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INTRODUCTION
Human metapneumovirus (hMPV), which was identified recently in the Netherlands in respiratory samples from children with acute respiratory symptoms, is enveloped non-segmented RNA virus belonging to genus Metapneumovirus, family Paramyxoviridae (1, 2). hMPV has been emerged as an important etiologic agent of upper and lower respiratory tract infections in individuals of all age, especially in young children (3-8). The clinical manifestations associated with hMPV ranges from upper respiratory tract infection including cough, rhinorrhea, to lower respiratory tract diseases such as severe bronchiolitis and pneumonia, similar to those of human respiratory syncytial virus (RSV) (1, 9). Although the prevalence of hMPV infection by using reverse transcriptase PCR (RT-PCR) has been reported in several countries including U.S.A., Canada, Australia, England, Japan, and Hong Kong (3, 4, 8, 10-12), it is still unclear in Korean children. The purpose of our study was to determine the frequency of hMPV in hospitalized children with acute respiratory tract disease in Korea.
MATERIALS AND METHODS
Respiratory samples and study subjects
Between December 2003 and February 2005, we collected nasal aspirates from 381 children under 15 yr (median age=19 months; range 0-144 months) who was hospitalized with respiratory infections in the Department of Pediatrics, Sanggye-Paik Hospital, Inje University, Seoul, Korea. The specimens were immediately transferred to the laboratory and each sample analyzed for RSV, adenovirus, influenza virus A and B, and parainfluenza virus by immunofluorescent assay using fluorescein-labelled monoclonal antibodies specific for each virus (IMAGEN; Dako Diagnostics Ltd., Cambridgeshire, U.K.). The specimens were stored at -70℃ until further testing. An informed consent was obtained at admission from patient's parents. The Ethics Committee of Inje University College of Medicine, approved the study.
RT-PCR
RNA was extracted using the QIAmp Viral RNA Mini Kit (Qiagen GmbH, Hilden, Germany). To synthesize cDNA, 0.2 µg of of RNA was incubated in a solution containing 5 µM random hexamer (Bioneer, Daejeon, Korea), 1 mM of each dNTP, 2 units of RNase inhibitor, and 10 units of reverse transcriptase (Bioneer, Daejeon, Korea) in a final volume of 20 µL at 42℃ for 60 min. The cDNA (1 µL) was subjected to PCR analysis to detect the hMPV F-gene. Published F-gene sequences of hMPV were used for PCR for the detection and sequencing of hMPV (13). The forward primer sequence was 5'-GCAACAATTGAA CTGATCTTCAGGAAAC-3'(AY304360-2; nucleotides 627 to 749) and the reverse primer sequence was 5'-GCAACATTGAACTGATCTTCAGGAAAC-3'(AY304360-2; nucleotides 1350 to 1376). When the first PCR was negative, a nested PCR was performed using forward primer [5'-ACATGCCAACATCTGCAGGACAAATAAAAC-3'(AY304360-2; nucleotides 698 to 727)] and the reverse primer [5'-ACAT GCTGTTCACCTTCAACTTTGC-3'(AY304360-2; nucleotides 1285 to 1307)]. Amplification was performed in a 20 µL reaction mixture containing the following: 0.2 µM of each primer; 200 µM dATP, dCTP, dTTP, and dGTP; 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2, 40 mM KCl; 1 unit of Taq polymerase (Bioneer, Daejeon, Korea); and 1 µL of cDNA sample (or the first round PCR product). Amplification was preformed in a DNA thermal cycler (iCycler Thermal Cycler, Bio-Rad Laboratories, Hercules, CA, U.S.A.). The amplification conditions were as follows: 95℃ for 15 min followed by 35 cycles of amplification (95℃ for 1 min, 55℃ for 1 min, 72℃ extension for 1 min) and a final extension at 72℃ for 3 min. The amplified DNA fragments were size-separated on a 2% agarose gel with ethidium bromide, and visualized with UV light (Fig. 1). To examine the sensitivity and specificity of RT-PCR, ten of hMPV-positive RNA samples (0.1 µg) and ten of RSV-positive samples were used. The amplified products were detected to 10 pg of RNA and no product was detected from RSV positive samples. cDNA from a clinical specimen was used as the positive control.
Phylogenic tree analysis
The PCR products were purified using Accuprep PCR Purification kit (Bioneer, Daejeon, Korea) and subsequently sequenced directly on both strands using BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster, CA, U.S.A.) with ABI prism 377 analyzer (Applied Biosystems). Phylogenic trees were constructed using MEGA version 3.0 (14). Sequences are available from GenBank under accession No. DQ092710-DQ092737.
RESULTS
Detection of hMPV by RT-PCR
In total 381 samples, negative samples in which any viral pathogen could not be identified by IFA were 231 cases (60.6%). Respiratory syncytial virus was detected in 129/381 children (33.8%). In addition, parainfluenza virus (n=9), influenza A (n=9), influenza B (n=2), and adenovirus (n=1) were identified (Fig. 1).
hMPV was detected using RT-PCR in 28 of 381 children (7.3%) and in 12.1% of children who were not infected with other common respiratory viruses. Of the 28 RT-PCR positive samples for hMPV, 25 cases (89.2%) were collected between November 2004 and February 2005 (Table 1).
