A molecular epidemiological study of respiratory viruses detected in Japanese children with acute wheezing illness
- Asako Fujitsuka1, 2,
- Hiroyuki Tsukagoshi3,
- Mika Arakawa4,
- Kazuko Goto-Sugai1, 2,
- Akihide Ryo5,
- Yoshimichi Okayama6,
- Katsumi Mizuta7,
- Atsuyoshi Nishina8,
- Masakazu Yoshizumi3,
- Yoichi Kaburagi1,
- Masahiro Noda9,
- Masato Tashiro10,
- Nobuhiko Okabe11,
- Masaaki Mori2,
- Shumpei Yokota2 and
- Hirokazu Kimura3, 11Email author
© Fujitsuka et al; licensee BioMed Central Ltd. 2011
Received: 9 February 2011
Accepted: 10 June 2011
Published: 10 June 2011
Recent studies strongly suggest that some respiratory viruses are associated with the induction of acute wheezing and/or exacerbation of bronchial asthma. However, molecular epidemiology of these viruses is not exactly known.
Using PCR technology, we attempted to detect various respiratory viruses from 115 Japanese children. Furthermore, the detected viruses were subjected to homology, pairwise distance, and phylogenetic analysis.
Viruses were detected from 99 (86.1%) patients. Respiratory syncytial virus (RSV) alone and human rhinovirus (HRV) alone were detected in 47 (40.9%) and 36 (31.3%) patients, respectively. Both RSV and HRV were detected in 14 (12.2%) patients. Human metapneumovirus (HMPV) alone and human parainfluenza virus (HPIV) alone were detected in 1 (0.9%) patient each, respectively. Homology and phylogenetic analyses showed that the RSV and HRV strains were classified into genetically diverse species or subgroups. In addition, RSV was the dominant virus detected in patients with no history of wheezing, whereas HRV was dominant in patients with a history of wheezing.
The results suggested that these genetically diverse respiratory viruses, especially RSV and HRV, might be associated with wheezing in Japanese children.
A range of respiratory viruses are known to cause acute respiratory infections (ARI), including the common cold, bronchiolitis, and pneumonia in humans . The major pathogens are potentially respiratory syncytial virus (RSV), human rhinovirus (HRV), human metapneumovirus (HMPV), human parainfluenza virus (HPIV), enterovirus (EV), influenza viruses (InfV), adenoviruses (AdV), and human bocavirus (HBoV) [2, 3]. Respiratory infections by RSV, HRV, and HPIV are implicated in the induction of wheezing and the exacerbation of asthma, although their mechanisms are not clearly known . The prevalence of asthma in developed countries is around 10 to 15% in children, while the prevalence is lower but increasing rapidly in developing countries . Accumulating evidence indicates that the etiology of most cases of asthma, namely virus-induced asthma, is linked to such respiratory virus infections [6–9]. In addition, other epidemiological studies suggest that about 70% of infants have experienced an RSV infection by the age of 1 year, and 100% by the age of 2 years; the host response to the virus varies greatly, but includes upper respiratory tract infections, typical bronchiolitis (with crepitations but no wheeze), and RSV-induced wheezy bronchitis [10, 11]. In addition, HRV includes over 100 serotypes and most of these are epidemic, although their epidemiology is not known . Similarly, most children are infected at least once with HPIV early in life, but reinfections occur throughout life . HBoV and HMPV are recently discovered agents of ARI, and these viruses are also associated with the common cold, bronchiolitis, and pneumonia . However, the relationships between these viruses and virus-induced wheezing are not exactly known.
Genetic analyses including sequence and phylogenetic analyses of various viruses enable detailed genetic characterization of these agents. With the use of these methods, detailed molecular epidemiological studies have been reported, even in non-culturable viruses such as HRV species C (HRV-C) or HBoV [15, 16]. However, molecular epidemiology of various respiratory viruses with regard to virus-induced asthma is not exactly known. From these backgrounds, we detected various respiratory viruses and performed a molecular epidemiological study of them in Japanese children with acute wheezing illness.
