a number of respiratory viruses including influenza viruses , respiratory syncytial virus ( rsv ) , parainfluenza viruses , adenovirus and the recently described human metapneumovirus ( hmpv ) play an important role in acute respiratory tract infections ( ari ) in children .
however , in a substantial portion of respiratory infections the aetiological agent is not known .
there has been renewed interest in human coronaviruses ( hcov ) as a cause of some of these infections .
coronaviruses are large enveloped singlestranded rna viruses that can infect both humans and a variety of domestic animals causing respiratory and enteric illness . until recently human coronavirus ( hcov )
229e and oc43 , identified in the 1960s , 
  were the only known coronaviruses to infect humans .
although primarily responsible for mild infections including the common cold 
  reports of more severe upper and lower respiratory tract infections associated with hcov229e and hcovoc43 have been documented . 
 , 
  the identification of a coronavirus , sarscov , as the causative agent of severe acute respiratory syndrome in 2003 
  has resulted in an increased interest in this group of viruses .
subsequently two new human coronaviruses , hcovnl63 
 , 
  and hcovhku1 , 
  have been described .
both infect young children , the elderly and immunocompromised and can lead to severe respiratory tract infections requiring hospitalisation .
the prevalence and clinical importance of hcovnl63 in the south african hospital setting is not known .
in this retrospective study 1055 nasopharyngeal , tracheal aspirate and bronchoalveoloar lavage samples were taken from children ( age 13  days to 5  years ) hospitalised with respiratory tract infections in 2003 and 2004 in the red cross war memorial children s hospital in cape town .
samples had previously been screened by using an indirect immunofluorecence assay ( light diagnostics , chemicon international , temecula , ca , usa ) for the common respiratory viruses including rsv , influenza viruses a and b , parainfluenza viruses 1 , 2 , 3 , adenovirus and cmv .
hmpv was also tested for using reversetranscriptionpcr ( rtpcr ) . in the south african setting , where the prevalence of hiv is high , all infant respiratory samples
are routinely screened for cmv as in our setting this virus is a major cause of pneumonia in hivinfected children . for the detection of hcovnl63
, rna was extracted from 200  l of sample using the seek viral rna kit according to the manufacturer s instructions ( talent , trieste , italy ) .
ten microlitres rna was reverse transcribed into cdna using random primers ( roche diagnostics gmbh , penzberg , germany ) and the iscript kit ( biorad , hercules , ca , usa ) .
pcr amplification of a region of the 1b gene of hcovnl63 was used for screening and positive samples were confirmed by amplification of a portion of the 1a gene . briefly
10  l of cdna was added to a 50  l of pcr mix containing 2  iu supertherm polymerase ( jmr holdings , kent , uk ) , 15  mm mgcl2 , 200  m each dntp and 02  m primers .
pcr was performed with two sets of outer primers , one set to the 1b gene and one targeting the 1a gene from the study of van der hoek et  al . 

  to improve sensitivity a nested pcr
was performed using 25  l outer product and inner primers described by smuts et  al . 

 
the 1a amplicons were sequenced directly and the nucleotide sequences were deposited into genbank ( eu477762eu477770 ) .
a respiratory virus was detected in 401/1055 ( 380% ) samples collected over the 2year period from 2003 to 2004 .
the detection rate was higher in 2004 , 248/559 ( 444% ) , compared with 2003 , 153/496 ( 308% ) .
cmv was most frequently found ( 158/1055 ; 150% ) followed by adenovirus ( n  =  65 ; 62% ) , rsv ( 42 ; 40% ) , parainfluenza 3 ( n  =  32 ; 30% ) , hmpv ( n  =  28 ; 26% ) , influenza virus a ( n  =  8 ; 076% ) , parainfluenza virus 1 ( n  =  6 ; 057% ) , parainfluenza virus 2 ( n  =  4 ; 038% ) and influenza virus b ( n  =  1 ; 009% ) . in 48 ( 45% )
samples a known respiratory virus was grown in shell vial culture but could not be further identified .
of cmvpositive respiratory samples 449% ( 71/158 ) were from hivinfected children , 240% ( 38/158 ) from hivnegative children and for the remainder the hiv status was unknown . in both hivpositive and hivnegative groups
the rates of coinfection with another known respiratory virus were similar , 12/71 ( 169% ) and 7/38 ( 184% ) respectively .
hcovnl63 was detected in 4/496 ( 081% ) and 5/559 ( 089% ) samples from 2003 and 2004 respectively .
all hcovnl63infected children , with the exception of one aged 30  months , were under 2  years ( table  1 ) .
the hiv status of only those children from 2004 was known and 4/5 hcovnl63infected children from this year were hivpositive . in two instances
in 2003 3/4 positive samples were collected in march ( autumn ) while in 2004 hcovnl63positive samples were found in march , may , august and september ( table  1 ) .
a diagnosis of pneumonia or lower respiratory tract infection was made in six ( 67% ) children .
two hcovnl63positive infants aged 50 and 71  days respectively , required admission to the intensive care unit ; both were hivpositive .  clinical data of hcov nl63infected children nk , not known ; ta , tracheal aspirate ; npa , nasopharyngeal aspirate ; bal , bronchoalveolar lavage ; ltri , lower respiratory tract infection ; hmpv , human metapneumovirus .
to determine the role hcovnl63 plays in respiratory illness in infants , respiratory samples that had previously been screened for rsv , influenza viruses a and b , parainfluenza viruses 1 , 2 , 3 , adenovirus , hmpv and cmv were also tested by rtpcr for hcovnl63 . due to limited resources ,
screening for other viruses such as enteroviruses , rhinoviruses and the other hcovs was not undertaken .
this may be considered a limitation of the study as there is accumulating evidence that these viruses , in particular rhinoviruses , may play a more significant role in lower respiratory tract infections than previously recognised . 
 
