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Journal of Medical Virology 81 1597 1604 2009 Detection of Four Human Coronaviruses in Respiratory Infections in Children A One Year Study in Colorado Samuel R Dominguez 1 Christine C Robinson 2 and Kathryn V Holmes 3 1 Department of Pediatrics The Children s Hospital University of Colorado Denver School of Medicine Aurora Colorado 2 Department of Pathology and Laboratory Medicine The Children s Hospital University of Colorado Denver School of Medicine Aurora Colorado 3 Department of Microbiology University of Colorado Denver School of Medicine Aurora Colorado Lower respiratory tract infections are the leading cause of death in children worldwide Studies on the epidemiology and clinical associations of the four human non SARS human coronaviruses HCoVs using sensitive polymerase chain reac tion PCR assays are needed to evaluate the clinical significance of HCoV infections world wide Pediatric respiratory specimens 1 683 submitted to a diagnostic virology laboratory over a 1 year period December 2004 November 2005 that were negative for seven respiratory viruses by conventional methods were tested for RNA of four HCoVs using sensitive RT PCR assays Coronavirus RNAs were detected in 84 5 0 specimens HCoV NL63 in 37 specimens HCoV OC43 in 34 HCoV 229E in 11 and HCoV HKU1 in 2 The majority of HCoV infections occurred during winter months and over 62 were in previously healthy children Twenty six 41 coronavirus positive patients had evidence of a lower respiratory tract infection LRTI 17 26 presented with vomiting and or diarrhea and 5 8 presented with meningoencephalitis or seizures Respiratory specimens from one immunocompromised patient were persistently positive for HCoV 229E RNA for 3 months HCoV NL63 positive patients were nearly twice as likely to be hospitalized P 0 02 and to have a LRTI P 0 04 than HCoV OC43 positive patients HCoVs are associated with a small but significant number at least 2 4 of total samples submit ted of both upper and lower respiratory tract illnesses in children in Colorado Our data raise the possibility that HCoV may play a role in gastrointestinal and CNS disease Additional studies are needed to investigate the potential roles of HCoVs in these diseases J Med Virol 81 1597 1604 2009 C223 2009 Wiley Liss Inc KEY WORDS coronavirus rhinovirus respi ratory infection children INTRODUCTION Respiratory infections are the most common infec tious disease worldwide The World Health Organiza tion WHO continues to rank lower respiratory tract infections as the leading cause of burden of disease in the world WHO 2008 The burden of respiratory tract infections in children is particularly high LRTIs are the leading cause of death in children younger than 5 years of age worldwide and in all age groups in low income countries WHO 2003 2008 Klig 2004 Klig and Shah 2005 In the United States acute respiratory infections ARIs are a significant cause of morbidity and hospitalization in children Fendrick et al 2003 Griffin et al 2004 Therefore defining the etiologies of respiratory infections is a public health priority In our institution the etiology of up to 40 of ARIs is unknown The first two human coronaviruses HCoVs HCoV 229E and HCoV OC43 were isolated in human cell cultures or fetal tracheal organ cultures in the 1960s They were associated with up to 15 30 of common colds and rarely with LRTIs Holmes 2001 Heikkinen and Jarvinen 2003 Interest in HCoVs was stimulated in 2002 2003 when the epidemic of severe acute respiratory syndrome SARS was found to be caused by a newly emerged HCoV SARS CoV Drosten et al 2003 Ksiazek et al 2003 Rota et al 2003 After the SARS outbreak had ended and SARS CoV was no Additional Supporting Information may be found in the online version of this article Grant sponsor TCH Research Institute Pilot Grant Grant sponsor NIH to S R D Grant number K08 AI 073525 Grant sponsor NIH to K V H Grant number PO1 AI 059576 Correspondence to Samuel R Dominguez