Subject data in this study
No. of patients
History of wheezing and/or asthma
No. of patients
No. of inpatients and outpatients
No. of cases of bronchitis and/or bronchiolitis
13.5 ± 21.0
7.1 ± 2.5
16.9 ± 23.9
20.8 ± 25.7
25.9 ± 28.9
18.1 ± 17.0
7.1 ± 1.2
28.5 ± 27.5*
35.7 ± 31.3
DNA/RNA extraction, PCR, and sequencing
Primers for PCR used in this study
Phylogenetic analysis and calculation of pairwise distances
We performed homology and phylogenetic analysis of the G gene of RSV, and the VP4/VP2 coding region of HRV, because these viruses were the most commonly detected strains. The nucleotide positions of the nucleotide positions of the G gene of RSV were 673-912 (240 bp, for subgroup A) or 670-963 (294 bp, for subgroup B), and the VP4/VP2 coding region of HRV were 623-1012 (390 bp). We used the CLUSTAL W program on the DNA Data Bank of Japan (DDBJ) homepage http://clustalw.ddbj.nig.ac.jp/top-j.html and TreeExplorer (Version 2.12) http://evolgen.biol.metro-u.ac.jp/TE/. Evolutionary distances were estimated using Kimura's two-parameter method, and phylogenetic trees were constructed using the neighbor-joining (NJ) method . The reliability of the tree was estimated using 1000 bootstrap replications. We selected the reference strains as previously described to construct the phylogenetic trees of RSV and HRV [17, 27]. Moreover, we calculated subgroup or species frequency distributions using pairwise genetic distances for each strain, as previously described .
Data were analyzed using SPSS software (SPSS for Windows, Version 10.0). All data are expressed as mean ± SD. We performed bivariate analyses using Pearson χ 2 and Fisher exact tests to compare the prevalence of respiratory viruses and other variables between the study groups. The Student's t-test was used to compare mean age in the study group. Statistical significance was set at the level of p< 0.05.
All samples were collected after written informed consent was obtained from the subjects' parents. The study protocol was approved by the Ethics Committee on Human Research of National Hospital Organization Yokohama Medical Center.
Viruses detected in the present subjects
Subtypes or species of detected viruses
No history of wheezing and/or asthma
Having history of wheezing and/or asthma
No. of strains
No. of strains
RSV/YOK/07/14(AB551036), RSV/YOK/07/22(AB551037), RSV/YOK/07/26(AB551038), RSV/YOK/07/43(AB551039), RSV/YOK/07/51(AB551040), RSV/YOK/07/52(AB551041), RSV/YOK/07/53(AB551042), RSV/YOK/07/66(AB551044), RSV/YOK/08/79(AB551046), RSV/YOK/08/83(AB551047), RSV/YOK/08/113(AB551049), RSV/YOK/08/122(AB551053), RSV/YOK/08/123(AB551054), RSV/YOK/08/125(AB551056), RSV/YOK/08/127(AB551057), RSV/YOK/08/128(AB551058), RSV/YOK/08/130(AB551059), RSV/YOK/08/133(AB551060), RSV/YOK/08/134(AB551061), RSV/YOK/08/141(AB551065), RSV/YOK/08/142(AB551066), RSV/YOK/08/146(AB551069), RSV/YOK/08/148(AB551071), RSV/YOK/08/150(AB551072), RSV/YOK/09/162(AB551075)
RSV/YOK/07/13(AB551078), RSV/YOK/07/16(AB551079), RSV/YOK/07/17(AB551080), RSV/YOK/07/21(AB551081), RSV/YOK/07/32(AB551083), RSV/YOK/07/33(AB551084), RSV/YOK/07/34(AB551085), RSV/YOK/07/38(AB551086), RSV/YOK/07/50(AB551092), RSV/YOK/07/56(AB551093), RSV/YOK/07/60(AB551095), RSV/YOK/07/62(AB551096), RSV/YOK/07/64(AB551097)
RSV/YOK/07/4(AB551076), RSV/YOK/07/59(AB551094), RSV/YOK/08/74(AB551102), RSV/YOK/08/80(AB551104), RSV/YOK/08/82(AB551105), RSV/YOK/08/84(AB551106), RSV/YOK/08/88(AB551107)
HRV/YOK/07/7(AB550346), HRV/YOK/07/61(AB550365), HRV/YOK/08/107(AB550377), HRV/YOK/08/110(AB550379), HRV/YOK/08/112(AB550380)
HRV/YOK/07/11(AB550348), HRV/YOK/07/15(AB550350), HRV/YOK/07/19(AB550352), HRV/YOK/07/24(AB550355), HRV/YOK/07/25(AB550356), HRV/YOK/07/36(AB550358), HRV/YOK/08/103(AB550374), HRV/YOK/08/131(AB550389), HRV/YOK/08/153(AB550396), HRV/YOK/08/167(AB550402), HRV/YOK/08/169(AB550403), HRV/YOK/08/171(AB550404)
