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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Clin Virol. Author manuscript; available in PMC 2010 September 1.
Published in final edited form as:
PMCID: PMC2759319

Human Rhinovirus C Associated with Wheezing in Hospitalized Children in the Middle East



Few studies have investigated the disease burden and genetic diversity of human rhinoviruses (HRV) in developing countries.


To assess the burden of HRV in Amman, Jordan and to characterize clinical differences between HRV groups.

Study Design

We prospectively studied children <5 years old hospitalized with respiratory symptoms and/or fever in Amman, Jordan. Viruses were identified by real-time RT-PCR. VP4/VP2 gene sequencing was performed on HRV-positive specimens.


Of 728 enrolled children, 266 (37%) tested positive for picornaviruses, 240 of which were HRV. Of the HRV-positive samples, 62 (26%) were of the recently identified group HRVC, 131 (55%) were HRVA, and 7 (3%) were HRVB. The HRVC strains clustered into at least 19 distinct genotypes. Compared with HRVA-infected children, children with HRVC were more likely to require supplemental oxygen (63% vs. 42%, p=0.007) and, when co-infections were excluded, were more likely to have wheezing (100% vs. 82%, p=0.016).


There is a significant burden of HRV-associated hospitalizations in young children in Jordan. Infection with the recently identified group HRVC is associated with wheezing and more severe illness.

Keywords: rhinovirus, HRVC, wheezing, hospitalized, children


Human rhinoviruses (HRV) are members of the family Picornaviridae first cultured in 1956, and approximately 100 serotypes have been identified 1. Although once thought to cause only the common cold, it is now known that HRV are associated with lower respiratory tract infections and asthma exacerbations in adults and children2-11. Twenty-six percent of hospitalizations for acute respiratory illness (ARI) among children younger than 5 years old were associated with HRV during one year of population-based surveillance in two US cities2.

The use of molecular diagnostics substantially improves the detection of HRV2, 12-15. Previously, HRV serotypes were classified into two species (HRVA and HRVB1, 16), but another group of HRV, HRVC, was recently identified by RT-PCR and partial sequence data, first in Australia and subsequently in other regions17-25. Little is known about the epidemiology of the recently identified HRVC group in developing countries, particularly in the Middle East.


We determined the frequency, clinical features, and genetic diversity of HRV infections during the peak winter months among children < 5 years of age who were hospitalized with ARI or fever in two major hospitals in Amman, Jordan.

Study Design

Prospective surveillance was conducted among children <5 years of age hospitalized with ARI/fever at Jordan University Hospital (JU) or Al-Basheer in Amman, Jordan from January 18 through March 29, 2007. Institutional Review Boards from Vanderbilt University, Jordan University, and the Jordan Ministry of Health approved this study.

Data collection

Study personnel consented and interviewed the parents or guardians to complete a standardized questionnaire that recorded clinical and demographic information. Medical records were reviewed. Data were entered into handheld PDA devices that were synchronized daily.

Virus testing and sequencing

Nasal/throat swabs were obtained and combined into a single tube of viral transport medium (Beckton-Dickinson). Lysis buffer was added (Roche MagNApure tNA) and samples were stored at -70°C until shipped on dry ice to Vanderbilt.

RNA was extracted from specimens in lysis buffer using an automated platform (MagNApure LC, Roche) as previously described26. Real-time RT-PCR for HRV was performed with the Smart Cycler II (Cepheid) using primers and probe sequences directed at a highly conserved HRV 5′-non-coding region (5′-NCR), allowing detection of all 100 HRV prototype strains27. Specimens were tested for hMPV, RSV, and influenza as described28-30.

RT-PCR was performed on HRV-positive samples using primers that amplify a ~548-bp fragment encompassing the VP4/VP2 region and the hypervariable region in the 5′-NCR31. Amplified fragments were sequenced bi-directionally on a 3730×l DNA Analyzer(Applied Biosystems). Sequences were aligned using MacVector (Accelrys) and phylogenetic analyses performed using MEGA 4.0.26, 32, 33 HRV87 was used as an outgroup for all phylogenetic analyses34.

