though the pathogens causing aris vary geographically and by season , globally viruses play a major role [ 13 ] . in a recent systematic review , the most common respiratory viruses causing acute lower respiratory tract infection ( lri ) in children under five years of age were respiratory syncytial virus ( rsv ) , influenza virus ( ifv ) , parainfluenza virus ( piv ) human metapneumovirus ( hmpv ) , and rhinovirus ( rv ) . besides this , 10%50% of children affected with ari develop secondary bacterial infections , namely , acute otitis media , sinusitis , or pneumonia . moreover , viruses are the most common pathogen associated with severe respiratory diseases ( e.g. , bronchiolitis ) , exacerbation of asthma , or pneumonia in early life and are leading cause of hospitalization in children under two [ 57 ] . although viral etiology of aris and their impact on health care are much studied in developed countries , there is a gap in knowledge regarding the same in developing countries including india . from the public health point of view , it is important to know the most common viral agents causing aris , their manifestations , how often they cause severe disease , and how severe aris can be prevented . in this study , we aimed to characterize the viral spectrum and pattern of upper and lower aris in children under five from eastern part of india . the present study was conducted in the pediatrics department of svppgip , scb medical college , a tertiary care teaching hospital in eastern india over 2-year period ( october 2011 to september 2013 ) . children aged 2 to 60 months with symptoms and signs of acute respiratory tract infections suggested by who were included . who classification of acute respiratory tract infection in children presenting with cough , difficult breathing , or both is as follows : pneumonia  respiratory rate per minute > 50 breaths ( 211 months of age ) or > 40 breaths ( 1259 months of age ) ; no lower chest indrawing ; severe pneumonia  symptoms of pneumonia , and lower chest indrawing with or without rapid breathing ; very severe disease  symptoms of severe pneumonia , inability to drink , convulsions , central cyanosis , being abnormally sleepy or difficulty to wake , stridor in calm child , or clinically severe malnutrition . those with suspected bacterial etiology ( e.g. , streptococcal sore throat , lobar pneumonia , pneumatocele , and empyema ) , underlying chronic conditions , hiv , illness lasting more than a week , severe malnutrition , and those hospitalized in last one month were excluded . nasal and throat swabs were collected and pooled into tubes containing 2  ml of virus transport medium ( vtm , copan , brescia , italy ) and kept in refrigerator at 2 to 8c . then the samples were transported over ice to the laboratory ( regional medical research centre , icmr ) situated at bhubaneswar , a grade 1 equipped virological laboratory . total nucleic acids ( including dna and rna ) were extracted from 200  l of each specimen using a qiaamp minelute virus spin kit ( qiagen , mississauga , on , canada ) according to the manufacturer 's instructions . for all collected specimens , pcr or rt - pcrs were performed to detect infection with rsv , ifv , piv , hmpv , adenovirus ( adv ) , human bocavirus ( hbov ) , human corona virus ( hcov ) , enterovirus ( ev ) , and rv . the method used for each virus is described in the following references : pivs 14 , ev , rv , ifv ( a , b , and c ) , and rsv ( a and b )   were detected by two multiplex nested rt - pcrs ; hcov   and hmpv   by two - step rt - pcr ; adv   by single - step pcr ; and hbov by touchdown pcr . rt - pcr was performed using a superscript ii one - step rt - pcr platinum taq kit ( invitrogen , carlsbad , ca , usa ) . typing for ifv , piv , and rsv was performed according to pcr product size . a total of 358 children of 260  mo age and acute respiratory infections were screened and finally 300 patients were included after meeting the eligibility criteria . following were the reasons for exclusion in 58 children : chest x - ray suggestive of bacterial pneumonia ( n = 19 ) , streptococcal sore throat ( n = 15 ) , underlying chronic conditions ( n = 10 ) , severe malnutrition ( n = 7 ) , illness lasting more than a week ( n = 3 ) , complicated pneumonia ( n = 2 ) , and being hospitalized in last one month ( n = 2 ) . the m  :  f ratio was 1.7  :  1 ( uri = 1.6  :  1 ; lri = 1.9  :  1 ) . out of 300 , 174 ( 58% ) were lri and 126 ( 42% ) were uri cases . month - wise recruitment of uri and lri cases has been shown in figure 1 . most lri cases occurred in the month of january , august , november , march , and july . most children with lri were in the age range of 212  mo ( 54% ) and with uri were in the age range of > 1260  mo ( 45.2% ) . chest x - ray findings were hyperinflation ( 93% ) , peribronchial cuffing ( 60% ) , interstitial infiltrates ( 48% ) , and patchy opacity ( 46% ) . of the 300 nasal and throat swabs , 248 cases were positive for viruses [ lri = 142/174 ( 81.6% ) , and uri = 106/126 ( 84.1% ) ] . month - wise recruitment of ari cases and samples positive for viruses has been shown in figure 2 . the maximum number of cases recruited and samples positive for viruses were in the months of january , december , and august . the virus positivity in ari was as follows : uri , 65 ( 61.3% ) samples were positive