2 Materials and methods

2.1 specimen collection

From April 2018 to March 2019, 1572 NPAs samples were collected from hospitalized children (aged < 14 years) with ARTIs in Beijing Friendship Hospital, and 233 samples were collected from September 2020 to August 2021. The study protocol was approved by the Ethical Committee of Beijing Friendship Hospital (number: IVDC2018-012). Informed consent has been received from the parent or guardian of the children participating in the study. ARTIs were defined as at least two of the following clinical manifestations during the previous week: fever, cough, nasal obstruction, expectoration, sneeze and dyspnoea. Patients with pneumonia diagnosed by chest radiography were also included, even if they did not exhibit the clinical features described above18. Collected samples were stored in virus preservation solution and placed at -80℃ for further processing.

2.2 Detection of HMPV

Total viral nucleic acids were extracted from 200ul specimen and eluted with 60ul water by using a QIAamp MinElute Kit (Qiagen, Germany) according to the manufacturer’s instructions. HMPV detection was performed by using a quantitative real-time reverse transcription PCR (qRT-PCR) assay with a One-step RT-PCR Kit (Ambion, USA). The HMPV qRT-PCR primer set was used as previously described, designed based on the conversed fragment of N gene 19: HMPV forward primer(5’- TTAARTTACAAAAAAACATGGGAC -3’), reverse primer (5’- AAAGAATATCTTTTCCTTCAGGG -3’), and probe (5’-FAM- AATTACTCATAATCATTTTGACTG -3’-TAMRA). Each 25 μL reaction mixture comprised 12.5 μL of RT-PCR 2×Buffer, 1 μL of each primer (10 pM), 0.3 μL of probe (10pM) , 7.2 μL of Nuclease-Free water, 1μL of 25×RT-PCR Enzyme Mix and 2.0 μL of the nucleic acid components extracted from each sample. The qRT-PCR cycling program was as follows: 45 °C for 10 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 45 s. Samples with a cycle threshold (Ct) < 38 were considered positive. Measure of viral load based on a standard curve that has been established in our laboratory20.

2.3 Detection of viral co‑infection in HMPV‑positive specimens

The HMPV-positive specimens were subsequently screened for 15 common respiratory viruses.: influenza virus types A, B, and C (IFV A/B/C), parainfluenza virus types 1–4 (HPIV), human coronaviruses HKU1/229E/OC43/NL63 (HCoV), respiratory syncytial virus (RSV), human rhinovirus (HRV), adenovirus (ADV), and human bocavirus (HBoV). All viruses were detected by real-time polymerase chain reaction21,22, DNA viruses were tested using TaqMan™ Gene Expression Master Mix (Thermo Fisher, USA), RNA viruses were tested using AgPath-ID™ One-Step RT-PCR Kit (Ambion, USA) in accordance the corresponding manufacture’s protocols.

2.4 HMPV genotyping

The viral RNA of HMPV positive samples was reverse transcribed into cDNA using a SuperScript™ IV First-Strand Synthesis System (Invitrogen, USA) in accordance with the manufacture’s protocols. The conserved sequence of F gene (610bp) was amplified using a nested PCR for genotyping as previously described23. The outer primers used were forward 5′- CAATGCAGGTATAACACCAGCAATATC -3′ and reverse 5′- GCAACAATTGAACTGATCTTCAGGAAAC -3′, and the internal primers were forward 5′- ACATGCCAACATCTGCAGGACAAATAAAAC -3′ and reverse 5′- ACATGCTGTTCACCTTCAACTTTGC -3′. Nested PCR was conducted in 25 μL volume comprising 2.5 μL of 1 0 × Ex Taq buffer, 2.0 μL of dNTP Mix, 1.0 μL(10 pM) of each primer, 0.4 μL of Ex Taq DNA polymerase, 2.0 μL of viral nucleic acid extract or first nested-PCR product, and 16.1 μL of double-distilled water. The Nested-PCR products were sequenced at Ruibio Co., Ltd (Beijing, China), and a Maximum Likelihood (ML) tree was constructed by the Tamura-Nei model in MEGA 7.0 with 1000 bootstrap replicates. Samples that failed to be amplified were classified as untyped.

2.5 Statistical analysis

SPSS 26.0 software was used for data analysis, and Categorical data were tested using χ2 test and Fisher’s exact test. Wilcoxon’s test and independent-samples t-test were used to analyze continuous variables. Two-sided P-value < 0.05 was considered statistically significant.