One hundred twenty-four nasopharyngeal swab specimens were collected from the enrolled cases with acute respiratory tract infections (ARTI) from the First Affiliated Hospital of Bengbu Medical College in hospitalized children in Bengbu from October 2021 to January 2022. These cases contained upper respiratory tract infection (URTI) and community-acquired pneumonia (CAP). A URTI was defined as fever (body temperature ≥ 38°C) accompanied by respiratory signs or symptoms (i.e., cough, sore throat, and rhinorrhea). CAP was defined in accordance with the guidelines for the management of community-acquired pneumonia in children in China (the revised edition of 20,130) (22). All cases were investigated by clinicians using a uniform questionnaire that included demographic data, epidemiological data, and clinical manifestations.

Total viral nucleic acids (RNA and DNA) were extracted from the viral transportation medium using the Fujian Baineng Medical Technology Co., Ltd. Thirteen respiratory viruses were simultaneously detected with real-time PCR, including RSV, influenza virus A (FluA), influenza B (FluB), human coronavirus (NL63, OC43, 229E, and HKU1), parainfluenza virus 1 to 3 (PIV), human metapneumovirus (HMPV), adenovirus (ADV), and human bocavirus (HBoV). The nucleic acids were stored at −80°C until further use.

RSV cDNA was obtained from the extract using MultiScribe reverse transcriptase and random hexamers. Total cDNA was used in a PCR (Century 2 × Es Taq MasterMix (Dye), CWBIO). The amplification follows conditions: 94°C for 2 min, followed by 40 cycles of 94°C for 30s, 56°C for 45 s, 72°C for 30s, and a final extension at 72°C for 5 min. The PCR products were sequenced using an ABI Prism 3730XL DNA Analyzer at Tsingke Co., Ltd. (Beijing, China). The sequences were edited using Sequencher software version 5.0 (Gene Codes, Ann Arbor, MI, United States). These sequences were deposited in GenBank with accession numbers from QQ933800 to QQ933847.

SeqMan program (DNASTAR 7.0, Inc., Madison, WI) was used for contigs assembling and obtaining the full length of the G gene. The sequences obtained in this study were aligned with representative sequences retrieved from GenBank using Clustal W. The phylogenetic tree was constructed using the maximum likelihood with HKY+ G and TN93+ G models for RSV-A and RSV-B. The reliability of the tree topology was evaluated by bootstrapping with 1,000 replications in Mega 7.0 software. Deduced amino acid sequences were translated with the standard genetic code using MEGA software version 7.0. The sequences of the RSV-A strains and RSV-B strains were aligned with the prototype strain ON67-1210A and BA4128/99B, respectively. RSV-A and RSV-B sequences were downloaded from the GenBank database, respectively.

Putative N-glycosylation sites were predicted using NetNGlyc 1.0 webserver¹ to identify the sequence motifs N-X-S/T (sequon), where X can be any amino acid except proline. Only the sites with scores higher than 0.5 were accepted as glycosylated.

The clinical data were entered using Epidata 3.0 and organized using Excel 2019. SAS 9.4 was used for the statistical analysis, and continuous variables were presented as median (interquartile range, IQR) and compared with the Kruskal–Wallis test and t-test between different groups; categorical variables were presented as number (%) and compared by chi-square test or Fisher’s exact test between different groups. A p-value of <0.05 was considered statistically significant. A flow chart about the methodology of sampling and analyses is given in Supplementary Figure S1.

From October 2021 to January 2022, a total of 124 samples with ARTI were collected from hospitalized pediatric patients in Bengbu, Anhui, China. Of 124 children, 117 cases had complete clinical information. In this study, the majority (36.75%, 43 of 117) of the patients were younger than 6 months, 30.77% (36 of 117) of cases were 6 months to 2 years old, 23.93% (28 of 117) of cases were 2 years old to 5 years old, and 8.55% (10 of 117) of children’s cases were over 5 years old (range from 1 month old to 16 years old). The male-to-female ratio was 1.74:1. Of these samples, RSV infection was the main infection, accounting for 41.94% (52 of 124), followed by ADV 5.65% (7 of 124), FluB 5.65% (7 of 124), HBOV 2.42% (3 of 124) and HCoVs 0.81% (1 of 124). Therefore, subsequent studies focused on analyzing the pathogenic and epidemiological characteristics of RSV.

Of the 52 RSV-positive cases, the proportion of RSV-A and RSV-B was 71.15% (37 of 52) and 21.15% (11 of 52), respectively. The other 7.69% (4 of 52) cases were unclassifiable. The median age of the RSV-infected patients was 1 year old (IQR: 0.3–2.5 years old), and RSV infection mainly affected children under 2 years old (79.17%, 38 of 48). Excluding four cases of missing clinical information, the proportion rates of RSV of the four types of cases mainly occurred in SCAP (60%), followed by NSCAP (41.8%), bronchiolitis (40.6%), and URTI (33.3%) (Figure 1). RSV-A was the dominant strain in this study.

