Human adenovirus among hospitalized children with respiratory tract infections in Beijing, China, 2017–2018

The nasopharyngeal aspirate (NPA) samples (1276) used in this study were collected from hospitalized children (< 14 years) with RTIs at Beijing Friendship Hospital between April 2017 and March 2018. Informed consent from the parents or guardians of the children enrolled in the study was received. RTIs was defined as an illness that presented with at least two of the following clinical presentations: fever, cough, nasal obstruction, expectoration, sneeze and dyspnoea during the previous week. Patients, who were diagnosed with pneumonia by chest radiography, were also included in the study, even if they did not show the clinical features described above [11]. All the specimens were stored at − 80 °C until further processing. Demographic and clinical data were obtained from the hospital’s database.

Total viral nucleic acids were extracted from 200 μL of each clinical NPA specimen using the QIAamp MinElute Kit (Qiagen, Germany) according to the manufacturer’s instructions and eluted with 60 μL of nuclease-free water. HAdV detection was performed using qPCR assay targeting the highly conserved gene region (132-bp) of the HAdV hexon. TaqMan Universal PCR Master Mix (Applied Biosystems, USA) was used to amplify HAdV hexon DNA using specific primers (Forward:5′- GCCCCAGTGGTCTTACATGCACATC-3′; Reverse: 5′-GCCACGGTGGGGTTTCT AAACTT-3′) and probe (5′-FAM-TGCACCAGACCCGGGCTCAGGTACTCCGA- 3′-TAMRA) [12]. Each 20 μL reaction mixture comprised 10 μL of 2 × TaqMan Gene Expression Master Mix, 1.8 μL of each primer (10 pM), 0.2 μL of probe (10 pM), 4.2 μL of sterile water, and 2.0 μL of the nucleic acid components extracted from each sample. The qPCR cycling program was as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. Samples with a cycle threshold (Ct) < 38 were regarded as positive. 10-fold serial dilutions of pMD18-T/Hexon plasmid from 10¹⁰ to 10⁰ copies per μL were prepared to establish a standard curve to measure the HAdV load. qPCR were performed using the Mx3005P qPCR System (Agilent Stratagene, USA). HAdV-positive samples were subsequently screened for the following pathogens: influenza A (FluA) and B (FluB) viruses, parainfluenza viruses (PIVs) 1–4, human metapneumovirus (HMPV), human rhinovirus (HRV), WU polyomavirus (WUPyV), respiratory syncytial virus (RSV) and human coronaviruses (HCoV) NL63, OC43, 229E, HKU1 and human bocavirus (HBoV), as described previously [13‐15]. Additionally, mycoplasma pneumonia is determined by the detection of mycoplasma pneumoniae IgM antibody. As for bacterial pneumonia, it was confirmed by sputum culture.

Nested PCR targeting the HAdV hexon gene’s hypervariable region was employed for genotyping. The outer primers used were forward 5′-GCCACCTTCTTCCCCATGGC.

− 3′ and reverse 5′-GTAGCGTTGCCGGCCGAGAA-3′, and the internal primers were forward 5′-TTCCCCATGGCCCACAACAC-3′ and reverse 5′-GCCTCGATGACGCC.

GCGGTG-3′. Nested PCR was conducted in 25 μL volume comprising 2.5 μL of 10 × ^EXTaq buffer, 1.0 μL (10 pM) of each primer, 2.0 μL of dNTP Mix, 0.5 μL of ^EXTaq DNA polymerase, 2.0 μL of viral nucleic acid extract or first nested-PCR product, and 16 μL of double-distilled water. The mixtures were amplified with an initial denaturation at 94 °C for 10 min, followed by 36 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min, and a 7 min extension at 72 °C. PCR products were analyzed on 1.50% agarose gels, purified, and then confirmed as authentic by sequencing. Samples that failed to amplify are defined herein as untyped.

All the hexon gene sequences obtained by nested PCR together with the 26 HAdV strain sequences available in GenBank were used for the phylogenetic analyses. The phylogenetic tree was constructed using molecular evolutionary genetics analysis (Mega) 5.0 and evaluated using 100 bootstrap replicates to verify the HAdV genotypes. Positive PCR products were named according to the corresponding serial numbers of the specimens.

In total, 1276 NPA specimens were obtained from 1276 children with RTIs at Beijing Friendship Hospital, among which 674 (52.82%) were male and 602 (47.18%) were female (Table 1), all children survived, and the age range was from 1 day to 14 years of age with a median age of 3 years. From them, 897 (70.30%) patients were younger than 5 years old.

HAdVs were detected in 5.64% (72/1276) of the hospitalized children. As shown in Table 1, among the 72 HAdV-positive patients, 45 (62.50%) were male and 27 (37.50%) were female (a male/female ratio of 1.6:1). No significant difference was observed in males and females in the HAdV-positive cases (P = 0.090). HAdV was detected among hospitalized children (< 14 years) with RTIs at Beijing Friendship Hospital between April 2017 and March 2018, and there were significant differences in HAdV detection rates among different age groups (P = 0.008). Children under 6 years old accounted for 86.11% (62/72) of the infections. The HAdV detection rate peaked in the > 5 to≤6 years age group (12.79%, 11/86), while those in the ≤1 years old group had a relatively low detection rate of 3.18% (10/314). HAdV was detected in every month throughout the study period from April 2017 to March 2018, peaking in summer. The HAdVs detection frequencies were 5.60% (18/321), 9.52% (30/315), 3.31% (10/302) and 4.14% (14/338) (χ2 = 16.06, P = 0.001) in the spring, summer, autumn and winter months, respectively. Additionally, the HAdV detection rate peaked at 10.91% (12/110) in August 2017 (Fig. 1).

Of the 72 HAdV-infected cases, 27 (37.50%) comprised co-infections with other respiratory viruses including 21 (77.78%, 21/27) with one other virus, 3 (11.11%, 3/27) with two other viruses, 2 (7.41%, 2/27) with three other viruses and 1 (3.70%, 1/27) with four other viruses. The most frequently identified mixed infection was PIV (12.50%, 9/72) and HRV (9.70%, 7/72), as shown in Table 2. Among the HAdV-positive specimens, the log number of HAdV genome copies ranged from 3.30 to 9.14 copies per mL of NPA, as determined by the qPCR assay measurements (Fig. 2). The log number for the HAdV genome copies was 6.14 ± 1.52 copies/mL of NPA in the children infected with HAdV only, which is slightly higher than the 5.70 ± 1.39 copies/mL of NPA from those co-infected with HAdV and other respiratory viruses; there was, however, no significant difference in the viral loads between the HAdV mono- and co-infections (P = 0.220).

Basing on the 956-bp fragment of the hexon gene amplified by nested-PCR, 66 HAdV-positive samples were sequenced and successfully genotyped. The phylogenetic analyses indicated that 41 cases belonged to species B (HAdV-B3, HAdV-B7, and HAdV-B35) and 25 belonged to species C (HAdV-C2, HAdV-C1, and HAdV-C5) (Table 3). Six of the samples failed to be typed. The above-mentioned results indicate that species B and C (at least 6 HAdV genotypes) circulated simultaneously in Beijing, and that HAdV-B3 was the most prevalent genotype, followed by HAdV-C2 (Fig. 3).