV-positive samples were part of the original screening dataset, the current study's genomic characterization was limited to H3N2-positive samples. Of these 110 positive IAV samples, 68 (61.8%) were identified as A/H1N1pdm09 and 42 (38.2%) as A/H3N2. Samples were categorized into two collection periods: 2024 and 2025. In 2024, A/H1N1pdm09 was predominant (80.8%) whereas A/H3N2 accounted for only 19.2%. However, A/H3N2 prevalence increased markedly in 2025, representing 52.4% of positive IAV cases. Of the total specimens, 176 (48.4%) were from males and 187 (51.5%) from females. A higher infection rate was recorded among females (34.7%) compared with males (25.5%, p < 0.05). Age distribution analysis revealed that individuals aged 15–64 years had the highest positivity rate (32.6%), which was significantly higher than the rates in the 0–4, 5–14, and ≥65-year age groups (p < 0.05). Detailed demographic and seasonal distributions are presented in Table 2. 3.2. Nucleotide and amino-acid variation in HA and NA Genes Complete HA (1,701 nt) and NA (1,410 nt) gene sequences were obtained from seven representative A/H3N2 isolates (three from 2024 and four from 2025) were compared with 100 globally circulating A/H3N2 strains retrieved from the GISAID and GenBank databases using the consensus sequence (A/New York/392/2004). (Table S1). The sequence analysis indicated that the nucleotide identity varied from 97.75 to 98.56% ( HA ), whereas the NA identity was observed to be between 97.23 and 98.65%. Comparison of the HA1 domain between the current vaccine strain A/Croatia/10136RV/2023 and the A/Riyadh isolates revealed four amino-acid differences (N145S, A186D, V223I, P239Q) (Figure 1A, 1B, 1C). On the other hand, the NA gene of the current vaccine strain A/Croatia/10136RV/2023 was homologous to the study isolates (Figure 2A, 2B, 2C, 2D). The aim of such comparison is to compare current circulating strains with vaccine to ensure the effectiveness of current vaccine strain against highly mutated gene of H3N2 of circulating strains. Also, since the vaccine are designed for highly conserved region of the virus, it is important to monitor these differences to reduce risk of transmutation, lowering the burden of influenza disease, and it can also help in predicting early pandemic of the virus in the country. Several identical sequences connected to antigenicity within the receptor-binding domain (RBD), including the 130-loop, 150-loop, 190-helix, and 220-loop, were observed when the HA1 of the Riyadh isolates was compared to the vaccine strains (A/Croatia/10136RV/2023) (Figure 1B). Most of the isolates in the study demonstrated a consistent change in the A186D and V223I amino acid changes within the 190-helix and 220-loop, respectively, in contrast to the vaccine strain (Figure 1B). The mutations mentioned above were observed not only in our A/H3N2 Riyadh strains but also in sequences obtained from various countries globally; however, they were absent in the H3N2 vaccine strain A/Croatia/10136RV/2023. In fact, these mutations in our strains were situated within or adjacent to recognized antigenic sites, which may influence antigenic drifts and the recognition of the virus by the vaccine. 3.3. Glycosylation-site analysis Analysis of the HA1 open reading frame predicted 10- 11 N-glycosylation sites (positions 8, 22, 38, 63, 94, 126, 133, 165, 246, and 285). O-glycosylation analysis revealed potential modification sites at positions 55–59 within the HA1 domain, similar to the vaccine strain (Figure 1A, 1B, 1C). The NA protein possessed nine N-glycosylation sites (positions 61, 70, 86, 146, 200, 233, 245, and 367) consistent with the vaccine strain. O-glycosylation sites ranged from 70–72, also comparable to those of the vaccine strain (Figure 2A, 2B, 2C, 2D). 3.4. Phylogenetic analysis Phylogenetic trees constructed for both the HA and NA genes demonstrated that all Riyadh A/H3N2 strains clustered within clade 2a.3a.1, the same clade as the WHO-recommended vaccine strain A/Croatia/10136RV/2023. This finding indicates high genetic similarity between the circulating and vaccine strains (Figure 3Aand 3B). Sequence homology with the vaccine strain was approximately 68%, supporting the phylogenetic inference of relatedness. The Riyadh isolates were distinct from earlier local strains (2014–2020) and (2020-2023) but closely related to recent international strains such as A/Georgia/14289/2023, A/Vasteras/SE23/14213/2023, and A/Auckland/17/2024. These patterns suggest recent introduction of genetically distinct viruses into the region, which is facilitated by international air travelling activity. 4. Discussion Influenza virus evolve rapidly and continuously, which requires close monitoring of these mutations in order to inform public health authority about vaccine content and lowering the disease burden. Therefore, the present study provides updated molecular data on the circulation, genetic variation, and clade distribution of influenza A/H3N2 viruses in Riyadh during the 2024–2025 season. Of 363 samples analyzed, 110 (30.3%) were IAV-positive, with A/H1N1pdm09 predominating in 2024 and A/H3N2 rising sharply in 2025. This shift mirrors previous regional reports showing alternating dominance between these two subtypes [ 15 , 18 , 22 ]. Similar transitions were described in the Middle East and North Africa, where A/H1N1pdm09 accounted for ≈ 50% of infections but was later overtaken by A/H3N2 [ 23 ]. Also, the transition was observed by our research group in Riyadh during recent years (2020–2023) [ 18 ]. The observed pattern likely reflects the high mutation rate and rapid antigenic drift of A/H3N2 viruses [ 24 ]. Consistent with prior Saudi studies [ 25 , 26 ], adults aged 15–64 years represented the most affected group, and females exhibited a higher infection rate than males. Despite similarities observed in term of the prevalence of A/H3N2 of our study with our recent study, the difference was showed by males and children ages 0–4 having most of cases [ 18 ]. Similar to other types, the HA glycoprotein of A/H3N2 is necessary for attaching to host cell receptors and permitting the release of viral RNA into the cell. [ 27 ]. To date, the traditionally described RBS, which is framed by the 130 and 220 loops and the 190-helix, has been found to contain important receptor-binding residues or contributors to binding specificity in almost all influenza HAs. The main structural components of HA1 that comprise the RBD are the 130-loop (residue number 134–138, H3 numbering), 150-loop (150–156), 190-helix (181–193), and 220-loop (221–228). Moreover, the highly conserved residues Tyr98 (Y98), Trp153 (W153), and His183 (H183) make up the base of the pocket [ 28 ]. This structure particularly targets host cell sialic acid residues [ 29 – 31 ]. Four significant classical epitopes on the globular HA head have been recognized by the human immune system: Ca (including Ca1 and Ca2), Cb, Sa, and Sb. Changes in HA protein associated with antigenic drift occur continuously throughout time as the virus grows to evade being inhibited by antibodies produced by vaccination and spontaneous infection [ 32 ]. Comparing the study isolates to the vaccine strains (A/Croatia/10136RV/2023), most of the isolates in the study demonstrated a consistent change in the A186D and V223I amino acid changes within the 190-helix and 220-loop, respectively, in contrast to the vaccine strain. Escape mutants of influenza viruses exhibit differences in the structure and biological roles of HA , especially in regard to the RBD. Human reinfection may result from these mutations [ 27 , 33 ]. Influenza vaccination is recognized as a highly effective strategy in public health for preventing infections. The level of protection afforded to both individuals and communities through vaccination depends on factors such as the vaccine coverage, the rate at which people receive the vaccine, and how well the strains included in the vaccine match those circulating in a given season. Studies have shown that the influenza vaccine can exhibit efficacy rates of up to 60% against infections caused by Types A and B viruses [ 34 ]. For the aim of increasing the protection and efficacy of the vaccine, the WHO offers over 28 vaccine strain for both Northern and Southeren hemisphere [ 17 ]. A number of these strains have been recommended by the Saudi Ministry of health for A/H3N2 subtype including; A/Singapore/Infimh/16/0019/2016, A/Cambodia/e0826360/2020, A/Darwin/6/2021, A/Thailand/8/2022, A/Massachusetts/18/2022, A/Croatia/10136RV/2023. All