C oronavirus, a genus of the Coronaviridae family, is an enveloped virus with a large plus- strand RNA genome 1. Human coronavirus (HCoV) 229E and OC43 are members of group I and II, respectively. HCoV infection is thought to be responsible for up to 30% of common cold cases in winter and occasionally causes acute lower respiratory tract disease in susceptible infants, elderly individuals, and immunocompromized adults 2-3. In 2003, the outbreak of severe acute respiratory syndrome (SARS) led to the preliminary identification of SARS- coronavirus, 4 which is currently considered to be a distinct member of the group 2 coronaviruses 5 or the first member of group 4 coronaviruses. In 2004, a new HCoV-NL63, was identified in clinical specimens from both infants and adults with acute respiratory tract infection (ARTI) in the Netherlands 1. Sequence analysis of the complete genome of HCoV-NL63 revealed that the virus was more closely related to HCoV- 229E than to the other human coronaviruses 1. Preliminary data suggested that HCoV-NL63 might be an important respiratory tract pathogen in children, similarly to respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) 1,6-9. The epidemilogy and clinical features of HCoV-NL63 infection are largely unknown. We prospectively detected the presence of HCoV-NL63 in children with acute lower respiratory tract infections (ALRTI) for two consecutive years. INDIAN PEDIATRICS825VOLUME 49_OCTOBER 16, 2012 Human Coronavirus NL63 in Hospitalized Children With Respiratory Infection: A 2-Year Study From Chongqing, China *CHEN XIN, *ZHANG ZHI YONG, LI YAN, *ZHAO XIAO DONG *Division of Nephrology and Immunology, Childrens Hospital of Chongqing Medical University, Chongqing, China; and National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada. Human coronavirus (HCoV) NL63, a newly discovered coronavirus, has been associated with acute lower respiratory tract infections (ALRTI). We detected HCoV-NL63 via reverse transcriptional PCR (RT-PCR) in eight out of 878 respiratory specimens freshly collected from hospitalized children with ALRTI between April 2006 and March 2008 in Childrens Hospital of Chongqing Medical University. Peak of HCoV-NL63 activity often appeared during the summer and autumn in Chongqing area. All children with HCoV-NL63 infection were 1 year of age. The diagnosis included bronchial pneumonia, bronchitis, interstitial pneumonia and bronchiolitis. All children recovered. Key words: Acute lower respiratory tract infections, Epidemiology, HCoV-NL63, Respiratory tract virus. Correspondence to: Dr Zhao Xiao Dong, Division of Nephrology and Immunology, Childrens Hospital of Chongqing Medical University, Chongqing 400014, China. . Received: January 13, 2012; Initial review: February 06, 2012; Accepted: April 10, 2012. METHODS All children hospitalized with a diagnosis of ALRTI, between April 2006 to March 2008 were enrolled. Nasopharyngeal aspirates (NPA) were collected following informed consent, on the day of admission according to a standard protocol and transported to the virology diagnostic laboratory within 4h. The supernatants and cells of NPA were separated by centrifugation 10 and the cell-free fluid was stored in aliquots at -80C until RNA extraction. Total RNA was extracted by using the QIAamp viral RNA mini kit (Qiagen, Germany) and complementary DNA (cDNA) was synthesized by using the PrimeScript RT Reagent Kit (TaKaRa, China). Primers used for HCoV-NL63 gene amplification have been previously described 1,11. The bulk PCR products were purified using a QIAquick Gel Extraction kit (Qiagen, Germany). Sequence analyses of the PCR products were performed on an ABI 3100 sequencer at core facility of nucleotide sequencing of Chongqing Medical University. The sequenced fragments of the HCoV-NL63 1a gene were assembled and analyzed with the SEQMAN, EDITSEQ, and MEGALIGN programs in the Lasergene program (DNASTAR, Madison, WI). Phylogenetic trees were generated by the neighbor-joining method using the MEGA4 program. Co-infection with other viruses. RSV and hMPV in R R R R R E E E E E S S S S S E E E E E A A A A A R R R R R C C C C C HHHHH B B B B B R R R R R I I I I I E E E E E F F F F F Published online: June 10, 2012. PII: S09747551200045 2 INDIAN PEDIATRICS826VOLUME 49_OCTOBER 16, 2012 XIN, et al.EPIDEMIOLOGY OF HCOV-NL63 INFECTION IN CHINA specimens were detected by traditional PCR and Real- time PCR, respectively. Positive samples for HCoV- NL63 were also subjected to direct immunofluorescence (DFA) (Diagnostic Hybrids, America) testing for common respiratory viruses, including influenza A and B viruses, para-influenza virus types 1-3, and adenovirus. Clinical evaluation and statistical analysis. The clinical diagnosis of ALRTI was based on the presence of cough, tachypnea, chest indrawing, or wheeze of 7 days duration, as per WHO standard protocol 12. The medical files of HCoV-NL63 infected children were writt