MD PhD Section of Infectious Diseases Department of Pediatrics The Children s Hospital B055 13123 E 16th Ave Aurora CO 80045 E mail samuel dominguez ucdenver edu Accepted 15 April 2009 DOI 10 1002 jmv 21541 Published online in Wiley InterScience C223 2009 WILEY LISS INC longer circulating in humans two additional HCoVs were discovered HCoV NL63 in 2004 and HCoV HKU1 in 2005 van der Hoek et al 2004 Woo et al 2005 Based on genome sequences HCoV 229E and HCoV NL63 are classified as group 1 CoVs and HCoV OC43 and HCoV HKU1 are classified as group 2a CoVs van der Hoek et al 2004 Woo et al 2005 RNAs of HCoV NL63 and HCoV HKU1 are found in respiratory speci mens throughout the world in association with both URTIs and LRTIs in children and adults Arden et al 2005 Bastien et al 2005 Chiu et al 2005 Ebihara et al 2005 Esper et al 2005 2006 Kaiser et al 2005 Moes et al 2005 Vabret et al 2005 2006 Sloots et al 2006 Gerna et al 2007 Kuypers et al 2007 Pyrc et al 2007 Ongoing investigations are uncovering other clinical associations for these HCoVs HCoV NL63 is associated with croup van der Hoek et al 2005 and HCoV HKU1 is associated with febrile seizures Lau et al 2006 HCoVs may also be implicated in enteric diseases Vabret et al 2006 To date most studies have not found enough HCoV positive samples to compare the clinical presentations of different HCoVs Lau et al 2006 Dare et al 2007 Gerna et al 2007 Vabret et al 2008 Furthermore the epidemiology and clinical associations of the four non SARS HCoVs in the Americas has been compared in only one cohort of patients Kuypers et al 2007 Therefore in order to compare the epidemiology and clinical associations of four HCoVs in children we used sensitive RT PCR assays to detect HCoV RNA in pediatric respiratory specimens submitted to the diag nostic virology laboratory at The Children s Hospital TCH Denver CO over a 1 year period 2004 2005 that were negative for other respiratory viruses PATIENTS MATERIALS AND METHODS As part of an ongoing investigation of viral respiratory infections at The Children s Hospital in Denver we archived at C0708C all 1 662 archived of 2 621 sub mitted nasopharyngeal washes NPWs submitted from December 2004 to November 2005 that tested negative for respiratory syncytial virus RSV influenza viruses A FLUAV and B FLUBV parainfluenza viruses 1 3 HPIV 1 2 3 and adenovirus HAdV by direct immunofluorescence DFA In addition during influenza season NPWs 225 archived of 891 submitted that were negative by a rapid immunoassay IA for FLUAV and FLUBV were also archived at C0708C Use of the banked specimens and clinical data was approved by the Colorado Multiple Institutional Review Board All samples were de identified and coded at the TCH Virology Laboratory and then transferred to the Holmes lab for blinded analysis of HCoV RNA RNA was extracted using Qiagen EZ1 Virus Mini Kits Valencia CA on a BioRobot EZ1 Extractor following the manufacturer s instructions All of the specimens were initially screened using a modified quantitative real time RT PCR qRT PCR assay that detects all four non SARS HCoVs Kuypers et al 2007 in which we added 10ml of RNA to 10ml of master mix containing an additional 1 3 mM MgCl Specimens positive in the consensus coronavirus qRT PCR assay were analyzed using virus specific conven tional RT PCR assays using Superscript III reverse transcriptase Invitrogen Carlsbad CA with random primers followed by PCR Primer sets from previously published studies or designed based on consensus sequences for each HCoV were used Supplemental Table I Primers bound to unique regions within the HCoV NL63 spike gene and nucleocapsid gene Bastien et al 2005 Moes et al 2005 HCoV HKU1 lb gene Woo et al 2005 HCoV OC43 nucleocapsid gene and HCoV 229E nucleocapsid gene All positive specimens were further analyzed using a pancoronavirus nested RT PCR assay de Souza Luna et al 2007 A specimen was considered to be HCoV positive if it were positive in at least two different PCR assays that amplified