of which were used as a reference in this study. To evaluate the effectiveness of the existing vaccine against the prevalent (A/H3N2) strains in Riyadh, a comparison was made between the seven strains studied and the vaccine strain A/Croatia/10136RV/2023. Notably, our A/H3N2 strains exhibited four amino acid substitutions in the HA while NA gene was homologous with the vaccine strain. Our previous study also detected 12 and nine amino acids changes for both HA and NA genes in comparison with the vaccine strain A/Singapore/Infimh/16/0019/2016 recommended during the year of study [ 17 ]. Similarly, in a more recent study, Alkubaisi et al 2025 reported the presence of nine amino acids substitutions in HA and NA genes that differentiated the studied strains from vaccine strains A/Croatia/10136RV/2023, A/Thailand/8/2022, and A/Darwin/6/2021 [ 18 ]. As a consequence, yearly influenza surveillance is crucial for gathering vital information needed for the yearly reformulations of influenza vaccination and for detecting any possible epidemic or pandemic [ 18 ]. In line with facts about ongoing antigenic drift of H3N2 strains that were historically documented [ 37 ], we compared our A/H3N2 strains with the reference strain A/New York/392/2004 and strains identified previously in Riyadh (2014–2020) and (2020–2023), for the aim to identify evolutionary kinetic of this virus in the same city. 108 and 60 mutations site for HA and NA gene were detected, respectively. Of these, twelve were unique for HA gene of our strains as compared to other local circulating strains in previous years and reference strain. While the analysis for NA gene revealed the presence of only five unique mutations compared to other local strains. Phylogenetic analysis demonstrated that all A/H3N2 isolates clustered within clade 2a.3a.1—the same clade as the WHO-recommended vaccine strain A/Croatia/10136RV/2023. This genetic alignment indicates a favorable match between circulating and vaccine strains. In our previous study, we found A/Saudi Arabia/VRG-02/2016, A/Saudi Arabia/VRG-03/2016, A/New Jersey/26/2014, A/Fiji/2/2015, and A/Canberra/7/2016 in root 3c.2a [ 17 ]. Thirteen more strains of subclade 3C.2a1b were identified by mutations of F159Y, K160T, N171K, N121K, K92R, and H311Q [ 38 , 39 ]. Among the 16 strains that comprised the 3c. clades/subclade, two members of clade 3c.2a (A/Saudi Arabia/VRG-2/2016 and A/Saudi Arabia/VRG-7/2016) were identified using the mutations F159Y and K160T [ 38 , 40 ]. Except for A/Saudi Arabia/VRG-01/2016, which belonged to clade 3c.2a3 and was not grouped with a vaccine strain. While our previous and recent study (2020–2023) identified that circulating strains were not clustering in any clade of recommended vaccine strains, but were grouped into two subclade 3c.2a1b.1a and 3c.2a1b.1b [ 18 ].The strains under investigation remained in the same clade as the vaccine strains. This might suggest global predominance of 2a.3a.1 clade in 2024–2025. The global predominance of H3N2 clade 2a. 3a.1 has important virological and public health implication, as recent surveillance reports from the CDC and ECDC indicate that nearly all genetically characterized H3N2 viruses detected across multiple continents during 2024–2025 season belong to 2a. 3a.1, reflecting a clear selective advantage and widespread transmission [ 41 , 42 ]. This dominance is consistent with the well -known capability of H3N2 to undergo rapid antigenic drift, particularly through amino-acids substitutions in key HA antigenic sites, which can reduce population immunity and facilitate immune escape [ 24 , 34 ]. Such drifted lineages belonging to clade 2a. 3a.1 have recently been observed to be associated with reduced vaccine effectiveness. Indeed, in a current report of WHO found that based on human serological tests, strains belonging to 2a. 3a.1 clade had a low antibody detection against the virus after vaccination compared to the recommended vaccine strain [ 43 ]. The emergence and global expansion of clade 2a.3a.1 therefore, raise concern about potential mismatch with current vaccine strain and highlight the necessity for continuous influenza surveillance to inform public health authority about vaccine update. furthermore, it might also raise concern about the possibility of pandemic occurrence in the future. The phylogenetic clustering identified in our study provides vital insight into the possible influence of international travel on the diversity of circulating H3N2 strains in Saudi Arabia. Several of our isolate correlated closely with globally circulating 2a.3a.1 lineage rather than with strains historically reported in the region, suggesting new viral introduction from outside the country. Saudi Arabia hosts millions of travelers annually for job, tourism, and religious pilgrimages, giving ongoing opportunities for the introduction of genetically different influenza viruses. By displacing or co-circulating with established strains, these introductions can change the local viral ecosystem by increasing selective pressure and influence of population immunity [ 44 , 45 ]. Therefore, the phylogenetic tree supports the idea that travelling abroad significantly influences the temporal and genetic variety of influenza viruses found locally. Nevertheless, phylogenetic relatedness alone does not guarantee antigenic equivalence, as minor HA or NA substitutions can substantially affect immune recognition and viral antigenicity [ 12 ]. Therefore, continued regional monitoring and, ideally, serologic confirmation through hemagglutination-inhibition (HI) assays remain priorities. The quantity, length, and types of N-linked glycosylation on the surface of HA significantly influence its antigenicity and the immunogenic response of the virus [ 46 ]. Indeed, N-linked glycosylation can diminish the efficacy of antibody-mediated cross-neutralization through antigen masking, a phenomenon observed with the 1918 influenza pandemic strain and the A(H1N1)pdm09 strain, where additional N-linked glycosylation sites were located at the 129 and 163 residues [ 47 ]. In contrast, insufficient glycosylation of glycoproteins can adversely affect HA by leading to improper folding and transport, as observed in a study conducted by Gallagher et al [ 48 ]. Moreover, the connection between variations in N-linked glycans (NLG) and the transmissibility of IAV must also be taken into account. Since the NLG on the globular head of hemagglutinin (HA) can alter receptor-binding specificity, modifications in NLG may influence the transmissibility of IAV. In a study using a guinea pig model, an H5N1 virus that was able to bind to both 3' sialic acid (SA) and 6' SA lost its ability to bind to 6' SA due to the A160T mutation, leading to a complete loss of transmissibility of the parental strain. Therefore, beyond changing receptor specificity, alterations in the NLG of the globular head of HA can significantly impact viral transmission [ 49 ]. Additionally, a novel N-linked glycosylation site in either HA or NA can serve as a marker for specific strains. For example, in the strains associated with the 1918 and 2009 influenza pandemics, the presence of a distinct 104 amino acid residue with an N-glycosylation site can help determine whether an influenza reassortment originated from the 1918 or 2009 pandemic strains. Overall, mutations in N-linked glycosylation sites in HA and NA influence the virulence, pathogenicity, and susceptibility of IAV to neutralizing antibodies [ 50 ]. While in terms of O-linked glycosylation found in the influenza surface glycoproteins HA and NA, O-linked glycan can act as glycan shield that mask antigenic sites from host neutralizing antibodies, thereby contributing to immune evasion and promoting antigenic drift [ 51 ]. For instance, Ming et al [ 52 ] showed that the addition of O-glycan near the receptor binding domain of HA diminished antibody recognition, facilitating escape from humoral immunity [ 53 ]. Furthermore, O-glycosylation influences the receptor-binding specificity of HA , where the presence or absence of specific O-glycan can modulate the affinity of HA for human-like (α2,6 linked) or avian-like (α2,3 linked) sialic acid receptors, affecting both tissue tropism and interspecies transmission [ 54 ]; this was demonstrated by Li et al [ 55 ], who observed that alteration in HA O- glycosylation impacted host specificity and adaptation. Additionally, O-glycan affect HA protein stability, cleavage efficiency, and membrane