Statistical analysis

Comparisons of demographic and clinical data between groups were made using Chi-square or Fisher's exact test for categorical variables and Wilcoxon rank sum test or Kruskal-Wallis test for continuous variables. All analyses were performed using R software (, v2.6.1).


Study Population

Of 803 eligible children hospitalized with respiratory symptoms and/or fever between January 18 and March 29, 2007, 743(92.5%) were enrolled. Analyses included the 728(98%) of enrolled children for whom clinical and laboratory data were available. Forty-four(6%) children were enrolled from Jordan University. The mean age of the subjects was 7.85 months, the median age was 4.34 months, and 58.4% were male. Of all children enrolled, 638/728 (88%) tested positive for at least one virus. There were 266/728(37%) patients who tested positive for picornaviruses: 240(33%) for HRV and 26(4%) for human enteroviruses(HEV). There were 467(64%) who tested positive for RSV, 44(6%) who tested positive for hMPV, and 6(0.8%) who tested positive for influenza A or B.

Demographic and Clinical Characteristics of Rhinovirus-infected Children

Demographic and presenting symptoms of children with HRV vs RSV vs no study virus are presented in Table 1. Compared to children with RSV, those with HRV were more likely to be older (5.4 vs 3.8 months, p=0.04) and were less likely to have poor appetite(64%vs77%, p=0.009), or require supplemental oxygen(43%vs74%, p<0.001). Children infected with HRV or RSV were more likely to have wheezing (HRV 87%, RSV 89%, no virus 73%, p<0.001) and nasal congestion than children who had no study virus detected (HRV 68%, RSV 74%, no virus 54%, p=0.001).

Table 1
Demographic and clinical characteristics of children with HRV vs RSV vs no virus detected, excluding coinfections.

The age distribution of children with HRV, RSV, and hMPV is illustrated in Figure 1. The age distribution of HRV and RSV were similar. Of the HRV-positive children, 64% were younger than 6 months old. Of the 240 HRV-positive children, 128 were coinfected with another virus studied: 110(46%) with RSV, 17(7%) with hMPV, and 1(0.4%) with influenza A. Compared with children infected with HRV alone, children coinfected with HRV and RSV were younger (2.5vs5.4 months, p<0.001), more likely to require supplemental oxygen (65%vs43%, p=0.001), more likely to vomit (55%vs38%, p=0.011), and more likely to stay an extra day in the hospital (5vs4 days, p=0.007) (Table 2).

Figure 1
Age distribution of subjects infected with respiratory syncytial virus (RSV), human metapneumovirus (MPV), or human rhinovirus (HRV).
Table 2
Demographic and clinical characteristics of children with HRV only versus HRV coinfection.

Two children with HRV died. The first was an 18-month-old male with low birth weight and a history of mental retardation/developmental delay. This child was infected with HRVA alone and was admitted with a diagnosis of ARI and pneumonia. The second fatal case was a 1.4-month-old male term infant with congenital heart disease. This child was coinfected with HRVC and RSV, required supplemental oxygen on admission, and died on the 11th day of hospitalization. Other bacterial or parasitic culture data on these two subjects were not available.

Clinical Features of HRVA Compared with HRVC

Of the 240 HRV positive samples, 62(26%) were HRVC, 131(55%) were HRVA, and 7(3%) were HRVB. We were unable to amplify sequences from the remaining 40(17%) specimens. In January, 52% of HRV-positive specimens were HRVA and 20% were HRVC; in February, 40% were HRVA and 36% were HRVC; and in March, 70% were HRVA and 18% were HRVC. The median ages of children infected with HRVA or HRVC did not differ. Children with HRVC were more likely to require supplemental oxygen than those with HRVA(63%vs42%, p=0.007) and were less likely to have otalgia(11%vs24%. p=0.047). Excluding coinfections, children with HRVC were still more likely to require supplemental oxygen than children with HRVA (57%vs34%, p=0.04). Compared to children infected with HRVA, those infected with HRVC alone were more likely to report wheezing/noisy breathing (100%vs82%, p=0.02). (Table 3).