different parts of the viral genome and the sequences of these amplicons confirmed that the bands were from HCoVs Because the initial DFA screens in the diagnostic laboratory did not test for human metapneumovirus HMPV human bocavirus HBoV or human rhino viruses HRVs we also screened all HCoV positive samples by RT PCR assays specific for these viruses Coiras et al 2004 Maertzdorf et al 2004 Lu et al 2006 The RT PCR assay used for detection of HRVs detected HRV groups A B and the newly discovered HRV clade C Dominguez et al 2008 Because samples submitted for IA were only tested for influenza viruses HCoV positive IA samples were also tested by multiplex RT PCR for RSV HPIV 1 3 and HAdV Heim et al 2003 Bellau Pujol et al 2005 to detect possible co infection of these viruses with HCoVs A table of all the primers used in this study is provided in Supplemental Table I After all specimens had been analyzed and confirmed in duplicate assays HCoV positive specimens were decoded and medical chart review performed using a standardized form Lower respiratory tract illness LRTI was defined as having one or more of the following requirement for supplemental oxygen or mechanical ventilation or a chest radiograph showing infiltrates Statistical computations were conducted with SAS software version 9 1 3 Significance was determined using the Wilcoxon Signed Rank test or the Fisher s Exact Test RESULTS Virus Detection Sufficient sample was available for analysis from 1 683 89 of the 1 887 archived pediatric respiratory specimens that had previous been shown to be negative for other viruses by either DFA or IA We detected HCoV RNA using the consensus qRT PCR assay in 84 5 0 of these 1 683 specimens In the 3 512 consecutive NPWs from children with respiratory symptoms submitted to the viral diagnostic laboratory during the same time period the prevalence of RSV was 14 5 parainfluenza J Med Virol DOI 10 1002 jmv 1598 Dominguez et al viruses 9 0 influenza virus type A 7 7 adenovi ruses 4 2 and influenza virus type B 2 8 The majority of the 84 HCoV positive infections occurred during the winter months with the maximum number 26 30 in February 2005 Fig 1 Of the 84 specimens positive for HCoV RNA 37 44 were HCoV NL63 34 40 were HCoV OC43 11 13 were HCoV 229E and 2 2 were HCoV HKU1 One specimen was positive for both HCoV NL63 and HCoV 229E Eight of the 84 HCoV positive specimens were collected at different times from three patients four specimens from one patient and two specimens each from two other patients PCR tests for other respiratory viruses showed that 5 of the 84 HCoV positive speci mens were also positive for HMPV RNA but none were positive for HBoV Of the HCoV positive samples that had only been tested by influenza IAs one was also positive for RSV RNA one for HPIV 1 RNA and for HAdV DNA We excluded from analysis all of the HCoV positive specimens found to be co infected with RSV HPIV 1 3 HAdV HMPV or another HCoVs and all but the first specimens from each of the three patients with multiple specimens Tables I IV Clinical data were available from 64 90 of the 71 remaining patients including 17 27 who were also positive for HRV RNA Because of the large percentage of specimens positive for both an HRV and an HCoV and to determine if co infection with HRVs affected the clinical presen tation of HCoV infections HCoV positive patients co infected with an HRV were included in the clinical analysis Clinical Epidemiology The demographic and clinical characteristics of the HCoV infected patients are shown in Table I Most of the patients were young median age 15 months previously healthy 62 children The most common clinical finding in the HCoV positive patients was fever 61 The majority of patients presented with upper respira tory tract symptoms of cough 53 rhinorrhea 47 and congestion 45 Twenty four 38 patients were hypoxic requiring admission for oxygen support and 26 41 patients had evidence of a LRTI Seventeen 27 HCoV positive patients had accompanying