Human adenovirus (HAdV), a non-enveloped dsDNA virus of the genus Mastadenovirus (family Adenoviridae), represents a significant global pathogen causing heterogeneous clinical manifestations across age groups. Pediatric populations exhibit heightened susceptibility, with 80% of infections occurring in children under 4 years of age due to waning maternal antibodies and immature adaptive immunity (Lynch and Kajon, 2016). The International Committee on Taxonomy of Viruses (https://talk.ictvonline.org/taxonomy) classifies more than 120 genotypes into seven species (A-G), with distinct tissue tropisms dictating clinical outcomes. Acute respiratory infections (ARIs) are commonly linked to species B (B3, B7, B14, B16, B21), C (C1, C2, C5, C6, C57), and E (E4) (Lynch and Kajon, 2016; Tian et al., 2021), manifested as pneumonia, bronchitis, upper respiratory tract infections (URTIs), or tonsillitis (Wen et al., 2021). Notably, approximately one-third of HAdV-associated pneumonia progresses to severe adenoviral pneumonia, characterized by radiographic consolidation and extrapulmonary complications (Lu et al., 2013; Wang et al., 2021). Despite this clinical burden, evidence remains limited regarding genotype-specific virulence determinants. This emphasizes the importance of rapid and accurate HAdV genotyping in clinical diagnosis and epidemiological investigation.

Conventional HAdV serotype determination relies on neutralization assays and hemagglutination inhibition tests, which are time-consuming and reagent-limited. The emergence of novel recombinant strains through homologous recombination complicates traditional serological typing, as evidenced by circulating variants containing discordant antigenic determinants. Modern molecular approaches, particularly multiplex PCR and real-time quantitative PCR (qPCR), demonstrate superior diagnostic utility with higher sensitivity for established genotypes (Ison and Hayden, 2016). However, partial genome sequencing strategies (e.g., Hexon hypervariable region analysis) carry inherent misidentification risks, as critical recombination breakpoints frequently occur outside targeted loci. To ensure accurate molecular epidemiology, the WHO HAdV Working Group (http://hadvwg.gmu.edu) recommends integrated analysis of three capsid genes (Hexon, Penton base, and Fiber) (Wu et al., 2022).

During the last decade, outbreaks of respiratory tract infections caused by HAdV have occurred frequently in many countries including China (Barnadas et al., 2018; Zhao et al., 2020; Huang et al., 2021; Pscheidt et al., 2021; Huang et al., 2023). While genotype-specific virulence has been hypothesized, only a few published pediatric studies incorporate molecular subtyping data, and existing evidence suggests a correlation between genotypes and clinical outcomes (Lin et al., 2017; Wang et al., 2021). Thus, the genotyping of HAdV is crucial for understanding local epidemiology, tracking virulence variants, and guiding vaccine development. Notably, the widespread implementation of non-pharmaceutical interventions (NPI) during the COVID-19 pandemic significantly altered the transmission patterns of respiratory viruses, including HAdV (Huang et al., 2023). In the post-pandemic era, the epidemiological characteristics, predominant serotype distribution, and associations with disease severity of HAdV have exhibited new dynamics, urgently requiring continuous surveillance and in-depth investigation (Yan et al., 2024; Zhao et al., 2024; Contes and Liu, 2025; Han et al., 2025). The aim of this study was to investigate the epidemiological, clinical, and molecular characteristics of HAdV infections among hospitalized children with ARIs in Tianjin from March 2022 to March 2024, aiming to inform future prediction and intervention strategies for HAdV-related diseases. The findings provide scientific evidence for the prevention and control of HAdV-related diseases.