for single virus and 41 ( 38.7% ) were positive for more than one virus , and lri , 86 ( 61% ) samples were positive for single virus and 56 ( 39% ) were positive for more than one virus . the coinfection pattern differed between acute uri and lri , and this has been detailed in table 2 . the most common viruses isolated from uri cases were rv ( 31.1% ) , adv ( 18.9% ) , rsv ( 17% ) , and ifv ( 17% ) . the most common viruses isolated from lri cases were rsv ( 30.3% ) , ifv ( 17.6% ) , rv ( 14.8% ) , and adv ( 13.4% ) . rsv , adv , rv , and ifv were the most prevalent viruses isolated from all the ari cases . ifv - a was predominant over ifv - b , and piv-3 was predominant over other subtypes of piv . rsv - a subtype dominated over rsv - b in uri cases , whereas rsv - b dominated over rsv - a in lri cases . the detection rates of viruses corresponding to different age groups are shown in table 1 . in case of uris , rv was the most prevalent virus ( 26  mo age = 2.8% ; > 612  mo age = 13.3% ; > 1260  mo age = 14.2% ) . rsv was the second most common virus detected in age groups of 26  mo and > 612  mo . adv was the second most common virus detected in age group of > 1260  mo . the least common viruses in all age groups were piv , hbov , and hmpv . hcov and ev were not detected in any age group . in case of lris , rsv was the most prevalent virus ( 26  mo age = 13.4% ; > 612  mo age = 11.3% ; > 1260  mo age = 5.6% ) . hmpv , hbov , and hcov were not detected up to 6  mo age . the least common viruses in all age groups were hbov , hcov , ev , and hmpv . rv was the most common virus detected in uris , but it was fourth most prevalent in lris . it was detected throughout the year , the maximum rate being in december , january , march , and august . rsv was the most common virus detected in lris , but it was third most prevalent in uris . it was detected throughout the year , the maximum rate being in december , january , and february . ifv was second most common virus detected in lris , but it was third most prevalent in uris ; maximum positivity rate was in august followed by in july , and minimum positivity rate was in november . piv had a higher prevalence in april to july , and a low prevalence from september to march . there detection rate was lower in lris . like hmpv , hbov was present throughout the year in lris . the clinical features , demographics , and risk factors of children among viral positive and virus negative group with lris were compared ( table 3 ) . it was observed that significantly higher number of children below 12  mo were virus positive ( p = 0.01 ) . children presenting with preceding bronchiolitis were significantly associated with total viral infections ( p = 0.003 ) . rhinorrhea was significantly present in the virus positive group ( p = 0.02 ) . among risk factors , ari in family was significantly associated with virus positivity ( p = 0.04 ) . in lris , out of 174 cases ( lri = 101 ; severe lri = 51 ; very severe lri = 22 ) , 161 were cured and 13 died . of 13 cases who died , 10 had coinfection with more than 1 respiratory virus ( ifv + rsv = 5 ; rsv + rv = 3 ; ifv + hcov43 + rv = 2 ) . in uris , in the present study , respiratory viruses were detected in 248 cases ( lri = 142 ; uri = 106 ) . rsv ( 25.8% ) , rv ( 21.9% ) , ifv ( 21% ) , and adv ( 12.5% ) were the most prevalent single viruses isolated from all the ari cases . the most common viruses isolated from acute uri cases were rv ( 32.6% ) , adv ( 19.6% ) , rsv ( 17.4% ) , and ifv - a ( 15.2 ) . the most common viruses isolated from acute lri cases were rsv ( 32.4% ) , ifv - a ( 19.6% ) , adv ( 18.6% ) , and rv ( 15.7% ) . most cases occurred in the month of january , december , and august . in uris , the most common age group affected was > 1260  mo , and in lris , the most common age group affected was 212  mo . present study results are similar to the previous study results from india . in the study by broor et al . , rsv , ifv - a , and piv-3 were important causes of ari in children under five in the rural indian community . in the study by singh et al . , rsv was the most frequently detected virus ( 21.3% ) from hospitalised children presenting as alri followed by ifv ( 9.0% ) , measles ( 8.5% ) , and adv ( 5.3% ) . in the study by bharaj et al . , rsv was the most frequently detected virus ( 58.1% ) from hospitalised children presenting as alri followed by piv ( 22% ) , hmpv ( 10.5% ) , and ifv - a ( 9.3% ) . in the study by maitreyi et al . , rsv was the most frequently detected virus ( 17% ) from hospitalised children presenting as alri followed by ifv ( 14.5% ) , piv ( 11.5% ) , and adv ( 1.5% ) . in the study by yeolekar et al . , rsv was the most frequently detected virus ( 26% ) from hospitalised children presenting as alri followed by ifv ( 5.4% ) , piv ( 2.07% ) , and adv ( 0.8% ) . panda et al . recently found rv as the most frequently detected pathogen ( 24.7% ) followed by rsv ( 4.22% ) , piv ( 2.11% ) , and hmpv ( 2.11% ) in children with ari from eastern india . in the present study , we found rv as the second most important agent to rsv causing ari in young infants and children . the present study findings are more or less similar to the recently published studies from other parts of the globe , though the most prevalent virus may be different [ 2127 ] . the viral uri and lri prevalence