Zhang et al. Virol J (2021) 18:89 https://doi.org/10.1186/s12985-021-01562-8 RESEARCH Open Access Etiology and clinical characteristics of SARS-CoV-2 and other human coronaviruses among children in Zhejiang Province, China 2017–2019 Yanjun Zhang1,2†, Lingxuan Su1†, Yin Chen2, Sicong Yu1, Dan Zhang3, Haiyan Mao2 and Lei Fang2* Abstract Background: A novel coronavirus (SARS-CoV-2) emerging has put global public health institutes on high alert. Little is known about the epidemiology and clinical characteristics of human coronaviruses infections in relation to infec- tions with other respiratory viruses. Methods: From February 2017 to December 2019, 3660 respiratory samples submitted to Zhejiang Children Hospi- tal with acute respiratory symptoms were tested for four human coronaviruses RNA by a novel two-tube multiplex reverse transcription polymerase chain reaction assays. Samples were also screened for the occurrence of SARS-CoV-2 by reverse transcription-PCR analysis. Results: Coronavirus RNAs were detected in 144 (3.93%) specimens: HCoV-HKU1 in 38 specimens, HCoV-NL63 in 62 specimens, HCoV-OC43 in 38 specimens and HCoV-229E in 8 specimens. Genomes for SARS-CoV-2 were absent in all specimens by RT-PCR analysis during the study period. The majority of HCoV infections occurred during fall months. No signifcant diferences in gender, sample type, year were seen across species. 37.5 to 52.6% of coronaviruses detected were in specimens testing positive for other respiratory viruses. Phylogenic analysis identifed that Zhejiang coronaviruses belong to multiple lineages of the coronaviruses circulating in other countries and areas. Conclusion: Common HCoVs may have annual peaks of circulation in fall months in the Zhejiang province, China. Genetic relatedness to the coronaviruses in other regions suggests further surveillance on human coronaviruses in clinical samples are clearly needed to understand their patterns of activity and role in the emergence of novel coronaviruses. Keywords: Human coronavirus, Hospitalized children, Phylogenic analysis, Acute respiratory infection, SARS-CoV-2 Introduction including two α-CoVs (HCoV-229E and HCoV-NL63) Human coronaviruses (HCoVs) are common viruses that and fve β -CoVs [HCoV-HKU1, HCoV-OC43, severe cause mild to severe respiratory infections. To date, there acute respiratory syndrome coronavirus (SARS-CoV) are seven CoVs have been confrmed to infect human, and SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV)] [1]. Te ongoing pandemic of COVID-19 ofers the most recent example of the trag- *Correspondence: [email protected] †Yanjun Zhang and Lingxuan Su contributed equally to this work. edy when highly pathogenic coronaviruses emerge in the 2 Zhejiang Provincial Center of Disease Control and Prevention, 3399 human population [2–4]. As of 23 March 2021, approxi- Bincheng Road, Hangzhou 310051, China mately 25 million cases have been reported to the WHO Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhang et al. Virol J (2021) 18:89 Page 2 of 11 accounting for a 2.2% case fatality rate. Over the last two Hospital of Zhejiang University from 2017 to 2019. Writ- decades, the emergence of Severe Acute Respiratory Syn- ten informed consent was obtained from the guardians drome Coronavirus (SARS-CoV) has been associated of all participants before specimens and data collection. with severe atypical pneumonia and caused 774 deaths Specimens were collected in accordance with Zhejiang [5]. In 2012, a man in Saudi Arabia who infected by Mid- Provincial CDC guidance and contained nasopharyngeal dle East Respiratory Syndrome Coronavirus (MERS- swab (n = 26), sputum (n = 3590) and bronchoalveolar CoV) died of acute pneumonia and renal failure [6]. Te lavage (n = 41). 