Rhinovirus Diversity

While several clinical differences were seen between patients infected with HRVA versus HRVC, there were also significant genetic differences among viruses. The VP4/VP2 sequence regions correlate with the serotype classification of HRV 1, 16, 31. HRVC strains were defined using a nucleotide identity of <90%, based on the calculated genetic relatedness of established serotypes as previously described26. Applying this criterion, 19 distinct HRVC genotypes were identified, comprising 63 distinct strains that did not cluster into either the HRVA or HRVB groups(Figure 2). The HRVC genotypes had a mean nucleotide identity of 72.7%(minimum 66.4% and maximum 91.4%), compared to HRVA (mean 77.9%, minimum 70.5% and maximum 91.3%) and HRVB (mean 80%, minimum 78.6% and maximum 82.9%). Thus, HRVC viruses exhibited greater diversity among themselves than did HRVA. Based on comparisons of nucleotide identity, 3 of the HRVC genotypes were >95% identical with strains HRVQPM, HRVNY, and HRVX, and likely represent the same viruses 19-23. To our knowledge, the other 16 HRVC genotypes we identified have not been reported. Of the 131 HRVA strains sequenced, 10 were <90% identical to previously identified HRVA, including 5 that were <85% identical; these may represent novel HRVA types. Two of these potentially novel HRVA types were detected in 9 and 7 distinct subjects, respectively, demonstrating that these were common viruses in the study population. All but one of the few HRVB viruses were ≥90% identical with previously described strains; the remaining strain was 88.8% identical.

Figure 2
Phylogenetic tree depicting relationships between known HRV serotypes and novel HRV. Previously described HRV serotypes are designated by “HRV” and a black circle. Novel sequences identified in this study are designated by numbers. The ...


In this study, we found a significant burden of HRV-related illness among young children hospitalized with ARI or fever in Amman, Jordan. Few studies in the Middle East have reported HRV infections. Rashid detected HRV in 13%(19/150) of UK pilgrims and 3%(3/110) of domestic pilgrims with URI in Mecca, Saudi Arabia35. Kaplan identified HRV in 11%(36/325) of hospitalized young children in Jordan 35, 36. We enrolled a larger cohort than these studies and discovered a greater burden of HRV-associated illness in young children, likely because we used highly sensitive real-time RT-PCR. In addition, we found a trend in children with HRV/RSV coinfection to have more severe disease compared with HRV alone, evidenced by increased use of supplemental oxygen and duration of hospitalization. Papadopoulos et al. reported that the presence of HRV increases the risk for disease severity in acute bronchiolitis by approximately 5-fold37.

Investigators have detected the newly described HRVC in several countries. Of the HRV-positive samples sequenced in this study, 26% were HRVC. These findings are the first report of HRVC disease in the Middle East. We observed several clinically significant differences between patients infected with HRVC compared to HRVA. Children with HRVC were more likely to require supplemental oxygen and less likely to report otalgia, suggesting that HRVC may be more virulent than HRVA. Excluding coinfections, children with HRVC were more likely to wheeze than children with HRVA. A recent multi-year population-based study of HRVC in two U.S. locations found that HRVC was associated with medically diagnosed wheezing and asthma 26. These data, as well as other reports, suggest that viruses in the HRVC group are more likely to be associated with wheezing 19-24.

