Human adenovirus in Taiwan, 2019–2023

The study protocol was approved by the Taiwan Centers for Disease Control Institutional Review Board (IRB 109107). The requirement to obtain informed consent was waived by the board.

Swab samples from patients with suspected respiratory viral infections were collected and sent to the contract laboratories of the Taiwan CDC for diagnosis and virus isolation between 1 January 2019 and 31 December 2023. To ensure anonymity, all sample data were depersonalized. Eight contract laboratories, located across northern, central, southern, and eastern Taiwan, conducted community virus surveillance and provided valuable data on epidemic trends. A total of 54,329 samples from patients with suspected respiratory viral infections were tested, with approximately 10,010 to 11,793 samples analyzed annually. Laboratory-confirmed HAdV infections were identified through viral isolation via culture. The isolated HAdVs were subsequently sent to the Taiwan CDC for PCR and genotype analysis.

Nucleic acid extraction was performed using a TANBead Maelstrom 4800 automated nucleic acid extractor (Taiwan Advanced Nanotech Inc., Taiwan) according to the manufacturer’s instructions, with 200 µL of each sample. Sterile water was used as a negative control for PCR. Viral DNA extracted from an HAdV strain was used as a positive control for PCR. The primers AdnU-20743F (5’-TTCCCCATGGCNCACAACAC-3’) and AdnU-21679R (5’-GCCTCGATGACGCCGCGGTG-3’) were used to amplify a 936-bp fragment of the hexon gene. PCR was performed using a one-step PCR kit (HotStarTaq Master Mix Kit, QIAGEN) according to the manufacturer’s protocol. Briefly, 5 µL of DNA was added to the PCR mixture, which included 12.5 µL of HotStarTaq Master Mix, 1 µL of 10 µM each primer, and 5.5 µL of diethylpyrocarbonate (DEPC)-treated water in a final volume of 25 µL. The PCR protocol was as follows: 95 °C for 15 min, followed by 30 cycles of PCR (94°C for 30 s, 55 °C for 45 s, and 72 °C for 1 min), with a final extension at 72 °C for 10 min. The PCR products were loaded on a 1.5% agarose gel to detect the amplified 936-bp fragment of the gene.

The PCR products were purified using a Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, California, USA). Nucleotide sequences were determined using an automated DNA sequencing kit and an ABI Prism 3730XL DNA sequencer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocols. Overlapping nucleotide sequences were combined for analysis and edited using the Sequencher software package (v5.4.6, Gene Codes Corporation, Ann Arbor, MI, USA). Nucleotide sequences were submitted to the GenBank database under the accession numbers PV147116-PV147123, PV224130-PV224314, PV231314-PV231315, PV235932-PV235939, PQ393377-PQ394015, PQ421864-PQ421958, and PQ430419-PQ431046.

Between 2019 and 2023, a total of 54,329 samples from patients with suspected respiratory virus infections were collected, among which 1,829 tested positive for HAdV, yielding an average positivity rate of 3.4%. The annual distribution of tested samples and positivity rates are presented in Fig. 1. HAdV infections were detected year-round, with the highest detection rate reaching 6.6% in 2023 – more than three to four times the rates observed from 2020 to 2022. The HAdV typing results from 203 randomly selected isolates from 2019, along with all available typing data from 2020 to 2023, are presented in Table 1. The analysis revealed six adenovirus species and 17 distinct types that circulated in Taiwan during this period. Among these species, HAdV-C was the most prevalent, accounting for 54.1% (846/1,565) of typed isolates, followed by HAdV-B (39.4%, 617/1,565) and HAdV-E (3.8%, 59/1,565).

At the type level, HAdV-B3 was the most frequently detected virus, accounting for 36.7% (574/1,565) of all sequenced viruses. Notably, no cases of HAdV-B3 infection were identified in 2021, but infections increased dramatically in 2023. HAdV-C2 was the second most prevalent type, representing 30.8% (482/1,565) of the sequenced viruses. HAdV-C2 was detected each year, with the number of cases increasing from 41 in 2019 to 169 in 2023, and HAdV-C2 was the dominant type circulating from 2020 to 2022. In contrast, some types were detected only sporadically. For example, HAdV-D212 was identified in a single case in 2020, whereas HAdV-B14 appeared exclusively in 2023.