fusion- key steps in viral entry and replication [ 55 ]. In particular, Schulze (1997) reported that the removal of certain O-linked glycans enhanced membrane fusion activity, leading to increased viral infectivity. These modifications not only play a role in the evolutionary dynamics of influenza viruses, as shift in glycosylation often occur alongside key antigenic mutations, highlighting the importance of monitoring O-linked glycosylation patterns for vaccine strain selection and molecular surveillance [ 56 ]. Therefore, from the above paragraphs about N and O- linked glycosylation, it is important to identify any new modification in these two types of glycosylation. In the current study, all predicted N- and O-linked glycosylation sites were conserved between vaccine and circulating strains which might indicate high similarities with vaccine strain. The data support that the 2024–2025 A/H3N2 vaccine component likely provided adequate genetic coverage for strains circulating in Riyadh. However, the identified amino-acid substitutions compared to vaccine strain highlight the virus’s ongoing evolution and the need for year-round molecular surveillance. Key limitations include the relatively small number of sequenced isolates and the absence of cross-HI testing, which restrict antigenic interpretation. Future work should incorporate larger multicenter sampling, complete-genome sequencing, and phenotypic assays to better evaluate vaccine efficacy and transmission dynamics within the Kingdom. 5. Conclusions This study provides updated molecular insights into the circulation and genetic diversity of influenza A/H3N2 viruses in Riyadh, Saudi Arabia, during the 2024–2025 influenza season. Among the 363 samples analyzed, A/H3N2 accounted for 42 (38.2%) of all IAV detections and increased in frequency during 2025. Phylogenetic analysis demonstrated that all local isolates belonged to clade 2a.3a.1, the same clade as the current WHO-recommended vaccine strain A/Croatia/10136RV/2023, indicating a high level of vaccine–strain compatibility. Nevertheless, four amino-acid substitutions in the HA gene and sequence homology of NA gene, underline the virus’s continuing molecular evolution. Although the overall vaccine match appears favorable, the observed mutations of our strains compared to vaccine strain warrant continued genomic surveillance and functional evaluation to assess their effects on antigenicity and vaccine performance. Expanding molecular surveillance programs across multiple Saudi regions and integrating serological assays such as HI tests will be essential for early detection of emerging variants and for supporting evidence-based updates of seasonal vaccine formulations. Declarations Acknowledgments: The authors thank the Ongoing Research Funding Program (ORF-2025-418), King Saud University, Riyadh, Saudi Arabia, for supporting this work. Author Contributions: Conceptualization, S.A.A., F.N.A, I.M.A., A.M.A. (Abdulhadi M. Abdulwahed); methodology, S.A.A., and I.M.A.; software, S.A.A., M.A.F., and I.M.A.; validation, S.A.A., I.M.A., and R.M.A.; formal analysis, S.A.A. and I.M.A.; investigation, S.A.A. and I.M.A., A.M.A. (Abdulhadi M. Abdulwahed), A.M.A. (Abdulaziz M. Almuqrin), and F.N.A.; resources, F.N.A.; data curation, S.A.A. and I.M.A.; writing—original draft preparation, S.A.A.; writing—review and editing, M.A.F., R.M.A, A.M.A. (Abdulhadi M. Abdulwahed), A.M.A. (Abdulaziz M. Almuqrin), and I.M.A.; visualization, S.A.A. and M.A.F.,; supervision, A.M.A. (Abdulhadi M. Abdulwahed), A.M.A. (Abdulaziz M. Almuqrin), and F.N.A.; project administration, F.N.A.; funding acquisition, F.N.A. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Ongoing Research Funding Program (ORF-2025-418), King Saud University, Riyadh, Saudi Arabia. Conflicts of Interest: the authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki and approved by the Research Ethics Committee at King Saud University, Riyadh, Saudi Arabia (Institutional Review Board Nos. E-24-9609 and E-25-9609, approved in December 2023 and May 2025, respectively). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. 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Supplementary Files Table1.docx Table2.docx Supplementary.docx Cite Share Download PDF Status: Published Journal Publication published 19 Feb, 2026 Read the published version in BMC Infectious Diseases → Version 1 posted Editorial decision: Revision requested 30 Jan, 2026 Reviews received at journal 28 Jan, 2026 Reviewers agreed at journal 12 Jan, 2026 Reviewers agreed at journal 11 Jan, 2026 Reviewers agreed at journal 10 Jan, 2026 Reviews received at journal 07 Jan, 2026 Reviewers agreed at journal 21 Dec, 2025 Reviewers agreed at journal 19 Dec, 2025 Reviewers invited by journal 19 Dec, 2025 Editor invited by journal 17 Dec, 2025 Editor assigned by journal 15 Dec, 2025 Submission checks completed at journal 15 Dec, 2025 First submitted to journal 13 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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00:24:21","extension":"xml","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":130999,"visible":true,"origin":"","legend":"","description":"","filename":"04961da17c0c4c4696c663aa8a51dc961structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8353173/v1/432569803e2dff278e50bc9a.xml"},{"id":99317008,"identity":"d6936975-dec2-4b66-adcb-b159149559f7","added_by":"auto","created_at":"2025-12-31 16:29:34","extension":"html","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144929,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8353173/v1/98e07e0427b005a0a0d0a781.html"},{"id":99189080,"identity":"0d7814ce-a482-4361-adbe-ce09b589b064","added_by":"auto","created_at":"2025-12-30 00:24:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":674287,"visible":true,"origin":"","legend":"\u003cp\u003eHA1 domain of \u003cem\u003eHA\u003c/em\u003e gene\u003cstrong\u003e: \u003c/strong\u003eAlignment and Comparison of A/Riyadh A/H3N2 Strains with the Vaccine Strain (A/Croatia/10136RV/2023, A/Thailand/8/2022, and A/Darwin/6/2021), and other local circulating strains. (\u003cstrong\u003eA\u003c/strong\u003e) amino acid residues from 1 to 118, (\u003cstrong\u003eB\u003c/strong\u003e) from 119 to 237, and (\u003cstrong\u003eC\u003c/strong\u003e) from 235 to 334. Identical amino acids are represented by colored dots, whereas variations in amino acids are denoted using capital letters. The enclosed red rectangle represents the 130-loop (residues 134–138); the enclosed blue rectangle represents the 150-loop (residues 150–156); the enclosed orange rectangle represents the 190-helix (residues 181–193); and the enclosed gray rectangle represents the 220-loop (residues 221–228). Red dots indicate conserved residues. Predicted N-linked glycosylation sites are enclosed in green rectangles. Small, filled black circles correspond to predicted O-linked glycosylation sites.\u003c/p\u003e","description":"","filename":"OnlineFigure1A.png","url":"https://assets-eu.researchsquare.com/files/rs-8353173/v1/c5a36e225299c36154115f38.png"},{"id":99316369,"identity":"7f608444-ff98-41f5-b74c-f2ba24c99ad5","added_by":"auto","created_at":"2025-12-31 16:28:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":704724,"visible":true,"origin":"","legend":"\u003cp\u003eDescribe the \u003cem\u003eNA\u003c/em\u003e gene sequence of study strains A/H3N2 in alignment and comparison with vaccine strain and other local circulating strains, (\u003cstrong\u003eA\u003c/strong\u003e) amino acid residues from 1 to 119, (\u003cstrong\u003eB\u003c/strong\u003e) from 120 to 237, (\u003cstrong\u003eC\u003c/strong\u003e) from 239 to 356, and (\u003cstrong\u003eD\u003c/strong\u003e) from 358 to 461. Identical amino acids are shown as colored dots, while variations in the amino acids are represented by uppercase letters. The locations of N-glycosylation sites are marked with green rectangles, and potential O-glycosylation sites are signified by black dots.\u003c/p\u003e","description":"","filename":"OnlineFigure2A.png","url":"https://assets-eu.researchsquare.com/files/rs-8353173/v1/84d2a08f5c65c985bbe5a377.png"},{"id":99316883,"identity":"524d43b5-9e9f-4d80-b8d1-495722254a21","added_by":"auto","created_at":"2025-12-31 16:29:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":253587,"visible":true,"origin":"","legend":"\u003cp\u003eA phylogenetic tree for A/H3N2 constructed from nucleotide sequences of the (\u003cstrong\u003eA\u003c/strong\u003e) \u003cem\u003eHA\u003c/em\u003e gene and (\u003cstrong\u003eB\u003c/strong\u003e) \u003cem\u003eNA\u003c/em\u003e gene. The study strains of A/H3N2 are highlighted in blue, green color showed vaccine strains, local circulating strains in previous years are denoted in red, and reference strains are shown in purple.