varied with age , uris being common in children of > 1260  mo and lris being common in infants of < 12  mo age . infants < 12  mo age were most commonly positive for rsv and less commonly for ifv , the latter occurring commonly beyond infancy in older children . this is because maternal antibody protection against rsv during infancy remains questionable , and as the child grows , the immunity builds up against rsv due to repeated exposures . ifv due to marked antigenic diversity evades the herd immunity and does not give long lasting immunity . therefore , the susceptibility to ifv increases as the child grows . adv is uncommon during first six months , where maternal antibody confers protection . previous indian studies [ 16 , 17 ] have compared the clinical , demographic , and risk factors for alri testing positive for respiratory viruses with those testing negative for respiratory viruses . one study observed that significantly higher number of children below 12  mo of age had rsv infection , those presenting with preceding bronchiolitis were significantly associated with virus positivity , and ari in family was significantly associated with virus positivity . in another study , patients testing positive for viruses showed significantly high percentage of cough , nasal discharge , and diarrhea . we found a significantly higher number of children below 12  mo being virus positive , those presenting with preceding bronchiolitis being significantly associated with virus positivity , rhinorrhea significantly presenting in the virus positive group , and ari in family being significantly associated with virus positivity . the respiratory illness exhibits a clear - cut seasonality , and all the published studies till date support this . most of the studies report increased incidence of acute respiratory infection ( both uri and lri ) during winter and fall season [ 2027 ] . this is because of the following sequence of events : decrease in air temperature causing decrease in the nasal airway temperature ( compromised cooling of the nasal airway ) leading to poor defence against infection ( because of decrease mucociliary clearance and phagocytosis ) . in the present study , the incidence of both uri and lri was higher in winter season ( as upper airway acts as an entry point to lower airway ) . though this can not be solely explained based on the above hypothesis , it may be because of an increase in the birth cohort during this period . in acute lris , severe clinical phenotypes ( severe and very severe pneumonia , acute respiratory distress syndrome ) the present study findings throw lights on the management of an individual case as well as the community . first , good hand hygiene , maintenance of warm temperature in the living place / avoidance of exposure to sudden cold temperature , avoidance of taking of freeze dried / cold food items , and wearing of the mask to prevent spread of the infection should be practiced . second , the nutritional status of the children in the community should be improved as malnutrition predisposes to increase risk of infection and severe illness . third , vaccines should be developed against respiratory viruses ( including against the new viruses ) to decrease the morbidity and mortality from respiratory illness . fourth , any child with ari and viral coinfection should be closely monitored for development of complications / severe illness . strengths of present study are adequate sample size and children being recruited over 2 consecutive years including all the seasons . limitations include children presenting to the health facility only were recruited , reinfection rate by the viruses were not considered , and simultaneous / secondary bacterial infections were not studied . rsv , adv , rv , and ifv were the most prevalent viruses isolated from the ari cases . the epidemiology of respiratory viruses needs to be studied further for vaccine development and implementation purpose . any child with ari and viral coinfection should be monitored for development of complications / severe illness .
background . acute respiratory infections ( aris ) 
 are important cause of mortality and morbidity in children under five in developing country . methods . 
 this observational study was conducted over two - year period in a tertiary care teaching hospital of eastern india . 
 nasal and throat swabs were collected , transported to the laboratory at 28c in viral transport media , and then processed for detection of viruses using mono / multiplex real - time polymerase chain reaction . 
 results . 
 a total of 300 children aged 260 months with aris were included . 
 the most common age group affected with lri was 212  mo and with uri was > 1260  mo . 
 viruses were detected in 248 cases . in uri , 
 77 were positive for single virus and 19 were positive for more than one virus ; in lri , 113 were positive for single virus and 12 were positive for more than one virus . 
 the most common viruses isolated from uri cases were rhinovirus and adenovirus . 
 the most common viruses isolated from lri cases were respiratory syncytial virus and influenza virus . 
 most cases occurred in the months of january , december , and august . 
 conclusion . 
 viruses constitute a significant cause of ari in children under five . 
 rsv , adv , rv , and ifv were the most prevalent viruses isolated .