HRV/YOK/07/5(AB550345), HRV/YOK/07/20(AB550356), HRV/YOK/07/41(AB550368), HRV/YOK/08/100(AB550379), HRV/YOK/09/163(AB550400), HRV/YOK/09/164(AB550401)
HRV/YOK/07/2(AB550343), HRV/YOK/07/10(AB550347), HRV/YOK/07/12(AB550349), HRV/YOK/07/18(AB550351), HRV/YOK/07/23(AB550353), HRV/YOK/07/55(AB550371), HRV/YOK/08/86(AB550377), HRV/YOK/08/106(AB550382), HRV/YOK/08/120(AB550386), HRV/YOK/08/126(AB550388), HRV/YOK/08/159(AB550398)
RSV/YOK/08/116(AB551050) + HRV/YOK/08/116(AB550381), RSV/YOK/08/145(AB551068) + HRV/YOK/08/145(AB550392)
RSV/YOK/07/1(AB551033) + HRV/YOK/07/1(AB550342), RSV/YOK/08/117(AB551051) + HRV/YOK/08/117(AB550382), RSV/YOK/08/119(AB551052) + HRV/YOK/08/119(AB550384)
RSV/YOK/08/140(AB551064) + HRV/YOK/08/140(AB550392)
RSV/YOK/07/3(AB551034) + HRV/YOK/07/3(AB550344)
RSV/YOK/07/42(AB551087) + HRV/YOK/07/42(AB550361), RSV/YOK/07/47(AB551090) + HRV/YOK/07/47(AB550363)
RSV/YOK/08/118(AB551108) + HRV/YOK/08/118(AB550363)
RSV/YOK/07/28(AB551082) + HRV/YOK/07/28(AB550365),
RSV/YOK/07/45(AB551088) + HRV/YOK/07/45(AB550405), RSV/YOK/07/46(AB551089) + HRV/YOK/07/46(AB550370),
RSV/YOK/07/67(AB551099) + HRV/YOK/07/67(AB550375)
Seasonal variations of detected viruses
Homology, phylogenetic analysis, and pairwise distances of RSV and HRV
Pairwise distances and homology of RSV and HRV strains based on nucleotide sequences
82.0 - 100
83.5 - 100
0.063 ± 0.043
0.035 ± 0.034
74.2 - 100
92.8 - 100
0.060 ± 0.040
0.029 ± 0.014
66.4 - 100
66.5 - 100
0.202 ± 0.031
0.200 ± 0.038
68.1 - 100
99.5 - 100
0.204 ± 0.039
0.002 ± 0.003
41.0 - 100
52.2 - 100
0.263 ± 0.069
0.254 ± 0.077
We detected and genetically analyzed major ARI viruses including RSV, HRV, HMPV, and HPIV in samples from 115 Japanese children with acute wheezing during a 17-month period (November 2007 and March 2009). These viruses were detected in over 80% of the patients. The dominant viruses were RSV and HRV, and both were detected in over 10% of the patients. In addition, these viruses were confirmed as belonging to various subgroups, genotypes, or species. All three species of HRV detected showed wide genetic diversity (more than 30% divergence). Interestingly, RSV was the dominant species detected in patients with no history of wheezing and/or asthma, while HRV was dominant in patients with a history of wheezing and/or asthma. The results suggested that RSV and HRV were major ARI viruses regarding virus-induced acute wheezing in the present study.
It is suggested that various respiratory viruses such as RSV, HRV, HMPV, HPIV, EV, InfV, AdV, and HBoV are detected in patients with lower respiratory tract infections including bronchiolitis and pneumonia [6, 7]. These viruses are also detected in cases of acute wheezing [6, 7]. Thus, they may be associated with both lower respiratory tract infection and acute wheezing in children [6, 7]. At present, this disease status is recognized by physician and pediatrician as virus-induced asthma [28, 29]. It may be important to address the genetic properties of ARI viruses associated with these diseases. However, few studies have been conducted into the genetic analysis of these viruses in acute wheezing. To better understand the relationships between viral properties and acute wheezing, it may be important to genetically analyze ARI viruses detected in the wheezy children. We studied the molecular epidemiology of these respiratory viruses detected in Japanese children with acute wheezing. To the best of our knowledge, the present study is the first to report the detection of RSV, and HRV-A, -B, -C with different genetic characteristics in Japanese children with acute wheezing.
