Etiology and clinical characteristics of SARS-CoV-2 and other human coronaviruses among children in Zhejiang Province, China 2017–2019

The studies involving human participants were reviewed and approved by Zhejiang Provincial Center for Disease Control and Prevention of ethics committee. Children’s parents/guardians have been notified the purpose of the study and the right to keep information confidential. Children’s parents/guardians provided their written informed consent to participate in this study.

A total of 3660 retrospective respiratory samples were collected from children (age, ≤ 18 years) with acute respiratory tract infections who admitted to Children’s Hospital of Zhejiang University from 2017 to 2019. Written informed consent was obtained from the guardians of all participants before specimens and data collection. Specimens were collected in accordance with Zhejiang Provincial CDC guidance and contained nasopharyngeal swab (n = 26), sputum (n = 3590) and bronchoalveolar lavage (n = 41). 2138 samples were from male and the remaining were from female. Clinical specimens were initially kept in sterile EP tube with 5 mL viral transport medium (Becton, Dickinson and Company, NJ) and then transferred to Zhejiang Provincial CDC within the same day for routine monitoring. All clinical samples were stored at − 80 °C prior to blinded analysis. Clinical data including symptoms, history of illness, severity of infection, demographic data, drug usage and laboratory examination were recorded for each patient and reviewed retrospectively when patients confirmed with HCoV infections.

Viral nucleic acid was extracted from 200 μL of clinical specimens using QIAGEN Cador Pathogen 96 QIAcube HT Kit (Qiagen, Hilden, German) and following manufacturer’s instructions. The extracts were eluted into 50 μL of DNase- and RNase-free water and stored at − 80 °C until used as a template for PCR assay.

A two-tube multiplex RT-PCR assay (a two-tube assay) was performed for the identification of sixteen respiratory virus using virus-specific primers as described previously [15]. Briefly, one tube was added with nine pairs of chimeric primers for the detection of nine viruses (HRV, FluA, FluB, 229E, OC43, HKU1, PIV1, PIV3 and ADV), and the other tube was added with eight pairs of chimeric primers to detect another eight viruses (RSVA, RSVB, NL63, PIV2, PIV4, H3N2, H1N1 and HBoV). The primer sequences, target genes, amplicon sizes and primer working concentrations were summarized in Additional file 1: Table 1. Qiagen one-step RT-PCR Kit (Qiagen, Hilden, German) was used for amplification. A final volume of 25 μL PCR mixture containing 2 μL of template, 1 μL of primer-mix (mixture was prepared according to the primer working concentration in Table 1), 1 μL of enzyme-mix, 1 μL of deoxinucleotidetriphospate (dNTP), 5 μL of 5X buffer, 1 μL of Taq polymerase was subjected to multiplex RT-PCR screening. The amplification cycle was initial denaturation at 50 °C for 30 min, 95 °C for 15 min, followed by 10 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s; 10 cycles of 95 °C for 30 s, 65 °C for 30 s, 72 °C for 30 s; 25 cycles of 95 °C for 30 s, 48 °C for 30 s, 72 °C for 30 s and one final extension at 72 °C for 3 min. The amplified fragment (15 μL) was separated on the QIAxcel automatic electrophoresis using QIAxcel DNA High-Resolution kit and confirmed as specific virus types following reference marker in the system.

The specificity of the two-tube assay was based on the automatic electrophoresis. All positive controls were observed and separated clearly from the other viral targets and no primer dimer was observed in either tube. The expected size of each virus type/subtype-specific amplicon was listed in Additional file 1: Table 1. The detection sensitivity in tube 1 and tube 2 was 2000 and 200 copies per reaction, respectively. The detection limit for HRV, PIV2, PIV3, RSVA, HBoV and ADV was 20 copies per reaction and the remaining 10 viruses had a detection limit of 200 copies per reaction.

SARS-CoV-2 nucleic acid detection kit (Daan, China) was applied to detect the ORF1ab gene and N gene of SARS-CoV-2 according to protocols as described previously [16]. Primers and probes used for SARS-CoV-2 were listed in Additional file 1: Table 1. The reaction was performed in a 25 μL reaction mixture containing 17 μL of reaction mixture A, 3 μL of reaction mixture B and 5 μL of RNA. The RT-qPCR reactions were performed with initial one cycle at 50 °C for 15 min and 95 °C for 15 min, following by 45 cycles of 94 °C for 15 s and 55 °C for 45 s. All RT-qPCRs were performed on the 7500 Fast Real-Time PCR system (Applied Biosystems) and all samples were analyzed in duplicate.

The S genes of HKU1, OC43, NL63 and 229E positive samples were amplificated using PrimeScript™ One Step RT-PCR Kit Ver.2 (TaKaRa 055RA). The volume of PCR mixture was 25 μL, containing buffer 12.5 μL, enzyme-mix 1 μL, forward primer 0.5 μL, reverse primer 0.5 μL, RNA 5 μL, and Rnase free water 5.5 μL. Their forward and reverse primers were listed in Additional file 1: Table 2. The amplification cycle was reverse transcription at 50 °C for 30 min and initial denaturation at 94 °C for 2 min, followed by 40 cycles of 94 °C for 45 s, 50–56 °C (depending on species) for 30 s, 72 °C for 45 s, and one final extension at 72 °C for 10 min at the end of 40 cycles. Amplified PCR products were sequenced by Sanger sequencing method (TsingKe Biological Technology).