Clinical findings of hMPV positive patients
Clinical manifestations of the 28 children with hMPV infection are presented in Table 2. The median age was 15 months (range 7-86 months) in the 28 patients. Symptoms included cough (92.8%), fever (60.2%), sputum (42.8%), rhinorrhea (39.2%), dyspnea (17.8%), diarrhea (7.2%), and vomiting (7.2%). Expiratory wheezing and crackles were observed in 21.4% and 25% of the patients, respectively. Abnormal infiltrates of chest radiograph were noted in 10 (35.7%) of the 28 patients. The hMPV-infected children were diagnosed as having pneumonia (35.7%), bronchiolitis (28.5%), bronchial asthma exacerbation (14.3%), croup (10.7%), and upper respiratory tract infection (7.1%). Six of 28 patients had a history of reactive airway disease. A 27-month-old girl who was admitted with respiratory distress expired of cardiorespiratory failure 2 days after admission without known bacteremia. Only one patient of the 28 patients had underlying medical problem, chronic lung disease of prematurity.
Phylogenetic analyses of hMPV strains
Amplified products corresponding to part of the hMPV F gene (750 bp) were visualized on 1.5% gels. The PCR products from each positive specimen were sequenced and were consistent with hMPV. The 28 hMPV strains detected in this study were classified into two distinct F lineages, 26 strains belonged to genogroup A (A2) and two strains to genogroup B (Fig. 2).
DISCUSSION
This study shows that hMPV is an important etiologic agents among Korean children hospitalized with acute respiratory tract infections. The prevalence of hMPV detected in nasopharyngeal samples from children with respiratory infections of unknown etiology has varied from 1.5% to 21% (1, 11, 15-18), but it has been unclear in Korea due to lack of published reports. The difference of prevalence in several studies may be explained by yearly variation in incidence, different group of patients and primers used in PCR assays for hMPV (15-18). Because we studied hospitalized children only, actual prevalence of hMPV infections may be higher. Some researcher suggested that hMPV cause lower respiratory tract infection in healthy children at a relatively high frequency (19).
It seems that hMPV shows a seasonal variation, with sporadic epidemics. In a study, hMPV was the most common virus isolates during the winter season 2002-2003 in children hospitalized for acute respiratory tract infection, but no specimen was found positive for hMPV in the following season (18). Maggi et al. (7) reported that the incidence of hMPV infection in infants varied from season to season over 3 yr period. The peak time of hMPV transmission in the Netherlands was in December, in Canada in April, and in Hong Kong in spring and early summer (1, 4, 12). Some reported that significant hMPV activity occurred in every month of the year with the peak incidence in the winter and spring seasons (19, 20). In our study, we detected 25 hMPV positive samples from November to February during the winter season of 2004-2005, with a peak in November. In the spring season of 2004, we also detected three hMPV positive samples. But, the exact prevalence of hMPV during the winter season of the 2003-2004 in this study is uncertain due to small number of available respiratory specimens.
The clinical features of children with hMPV positive samples observed in our study were similar to those of previous reports (8, 20). Exacerbation of asthma was observed in 14% (4/28) of hMPV only positive patients in this study. Like other viruses, such as hRSV and rhinoviruses, which have been suggested as important triggers of asthma exacerbation in children, an association between hMPV and asthma exacerbation has been implicated. In a Finnish study, the positive rate of hMPV was 32% (10/31) in hospitalized children with acute wheezing during the period of peak of hMPV infection (21). Although it may be possible that asthmatic bronchitis is triggered by hMPV, it is not certain to conclude definitely the association between and bronchial asthma (22). hMPV has been suggested as a common and frequent etiologic agent of bronchiolitis in young children (8, 23, 24). Some reported that co-infection with RSV and hMPV may be a possible cause of increasing clinical severity in hospitalized children (25, 26), but others reported different results (7). The limitations of this study are relatively short study period, small number of samples in the first half of the 2004, which is not sufficient to know the exact epidemiologic characteristics. The data of combined infections of hMPV with other common respiratory viruses are also lacking.
RT-PCR is more useful method than cell culture, which has characteristics of slow growth and mild cytopathic effect of hMPV, and is now used commonly in epidemiologic studies in various populations (27, 28). The strains of hMPV can be divided into two major genetic lineages (1, 3). Variability of hMPV genes may affect the sensitivity of study due to limited available sequence information. We have performed the analysis of F-gene as van den Hoogen et al. described (13), which is known to have highly conserved sequence and allow the differentiation of all four sublineages in several studies (5, 8, 9, 13). Phylogenetic analysis of F gene sequences of our strains showed highly nucleotide identity with viruses belonging to genogroup A during the winter season of 2004-2005, all belonged to A2. Otherwise, three hMPV positive samples detected in spring of 2004 belonged to genogroup B2 in two, genogroup A1 in one. An Italian study reported striking variation in the overall circulation rates and co-circulation of multiple strains in the same area in different years (18).
This study suggests that hMPV may be an important etiologic agent of respiratory tract infection requiring hospitalization in children and two distinct groups of hMPV are co-circulating in Korea.
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