In total, 40 full-length G genes (30 RSV-A and 10 RSV-B) and 8 HVR2 sequences of G gene (7 RSV-A and 1 RSV-B) were obtained by PCR amplification for subsequent analysis. Phylogenetic analysis showed that all RSV-A strains and RSV-B strains were of the ON1 and BA9 genotypes, respectively (Figures 2A,B). Thirty-seven ON1 genotype was further divided into two branches in this study. Thirty-three strains were only clustered with reference sequences from China in clade 1, and 4 strains were clustered with reference sequences from Portugal, China, Italy, Brazil, Kenya, the United States, and 13 other countries or regions in clade 2 (Figure 2A). In this study, the BA9 genotype of clade 1 was mainly clustered with the Chinese BA9 genotype sequences (Figure 2B). The nucleotide homology of HVR2 of ON1 and BA9 genotypes was 92.5%–100% and 94.8%–100%, respectively. The calculated overall mean distance was 0.031 for RSV-A and 0.033 for RSV-B.

Subsequently, the diversity of amino acid mutations of ON1 and BA9 was analyzed. The most common amino acid substitutions of the G glycoprotein gene of RSV stains were identified in mucin-like regions 1 and 2 compared to the prototype ON1 (JN257693) strains and BA1 strains (AY333364) (Figures 3, 4). The most common substitutions of the G protein gene of ON1 strains were T113I, V131D, N178G, T245A, H258Q, H266L, and L274P in this study (Figure 3A). Additionally, the majority of ON1 strains with T113I, V131D, N178 G, and H258Q mutations formed a unique genetic cluster. N178 G was located near the CX3C motif binding to CX3CR1 to initiate infection. H258Q was observed within the 24aa duplication region of G glycoprotein. Moreover, eight amino acid substitutions occurred in the majority of BA9 strains, including R98M, N121S, T254I, T270I, V271A, N296Y, T302I, and N178S. Similarly, N178S substitution was also found in the BA9 strain (Figures 3B, 4B).

To predict N-linked glycosylation sites, amino acid sequences of G protein were submitted to online bioinformatics tools (NetNGlyc). For ON1 genotypes, three major putative N-glycosylation sites, N103, N135, and N237 were identified (Figure 3A, gray shading). N-glycosylation site N239 was also identified in the G gene of 23% ON1. For BA9 genotype, the N-glycosylation site N310, N318, N86 (91%), N114 (36%), and N296 (45%) were also predicted in this study that were similar with BA prototype strain (Figure 3B, gray shading). In addition, one of the BA9 strains carried one N-glycosylation site at aa 230 due to the P231S substitution (Figure 3).

To understand the clinical symptoms of children infected with RSV, the differences in clinical symptoms, laboratory, radiographic findings, treatments, and outcomes were analyzed between RSV-infected and non-infected children. The results showed that the most common clinical manifestations in RSV-infected cases were cough (83.33%), pulmonary rales (81.25%), sputum (70.83%), fever (62.5%), and wheezing (60.42%) (Table 1). The median age of RSV-infected children was lower than that of RSV-uninfected children (0.45 years old vs. 1.2 years old p < 0.01) (Table 1). Compared to patients without the RSV infection, the RSV-infected patients were more likely to produce sputum (70.83%, 34/48, p = 0.030), wheezing (60.42%, 29/48, p < 0.01), pulmonary rales (81.25%, 39/48, p < 0.01), and chest shadow (26.32%, 10/38, p = 0.048) (Tables 1, 2). In addition, patients with RSV infection were more likely to require mechanical ventilation (35.42%, 17 of 48, p < 0.01) (Table 2), in which children infected with RSV-B need more mechanical ventilation than those with RSV-A (63.64% VS 26.47%, p = 0.035) (Supplementary Tables S1, S2).

To avoid overfitting in the model, a multiple logistic regression analysis was performed to understand the association between using a ventilator and RSV infection. Based on the results of univariable results, six variables (age groups, gender, RSV infection, coinfections, comorbidity, and SCAP) were chosen for multivariable analysis (Table 3). RSV infection (OR = 9.55, 95% CI 1.87–48.64) was significantly higher in ventilator-used groups compared with non-ventilator-used groups in pediatric patients (p = 0·007). This indicates RSV infection is an independent risk factor for ventilator use. Furthermore, multiple logistic regression analysis showed that comorbidity was significantly associated with ventilator use (OR = 13.73, 95% CI 4.16–45.35), p < 0.001 (Table 3).