\u003c/p\u003e","description":"","filename":"OnlineFigure3A.png","url":"https://assets-eu.researchsquare.com/files/rs-8353173/v1/41e6f98d31ca8c132b893e9f.png"},{"id":103251500,"identity":"64a3d0f5-b31a-453d-8589-0f6dea8c1551","added_by":"auto","created_at":"2026-02-23 16:09:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1824252,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8353173/v1/161a3a04-9dec-4ea5-b946-60fb99ade788.pdf"},{"id":99316252,"identity":"00e0d84e-ba8e-4313-84bd-fa885dc96aa9","added_by":"auto","created_at":"2025-12-31 16:27:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":27054,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8353173/v1/c756f709d67733e4425ba376.docx"},{"id":99189077,"identity":"91227e18-7110-4bdb-a0f3-981958a8ff7d","added_by":"auto","created_at":"2025-12-30 00:24:21","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":22437,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8353173/v1/e6042fb928dcca05be60eece.docx"},{"id":99317812,"identity":"67a4dbd2-3401-4693-abaa-0963f3b21df5","added_by":"auto","created_at":"2025-12-31 16:30:44","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":30618,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8353173/v1/393fc54eaca7ead693c726be.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genetic Characterization of Influenza A (A/H3N2) Viruses Reveals Antigenic Drift in Receptor Binding Domain and Possible Vaccine Mismatch in Strains Circulating in Riyadh, Saudi Arabia, 2024-2025","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eInfluenza viruses are highly contagious pathogens and the leading cause of acute febrile respiratory illness worldwide. They impose a major public-health and economic burden, triggering seasonal epidemics and, at times, devastating pandemics [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Several subtypes of influenza A virus (IAV) have caused global outbreaks in the past century, including the 1918 H1N1, 1957 H2N2, 1968 H3N2, and 2009 H1N1 pandemics [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The ability of IAV to cross species barriers and to evolve continuously through genetic change has enabled the emergence of novel antigenic variants such as H5N1, H7N9, H9N2, H5N8, and H7N7 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTwo main mechanisms drive the evolutionary of IAV: antigenic drift and antigenic shift. Antigenic drift arises from point mutations that accumulate gradually within the viral genome, particularly at the antigenic sites of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Because the viral RNA-dependent RNA polymerase lacks proofreading activity, these mutations occur frequently and can alter viral tropism, virulence, replication efficiency, and antigenicity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In contrast, antigenic shift results from reassortment of the segmented viral RNA during co-infection, generating new genome arrangements with unique antigenic properties and the potential to cause pandemics [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong IAV subtypes, A/H3N2 viruses have demonstrated remarkable antigenic drift since their first detection in 1968 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The World Health Organization (WHO) has consequently updated the A/H3N2 vaccine component more than 28 times [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Despite these efforts, vaccine effectiveness often remains suboptimal; for example, efficacy during the 2016\u0026ndash;2017 season was only 28\u0026ndash;42% across age groups [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Such limited protection underscores the need for continuous genetic and antigenic monitoring to guide timely vaccine reformulation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSaudi Arabia represents a unique setting for respiratory-virus transmission owing to its annual mass gatherings (Hajj and Umrah) and the movement of millions of foreign workers [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These dynamics can accelerate viral importation, circulation, and mutation. Although several local studies have described influenza A/H3N2 activity in previous years [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], information on its molecular epidemiology and genetic diversity remains limited\u0026mdash;particularly for the 2024\u0026ndash;2025 season. Our previous surveillance in Riyadh (2014\u0026ndash;2020) showed that 48.8% of IAV isolates were A/H3N2 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Also, our recently and a more recent surveillance in Riyadh (2020\u0026ndash;2023) revealed that A/H3N2 subtype was found in 9.21% of IAV isolates, which overcame the number of A/H1N1 isolates 7.89% [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, comprehensive molecular characterization of circulating strains during the recent 2024\u0026ndash;2025 season remains absent, creating a critical knowledge gap for evidence-based vaccine policy.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eThis study specifically aimed to (1) determine the prevalence of A/H3N2 viruses in Riyadh during 2024\u0026ndash;2025; (2) characterize the complete \u003cem\u003eHA\u003c/em\u003e and \u003cem\u003eNA\u003c/em\u003e gene sequences of circulating strains; (3) assess phylogenetic relationships and clade classification; (4) identify amino-acid substitutions within key antigenic sites; (5) evaluate N-glycosylation patterns; and (6) determine vaccine-strain compatibility for the strains identified from Saudi population. Understanding these molecular patterns will provide critical insight for national vaccination policy, future vaccine-strain selection, and preparedness for potential influenza epidemics in Saudi Arabia.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e2.1. Ethical approval\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki and approved by the Research Ethics Committee at King Saud University, Riyadh, Saudi Arabia (Institutional Review Board Nos. E-24-9609 and E-25-9609, approved in November 2023 and May 2025, respectively). All NPAs that tested positive for IAV were handled in accordance with ethical standards, ensuring patient confidentiality and appropriate medical follow-up as per King Saud University regulations.\u003c/p\u003e\n\u003cp\u003e2.2. Acquisition of clinical samples \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA total of 363 NPAs were collected from individuals presenting with influenza-like illness (ILI) at King Khalid University Hospital, Riyadh, between September 2024 and February 2025. Patients exhibiting symptoms such as fever, cough, sore throat, runny nose, muscle or body aches, headache, or fatigue were enrolled after providing informed consent. Individuals who had been vaccinated with seasonal influenza vaccines were excluded from the study. Each specimen was mixed with 2 mL of virus minimum essential medium (MEM) transport medium (Gibco, Invitrogen, Grand Island, NY, USA) and transported under refrigerated conditions to the Virology Research Laboratory, College of Science, King Saud University. Samples were briefly vortexed for 15 s, centrifuged at 1,000\u0026times; g for 10 min at 4 \u0026deg;C, aliquoted, and stored at \u0026minus;80 \u0026deg;C until further analysis.\u003c/p\u003e\n\u003cp\u003e2.3. Typing, detection and sequencing of IAV\u003c/p\u003e\n\u003cp\u003e2.3.1. Detection and typing\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eViral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer\u0026apos;s instructions. Detection and subtyping of IAV were carried out using the One-Step Ahead RT-PCR Kit with Taq High-Fidelity DNA Polymerase (Qiagen, Hilden, Germany; Cat. No. 220213). The reaction was performed on a GeneAmp 9700 Thermal Cycler (Applied Biosystems, USA) under the following cycling conditions: reverse transcription at 50 \u0026deg;C for 30 min, initial denaturation at 95 \u0026deg;C for 15 min, followed by 35 cycles of denaturation at 94 \u0026deg;C for 15 s, annealing at 52 \u0026deg;C for 30 s, and extension at 72 \u0026deg;C for 2 min. A final extension was performed at 72 \u0026deg;C for 10 min. PCR products were visualized by electrophoresis on a 1% agarose gel stained with ethidium bromide and compared against a 100 bp DNA ladder (Qiagen, Germany).