the case  control study was conducted in metropolitan atlanta , georgia , usa , during march 2003february 2004 , among children with asthma who were > 2 years of age ( 10 ) .
case - patients were defined as patients with asthma exacerbation ; controls were defined as patients with stable asthma .
the definitions , epidemiologic and laboratory methods , and clinical description of patients are available from table 1 and the previously published report ( 10 ) .
pcr ( rt - pcr ) targeting the 5-noncoding region ( ncr ) ( 10 ) . for further genetic characterization ,
hrv - positive samples were extracted from a previously unopened aliquot and amplified by using a nested rt - pcr that targeted the virus capsid protein 1 ( vp1 ) gene at positions 24322781 , based on hrv 1b ( genbank accession no .
d00239 ) for species a and positions 25312799 , based on hrv 14 ( genbank accession no .
nc_001490 ) for species b. we used sequencher 3.1.1 software ( gene codes , ann arbor , mi , usa ) for sequence assembly and editing .
nucleotide and predicted amino acid sequences were aligned with previously published hrv vp1 sequences ( genbank accession nos .
ay355180ay3552831 , ef186077 , ef077279 , ef077280 , ef582385ef582387 ) by using clustalw as implemented in bioedit ( version 7.0.5 ) ( www.mbio.ncsu.edu/bioedit/bioedit.html ) .
phylogenetic trees were constructed by using the neighbor - joining algorithm implemented in paup * version 4.0.d10 ( 11 ) .
partial vp1 sequences for the novel hrv strains were submitted to genbank ( accession nos .
as reported previously ( 10 ) , hrvs were detected by a 5-noncoding region seminested rt - pcr in 53 ( 37% ) of 142 children with asthma , including 39 ( 60% ) of 65 case - patients and 14 ( 18% ) of 77 controls .
of these , the hrvs from 29 ( 55% ) ( 24 [ 62% ] of the 39 hrv - positive case - patients and 5 [ 36% ] of the 14 hrv - positive controls ) were subsequently genotyped .
vp1 sequences from the remaining 24 hrv - positive specimens could not be obtained because of low amplicon yield ( table 2 ) .
specimens from patients with symptoms of acute viral respiratory infection ( table 1 ) were more likely than those from patients without viral symptoms to yield sufficient vp1 amplicon for genotyping ( percent genotyped 85% and 36% , respectively ; odds ratio [ or ] 9.1 ; 95% confidence interval [ ci ] 2.150.0 ; p<0.05 ) .
* hrv , human rhinovirus ; case - patients , asthma patients with exacerbations ; controls , asthma patients without exacerbation . 
serotype designation based on > 90% vp1 amino acid sequence identity with respective prototype strains . 
 genogroup c hrvs form a clade phylogenetically distict from species a and b hrvs . of the 29 hrvs
successfully genotyped , species a accounted for 18 ( 62% ) strains , species b accounted for 3 ( 10% ) , whereas 8 ( 28% ) strains formed a phylogenetically distinct clade , which we provisionally named  genogroup c  ( table 2 , figure ) .
of the 18 hrv - a strains , 17 showed close genetic relatedness ( 80.7%93.8% nucleotide and 89.6%98.8% predicted amino acid sequence identity ) to hrv prototype strains .
one hrv - a strain ( ga23584 ) was highly divergent from the closest prototype , hrv80 ( 73.2% nucleotide and 73.0% amino acid sequence identity ) , which suggests that it could represent a distinct previously undescribed hrv . the 3 hrv - b strains were closely related to prototype strains ( 84.0%88.6% nucleotide and 89.7%93.4% predicted amino acid sequence identity ) .
phylogenetic tree of partial virus capsid protein 1 ( vp1 ) amino acid sequences of human rhinoviruses ( hrvs ) identified in 29 hrv - positive pediatric asthma patients , march 2003february 2004 , atlanta , georgia , usa ( designated * ) , previously published sequences of strains qpm ( genbank accession no .
the partial vp1 sequences of genogroup c strains were phylogenetically distinct from hrv species a and b and showed a substantial intragroup diversity ( figure ) .
vp1 sequence identity of these viruses with the closest match within the same genogroup ranged from 68.4% to 74.6% for nucleotide and from 68.5% to 85.5% for amino acid sequences .
