Worldwide, acute respiratory infections (ARIs) are a leading cause of morbidity and mortality, particularly in vulnerable populations such as children, immunocompromised cases and the elderly (1). HAdV, as one of the most prevalent pathogens, is responsible for 5%–10% of ARIs in children and 1%–7% in elderly patients (2–5).

HAdVs, first isolated from human adenoids in 1953, are nonenveloped viruses with a linear, double-stranded DNA of ∼36,000 base pairs (6). They also include nine transcription units E1–E4 and L1–L5, with the hexon gene located in L3 (7). The hexon protein, a major capsid protein with type-specific antigenic epitopes, plays a vital role in cell entry and host infection, making it crucial in the pathogenesis of HAdV infection (7, 8).

Currently, 111 different types of HAdV have been identified and classified into seven species designated HAdV A–G (http://hadvwg.gmu.edu). New types are expanding and derive primarily from homologous recombination within the same HAdV species (9). Different HAdV types cause distinct clinical symptoms due to different specific tissue tropisms (10). ARI is typically associated with species B (type 3, 7, 11, 14, 21), C (type 1, 2, 5, 6) and E (type 4) (4, 11). In addition to ARI, HAdV can also lead to other self-limiting diseases such as gastroenteritis, conjunctivitis and urinary tract infection; however, in immunocompromised patients (e.g., in oncology units and undergoing stem cell transplantation), it can cause severe complications, including hepatitis, cardiomyopathy and encephalitis, with a mortality rate up to 32.9% (10, 12–15).

Systematic reviews and meta-analyses have shown that the average prevalence rate of HAdV is 15.91% in China, 12.82% in Middle Eastern countries, and 12.58% in North African countries (16, 17). The epidemiology of HAdV is complex, with various types circulating and causing regional epidemics of respiratory infections (18). Therefore, it is essential to monitor the epidemiological features and investigate the molecular evolution process.

In this study, we focused on pediatric patients diagnosed with ARIs. ARI in children displays seasonal variation, with a significant increase during winter and spring (19). Hence, we selected the period from November 2020 to March 2021, the first period with a high incidence after the COVID-19 pandemic, to study the prevalence of HAdV in Hangzhou.

During the COVID-19 epidemic, various Non-Pharmacological Interventions (NPIs), such as party cancellations, closure of educational facilities and restrictions on personal movement, were implemented in China to control the spread of COVID-19 (20). Despite these measures, the positive rate of HAdV remained high. Our study revealed that between November 2020 and March 2021, 26.77% of children with ARIs in Hangzhou were HAdV-positive. This rate is higher than in previous surveys conducted in the same region (6.9%) from 2018 to 2019 (21), in Wenzhou (3.5%) from 2018 to 2019, and in Huzhou (7.08%) from 2017 to 2019 (22, 23), but lower than southern Brazil (35.8%) from 2004 to 2018 (24). These findings suggest that the prevalence rate of HAdV varies based on geographical location, sample collection time, and different study populations.

Our study revealed that the HAdV-positive rate was affected by gender and age (p < 0.05). Males had a slightly higher rate than females, which was inconsistent with previous studies (25, 26). This discrepancy could potentially be attributed to the fact that girls may have exhibited better adherence to NPIs such as mask-wearing and frequent handwashing. However, further investigation with a larger sample size would be required to confirm this hypothesis. Additionally, we observed that the lowest positive rate of HAdV was found in children aged 0–1 year (20.00%), whereas the highest rate occurred in children aged 6–18 years (31.49%). This finding contradicted a previous study conducted at the same hospital, which reported the highest rate in children aged 0.5–2 years from 2018 to 2019 (21). One possible explanation for this discrepancy is that during the COVID-19 pandemic, there was an emphasis on wearing masks and practicing good hand hygiene, which potentially reduced the risk of respiratory tract infections in adults and subsequently led to lower infection rates in younger children. As a result, children aged 0–1 year with maternal antibodies and limited activities, had the lowest rate. While children aged 6–18 years had the highest rate, possibly because primary and secondary schools returned to normal instruction earlier than kindergartens after the COVID-19 pandemic. These results show that the prevalence of HAdV also differs by gender and age during the COVID-19 pandemic. Therefore, continuous and unified epidemiological surveillance is urgently needed (27).

Moreover, our study found that HAdV-positive outpatients were more prevalent than inpatients (28.72%, 361/1,257 vs. 13.51%, 25/185; p = 0.000). This confirms the statement that most HAdV infections are mild to moderate and self-limiting, requiring only outpatient treatment (28, 29). Similar to our study, fever and cough were the most common symptoms in HAdV-positive patients (30). However, comparable clinical symptoms were found in other viral respiratory infections including HRSV, HPIV and HMPV. Therefore, the identification of pathogens is crucial for epidemiological monitoring and clinical diagnosis.