The monthly distribution of HAdV infection cases from 2019 to 2023 is shown in Fig. 2A. The lowest detection rate was recorded in June 2022 (0.02%), whereas the highest detection rate exceeded 15% in December 2023. Outbreaks peaked in January-February 2019, followed by another surge from October 2019 to January 2020. In 2021, the number of cases peaked between February and March. In 2023, the number of infections began to rise in March, followed by a sharp increase from August onward, with the highest number of positive cases and detection rates recorded in December. The monthly distribution of HAdV species among the 1,565 sequenced isolates from 2019 to 2023 is presented in Fig. 2B. HAdV-B was detected from January 2019 to April 2020 and reemerged as the dominant species from July to December 2023. In contrast, HAdV-C was the predominant species from May 2020 to June 2023. Notably, HAdV-F was detected only sporadically, with single cases identified in June 2020 and June 2023 (Supplementary Table S1). Additionally, HAdV-D212 was detected only once, in September 2020 (Supplementary Table S2).

HAdV infections were observed across all age groups. Of the infected individuals, 53.9% were male, with no significant difference in prevalence between sexes. However, a significant difference was observed between the age groups (P < 0.05). The median age of the infected patients was 3 years (range: 0–92 years), with a mean age of 5.4 years. The age distribution of HAdV infections and positivity rates for HAdV in all patients with suspected respiratory viral infections tested from 2019 to 2023 are presented in Fig. 3A. Most infections (91.9%) occurred in children aged 0–9 years, with infection rates decreasing in older age groups. One-year-old children had the highest number of HAdV cases, while positivity rates were above 7% in children aged 3–6 years, as highlighted in Fig. 3B. The distribution of HAdV species by age is shown in Fig. 3C. All six HAdV species were detected in 1-year-old children. HAdV-C was the predominant species detected in children under 2 years of age, whereas HAdV-B became the most common species detected in children over 2 years of age. Notably, the infection rate of HAdV-B reached 85.9% in children under 7 years of age. The age distribution of patients infected with specific HAdV types is presented in Table 2. Most HAdV-A31 infections (90.9%, 10/11) occurred in children under 3 years of age. HAdV-B7, HAdV-D (types 8, 53, and 64), and HAdV-F41 were detected exclusively in children under 7 years of age. In contrast, HAdV-B55 was identified in three males aged 15–64 years, whereas HAdV-D212 was detected in an 80-year-old patient.

The clinical symptoms associated with infections with different HAdV types are presented in Table 3. Fever was the most common symptom, reported in 80.0% of cases, followed by rhinorrhea (43.3%), myalgia (40.7%), headache (38.1%), and acute tonsillitis (30.3%). Severe symptoms, such as pneumonia and CNS involvement, were observed in patients infected with HAdV-B3, B55, C1, C2, and C5. Conjunctivitis was reported in cases involving HAdV-B3, D37, D54, and E4. Additionally, patients infected with HAdV-D212 experienced fever, rhinorrhea, acute tonsillitis, and acute bronchitis.

Data from 34 patients in whom HAdV was codetected with other respiratory pathogens, including influenza virus, respiratory syncytial virus (RSV), coxsackievirus, parainfluenza virus, metapneumovirus, echovirus, herpes simplex virus, and cytomegalovirus, are presented in Table 4. Among these viruses, RSV was the most frequently codetected virus, accounting for 23.5% (8/34) of the cases, followed by coxsackievirus at 20.6% (7/34). Notably, two cases involved coinfection with three respiratory pathogens: HAdV-B3, parainfluenza virus 2, and parainfluenza virus 3. Statistical analysis revealed no significant correlation between HAdV coinfection and the severity of clinical syndromes.