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adenoviruses , coronaviruses , human enteroviruses ( hev ) , human rhinoviruses ( hrv ) ,   influenza viruses , parainfluenza viruses ( piv ) , and respiratory syncytial viruses ( rsv ) are well - known causes of acute respiratory tract infections ( arti ) in both industrialized and developing countries .   over the last decade , modern molecular techniques have led to the discovery of several previously unknown respiratory tract viruses , including human metapneumovirus ( hmpv ) , two new human coronavirus types [ 2 , 3 ] , human bocavirus ( hbov ) , and two new human polyomaviruses [ 5 , 6 ] .
the significance of these novel viruses has been reviewed recently [ 7 , 8 ] .
it is widely accepted that common cold is almost always caused by viruses , most frequently by hrv , and viral infections are considered to contribute to the generation of complications of common cold , such as acute otitis media and sinusitis . moreover , different viruses , including influenza viruses and rsv , are also frequently detected in samples obtained from patients with lower respiratory tract infection ( lrti ) , either alone or together with pathogenic bacteria .  
several recent reports , including some from africa , suggest viruses as potential etiologic agents in pneumonia in children [ 1013 ] , or exacerbations of asthma [ 1416 ] .  
several studies underscore the importance of respiratory tract viruses in nigerian patients , but these studies were carried out before the introduction of modern molecular diagnostic techniques [ 14 , 1719 ] .   the present study was designed to identify viral agents associated with respiratory infections among young children in nigeria using modern , validated molecular techniques .
we wanted to explore the presence of different virus groups , including some of the newer ones detected by only molecular techniques .
the study was approved by the ethical committee of the oyo state ministry of health .  
participation of children in the study was voluntary and required informed consent from the parents .
inclusion criteria were recent onset of symptoms suggestive for respiratory tract infection , such as cough , coryza , repeated sneezing , and/or difficulty in breathing .
patients were recruited between february and may , 2009 , and included hospitalized patients , children seen at emergency departments , and outpatient clinics at 3 different children 's hospitals in ibadan , oyo state .
demographic and clinical information , including age , sex and clinical symptoms , was recorded during the medical visit by means of a structured questionnaire .  
a nasal swab sample was obtained from children by inserting a sterile nylon swab ( regular flocked swab , cat .
murrieta , calif usa ) into the nostril to a depth of 24  cm , and retracting it with a rotating motion , in order to trap epithelial cells in the swab . with a second swab ,
a throat specimen was collected by rubbing the tonsils and the posterior wall of the pharynx .  
the 2 swabs were then placed in a vial containing 2  ml of rnalater solution ( rnalater tissue collection , applied biosystems , espoo , finland ) .
the specimens were transported to the laboratory on the same day in an ice pack and stored at 70c until further processing .
viral nucleic acids were extracted from 200  l of sample using a commercial kit ( rneasy mini kit , qiagen , hilden , germany ) , and viral rna was reverse transcribed into cdna with random hexamer primers ( roche , mannheim , germany ) and revertaid reverse transcriptase enzyme ( fermentas , st .
for the detection of influenza a and b viruses , rsv , piv 1 , 2 , and 3 , and cdna were amplified in two separate real - time multiplex pcrs   with minor modifications .
other , slightly modified real - time pcrs were used for the detection of influenza a subtypes h1 and h3 ( r. fouchier , rotterdam , the netherlands , personal communication ) , influenza c ( l. p. nielsen , copenhagen , denmark , personal communication ) , bocavirus , metapneumovirus , and adenovirus .
real - time rt - pcr for the detection of hrv was performed as described   and of enteroviruses ( hev ) according to the method by centers for disease control and prevention , atlanta , usa ( w.a . nix and d.r .
we know that the hev test is not 100% specific but also reacts with some hrv strains ( savolainen - kopra et al .
therefore , specimens yielding a positive result in the hev test were divided into two groups , and a designated  true hev 
result was scored only for those hev - positive specimens that were negative in the hrv test .
more than half of the patients ( 132/246 , 53.6% ) were less than one year of age , and almost one half of the remaining children , whose age was recorded ( 40/83 , 48.1% ) , were under two years of age ( table 1 ) .
the overall rate of virus positivity was similar in children less than 1 year of age ( 102/132 , 77.3% ) , between 1 and 2 years ( 32/40 , 80.0% ) , and above 2 years of age ( 33/43 , 76.7% ) and slightly lower among the 13% of children whose age was not recorded ( 22/31 , 71.0% ) .
the most frequently detected virus groups , both found in about one third of the children each , were hrv and piv , with type 3 of piv being the most prevalent serotype in the latter group ( table 1 ) . adenoviruses , influenza virus c , hmpv , and hbov were also found in considerable numbers .