gastrointes tinal symptoms Table I and 5 8 presented with meningoencephalitis or seizures Table II Of the patients with CNS disease we were able to locate frozen CSF on two of these patients The CSF was negative for coronavirus RNA by RT PCR on both these samples The diagnoses given HCoV positive patients by their treat ing physicians are shown in Table II Although there J Med Virol DOI 10 1002 jmv Fig 1 Number of coronavirus positive respiratory specimens n 84 detected each month during a 1 year period TABLE I Demographic and Clinical Characteristics of Patients With a Human Coronavirus Positive Respiratory Specimen Total N 64 Median age in days range 462 9 6 856 Male 39 60 9 Hospitalized 40 62 5 Admitted to PICU 14 21 9 Median length of stay range 4 0 47 Antibiotics 30 46 9 Albuterol 9 14 1 Steroids 8 12 5 Positive chest X ray 11 17 2 Fever 39 60 9 Hypoxia 24 37 5 Wheezing 4 6 3 Crackles 6 9 4 Retractions 9 14 1 Cough 34 53 1 Rhinorrhea 30 46 9 Congestion 29 45 3 Conjunctivitis 4 6 3 Pharyngitis 4 6 3 Rash 6 9 4 Vomiting 17 26 6 Diarrhea 7 10 9 Apnea 4 6 3 Seizures 5 7 8 Underlying condition a 24 37 5 Immunocompromised 8 12 5 LRTI 26 40 6 PICU pediatric intensive care unit LRTI lower respiratory tract infection a Underlying disease conditions were defined as significant pulmonary cardiac genetic central nervous system renal and or hepatic conditions malignancy or prematurity defined as gestational age 37 weeks TABLE II Most Common Clinical Diagnoses of Patients With Human Coronavirus Positive Respiratory Specimens Diagnosis Number n 64 Viral syndrome or URI 13 20 0 Pneumonia 12 18 5 ALTE or apnea 6 9 2 Croup 4 6 2 Fever and neutropenia 4 6 2 Meningoencephalitis 3 4 6 Fever in young infant 3 4 6 Bronchiolitis 3 4 6 Asthma exacerbation 2 3 1 Seizures 2 3 1 URI upper respiratory tract infection ALTE acute life threatening event Human Coronavirus Infections in Children 1599 J Med Virol DOI 10 1002 jmv TABLE III Comparison of Clinical and Demographic Characteristics of Patients with Respiratory Specimens Positive for a Single Coronavirus and No Other Detectable Viruses Versus Those Positive for Both a Coronavirus and a Rhinovirus Coronavirus only N 47 Coronavirus HRV N 17 P value a Median age in days range 550 9 6856 438 22 5 250 0 44 b Male 31 66 0 8 47 1 0 14 Hospitalized 29 61 7 11 64 7 0 22 Admitted to PICU 12 25 5 2 11 8 0 15 Median length of stay range 4 0 47 4 2 16 0 29 b Antibiotics 22 46 8 8 47 1 0 22 Albuterol 5 10 6 4 23 5 0 13 Steroids 5 10 6 3 17 6 0 24 Positive chest X ray 8 17 0 3 17 6 0 29 Fever 30 63 8 9 52 9 0 17 Hypoxia 17 36 2 7 41 2 0 21 Wheezing 2 4 3 2 11 8 0 23 Crackles 4 8 5 2 11 8 0 32 Retractions 6 12 8 3 17 6 0 27 Cough 23 48 9 11 64 7 0 13 Rhinorrhea 20 42 6 10 58 8 0 13 Congestion 19 40 4 10 58 8 0 11 Conjunctivitis 3 6 4 1 5 9 0 43 Pharyngitis 2 4 3 2 11 8 0 24 Rash 6 12 8 0 0 0 0 14 Vomiting 10 21 3 7 41 2 0 10 Diarrhea 3 6 4 4 23 5 0 08 Underlying condition 19 40 4 5 29 4 0 31 Immunocompromised 7 14 9 1 5 9 0 24 LRTI 19 40 4 7 41 2 0 59 HRV rhinovirus PICU pediatric intensive care unit LRTI lower respiratory tract infection a Fisher s exact T test unless otherwise noted b Wilcoxon signed rank test TABLE IV Comparison of Patients With Respiratory Specimens Positive for HCoV NL63 Versus HCoV OC43 HCoV NL63 N 26 HCoV OC43 N 29 P value a Median age in days range 285 21 6631 455 9 6856 0 20 b Male 17 65 4 17 58 6 0 41 Hospitalized 19 73 1 13 44 8 0 02 Admitted to PICU 3 11 5 8 27 6 0 09 Median length of stay range 4 0 21 3 5 2 23 0 40 b Antibiotics 15 57 7 10 34 5 0 05 Albuterol 4 15 4 4 13 8 0 29 Steroids 2 7 7 5 17 2 0 19 Positive chest X ray 6 23 1 3 10 3 0 13 Fever 15 57 7 19 65 5 0 18 Hypoxia 12 46 2 8 27 6 0 08 Wheezing 1 3 8 2 6 9 0 40 Crackles 2 7 7 3 10 3 0 34 Retractions 4 15 4 5 17 2 0 28 Cough 11 42 3 20 69 0 0 02 Rhinorrhea 9 34 6 19 65 5 0 01 Congestion 10 38 5 18 62 1 0 04 Conjunctivitis 3 11 5 1 3 4 0 23 Pharyngitis 2 7 7 2 6 9 0 39 Rash 4 15 4 1 3 4 0 13 Vomiting 8 30 8 7 24 1 0 47 Diarrhea 3 