HAdV infection represents a significant etiology of ARIs in children, accounting for approximately 4%-10% of global pediatric ARIs cases (Yu et al., 2021; Zhu et al., 2021). Our surveillance in Chinese cities revealed substantial spatiotemporal variation in HAdV detection. Among hospitalized pediatric ARIs patients in Tianjin, the HAdV positive rate was 8.9%, significantly higher than Beijing (2.89%, 2015-2021) but lower than Hangzhou (26.77%, 2020-2021) (Huang et al., 2023; Wang et al., 2024).Longitudinal analysis showed cyclical fluctuations in Tianjin, peaking at 12.22% in 2019, aligning with the national trend (Liu et al., 2022; Wang et al., 2024). The implementation of NPI measures from 2020 to 2022 substantially suppressed HAdV transmission (declining to 7.82% in 2020, 172/2199). However, a resurgence was observed in 2021 as social activities resumed (9.69%, 897/9253) ( Supplementary Figure S1 ). Parallel trends were documented in other urban centers (Wang et al., 2024). During the NPI period, the HAdV positivity rate in Tianjin was 8.78% (1,270/1,4471). Following the lifting of the NPI, there was a significant increase in both the number of ARIs patients and the absolute number of HAdV cases. However, the HAdV positivity rate remained stable at 9.12% (2,283/25,041), in contrast to the upward trend observed in Beijing and Hebei (Zhao et al., 2024; Han et al., 2025). This ‘asynchronous change’ highlights the complexity of transmission dynamics in the context of co-circulation of multiple pathogens (such as RSV) (Lyu Y and Zhang, 2024).

HAdV positivity exhibited significant differences by age, gender, and season in Tianjin. Infections peaked in autumn and winter (December-January), consistent with seasonal patterns in northern temperate regions (Huang et al., 2021; Yu et al., 2021), with HAdV-3 predominating (53.4%). Contrary to previous studies reporting higher prevalence in children under 5 (Barnadas et al., 2018; Zhao et al., 2020; Huang et al., 2021; Pscheidt et al., 2021), our study found the lowest rate in 0–1 year-olds (6.25%) and the highest in 6–18 year-olds (10.4%). This shift may relate to reduced intra-family transmission during NPIs (decreasing infant exposure) and increased transmission within schools post-reopening (Huang et al., 2023; Yan et al., 2024). Gender difference analysis showed that there were more hospitalized male ARIs patients in this study, which may be related to biological factors (boys with relatively narrower airway diameters are more prone to severe lung infections (Pagtakhan et al., 1984)) and behavioral factors (girls have higher compliance with mask wearing during NPI (Huang et al., 2023)). The HAdV positivity rate was significantly higher in males (9.2% vs. 8.4%; χ² = 5.496, P = 0.019), consistent with some studies (Pscheidt et al., 2021; Huang et al., 2023) but not others (Wang et al., 2021; Liu et al., 2022). The reasons for gender differences warrant further investigation. Clinically, male HAdV patients may require heightened vigilance for severe symptoms and pneumonia progression. Molecular epidemiology showed a shift in predominant types: HAdV-C dominated during NPIs (typically milder), rapidly replaced by HAdV-B (78.2%) post-NPIs, often associated with more severe illness (Huang et al., 2023; Wang et al., 2024). This indicates NPIs not only affect transmission but also viral type composition and community disease severity, underscoring the need for long-term subtype surveillance and early warning.

HAdV-C infections were predominantly observed in children under 3 years old (206/395, 52.2%), whereas HAdV-B3/B7 infections were more prevalent among school-aged children. In addition to the age-specific distribution, adenovirus serotypes significantly influenced disease phenotypes and severity. HAdV-3 predominated across all disease categories, especially pneumonia and bronchitis. Previous study revealed that HAdV-C frequently resides latently in adenoid tissues (Alkhalaf et al., 2013); thus, it is the second most common serotype observed in tonsillitis patient samples. Although HAdV-3 displayed higher overall prevalence, children infected with HAdV-7 more commonly progressed to severe symptoms, which may be attributed to its replication advantages: in vitro experiments demonstrated that HAdV-7 exhibits a higher viral load compared to HAdV-3 and induces stronger cytokine responses. Additionally, infections with HAdV-7 were more frequently associated with complications such as myocardial injury and toxic encephalopathy or encephalitis. Patients infected with this serotype exhibited a higher requirement for bronchoscopic interventions and immunoglobulin therapy. These findings suggest that HAdV-7 infections should be incorporated into pediatric severe case warning systems, prioritizing the allocation of intensive care resources for such cases (Lin et al., 2017; Fu et al., 2019; Zhao et al., 2020; Liu et al., 2022).