\u003c/p\u003e\n\u003cp\u003e2.3.2. Amplification of full-length HA and NA genes \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eHA\u0026nbsp;\u003c/em\u003eand \u003cem\u003eNA\u003c/em\u003e genes of A/H3N2 strains were amplified using the same kit and thermal protocol described above, with two sets of overlapping primers to obtain the complete gene sequences. Primer sequences and PCR conditions for \u003cem\u003eHA\u003c/em\u003e and \u003cem\u003eNA\u003c/em\u003e gene amplification are detailed in Table 1. All primers were designed based on conserved regions and validated using reference strains before clinical application. Seven representative A/H3N2 isolates (n = 7) were selected for sequencing based on sample quality and temporal distribution across the study period (2024 and 2025). Sequencing was performed by Macrogen Inc. (Seoul, South Korea). Raw sequence data were edited using BioEdit v7.0 (Ibis Biosciences, Carlsbad, CA, USA) and assembled using the EditSeq and MegAlign modules of Lasergene software v3.18 (DNAStar, Madison, WI, USA). All generated sequences were submitted to GenBank under accession numbers EPI_ISL_653577, EPI_ISL_653578, EPI_ISL_653579, EPI_ISL_653580, EPI_ISL_653581, EPI_ISL_653582, EPI_ISL_653583 for HA gene. EPI_ISL_653552, EPI_ISL_653553, EPI_ISL_653554, EPI_ISL_653555, EPI_ISL_653556, EPI_ISL_653557, EPI_ISL_653558.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.4. Sequence and phylogenetic analysis \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMultiple sequence alignment of complete \u003cem\u003eHA\u003c/em\u003e and \u003cem\u003eNA\u003c/em\u003e genes were performed using the ClustalW algorithm implemented in the MegAlign program. The analysis compared the A/H3N2 Riyadh strains with 100 local and international reference and vaccine strains retrieved from GISAID and GenBank databases (see Supplementary Table S1). Sequence variations including; amino-acid substitutions, indels, nucleotide divergences were considered in the analysis. In addition, potential N- and O-glycosylation sites were identified using NetNGlyc 1.0 \u003cu\u003e[\u003c/u\u003e\u003ca href=\"#_ENREF_20\" title=\"Gupta, 2004 #20\"\u003e20\u003c/a\u003e\u003cu\u003e]\u003c/u\u003e and NetOGlyc 3.1\u003cu\u003e[\u003c/u\u003e\u003ca href=\"#_ENREF_21\" title=\"Julenius, 2005 #21\"\u003e21\u003c/a\u003e\u003cu\u003e]\u003c/u\u003e. Phylogenetic trees were constructed by the neighbor-joining method using MEGA v11 (Pennsylvania State University, University Park, PA, USA), with 1,000 bootstrap replicates to assess branch reliability.\u003c/p\u003e\n\u003cp\u003e2.5. Statistical analysis:\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using IBM SPSS Statistics v26.0 (IBM Corp., Armonk, NY, USA). Categorical variables were analyzed using Fisher\u0026apos;s exact test, and post-hoc comparisons were conducted using the Z-test with Bonferroni correction. A p-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1. Detection and typing of influenza A/H3N2\u003c/p\u003e\n\u003cp\u003eAmong the 363 NPAs analyzed, 110 (30.3%) were positive for IAV, and 68 (18.7%) were confirmed cases of influenza B (IBV). Although these IBV-positive samples were part of the original screening dataset, the current study\u0026apos;s genomic characterization was limited to H3N2-positive samples.\u003c/p\u003e\n\u003cp\u003eOf these 110 positive IAV samples, 68 (61.8%) were identified as A/H1N1pdm09 and 42 (38.2%) as A/H3N2. Samples were categorized into two collection periods: 2024 and 2025. In 2024, A/H1N1pdm09 was predominant (80.8%) whereas A/H3N2 accounted for only 19.2%. However, A/H3N2 prevalence increased markedly in 2025, representing 52.4% of positive IAV cases.\u003c/p\u003e\n\u003cp\u003eOf the total specimens, 176 (48.4%) were from males and 187 (51.5%) from females. A higher infection rate was recorded among females (34.7%) compared with males (25.5%, p \u0026lt; 0.05). Age distribution analysis revealed that individuals aged 15\u0026ndash;64 years had the highest positivity rate (32.6%), which was significantly higher than the rates in the 0\u0026ndash;4, 5\u0026ndash;14, and \u0026ge;65-year age groups (p \u0026lt; 0.05). Detailed demographic and seasonal distributions are presented in Table 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.2. Nucleotide and amino-acid variation in HA and NA Genes\u003c/p\u003e\n\u003cp\u003eComplete \u003cem\u003eHA\u003c/em\u003e (1,701 nt) and \u003cem\u003eNA\u003c/em\u003e (1,410 nt) gene sequences were obtained from seven representative A/H3N2 isolates (three from 2024 and four from 2025) were compared with 100 globally circulating A/H3N2 strains retrieved from the GISAID and GenBank databases using the consensus sequence (A/New York/392/2004). (Table S1). The sequence analysis indicated that the nucleotide identity varied from 97.75 to 98.56% (\u003cem\u003eHA\u003c/em\u003e), whereas the \u003cem\u003eNA\u003c/em\u003e identity was observed to be between 97.23 and 98.65%.\u003c/p\u003e\n\u003cp\u003eComparison of the HA1 domain between the current vaccine strain A/Croatia/10136RV/2023 and the A/Riyadh isolates revealed four amino-acid differences (N145S, A186D, V223I, P239Q) (Figure 1A, 1B, 1C). On the other hand, the NA gene of the current vaccine strain A/Croatia/10136RV/2023 was homologous to the study isolates (Figure 2A, 2B, 2C, 2D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe aim of such comparison is to compare current circulating strains with vaccine to ensure the effectiveness of current vaccine strain against highly mutated gene of H3N2 of circulating strains. Also, since the vaccine are designed for highly conserved region of the virus, it is important to monitor these differences to reduce risk of transmutation, lowering the burden of influenza disease, and it can also help in predicting early pandemic of the virus in the country.\u003c/p\u003e\n\u003cp\u003eSeveral identical sequences connected to antigenicity within the receptor-binding domain (RBD), including the 130-loop, 150-loop, 190-helix, and 220-loop, were observed when the HA1 of the Riyadh isolates was compared to the vaccine strains (A/Croatia/10136RV/2023) (Figure 1B). Most of the isolates in the study demonstrated a consistent change in the A186D and V223I amino acid changes within the 190-helix and 220-loop, respectively, in contrast to the vaccine strain (Figure 1B).\u003c/p\u003e\n\u003cp\u003eThe mutations mentioned above were observed not only in our A/H3N2 Riyadh strains but also in sequences obtained from various countries globally; however, they were absent in the H3N2 vaccine strain A/Croatia/10136RV/2023. In fact, these mutations in our strains were situated within or adjacent to recognized antigenic sites, which may influence antigenic drifts and the recognition of the virus by the vaccine. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;3.3. Glycosylation-site analysis\u003c/p\u003e\n\u003cp\u003eAnalysis of the HA1 open reading frame predicted 10- 11 N-glycosylation sites (positions 8, 22, 38, 63, 94, 126, 133, 165, 246, and 285). O-glycosylation analysis revealed potential modification sites at positions 55\u0026ndash;59 within the HA1 domain, similar to the vaccine strain (Figure 1A, 1B, 1C).\u003c/p\u003e\n\u003cp\u003eThe NA protein possessed nine N-glycosylation sites (positions 61, 70, 86, 146, 200, 233, 245, and 367) consistent with the vaccine strain. O-glycosylation sites ranged from 70\u0026ndash;72, also comparable to those of the vaccine strain (Figure 2A, 2B, 2C, 2D).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4. Phylogenetic analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePhylogenetic trees constructed for both the \u003cem\u003eHA\u003c/em\u003e and \u003cem\u003eNA\u003c/em\u003e genes demonstrated that all Riyadh A/H3N2 strains clustered within clade 2a.3a.1, the same clade as the WHO-recommended vaccine strain A/Croatia/10136RV/2023. This finding indicates high genetic similarity between the circulating and vaccine strains (Figure 3Aand 3B).\u003c/p\u003e\n\u003cp\u003eSequence homology with the vaccine strain was approximately 68%, supporting the phylogenetic inference of relatedness. The Riyadh isolates were distinct from earlier local strains (2014\u0026ndash;2020) and (2020-2023) but closely related to recent international strains such as A/Georgia/14289/2023, A/Vasteras/SE23/14213/2023, and A/Auckland/17/2024. These patterns suggest recent introduction of genetically distinct viruses into the region, which is facilitated by international air travelling activity.