11 5 3 10 3 0 61 Underlying condition 10 38 5 7 24 1 0 20 Immunocompromised 7 26 9 1 3 4 0 02 LRTI 14 53 8 8 27 6 0 04 HRV positive 6 23 1 6 20 7 0 54 Bold font indicates a statistically significant association PICU pediatric intensive care unit HRV human rhinovirus LRTI lower respiratory tract infection a Fisher s exact T test unless otherwise noted b Wilcoxon signed rank test 1600 Dominguez et al were no statistically significant clinical differences between patients infected with a single HCoV and those co infected with a HRV none of the co infected patients presented with rash Table III The majority of the HCoV positive specimens were positive for either the group 1 virus HCoV NL63 26 41 or the group 2 virus HCoV OC43 29 45 which permitted comparison between the clinical pre sentations associated with these two viruses Table IV HCoV NL63 positive patients were almost twice as likely as HCoV OC43 positive patients to be hospital ized P 0 02 have evidence of a LRTI P 0 04 or receive antibiotics P 0 05 and nine times more likely to be immunocompromised P 0 02 Table IV HCoV OC43 positive patients were more likely to have URTI symptoms of cough P 0 02 rhinorrhea P 0 01 and congestion P 0 04 Table IV Except for the clinical symptom of congestion these findings remained stat istically significant even when the 17 specimens co infected with HRV were excluded from the analysis data not shown Because of the small number of cases in our data set we did not have enough power to conduct a highly informative multivariate analysis Neverthe less a binary logistic regression analysis showed that rhinorrhea URTI symptoms was independently corre lated with a higher likelihood of HCoV OC43 infection data not shown Because more HCoV NL63 patients than HCoV OC43 patients were immunocompromised P 0 02 we per formed a separate analysis on patients who were not immunocompromised to see if this could account for the differences in clinical presentations Limiting our analysis to immunocompetent children did not affect the results of our analysis except that the use of antibiotics became nonsignificant data not shown Three immunocompromised patients in our study had multiple serial respiratory samples positive for the same HCoV The first was a 4 month old male with hepatoblastoma who presented in January 2005 with a 1 day history of fever fussiness rhinorrhea and congestion He was admitted for 2 days with a diagnosis of fever and neutropenia He was readmitted 22 days later for 4 days with a second episode of fever and neutropenia NPWs collected on both admissions were positive for HCoV NL63 RNA The second was a 9 year old female with severe aplastic anemia who had received a bone marrow transplant in September 2004 In January 2005 due to persistent respiratory symptoms she had two NPW collected 12 days apart both of which were positive for HCoV NL63 RNA The third patient was a 4 year old male with a rhabdoid brain tumor who underwent an autologous bone marrow transplant in March 2005 He was admitted to the hospital for 16 days in April 2005 due to severe mucositis fever diarrhea and increased oxygen requirement He was readmitted to the hospital in mid May for 43 days On day 6 of that admission he developed fevers and increasing respira tory compromise and a chest CT revealed bilateral pulmonary infiltrates He was transferred to the PICU for a diagnostic bronchoalveolar lavage BAL and remained intubated for 13 days Four respiratory speci mens collected from late April to early June a total of 37 days were persistently culture positive for HRV He was also persistently positive for HCoV 229E RNA with four positive respiratory specimens three NPWs and one BAL over a 77 day period DISCUSSION In this study coronavirus RNA was detected in 5 of 1 683 specimens from children with acute respira tory illnesses submitted to our diagnostic virology laboratory in 2004 2005 that were negative for other respiratory pathogens by conventional clinical labora tory methods This is