these novel viruses were related to other recently described hrvs : hrv  qpm detected in specimens from australia ( 4 ) , c024c026 detected in specimens from hong kong ( 6 ) , and nat001 and nat045 detected in specimens from california ( 8) ( figure ) .
their identity scores compared with hrv  qpm were 66.0%82.7% for nucleotide and 65.2%86.9% for amino acid sequences .
one of the strains ( ga23592 ) was almost identical in partial vp1 sequence to c026 ( figure ) .
the degree of genetic diversity among the genogroup c viruses far exceeded that between hrvs defined as distinct serotypes by classical serologic methods , which suggests that at least 7 of 8 of these viruses are antigenically distinct from each other rather than minor variants of the same serotype .
the genogroup c hrv identity scores were substantially lower when compared with their closest matches from species a and b : 48.2%51.1% for nucleotide and 38.5%49.8% for amino acid sequences , and 35.9%42.8% for nucleotide and 29.3%35.8% for amino acid sequences , respectively .
in our study , the association of asthma exacerbations with hrv infection appeared to be largely driven by the novel genogroup c , which was found exclusively in case - patients , and species a. the association was statistically significant for species a ( detected in 15 [ 23% ] of 65 case - patients vs. 3 [ 4% ] of 77 controls ; or 7.4 ; 95% ci 1.943.1 ; p<0.001 ) and for genogroup c ( detected in 8 [ 12% ] case - patients vs. 0 controls ; or undefined ; p<0.010 ) but not for infrequently identified species b ( detected in 1 [ 2% ] case - patient vs. 2 [ 3% ] controls , p>0.05 ) or for hrvs that could not be genotyped ( 15 [ 23% ] cases vs. 9 [ 12% ] controls ; p>0.05 ) .
the distribution of hrvs between case - patients and controls still differed when the analysis was limited to the hrv - positive group ( p = 0.05 ) or to genotyped hrvs only ( p<0.05 ) . the results of the only other study that reported novel hrvs in asthma patients ( 2 of which , nat001 and nat045 , were related to genogroup c viruses in our study ) are difficult to interpret because that study of adults with  cold  symptoms showed an unexpected lack of association of hrvs with asthma exacerbation ( 8) .
patients infected with genogroup c hrvs had lower forced expiratory volumes during the first second ( fev1 ) than did those infected with other hrvs ( median 58.5% vs. 93% ; p = 0.01 ) , but the distribution of demographic and other clinical variables did not differ significantly between the 2 groups .
lower fev1 with genogroup c infection than with other hrvs suggests a potentially greater severity of asthma exacerbation in patients infected with these hrvs . when one considers the great variation among hrv serotypes in levels of sensitivity to candidate antiviral compounds ( 12,13 ) , genogroup - related differences in associated disease patterns have implications for clinical management of hrv infections in asthma patients and for development of antiviral drugs against hrvs .
preliminary data suggest that hrv - qpm and related hrv - c strains from hong kong share certain vp1 sequence characteristics with hrvs that are resistant to a candidate antipicornavirus drug , pleconaril ( 4,6,13 ) .
these data raise the possibility that these novel hrvs might also be resistant to this compound .
the hrv - positive specimens from which vp1 gene sequences could not be obtained derived predominantly from patients without symptoms of acute respiratory viral illness .
the absence of symptoms in hrv - infected persons likely reflects subclinical , asymptomatic infection , which is common for hrvs ( 14 ) , or hrv persistence after a recently resolved infection ( 15 ) , both of which are likely associated with lower viral loads ( as opposed to acute symptomatic infections ) , thus leading to lower detection rates in a vp1 assay that uses highly degenerate primers . in conclusion
, we found a striking genetic diversity of hrvs among children with asthma and confirmed the existence and wide geographic distribution ( usa , australia , hong kong ) of hrvs distinct from both previously recognized hrv species , a and b. our finding supports the role of the novel hrvs as human pathogens .
additional studies are needed to further explore clinical and public health implications of these findings .
 to determine links between human rhinoviruses ( hrv ) and asthma , we used data from a case  control study , march 2003february 2004 , among children with asthma . molecular characterization 
 identified several likely new hrvs and showed that association with asthma exacerbations was largely driven by hrv - a and a phylogenetically distinct clade of 8 strains , genogroup c. 
The Study
Conclusions