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eInfluenza virus evolve rapidly and continuously, which requires close monitoring of these mutations in order to inform public health authority about vaccine content and lowering the disease burden. Therefore, the present study provides updated molecular data on the circulation, genetic variation, and clade distribution of influenza A/H3N2 viruses in Riyadh during the 2024–2025 season.\u003c/p\u003e\u003cp\u003eOf 363 samples analyzed, 110 (30.3%) were IAV-positive, with A/H1N1pdm09 predominating in 2024 and A/H3N2 rising sharply in 2025. This shift mirrors previous regional reports showing alternating dominance between these two subtypes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Similar transitions were described in the Middle East and North Africa, where A/H1N1pdm09 accounted for ≈ 50% of infections but was later overtaken by A/H3N2 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Also, the transition was observed by our research group in Riyadh during recent years (2020–2023) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The observed pattern likely reflects the high mutation rate and rapid antigenic drift of A/H3N2 viruses [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Consistent with prior Saudi studies [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], adults aged 15–64 years represented the most affected group, and females exhibited a higher infection rate than males. Despite similarities observed in term of the prevalence of A/H3N2 of our study with our recent study, the difference was showed by males and children ages 0–4 having most of cases [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSimilar to other types, the HA glycoprotein of A/H3N2 is necessary for attaching to host cell receptors and permitting the release of viral RNA into the cell. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To date, the traditionally described RBS, which is framed by the 130 and 220 loops and the 190-helix, has been found to contain important receptor-binding residues or contributors to binding specificity in almost all influenza HAs. The main structural components of HA1 that comprise the RBD are the 130-loop (residue number 134–138, H3 numbering), 150-loop (150–156), 190-helix (181–193), and 220-loop (221–228). Moreover, the highly conserved residues Tyr98 (Y98), Trp153 (W153), and His183 (H183) make up the base of the pocket [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This structure particularly targets host cell sialic acid residues [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e–\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Four significant classical epitopes on the globular HA head have been recognized by the human immune system: Ca (including Ca1 and Ca2), Cb, Sa, and Sb. Changes in HA protein associated with antigenic drift occur continuously throughout time as the virus grows to evade being inhibited by antibodies produced by vaccination and spontaneous infection [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Comparing the study isolates to the vaccine strains (A/Croatia/10136RV/2023), most of the isolates in the study demonstrated a consistent change in the A186D and V223I amino acid changes within the 190-helix and 220-loop, respectively, in contrast to the vaccine strain. Escape mutants of influenza viruses exhibit differences in the structure and biological roles of \u003cem\u003eHA\u003c/em\u003e, especially in regard to the RBD. Human reinfection may result from these mutations [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eInfluenza vaccination is recognized as a highly effective strategy in public health for preventing infections. The level of protection afforded to both individuals and communities through vaccination depends on factors such as the vaccine coverage, the rate at which people receive the vaccine, and how well the strains included in the vaccine match those circulating in a given season. Studies have shown that the influenza vaccine can exhibit efficacy rates of up to 60% against infections caused by Types A and B viruses [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. For the aim of increasing the protection and efficacy of the vaccine, the WHO offers over 28 vaccine strain for both Northern and Southeren hemisphere [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. A number of these strains have been recommended by the Saudi Ministry of health for A/H3N2 subtype including; A/Singapore/Infimh/16/0019/2016, A/Cambodia/e0826360/2020, A/Darwin/6/2021, A/Thailand/8/2022, A/Massachusetts/18/2022, A/Croatia/10136RV/2023. All of which were used as a reference in this study. To evaluate the effectiveness of the existing vaccine against the prevalent (A/H3N2) strains in Riyadh, a comparison was made between the seven strains studied and the vaccine strain A/Croatia/10136RV/2023. Notably, our A/H3N2 strains exhibited four amino acid substitutions in the \u003cem\u003eHA\u003c/em\u003e while \u003cem\u003eNA\u003c/em\u003e gene was homologous with the vaccine strain. Our previous study also detected 12 and nine amino acids changes for both \u003cem\u003eHA\u003c/em\u003e and \u003cem\u003eNA\u003c/em\u003e genes in comparison with the vaccine strain A/Singapore/Infimh/16/0019/2016 recommended during the year of study [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Similarly, in a more recent study, Alkubaisi et al 2025 reported the presence of nine amino acids substitutions in \u003cem\u003eHA\u003c/em\u003e and \u003cem\u003eNA\u003c/em\u003e genes that differentiated the studied strains from vaccine strains A/Croatia/10136RV/2023, A/Thailand/8/2022, and A/Darwin/6/2021 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. As a consequence, yearly influenza surveillance is crucial for gathering vital information needed for the yearly reformulations of influenza vaccination and for detecting any possible epidemic or pandemic [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn line with facts about ongoing antigenic drift of H3N2 strains that were historically documented [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], we compared our A/H3N2 strains with the reference strain A/New York/392/2004 and strains identified previously in Riyadh (2014–2020) and (2020–2023), for the aim to identify evolutionary kinetic of this virus in the same city. 108 and 60 mutations site for \u003cem\u003eHA\u003c/em\u003e and \u003cem\u003eNA\u003c/em\u003e gene were detected, respectively. Of these, twelve were unique for \u003cem\u003eHA\u003c/em\u003e gene of our strains as compared to other local circulating strains in previous years and reference strain. While the analysis for \u003cem\u003eNA\u003c/em\u003e gene revealed the presence of only five unique mutations compared to other local strains.\u003c/p\u003e\u003cp\u003ePhylogenetic analysis demonstrated that all A/H3N2 isolates clustered within clade 2a.3a.1—the same clade as the WHO-recommended vaccine strain A/Croatia/10136RV/2023. This genetic alignment indicates a favorable match between circulating and vaccine strains. In our previous study, we found A/Saudi Arabia/VRG-02/2016, A/Saudi Arabia/VRG-03/2016, A/New Jersey/26/2014, A/Fiji/2/2015, and A/Canberra/7/2016 in root 3c.2a [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Thirteen more strains of subclade 3C.2a1b were identified by mutations of F159Y, K160T, N171K, N121K, K92R, and H311Q [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Among the 16 strains that comprised the 3c. clades/subclade, two members of clade 3c.2a (A/Saudi Arabia/VRG-2/2016 and A/Saudi Arabia/VRG-7/2016) were identified using the mutations F159Y and K160T [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Except for A/Saudi Arabia/VRG-01/2016, which belonged to clade 3c.2a3 and was not grouped with a vaccine strain. While our previous and recent study (2020–2023) identified that circulating strains were not clustering in any clade of recommended vaccine strains, but were grouped into two subclade 3c.2a1b.1a and 3c.2a1b.1b [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].The strains under investigation remained in the same clade as the vaccine strains. This might suggest global predominance of 2a.3a.1 clade in 2024–2025. The global predominance of H3N2 clade 2a. 3a.1 has important virological and