We identified two HAdV-C suspected recombinant samples but found no evidence of recombination in the B/E subgroups. Previous studies have shown that HAdV-C establishes persistent infections after primary infection, characterized by intermittent shedding in the host (Alkhalaf et al., 2013). Such persistence creates conditions conducive to mixed infections with different serotypes within this subgroup, thereby promoting homologous recombination between distinct viral types (Wang et al., 2024). Notably, new recombinant HAdV strains may exhibit significant alterations in pathogenicity, tissue tropism, and clinical manifestations compared to their prototype strains (Wang et al., 2021; Wu et al., 2022), highlighting the importance of conducting continuous surveillance on emergent recombinant strains. Phylogenetic analysis revealed that all types exhibited complete segregation across the three gene fragments (Bootstrap >90%), except for HAdV-C in the Penton tree (Bootstrap <70%). The observed high heterozygosity in Hexon and Fiber gene sequences indicates that these proteins may be under significant immune pressure. The sequence diversity of the HAdV-C Penton gene is notably lower than that observed for HAdV-B and HAdV-E. However, Mao et al. ‘s (Mao et al., 2019) research demonstrated that the HAdV-C Penton phylogenetic tree could be divided into distinct clades with robust bootstrap support, implying a greater degree of genetic diversity in the Penton gene than previously recognized.

High nucleotide consistency across HAdV types was observed, clustering with strains circulating widely in China, reflecting the slow evolutionary rate of this DNA virus—a positive factor for vaccine development. Among the 14 HAdV-3 isolates analyzed, all exhibited >98.0% nucleotide identity across three core structural genes relative to the prototype strain. This is attributed to the combined effects of HAdV DNA polymerase’s high-fidelity replication, which enables HAdV-3 to maintain genomic stability for at least 50 years (Mahadevan et al., 2010). Notably, The 7 HAdV-7 strains isolated in this study exhibited low nucleotide consistency in the Hexon gene fragment, which comprises 28 sites of amino acid mutation. Research has shown that Hexon gene fragments contain specific antigenic epitopes that can stimulate the production of neutralizing antibodies in the host. Mutations in this region may result in immune escape (Lee et al., 2025). While genomic variation in HAdV-7 shows a high correlation with enhanced pathogenicity, establishing the causative relationship between specific mutation sites and the high-virulence phenotype observed in contemporary pediatric HAdV-7 strains requires further experimental data support. There are few reports of sporadic infections with HAdV-14. The singular HAdV-14 isolate showed >99.0% genomic conservation across all three genes compared to the prototype strain, and the fiber fragment contained three amino acid deletions. Studies have indicated that the fiber fragment contains type-specific antigenic epitopes that determine viral tropism, and that mutations in this region may result in different tissue tropism (Wang et al., 2014). In the HAdV-21 cohort (n=23), all except TJ2024–749 exhibited complete Penton sequence identity with GZ2019 (MW091531.1), classifying them as 21a subvariants. Due to the presence of 15 consecutive amino acid deletion variants on the Penton fragment (residues 287-301), the amino acid consistency is low, which may affect the RGD motif mediated viral endocytosis function on Penton. This deletion results in a truncated RGD loop, exhibiting structural homology to the naturally shorter loops found in HAdV-B3 and HAdV-B7. Research suggests that this conserved deletion pattern may compromise viral internalization mediated by the RGD motif and potentially contribute to the severe lower respiratory tract tropism associated with 21a infections (Hage et al., 2014).

Although our investigation provided crucial molecular insights into the epidemiological and clinical characteristics of HAdV infections in Tianjin, China, several limitations warrant consideration. Firstly, focusing solely on hospitalized pediatric ARIs patients may introduce selection bias, potentially underestimating HAdV-C prevalence (milder cases) and skewing subtype analysis towards more severe variants like HAdV-B7. Secondly, clinical manifestations could be influenced by co-infections. Thirdly, cost constraints limited Sanger sequencing to 100 randomly selected HAdV-positive samples, reducing statistical power for nucleotide diversity analysis of some serotypes. Fourthly, recombination analysis is based on partial gene fragments (Penton, Hexon, Fiber), and there may be additional recombination events in the unanalyzed regions, which may affect the precise localization of recombination breakpoints. Finally, sampling from a single institution may limit generalizability to the broader Tianjin population. These factors may lead to underestimation of HAdV prevalence and incomplete characterization of type distribution.

This two-year study systematically characterized HAdV epidemiology among pediatric inpatients with ARIs in Tianjin, revealing key insights into prevalence, age-specific susceptibility, seasonal dynamics, and evolving genotype distributions. Significant heterogeneity in genotype distributions across clinical phenotypes was observed. These findings are crucial for optimizing diagnosis, antiviral stewardship, and region-specific vaccine development.