Many studies suggest that RSV is a major candidate as an inducer of acute wheezing [4, 10, 11] and it may infect all children under the age of 2 years [10, 11]. Furthermore, some of these children may develop bronchiolitis and/or pneumonia with acute wheezing . Sugai-Goto et al. demonstrated that genotypes and the major genes (F, G, and N) of RSV isolated from hospitalized children with bronchiolitis or bronchopneumonia accompanied by acute wheezing were not significantly different when compared with RSV strains detected from upper respiratory tract infections . These viruses belong to subgroup A, genotype GA2 and subgroup B, genotype BA . Furthermore, Nakamura et al. showed similar genetic data from various acute respiratory infections in Okinawa, Japan . Our findings regarding the properties of G gene in the RSV strains detected were comparable with the above-mentioned reports. In contrast, it has been suggested that a specific genotype, GA3 type virus, might be associated with a significantly greater severity of illness . Riccetto et al. demonstrated that the severity of illness of RSV infection in infants can be associated with other factors such as body weight and prematurity , and any association between the viral properties and pathogenicity of RSV has yet to be elucidated. Another report suggested that host immunity such as TLR4 polymorphism is linked to symptomatic RSV . Thus, both the antigenicity of the viruses and host immune conditions may play important roles in the pathophysiology of severe respiratory infections such as bronchiolitis, pneumonia, and virus-induced asthma [1, 34].
For a long time, HRV was simply thought to be causative agents of the mild common cold . In general, this acknowledgement may not be incorrect in non-asthmatic people . However, it is suggested that HRV induces wheezing and exacerbation of symptoms in most asthmatics . However, the molecular epidemiology of each HRV species is not yet known, because HRV is relatively difficult to isolate and detect. Thus, non-culturable HRV-C was only recovered a few years ago. Very recently, Mizuta et al. demonstrated that HRV-A isolates showed wide genetic diversity, and some viruses belonging to specific clusters of the phylogenetic tree of HRV-A isolates might be associated with bronchiolitis . In addition, a new study suggested that HRV-C has a stronger link to virus-induced asthma than HRV-A and -B strains . However, our results did not reveal a similar tendency, although the reasons for this are unknown.
In this study, both RSV and HRV were detected from over 10% of patients with acute wheezing. Chung et al. demonstrated that both RSV and HRV were detected in 3.9% of Korean children with acute wheezing . Thus, our data and that of other studies may be comparable, although the percentages of virus detection differ. We additionally compared the severity of clinical symptoms between dual virus-detected patients and those in whom RSV or HRV was detected alone. However, there were no significant findings. In addition, RSV, HRV, HMPV, HPIV, EV, InfV, AdV, and HBoV were not detected in over 10% of patients. Although we were unable to explain why, it might be that other respiratory viruses and bacteria were involved.
It should be noted that some respiratory viruses might be detected in healthy children [1, 38–40]. As mentioned above, various species of HRV have been relatively frequently detected in healthy children (around 10-20%) [39, 40], although RSV was detected less frequently in healthy and asymptomatic persons . Thus, to better understand the etiology of these viruses, it may be important to determine the prevalence of these viruses in healthy children. A limitation of this study is that we did not examine such prevalence in healthy children and instead focused mainly on detailed molecular epidemiological analysis of various respiratory viruses detected in children with acute wheezing. Additional molecular epidemiological studies of viruses detected in wheezy and healthy children would be of value.
In the present study, HMPV and HPIV were detected in samples from the subjects, albeit rarely (each virus was detected in one of only two patients). It is suggested that HMPV and HPIV are also associated with bronchiolitis and bronchopneumonia . However, it is not known how these viruses are linked to the induction of wheezing and exacerbation of asthma . A previous study suggested that sputum from HPIV infection contains tryptase due to activation (degranulation) of mast cells, and this activation may strongly induce an asthmatic attack . Thus, HPIV infection may induce asthmatic conditions . Additional studies regarding the relationships between HPIV and HMPV infection and virus-induced asthma are warranted.
Our data suggested that both RSV and HRV with various genetic characteristics were associated with acute wheezing illness in Japanese children. In particular, HRV shows widely genetic diversity. Larger studies to examine the detailed genetic characteristics of the various respiratory viruses detected in wheezy and healthy children may be needed.
respiratory syncytial virus
human parainfluenza virus
DNA Data Bank of Japan
We thank Drs. Hirotaka Motoi and Yoshinori Kobayashi for constructive discussion. This work was supported by a Grant-in-Aid from the Japan Society for Promotion of Science and for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labour and Welfare.
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