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.