DNA sequences were concatenated manually and aligned by Clustal W and downloaded to MEGA 7.0 to generate a phylogenetic tree constructed by neighbor-joining algorithm and jukes-cantor model with 1000 bootstrap. Genome sequences of other coronaviruses were retrieved from GeneBank and included in phylogenetic analysis for comparison.

To survey human coronaviruses (HCoVs) in Zhejiang province, nasopharyngeal swabs (n = 26), sputum (n = 3593) and bronchoalveolar-lavage fluid (n = 41) specimens were collected periodically between February 2017 and December 2019 from children with respiratory symptoms. Overall, all clinical specimens were negative for SARS-CoV-2 RNA by RT-PCR analysis. 38 (1.04%) were positive for HCoV-HKU1, 62 (1.69%) for HCoV-NL63, 8 (0.22%) for HCoV-229E and 38 (1.04%) for HCoV-OC43 (Table 1). Comparison of the percentage of positive samples for multiple respiratory viruses was used to infer prevalence (Fig. 1). Respiratory syncytial viruses (RSVA + RSVB, 14.51%), human rhinovirus (HRV, 13.74%), parainfluenza (PIVs 1–4, 10.96%) and influenza (FluA + FluB, 6.61%) were the four dominant viruses in Zhejiang area, which were detected more frequently than HCoVs (P < 0.001).

Overall, HCoVs dominant circulation season was in the transition of summer to fall, especially August to October (Fig. 2). The percent of HCoV positive tests varied seasonally throughout each year and the percent positive also varied annually by HCoV species (Fig. 3a). The detection rates of HCoVs were 2.18%, 3.94%, 4.51%, 5.22% of winter, spring, summer and autumn, respectively (Fig. 3b). HCoV-HKU1 demonstrated a distinct peak each of the three years, with a relatively large peak in Spring 2018. For HCoV- NL63, in contrast to the pattern described for HCoV-HKU1, the peak detection frequency occurred in the third season. 51.61% of positive HCoV- NL63 samples were detected between September to November. HCoV-OC43 also demonstrated a consistent detection frequency between seasons, with the testing peak occurring each year in spring. The exception was HCoV-229E, as 87.50% of detectable HCoV-229E were found in the spring and summer of 2019. To be noted, HCoV-229E detected only in spring and summer through the study period, and no HCoV-229E was detected in 2018.

Almost all HCoV-positive patients (143/144, 99.31%) had lower respiratory tract infections. The incidence of lower respiratory tract infections in patients with HCoVs (3.94%) infection was significantly lower than that of patients infected with RSV (14.51%), PIV (10.96%), or HRV (13.85%). In addition, only one patient infected with HCoV-NL63 was diagnosed with an upper respiratory tract infection from nasopharyngeal swab in this study. The positive rate of HCoV infections was 3.85% (1/26) for nasopharyngeal swab, 4.88% (2/41) for bronchoalveolar lavage and 3.93% (141/3590) for sputum (P > 0.05) (Table 2). The four investigated HCoV subtypes were detected in sputum samples, while only HCoV-NL63 infections were positive in nasopharyngeal swab and bronchoalveolar lavage specimens.

Fever, cough, expectoration and sore throat were the common clinical presentations with HCoV infections, among which cough (47.22%), sore throat (40.97%) and fever (23.61%) were the top three manifestations (Fig. 4a). Difficulty in breathing and respiratory failure were seen in patients who infected with HCoV-OC43. Slightly more patients with HCoV infections were males but no significant difference was in the sex distribution between the four species (P > 0.2) (Table 1). Age distribution of patients differed between HCoV species and infants (< 1 year old) with weak respiratory immunity were most susceptible to four types of HCoVs infections (Fig. 4b). To be noted, HCoV-229E had a higher detection proportion in children between 2 and 18 years old compared to HCoV-OC43, HCoV-NL63, and HCoV-HKU1.

High rates of coinfection with other respiratory viruses were observed for all coronaviruses, with the coinfection rates of HCoV-HKU1, HCoV-NL63, HCoV-OC43 and HCoV-229E as 44.74% (17/38), 43.55% (27/62), 52.63% (20/38) and 37.50% (3/8), respectively (Table 3). Among the 146 positive detections, 54.11% reported a single HCoV species detection only, 1.37% reported two HCoV species, and 44.52% detected another respiratory virus. The most common HCoV co-infections were HCoV-HKU1 with HCoV-229E (2 specimens, 1.37%). Of all samples testing positive for coronavirus, 40% samples were coinfected with HRV. The most common co-detected non-HCoVs were human adenovirus (40.71%), human rhinovirus/bocavirus (37.57%), and human metapneumovirus (32.43%).

Phylogenetic trees were further constructed based on the sequencing of HCoV spike genes. A representative subset of HCoV-OC43 (n = 13), HCoV-NL63 (n = 8) and HCoV-NL63 (n = 8) were included in the phylogenetic analysis and compared to the HCoVs from clinical sources worldwide. A high level of genetic diversity was observed among those HCoVs. The OC43 coronaviruses were clustered into genotype G (9, 69.23%) and genotype B (4, 30.77%) and related to the sequences detected in Malaysia and Beijing, respectively (Fig. 5a). No significant differences were observed between HCoV-OC43 strains identified in different years. Similarly, HCoV-NL63 strains in this study were clustered into genotype B (5, 62.5%) and genotype C (3, 37.5%) and shown close identity to the viruses isolated from China and USA (Fig. 5b). Moreover, the phylogenetic tree of the HCoV-HKU1 isolates shown the existence of two major clusters that contained both Hong Kong and France strains of HCoV-HKU1 (Fig. 5c). All HCoV-HKU1 subtypes isolated from 2017 were grouped into genotype B.