In prior studies 24, 26, HRVC accounted for about half of the sequenced HRV, compared with the 26% reported here. This discrepancy may be attributable to the apparent seasonality of HRVC: HRV circulate year-round with peaks in the spring and fall 38. In our previous study, HRVC was significantly more prominent in the fall months compared with HRVA, which predominated in the spring. Lau et al also identified an HRVC peak in the fall/winter, although they only detected 5 HRVC strains 23. Thus, in this study during three winter months, we may have underestimated the true burden of illness of HRVC. Almost 70% of HRV detected in March were HRVA, consistent with our prior study. These seasonal variations underscore the importance of conducting studies over multiple seasons in diverse locations to understand fully the epidemiology of HRV.

We found a surprising amount of genetic diversity among the 63 HRVC strains detected with clustering into 19 genotypes. Although these are genotypes and do not prove distinct serotypes, phylogenetic analysis of the VP4/VP2 protein-coding region correlates with serotyping for known HRV 31. The genetic diversity seen amongst HRVC strains was greater than that seen amongst HRVA strains when analyzed by percent sequence identity or phylogenetic distance, consistent with other studies to date. All of the recently described HRV for which the VP4/VP2 sequences are available fell into the same group, including the QPM strains from Queensland that were originally classified as a subgroup of HRVA 17-26.

Study Limitations

Our study has some limitations. First, we did not test concurrent healthy controls. Previous reports suggest that the rate of HRV detection in asymptomatic individuals varies between 4-18% and thus is lower than the rate of HRV we detected in hospitalized children 9, 39. Second, we defined wheezing by parental report. Because other clinical symptoms may be confused with “wheezing”, this could lessen the strength of association of HRVC with wheezing. However, other studies support this association 19-24 18. Third, we only evaluated samples during the peak winter months; therefore, we may have underestimated the overall burden of HRVC. We did not collect data on bacterial co-infections or antibiotic usage. However, rates of antibiotic use are high in this population, one study showed that over 60% of RSV-infected children were treated with antibiotics 40. Thus, improved viral diagnostic capabilities might limit inappropriate antibiotic use.

In conclusion, there is a significant burden of HRV-associated illness in young children hospitalized during the peak winter months with fever or ARI in Amman, Jordan. The HRVC group is responsible for many of these infections. HRVC is associated with wheezing and increased severity of illness. Further studies are required to confirm these observations and better understand the relationship between HRVC and asthma.


The authors thank the children and families enrolled in the study, Ms. Manar Dweik and Ms. Ghadeer Azizi for data collection and specimen processing, and Mr. Omar Mansour for assisting with recruitment. We thank Ms. Amy Podsiad for assistance with real-time RT-PCR assays. The Vanderbilt Clinical and Translational Research Scholars Award and a Thrasher grant fund Dr. Miller's work. Dr. Williams has served as a consultant for MedImmune and Novartis. Dr. Halasa receives grant support from MedImmune and sanofi Pasteur, and she has served as a consultant for Novartis.


human rhinovirus
human metapneumovirus
respiratory syncytial virus
reverse transcriptase polymerase chain reaction
Jordan University
acute respiratory illness


Conflicts of Interest: Otherwise, the authors have no conflicts of interest to disclose.