the role and clinical significance of the recently identified human bocavirus 
  and polyomaviruses 
 , 
  in respiratory disease is still under investigation .
ideally comprehensive screening of respiratory samples for most if not all respiratory viruses and relevant respiratory bacteria should be undertaken in order to obtain greater insight into the epidemiological significance of these pathogens in respiratory disease in the local setting .
further this would be beneficial to the clinical management of patients , including the administration of appropriate antiviral drugs and antibiotics .
a postmortem study of hivinfected children in kwazulunatal , south africa showed frequent ( 52% ) cmv detection in lung tissue compared with uninfected controls ( 4% ) . 
 
the significance of cmv detection in respiratory samples from uninfected children is not known but probably represents viral shedding from either a recently acquired primary infection or reactivation .
this study is the first to report the presence of hcovnl63 in children hospitalised with respiratory illness in africa .
hcovnl63 was found to circulate in infants and young children in both 2003 and 2004 with similar low prevalence rates of 08% .
this finding is lower than that reported in previous studies where detection of hcovnl63 ranged from 1% to 73% 
 , 
 , 
 , 
 , 
 , 
 , 
 , 
  although higher prevalences of 88% and 93% have also been reported . 

, 
  most hcovnl63positive children ( 75% ) were under 6  months , indicating that this younger age group may be more susceptible to severe infections requiring hospitalisation ; a finding supported by other studies where 38/76 ( 50% ) , 4/5 ( 80% ) and 11/12 ( 92% ) of hcovnl63 infections occurred in this age group respectively . 

, 
 , 
  further , immunosuppression may also contribute to increased susceptibility to severe hcovnl63 infection . in this study 4/5
all four were < 4  months of age and two required admission to icu .
it is not known whether maternal antibodies , if present , would provide protection or reduce the severity of infection .
this protection is likely to be most effective in infants under 2  months . in this study
this observation is supported by the findings of other studies . 
 , 
 , 
 , 
  in a very recent study by dijkman et al .
these antibodies disappeared within 3 months providing further evidence of their possible protective role in early life . in a recently published study 
  of ambulatory children presenting with acute wheezing at the outpatient s department of the same hospital in 2004 ,
this is significantly different ( p  =  00186 ) from the prevalence in the hospitalised group .
this indicates that the virus is circulating in the community , probably causing mild symptoms which may trigger a wheezing episode requiring medical attention .
in contrast the lower rate of hcovnl63 infection identified in the hospitalised children suggests that the virus rarely causes severe lower respiratory tract infections .
however , all hcovinfected children in the ambulatory group were over 12  months of age supporting the possibility that younger children are more susceptible to severe infections requiring hospitalisation .
no hcovnl63 infection was detected during the winter months in either 2003 and 2004 , a finding also noted in the ambulatory population 
  this pattern differs from that previously reported where hcovnl63 infection was predominantly found during the winter season . 
 , 
 , 
 , 
 , 
 , 
 , 
 , 
  detection during early spring and summer indicates that hcovnl63 may circulate at low levels throughout the year . in this
study the clinical symptoms of hcovnl63infected infants were similar to those previously reported with lower respiratory tract infections including pneumonia predominating in this group . 

, 
 , 
 , 
 , 
 , 
  in this study only specimens from hospitalised children were examined resulting in a bias towards children with more severe respiratory illness .
mild hcovnl63 symptoms have also been reported 
 , 
 , 
  indicating that hcovnl63 infections are probably more severe in the very young and immunocompromised .
the role of hcovnl63 in enteric disease has not been established but reports of gastroenteritis associated with coronavirus infection have been documented with frequencies ranging from 6% to 33% . 
 , 
 , 
 , 
  in this study one child had a history of gastroenteritis and vomiting . a significant association with hcovnl63
infection and croup has been made 
  but in this study it could not be determined if any of the samples were from children with croup . in conclusion
these findings suggest that although hcovnl63 is circulating in the community it plays a minor role in severe respiratory tract infections in young children who require hospitalisation .
ethical approval ( 018/2004 ) was granted by the research ethics committee of the faculty of health sciences , university of cape town , south africa .
 
 background   human coronavirus nl63 ( hcovnl63 ) is a novel respiratory virus which is associated with respiratory tract infections in children . 
 
 objective   to determine the role of hcovnl63 in infants and young children hospitalised with acute respiratory tract infections ( ari ) in cape town , south africa . 
 
 methods   respiratory specimens were collected from 1055 infants and young children hospitalised with ari in 20032004 . 
 samples were screened by rtpcr to detect hcovnl63 and human metapneumovirus ( hmpv ) . 
 standard shell vial culture and immunofluoresence was used to detect the common respiratory viruses including rsv , influenza a and b viruses , parainfluenza viruses 1 , 2 , 3 , adenovirus and cmv . 
 
 results   a respiratory virus was found in 401/1055 ( 380% ) samples . 
 hcovnl63 was detected in 9/1055 ( 085% ) with peak activity during autumn ( 67% ) . 
 most patients had a diagnosis of pneumonia or lower respiratory tract infection ( 6/9 ; 67% ) . 
 
 conclusions   this is the first report of hcovnl63 infections in hospitalised children in africa . during the 2year period hcovnl63 played a minor role in ari in children . 
Introduction
Methods
Results
Discussion
Ethical approval