Several studies have reported co-infection of HAdV with other respiratory viruses (4, 31). Among our 130 co-infection patients, they exhibited more severe clinical symptoms than those with HAdV single-infection. Therefore, pediatricians should be aware of potential co-infection, leading to exacerbation of the disorder, increased hospitalization and more complicated therapy. As for severely symptomatic and immunocompromised patients, close monitoring of HAdV infection is required especially at an early stage due to the high mortality rate (14).

The hexon protein of HAdV comprises seven HVRs, which locate on the outer surface of the virion and contain type-specific residues responsible for viral serum neutralization (32, 33). The HVR is conserved for a given type, but varies between types, enabling type discrimination and phylogenetic analysis (34, 35). Previous studies have shown that HAdV-B (type 3, 7) were the most frequent strains in China (21–23, 25, 28, 36, 37). However, our phylogenetic tree showed that HAdV-C (type 1, 2, 6) were the predominant circulating strains in Hangzhou during the COVID-19 pandemic from November 2020 to March 2021. Previous studies have consistently associated HAdV-B (3, 7) with a higher incidence of severe respiratory disorders than HAdV-C (38–40). This change in the predominant strain from B to C during this period suggests that NPIs may have influenced the transmission of HAdV in the community, resulting in a tendency toward mild HAdV infection severity.

Furthermore, our analysis showed that C1 and C2 strains shared high nucleotide homology with AB601012 from Japan and KY969047 from Thailand, respectively. While C5 and C6 strains had high nucleotide homology with Jiangsu strains from China. These findings confirm that major strains vary geographically over time and cross provincial or national boundaries (18, 41).

Mutations are the foundation of molecular evolution. Our AA mutation results combined with the phylogenetic tree suggested that C1 and C5 were closer in the evolutionary relationship while C2 and C6 were closer among these Hangzhou HAdV-C strains. Two AA mutations (I677M and M788I) led to the splitting of C1 and C5 into two subclades, while C2 and C6 were split into another two subclades based on four AA mutations between C2 and C1 (L695F, M788I, D825E, F858V) and between C6 and C1 (L695F, M788I, D825E/L832I, F858V). Furthermore, C6 was further divided into two clusters based on one of these four mutations, namely D825E (17 strains) and L832I (12 strains). These six AA mutations between different C types confirm that the HVR is conserved for a given type but varies between types (34, 35). In addition to them, eleven mutations were also identified: D652A (AdV1258), E756G (AdV1319), N787H (AdV1335), Q827H (AdV1335), D825G (AdV1431), N746D (AdV1359), T650P (AdV1256), Y658F (AdV1256), N663T (AdV1256), M848V (AdV1203) and L862F (AdV1348). All these mutations suggest that there are differences between types during the evolution of HAdV, which may be explained by ongoing evolutionary process and possible antigenic drift. Over time, these differences may even give rise to new types (42). An in-depth exploration of these mutations may lead to a better understanding of the evolutionary mechanisms and pathogenesis of HAdV.

Adenoviral pneumonia accounts for about 10% of childhood pneumonia (43, 44). HAdV-B (type 3, 7, 21) are the most common pathogenic types of adenovirus pneumonia among children aged 0.5–5 years (45), as confirmed by the present result - the only B3-infected child was diagnosed with pneumonia. HAdV-C infections are most often subclinical or mild (46). However, the pathogenicity of pneumonia by different C types is rarely reported. Our analysis showed that children infected with C2, C5, and C6 were more likely to have pneumonia, while those infected with C1, C57 were not. This may be explained by our AA mutation results, as C1 and C57 have identical AA sequences, and compared to them, C2, C5, and C6 have 2 or 4 AA mutations, showing susceptibility to pneumonia. Whether these mutations affect the pathogenicity and virulence of viruses, larger sample sizes and more protein structure studies are needed to further confirm.

The main limitation of our study was that it was a short-period single-center study and didn't include the various known HAdVs. To address these limitations and enhance our understanding of HAdV dynamics over time, future research should implement a long-term and multi-center surveillance approach. This would involve monitoring HAdV infections and their associated factors across different geographical locations and over an extended period. By doing so, we can obtain more comprehensive and representative data that can contribute to a broader understanding of HAdV epidemiology.

In conclusion, our study provides valuable epidemiological and molecular data that will aid in epidemiological surveillance, antiviral therapies and specific types of vaccine development, leading to improve public health.