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1. Savolainen C, Blomqvist S, Mulders MN, Hovi T. Genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70. J Gen Virol. 2002 Feb;83(Pt 2):333–340. [PubMed]
2. Miller EK, Lu X, Erdman DD, et al. Rhinovirus-associated hospitalizations in young children. J Infect Dis. 2007 Mar 15;195(6):773–781. [PubMed]
3. Hayden FG. Rhinovirus and the lower respiratory tract. Rev Med Virol. 2004 Jan-Feb;14(1):17–31. [PubMed]
4. Gern JE. Rhinovirus respiratory infections and asthma. Am J Med. 2002 Apr 22;112 6A:19S–27S. [PubMed]
5. Jartti T, Lehtinen P, Vuorinen T, et al. Respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children. Emerg Infect Dis. 2004 Jun;10(6):1095–1101. [PMC free article] [PubMed]
6. Venarske DL, Busse WW, Griffin MR, et al. The relationship of rhinovirus-associated asthma hospitalizations with inhaled corticosteroids and smoking. J Infect Dis. 2006 Jun 1;193(11):1536–1543. [PubMed]
7. Lemanske RF, Jr, Jackson DJ, Gangnon RE, et al. Rhinovirus illnesses during infancy predict subsequent childhood wheezing. J Allergy Clin Immunol. 2005 Sep;116(3):571–577. [PubMed]
8. Martinez FD, Wright AL, Taussig LM, Holberg CJ, Halonen M, Morgan WJ. Asthma and wheezing in the first six years of life. The Group Health Medical Associates. N Engl J Med. 1995 Jan 19;332(3):133–138. [PubMed]
9. Heymann PW, Carper HT, Murphy DD, et al. Viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing. J Allergy Clin Immunol. 2004 Aug;114(2):239–247. [PubMed]
10. Peltola V, Waris M, Osterback R, Susi P, Hyypia T, Ruuskanen O. Clinical effects of rhinovirus infections. J Clin Virol. 2008 Dec;43(4):411–414. [PubMed]
11. Johnston NW, Johnston SL, Norman GR, Dai J, Sears MR. The September epidemic of asthma hospitalization: school children as disease vectors. J Allergy Clin Immunol. 2006 Mar;117(3):557–562. [PubMed]
12. Griffin MR, Walker FJ, Iwane MK, Weinberg GA, Staat MA, Erdman DD. Epidemiology of respiratory infections in young children: insights from the new vaccine surveillance network. Pediatr Infect Dis J. 2004 Nov;23(11 Suppl):S188–192. [PubMed]
13. Iwane MK, Edwards KM, Szilagyi PG, et al. Population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children. Pediatrics. 2004 Jun;113(6):1758–1764. [PubMed]
14. Mullins JA, Erdman DD, Weinberg GA, et al. Human metapneumovirus infection among children hospitalized with acute respiratory illness. Emerg Infect Dis. 2004 Apr;10(4):700–705. [PMC free article] [PubMed]
15. Weinberg GA, Erdman DD, Edwards KM, et al. Superiority of reverse-transcription polymerase chain reaction to conventional viral culture in the diagnosis of acute respiratory tract infections in children. J Infect Dis. 2004 Feb 15;189(4):706–710. [PubMed]
16. Ledford RM, Patel NR, Demenczuk TM, et al. VP1 sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds. J Virol. 2004 Apr;78(7):3663–3674. [PMC free article] [PubMed]
17. Mackay IM, Lambert SB, McErlean PK, et al. Prior evidence of putative novel rhinovirus species, Australia. Emerg Infect Dis. 2008 Nov;14(11):1823–1824. author reply 1824-1825. [PMC free article] [PubMed]
18. Khetsuriani N, Lu X, Teague WG, Kazerouni N, Anderson LJ, Erdman DD. Novel human rhinoviruses and exacerbation of asthma in children. Emerg Infect Dis. 2008 Nov;14(11):1793–1796. [PMC free article] [PubMed]
19. Arden KE, McErlean P, Nissen MD, Sloots TP, Mackay IM. Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections. J Med Virol. 2006 Sep;78(9):1232–1240. [PubMed]
20. Lamson D, Renwick N, Kapoor V, et al. MassTag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in New York State during 2004-2005. J Infect Dis. 2006 Nov 15;194(10):1398–1402. [PubMed]