public health implication, as recent surveillance reports from the CDC and ECDC indicate that nearly all genetically characterized H3N2 viruses detected across multiple continents during 2024–2025 season belong to 2a. 3a.1, reflecting a clear selective advantage and widespread transmission [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This dominance is consistent with the well -known capability of H3N2 to undergo rapid antigenic drift, particularly through amino-acids substitutions in key \u003cem\u003eHA\u003c/em\u003e antigenic sites, which can reduce population immunity and facilitate immune escape [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Such drifted lineages belonging to clade 2a. 3a.1 have recently been observed to be associated with reduced vaccine effectiveness. Indeed, in a current report of WHO found that based on human serological tests, strains belonging to 2a. 3a.1 clade had a low antibody detection against the virus after vaccination compared to the recommended vaccine strain [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The emergence and global expansion of clade 2a.3a.1 therefore, raise concern about potential mismatch with current vaccine strain and highlight the necessity for continuous influenza surveillance to inform public health authority about vaccine update. furthermore, it might also raise concern about the possibility of pandemic occurrence in the future.\u003c/p\u003e\u003cp\u003eThe phylogenetic clustering identified in our study provides vital insight into the possible influence of international travel on the diversity of circulating H3N2 strains in Saudi Arabia. Several of our isolate correlated closely with globally circulating 2a.3a.1 lineage rather than with strains historically reported in the region, suggesting new viral introduction from outside the country. Saudi Arabia hosts millions of travelers annually for job, tourism, and religious pilgrimages, giving ongoing opportunities for the introduction of genetically different influenza viruses. By displacing or co-circulating with established strains, these introductions can change the local viral ecosystem by increasing selective pressure and influence of population immunity [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Therefore, the phylogenetic tree supports the idea that travelling abroad significantly influences the temporal and genetic variety of influenza viruses found locally.\u003c/p\u003e\u003cp\u003eNevertheless, phylogenetic relatedness alone does not guarantee antigenic equivalence, as minor \u003cem\u003eHA\u003c/em\u003e or \u003cem\u003eNA\u003c/em\u003e substitutions can substantially affect immune recognition and viral antigenicity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, continued regional monitoring and, ideally, serologic confirmation through hemagglutination-inhibition (HI) assays remain priorities.\u003c/p\u003e\u003cp\u003eThe quantity, length, and types of N-linked glycosylation on the surface of \u003cem\u003eHA\u003c/em\u003e significantly influence its antigenicity and the immunogenic response of the virus [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Indeed, N-linked glycosylation can diminish the efficacy of antibody-mediated cross-neutralization through antigen masking, a phenomenon observed with the 1918 influenza pandemic strain and the A(H1N1)pdm09 strain, where additional N-linked glycosylation sites were located at the 129 and 163 residues [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In contrast, insufficient glycosylation of glycoproteins can adversely affect \u003cem\u003eHA\u003c/em\u003e by leading to improper folding and transport, as observed in a study conducted by Gallagher et al [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMoreover, the connection between variations in N-linked glycans (NLG) and the transmissibility of IAV must also be taken into account. Since the NLG on the globular head of hemagglutinin (HA) can alter receptor-binding specificity, modifications in NLG may influence the transmissibility of IAV. In a study using a guinea pig model, an H5N1 virus that was able to bind to both 3' sialic acid (SA) and 6' SA lost its ability to bind to 6' SA due to the A160T mutation, leading to a complete loss of transmissibility of the parental strain. Therefore, beyond changing receptor specificity, alterations in the NLG of the globular head of \u003cem\u003eHA\u003c/em\u003e can significantly impact viral transmission [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAdditionally, a novel N-linked glycosylation site in either \u003cem\u003eHA\u003c/em\u003e or \u003cem\u003eNA\u003c/em\u003e can serve as a marker for specific strains. For example, in the strains associated with the 1918 and 2009 influenza pandemics, the presence of a distinct 104 amino acid residue with an N-glycosylation site can help determine whether an influenza reassortment originated from the 1918 or 2009 pandemic strains. Overall, mutations in N-linked glycosylation sites in \u003cem\u003eHA\u003c/em\u003e and \u003cem\u003eNA\u003c/em\u003e influence the virulence, pathogenicity, and susceptibility of IAV to neutralizing antibodies [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile in terms of O-linked glycosylation found in the influenza surface glycoproteins HA and NA, O-linked glycan can act as glycan shield that mask antigenic sites from host neutralizing antibodies, thereby contributing to immune evasion and promoting antigenic drift [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. For instance, Ming et al [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] showed that the addition of O-glycan near the receptor binding domain of \u003cem\u003eHA\u003c/em\u003e diminished antibody recognition, facilitating escape from humoral immunity [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Furthermore, O-glycosylation influences the receptor-binding specificity of \u003cem\u003eHA\u003c/em\u003e, where the presence or absence of specific O-glycan can modulate the affinity of \u003cem\u003eHA\u003c/em\u003e for human-like (α2,6 linked) or avian-like (α2,3 linked) sialic acid receptors, affecting both tissue tropism and interspecies transmission [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]; this was demonstrated by Li et al [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], who observed that alteration in \u003cem\u003eHA\u003c/em\u003e O- glycosylation impacted host specificity and adaptation. Additionally, O-glycan affect HA protein stability, cleavage efficiency, and membrane fusion- key steps in viral entry and replication [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In particular, Schulze (1997) reported that the removal of certain O-linked glycans enhanced membrane fusion activity, leading to increased viral infectivity. These modifications not only play a role in the evolutionary dynamics of influenza viruses, as shift in glycosylation often occur alongside key antigenic mutations, highlighting the importance of monitoring O-linked glycosylation patterns for vaccine strain selection and molecular surveillance [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTherefore, from the above paragraphs about N and O- linked glycosylation, it is important to identify any new modification in these two types of glycosylation. In the current study, all predicted N- and O-linked glycosylation sites were conserved between vaccine and circulating strains which might indicate high similarities with vaccine strain.\u003c/p\u003e\u003cp\u003eThe data support that the 2024–2025 A/H3N2 vaccine component likely provided adequate genetic coverage for strains circulating in Riyadh. However, the identified amino-acid substitutions compared to vaccine strain highlight the virus’s ongoing evolution and the need for year-round molecular surveillance. Key limitations include the relatively small number of sequenced isolates and the absence of cross-HI testing, which restrict antigenic interpretation. Future work should incorporate larger multicenter sampling, complete-genome sequencing, and phenotypic assays to better evaluate vaccine efficacy and transmission dynamics within the Kingdom.