2138 samples were from male and the continuing spread of MERS-CoV resulted in unprece- remaining were from female. Clinical specimens were dented outbreaks and high case-fatality rates. initially kept in sterile EP tube with 5 mL viral trans- HCoV-NL63, HCoV-HKU1, HCoV-229E, and HCoV- port medium (Becton, Dickinson and Company, NJ) and OC43 circulate worldwide and cause a range of respira- then transferred to Zhejiang Provincial CDC within the tory symptoms [7]. Tese four HCoVs result in 30% of same day for routine monitoring. All clinical samples common colds and acute respiratory infections such as were stored at − 80 °C prior to blinded analysis. Clinical pneumonia and bronchiolitis, especially in immuno- data including symptoms, history of illness, severity of compromised children [8, 9]. Lower respiratory tract infection, demographic data, drug usage and laboratory infections are considered as the most common cause of examination were recorded for each patient and reviewed death among children and are responsible for 15% mor- retrospectively when patients confrmed with HCoV tality rate of children under 5 years of age globally [10, infections. 11]. HCoVs pose serious threats to children health as they are associated with acute upper or lower respiratory tract Nucleic acid extraction infections [12, 13]. Although HCoVs are distributed glob- Viral nucleic acid was extracted from 200 μL of clinical ally, the predominant species may vary by region or year. specimens using QIAGEN Cador Pathogen 96 QIAcube Moreover, a notable feature of COVID-19 pandemic is HT Kit (Qiagen, Hilden, German) and following manu- the relative paucity of published report that describes the facturer’s instructions. Te extracts were eluted into 50 potential impact of SARS-CoV-2 on children with acute μL of DNase- and RNase-free water and stored at − 80 °C respiratory infection [14]. However, the prevalence and until used as a template for PCR assay. clinical profles of HCoVs have been largely underdeter- mined and no studies described circulation of SARS- Two‑tube multiplex reverse transcription PCR (RT‑PCR) CoV-2 and four common HCoVs across multiple years in assay Zhejiang province, China. A two-tube multiplex RT-PCR assay (a two-tube assay) Considering the discovery of novel SARS-CoV-2, we was performed for the identifcation of sixteen respira- retrospectively investigated the role of SARS-CoV-2 and tory virus using virus-specifc primers as described previ- common human coronaviruses (HCoV-229E, HCoV- ously [15]. Briefy, one tube was added with nine pairs of OC43, HCoV-NL63 and HCoV-HKU1) in childhood chimeric primers for the detection of nine viruses (HRV, respiratory infections in Zhejiang province, China, and FluA, FluB, 229E, OC43, HKU1, PIV1, PIV3 and ADV), results were compared to data for other viral respiratory and the other tube was added with eight pairs of chimeric pathogens including infuenza A and B, parainfuenza, primers to detect another eight viruses (RSVA, RSVB, respiratory syncytial virus, rhinovirus, and adenovirus. NL63, PIV2, PIV4, H3N2, H1N1 and HBoV). Te primer sequences, target genes, amplicon sizes and primer Materials and methods working concentrations were summarized in Additional Ethical approval statement fle 1: Table 1. Qiagen one-step RT-PCR Kit (Qiagen, Te studies involving human participants were reviewed Hilden, German) was used for amplifcation. A fnal vol- and approved by Zhejiang Provincial Center for Disease ume of 25 μL PCR mixture containing 2 μL of template, Control and Prevention of ethics committee. Children’s 1 μL of primer-mix (mixture was prepared according to parents/guardians have been notifed the purpose of the primer working concentration in Table 1), 1 μL of the study and the right to keep information confden- enzyme-mix, 1 μL of deoxinucleotidetriphospate (dNTP), tial. Children’s parents/guardians provided their written 5 μL of 5X bufer, 1 μL of Taq polymerase was subjected informed consent to participate in this study. to multiplex RT-PCR screening. Te amplifcation cycle was initial denaturation at 50 °C for 30 min, 95 °C for Sample collection 15 min, followed by 10 cycles of 95 °C for 30 s, 55 °C for A total of 3660 retrospective respiratory samples were 30 s, 72 °C for 30 s; 10 cycles of 95 °C for 30 s, 65 °C for collected from children (age, ≤ 18 years) with acute res- 30 s, 72 °C for 30 s; 25 cycles of 95 °C for 30 s, 48 °C for piratory tract infections who admitted to Children’s 30 s, 72 °C for 30 s and one fnal extension at 72 °C for Zhang et al. Virol J (2021) 18:89 Page 3 of 11 Table 1 Distribution of 16 respiratory virus in children according to gender and age Total, n (%) Gender,