21. McErlean P, Shackelton LA, Lambert SB, Nissen MD, Sloots TP, Mackay IM. Characterisation of a newly identified human rhinovirus, HRV-QPM, discovered in infants with bronchiolitis. J Clin Virol. 2007 Jun;39(2):67–75. [PubMed]
22. Kistler A, Avila PC, Rouskin S, et al. Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity. J Infect Dis. 2007 Sep 15;196(6):817–825. [PubMed]
23. Lau SK, Yip CC, Tsoi HW, et al. Clinical features and complete genome characterization of a distinct human rhinovirus (HRV) genetic cluster, probably representing a previously undetected HRV species, HRV-C, associated with acute respiratory illness in children. J Clin Microbiol. 2007 Nov;45(11):3655–3664. [PMC free article] [PubMed]
24. Lee WM, Kiesner C, Pappas T, et al. A diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants. PLoS ONE. 2007;2(10):e966. [PMC free article] [PubMed]
25. Briese T, Renwick N, Venter M, et al. Global distribution of novel rhinovirus genotype. Emerg Infect Dis. 2008 Jun;14(6):944–947. [PMC free article] [PubMed]
26. Miller EK, Edwards KM, Weinberg GA, et al. A novel group of rhinoviruses is associated with asthma hospitalizations. J Allergy Clin Immunol. 2008 Nov 20; [PubMed]
27. Lu X, Holloway B, Dare RK, et al. Real-time reverse transcription-PCR assay for comprehensive detection of human rhinoviruses. J Clin Microbiol. 2008 Feb;46(2):533–539. [PMC free article] [PubMed]
28. Williams JV, Wang CK, Yang CF, et al. The role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience. J Infect Dis. 2006 Feb 1;193(3):387–395. [PMC free article] [PubMed]
29. Mentel R, Wegner U, Bruns R, Gurtler L. Real-time PCR to improve the diagnosis of respiratory syncytial virus infection. J Med Microbiol. 2003 Oct;52(Pt 10):893–896. [PubMed]
30. Mehlmann M, Bonner AB, Williams JV, et al. Comparison of the MChip to viral culture, reverse transcription-PCR, and the QuickVue influenza A+B test for rapid diagnosis of influenza. J Clin Microbiol. 2007 Apr;45(4):1234–1237. [PMC free article] [PubMed]
31. Savolainen C, Mulders MN, Hovi T. Phylogenetic analysis of rhinovirus isolates collected during successive epidemic seasons. Virus Res. 2002 Apr 23;85(1):41–46. [PubMed]
32. Felsenstein J. PHYLIP (Phylogeny Inference Package) version 3.67 Distributed by the author. Department of Genome Sciences, University of Washington; Seattle: 2004.
33. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 2007 Aug;24(8):1596–1599. [PubMed]
34. Ishiko H, Miura R, Shimada Y, et al. Human rhinovirus 87 identified as human enterovirus 68 by VP4-based molecular diagnosis. Intervirology. 2002;45(3):136–141. [PubMed]
35. Rashid H, Shafi S, Haworth E, et al. Viral respiratory infections at the Hajj: comparison between UK and Saudi pilgrims. Clin Microbiol Infect. 2008 Jun;14(6):569–574. [PubMed]
36. Kaplan NM, Dove W, Abd-Eldayem SA, Abu-Zeid AF, Shamoon HE, Hart CA. Molecular epidemiology and disease severity of respiratory syncytial virus in relation to other potential pathogens in children hospitalized with acute respiratory infection in Jordan. J Med Virol. 2008 Jan;80(1):168–174. [PubMed]
37. Papadopoulos NG, Moustaki M, Tsolia M, et al. Association of rhinovirus infection with increased disease severity in acute bronchiolitis. Am J Respir Crit Care Med. 2002 May 1;165(9):1285–1289. [PubMed]
38. Monto AS. The seasonality of rhinovirus infections and its implications for clinical recognition. Clin Ther. 2002 Dec;24(12):1987–1997. [PubMed]
39. Jartti T, Lehtinen P, Vuorinen T, Koskenvuo M, Ruuskanen O. Persistence of rhinovirus and enterovirus RNA after acute respiratory illness in children. J Med Virol. 2004 Apr;72(4):695–699. [PubMed]
40. Otoom S, Batieha A, Hadidi H, Hasan M, Al-Saudi K. Evaluation of drug use in Jordan using WHO prescribing indicators. East Mediterr Health J. 2002 Jul-Sep;8:537–43. [PubMed]