seven specimens tested positive for hev and negative for hrv (  true hev  ) , whereas 16 other additional specimens yielded a positive result by the hev and the hrv test . while the overall proportion of virus - positive specimens was similar in children aged under or over two years , as well as in the group with unrecorded age , all adenovirus , influenza virus a , rsv , and all but one hbov and  true hev  detections were in the youngest age group ( table 1 )
. altogether 224 virus findings were obtained from the 246 children , if only those hev - positive results were accounted where the same specimen was negative in the hrv test (  true hev  ) .
the number of findings was 240 if all positive test results for hev were included .
twenty - nine specimens contained two different viruses and another two specimens three different viruses .  
no obvious pattern could be seen in the mutual associations of two or three viruses ( table 2 ) .  
however , there were more multiple infections in children older than 2 years than in the younger than 2 years group ( 33% versus 9% of children , p = 0.0001 ( 2-sample t - test for equality of proportions ) , data not shown .
this study revealed that all major respiratory virus groups tested for can be detected in nigerian children with respiratory tract infection .
all viruses investigated , including the more recently discovered hmpv and hbov , were present in at least some of the specimens .
this wide variety is somewhat surprising because the specimens have been collected only during a four - month period , mostly in the dry season of the year .
it is known from the previous studies that circulation of some viruses is at a low level during the dry season [ 2527 ] .
seventy - seven percent of the children harboured at least one viral pathogen in their samples .  
this figure is relatively high taking in account that we did not have the possibility to test the specimens for human coronaviruses , and comparable to   or higher than in some recent similar studies performed elsewhere [ 2931 ] .
simultaneous infections with two or even three viruses were also found , similar to observations by others in comparable studies [ 9 , 25 ] .   in individual cases ,
a demonstration of viral nucleic acids in a nasal or throat swab does not necessarily prove an etiological role of this pathogen in the concurrent disease as positive test results have also been obtained from a proportion of healthy individuals .
etiological diagnosis of lrti is especially difficult without invasive procedures , but when searched for , for example , rhinovirus has been found in alveolar lavage specimens from pneumonia patients   establishing the role of hrv as a significant respiratory pathogen beyond being the major causative agent of common cold .
previous studies on respiratory tract viruses in nigeria have not tested for the presence of rhinoviruses .   in our study , hrv accounted for 39% of the virus findings in the study population reiterating the observations done elsewhere [ 9 , 29 , 30 , 34 ] .
our test for the related hev revealed a positive result in about 10% of the specimens , but two thirds of these specimens also tested positive for hrv .   because of the well - established cross - reactions , these double - reactive specimens were recorded as hrv , and only those 7 hev - positive specimens that were hrv negative were scored as  true hev positives .
 however , we can not exclude concomitant infection with both hev and hrv in these double - reactive cases .
a high incidence of parainfluenza viruses was found in our study population with piv3 being the most frequent of the three serotypes tested for .  
interestingly , piv3 has also been one major agent in some previous studies done in nigeria [ 14 , 17 ] and one in cambodia .
respiratory syncytial virus was a rare finding in our study , likely due to the seasonal occurrence of this virus .
our specimens were collected in february - may which has been a low season for rsv in nigeria in previous studies [ 26 , 27 ] .
little was known about the epidemiology of influenza viruses in west africa until very recently . a report from senegal   published , while this manuscript was in preparation ,   showed a september - october peak of influenza a and rsv .
hence , seasonality might have again been contributing to the paucity of influenza virus a and b findings among our patients . the two influenza a viruses further characterized were of the h3n2 subtype , that is , the same subtype that was predominant during the 2008 - 2009 influenza season ( http://apps.who.int/globalatlas/dataquery/default.asp ) .
in contrast , influenza virus type c was found surprisingly often . with an incidence of almost 5% ,
double or triple infections were detected in 16% of the virus - positive children , which is comparable to that observed by others [ 31 , 34 ] .  
we do not have any explanation for the observed relatively higher frequency in the older children .  
one could speculate about increasing number of contacts by age in the community , but we did not collect information enabling further analysis of this possibility . in conclusion
, the findings of this study emphasize the presence of all major groups of viruses in association with respiratory illnesses in young children of west africa .
rhinoviruses and parainfluenza viruses were the most prevalent virus groups while influenza a and b viruses , as well rsv were rarely detected , possibly due to low season of these viruses during the time of sample collection .
occurrence of different viruses in acute respiratory tract infections of nigerian children was examined . 
 respiratory swabs were collected from 246 children referred to hospital clinics because of acute respiratory symptoms from february through may 2009 . validated real - time rt - pcr techniques revealed nucleic acids of at least one virus group in 189 specimens ( 77% ) . 
 human rhinoviruses and parainfluenza viruses were present each in one third of the children . 
 adenoviruses , enteroviruses , human metapneumovirus , human bocavirus , and influenza c virus were also relatively common . possibly due to their seasonal occurrence , 
 influenza a and b virus , and respiratory syncytial virus were detected rarely . 
 we conclude that all major groups of respiratory tract viruses are causing illness in nigerian children .