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study provides updated molecular insights into the circulation and genetic diversity of influenza A/H3N2 viruses in Riyadh, Saudi Arabia, during the 2024–2025 influenza season. Among the 363 samples analyzed, A/H3N2 accounted for 42 (38.2%) of all IAV detections and increased in frequency during 2025. Phylogenetic analysis demonstrated that all local isolates belonged to clade 2a.3a.1, the same clade as the current WHO-recommended vaccine strain A/Croatia/10136RV/2023, indicating a high level of vaccine–strain compatibility. Nevertheless, four amino-acid substitutions in the \u003cem\u003eHA\u003c/em\u003e gene and sequence homology of \u003cem\u003eNA\u003c/em\u003e gene, underline the virus’s continuing molecular evolution.\u003c/p\u003e\u003cp\u003eAlthough the overall vaccine match appears favorable, the observed mutations of our strains compared to vaccine strain warrant continued genomic surveillance and functional evaluation to assess their effects on antigenicity and vaccine performance. Expanding molecular surveillance programs across multiple Saudi regions and integrating serological assays such as HI tests will be essential for early detection of emerging variants and for supporting evidence-based updates of seasonal vaccine formulations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThe authors thank the Ongoing Research Funding Program (ORF-2025-418), King Saud University, Riyadh, Saudi Arabia, for supporting this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization, S.A.A., F.N.A, I.M.A., A.M.A. (Abdulhadi M. Abdulwahed); methodology, S.A.A., and I.M.A.; software, S.A.A., M.A.F., and I.M.A.; validation, S.A.A., I.M.A., and R.M.A.; formal analysis, S.A.A. and I.M.A.; investigation, S.A.A. and I.M.A., A.M.A. (Abdulhadi M. Abdulwahed), A.M.A. (Abdulaziz M. Almuqrin), and F.N.A.; resources, F.N.A.; data curation, S.A.A. and I.M.A.; writing\u0026mdash;original draft preparation, S.A.A.; writing\u0026mdash;review and editing, M.A.F., R.M.A, A.M.A. (Abdulhadi M. Abdulwahed), A.M.A. (Abdulaziz M. Almuqrin), and I.M.A.; visualization, S.A.A. and M.A.F.,; supervision, A.M.A. (Abdulhadi M. Abdulwahed), A.M.A. (Abdulaziz M. Almuqrin), and F.N.A.; project administration, F.N.A.; funding acquisition, F.N.A. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was funded by the Ongoing Research Funding Program (ORF-2025-418), King Saud University, Riyadh, Saudi Arabia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003ethe authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThe study was conducted in accordance with the Declaration of Helsinki and approved by the Research Ethics Committee at King Saud University, Riyadh, Saudi Arabia (Institutional Review Board Nos. E-24-9609 and E-25-9609, approved in December 2023 and May 2025, respectively).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eInformed consent was obtained from all subjects involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. The sequence data reported in this study have been deposited in GenBank under accession numbers PV653577\u0026ndash;PV653583 for the \u003cem\u003eHA\u0026nbsp;\u003c/em\u003egene and PV653652\u0026ndash;PV653658 for the \u003cem\u003eNA\u003c/em\u003e gene.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eUyeki TM, Hui DS, Zambon M, Wentworth DE, Monto. AS Influenza Lancet. 2022;400:693\u0026ndash;706.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorens DM, Folkers GK. Fauci AS.The challenge of emerging and re-emerging infectious diseases.Nature. 2004; 430: 242\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDunning J, Thwaites RS, Openshaw PJ. Seasonal and pandemic influenza: 100 years of progress, still much to learn.Mucosal immunology. 2020; 13: 566\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalese P. 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D.Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics. J Virol. 2000;74:6316\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Liu D, Wang Y, Su W, Liu G, Dong W. The importance of glycans of viral and host proteins in enveloped virus infection.Frontiers in Immunology. 2021; 12: 638573.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchulze IT. Effects of glycosylation on the properties and functions of influenza virus hemagglutinin. J Infect Dis. 1997;176:S24\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"vaccine compatibility, IAV, phylogenetic analysis, glycosylation sites, molecular surveillance","lastPublishedDoi":"10.21203/rs.3.rs-8353173/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8353173/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction:\u003c/strong\u003e Influenza A/H3N2 viruses undergo continuous antigenic evolution, necessitating ongoing surveillance for informed vaccine strain selection. This study characterized the molecular epidemiology of H3N2 viruses circulating in Riyadh, Saudi Arabia, during the winter season of 2024-2025 and assessed their compatibility with current vaccine strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Nasopharyngeal samples (NPAs) (n=363) were collected from patients presenting with influenza-like illness at King Khalid University Hospital in Riyadh. Influenza A/H3N2 detection and subtyping were performed using RT-PCR. Complete hemagglutinin (HA) and neuraminidase (NA) gene sequencing was conducted on confirmed A/H3N2 strains (n=7), followed by phylogenetic analysis, amino acid substitution mapping, and glycosylation site prediction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Of 363 samples tested, 110 (30.3%) were positive for influenza A, with 42 (38.2%) identified as A/H3N2 and 68 (61.8%) as A/H1N1pdm09. Phylogenetic analysis revealed that all seven sequenced A/H3N2 strains belonged to clade 2a.3a.1, which is identical to the current vaccine strain clade. However, molecular analysis identified six amino acid substitutions in the \u003cem\u003eHA\u003c/em\u003e gene and four in the \u003cem\u003eNA\u003c/em\u003egene that distinguished circulating strains from the A/H3N2 vaccine strain A/Thailand/8/2022. Notably, all study strains showed deletion of an N-glycosylation site (N122S) that is present in the vaccine strain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e While phylogenetic clade compatibility suggests potential vaccine effectiveness, the observed amino acid differences and glycosylation site deletion highlight the importance of continued molecular surveillance to monitor antigenic drift and assess vaccine performance in the Saudi Arabian population.\u003c/p\u003e","manuscriptTitle":"Genetic Characterization of Influenza A (A/H3N2) Viruses Reveals Antigenic Drift in Receptor Binding Domain and Possible Vaccine Mismatch in Strains Circulating in Riyadh, Saudi Arabia, 2024-2025","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 00:24:15","doi":"10.21203/rs.3.rs-8353173/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-30T07:59:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-28T20:07:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1331123101490695375342951142250917440","date":"2026-01-12T22:16:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277736754739152763523350438670058801007","date":"2026-01-12T02:47:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"231535825969695955710820156888841185163","date":"2026-01-10T17:06:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-07T09:08:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7228549372627798169584505234943744921","date":"2025-12-21T18:05:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"109294216098724178895926004781936911933","date":"2025-12-19T14:21:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-19T13:37:45+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-17T07:07:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-15T23:19:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-15T23:18:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Infectious Diseases","date":"2025-12-13T13:39:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ba6fa61a-b709-41d6-bc9b-ea022afd22f0","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:06:17+00:00","versionOfRecord":{"articleIdentity":"rs-8353173","link":"https://doi.org/10.1186/s12879-026-12928-0","journal":{"identity":"bmc-infectious-diseases","isVorOnly":false,"title":"BMC Infectious Diseases"},"publishedOn":"2026-02-19 15:59:15","publishedOnDateReadable":"February 19th, 2026"},"versionCreatedAt":"2025-12-30 00:24:15","video":"","vorDoi":"10.1186/s12879-026-12928-0","vorDoiUrl":"https://doi.org/10.1186/s12879-026-12928-0","workflowStages":[]},"version":"v1","identity":"rs-8353173","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8353173","identity":"rs-8353173","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}