detection and genetic diversity of human metapneumovirus in
TRANSCRIPT
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Detection and Genetic Diversity of Human Metapneumovirus in 1
Hospitalized Children with Acute Respiratory Infections in Southwest 2
China 3
Cui Zhang﹟, Li-na Du﹟, Zhi-yong Zhang﹟, Xian Qin, Xi Yang, Ping Liu, Xin Chen, Yao Zhao, En-mei 4
Liu, Xiao-dong Zhao* 5
6
Corresponding to: 7
Xiaodong Zhao, MD., Ph.D 8
Division of Immunology, Children’s Hospital of Chongqing Medical University 9
Ministry of Education Key Laboratory of Child Development and Disorders 10
Key Laboratory of Pediatrics in Chongqing 11
Chongqing International Science and Technology Cooperation Center for Child Development and 12
Disorders 13
Chongqing 400014, China 14
Tel: 86 23 6362 2554 15
Fax: 86 23 6360 2136 16
Email: [email protected] 17
18
﹟ these authors contributed equally to the article 19
20
Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00809-12 JCM Accepts, published online ahead of print on 12 June 2012
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ABSTRACT 21
Human metapneumovirus (hMPV) is a main pathogen causing respiratory tract infection in 22
susceptible population, particularly in children and the elderly. Specimens were collected from 23
hospitalized children with acute lower respiratory tract infections (ALRTI) and the hMPV was 24
detected by using real-time RT-PCR. The full length G gene of hMPV was amplified by reverse 25
transcription PCR (RT-PCR). A total of 1410 nasopharyngeal aspirates (NPAs) were collected from 26
April 2008 to March 2011, and 114 (10.2%) were positive for hMPV. Most of hMPV positive children 27
were aged <5 years. The hMPV infection rate peaked in spring-summer season of 2008-2009 and 28
2009-2010, while hMPV circulated predominantly during winter-spring season of 2010-2011. The 29
full length G gene of 23 hMPV strains was amplified and group A and B viruses accounted for 30
95.7% (22/23) and 4.3% (1/23), respectively. Genotype A2b of hMPV appeared to be predominant 31
during the study period. Three genotypes (A2b, A1 and B1) were prevalent in the epidemic season 32
of 2008-2009, and only genotype A2b was identified in the other two seasons (2009-2010 and 33
2010-2011). The G gene of hMPV was predicted to encode proteins with four different lengths, in 34
which one with 210 amino acids was firstly identified in China. These findings suggest that hMPV 35
was an important pathogen of ALRTI in pediatrics, especially those aged <5 years. Group A2b of 36
hMPV likely predominates in Southwest China, and other genotypes also circulate. 37
38
Keywords: human metapneumovirus; genetic variability; epidemiology; attachment protein 39
40
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INTRODUCTION 42
Human metapneumovirus (hMPV) was first isolated in 2001 from nasopharyngeal aspirates (NPAs) 43
of children with acute lower respiratory tract infections (ALRTI) in the Netherlands (27). 44
Subsequently it has been identified all over the world (2, 20, 21, 24). Children aged <5 years, elderly 45
adults and immunocompromised patients are at an increased risk for severe hMPV infection (23, 46
24). Morphologically, hMPV consists of a negative-sense, single stranded and non-segmented RNA 47
that encodes at least 9 distinct proteins (26). Among them, the two major transmembrame 48
glycoproteins, G and F proteins, can stimulate the production of protective immune responses and 49
therefore are antigenically significant (11). Unlike the relatively conserved F protein (95% homology 50
at the amino acid level between group A and B hMPV), the G protein is highly variable with only 51
53% amino acid homology between group A and B（3）. 52
53
Similar to respiratory syncytial virus (RSV), hMPV strains vary genetically and antigenically and 54
have been classified into two broad groups: group A and group B (4, 26), with each group divided 55
into genetic subgroups 1 and 2 (12). More recently, phylogenetic analysis showed a further 56
bipartition of subgroup A2 into two new genetic clusters designated A2a and A2b (3). Both antigenic 57
group A and group B were noted to co-circulate in the same city during the epidemic periods and 58
had various patterns of predominance. 59
60
Among all the sub-lineages of hMPV, the A2 sub-lineage shows the greatest diversity. Antigenic 61
variability is thought to contribute to re-infection throughout the life and may pose a challenge to 62
vaccine development. Future planning for vaccine development requires in depth understanding of 63
genetic composition of the hMPV strains prevalent in target population. 64
65
However, less information is available on the distribution pattern of hMPV strains in Southwest 66
China. We had conducted a two-year epidemiological study on hMPV in Chongqing area from April 67
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2006 to March 2008 and genotype A2 was found to be the most predominant one during the study 68
period (6). Thus, ongoing epidemiological surveillance in a consecutive manner will help assess the 69
disease burden and genetic diversity of hMPV. The aim of the present study was to evaluate the 70
genetic diversity of the G protein gene of hMPV in hospitalized infants and young children with 71
ALRTI in the past three epidemic periods in Chongqing. Information on distribution of hMPV 72
genotypes in China will be beneficial for the development of hMPV vaccines. 73
74
MATERIALS AND METHODS 75
Collection of specimens 76
From April 2008 to March 2011, NPAs were obtained on three fixed days of each week from all 77
children admitted to Chongqing Children’s hospital due to ALRTI. ALRTI was diagnosed according 78
to the criteria developed by World Health Organization. The specimens were immediately placed at 79
4°C in tubes containing 3 ml of cold viral transport medium (PBS, 100 U/µL, penicillin and 100 80
µg/mL streptomycin) and transported to the Department of Virology within 4~6 h. Specimens were 81
vortexed and centrifuged at 1500 g for 10 min at 4°C to separate the cells for direct 82
immunofluorescence assay (DFA) to detect seven common viral respiratory pathogens ( respiratory 83
syncytial virus, parainfluenza virus 1, 2 and 3, influenza virus A and B and adenovirus) on the same 84
day. Cell pellet was washed with PBS for three times and added to the cytospun slides after being 85
adjusted to suitable concentration. Subsequently cells were fixed with acetone for 10 minutes after 86
being dried in room temperature. A kit from Chemicon was used to stain the cells and the 87
manufacturer’s instructions were followed. The supernatants were stored at -70 °C for use. This 88
study was approved by the Ethics and Research Council of Chongqing Children’s Hospital and 89
informed consent was obtained from their parents or guardians. 90
91
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RNA extraction and cDNA synthesis 93
RNA was extracted from 140 µl of frozen specimens with an RNeasy mini kit (QIAGEN, Germany) 94
according to the manufacturer’s instructions. The dried RNA was dissolved in 50 µl of DNAse-free, 95
RNAse-free water. For cDNA synthesis, 100 ng of total RNA was used as a template for reverse 96
transcription polymerase chain reaction (RT-PCR) with random primers. 97
98
Real-time PCR assays 99
All NPAs were tested for hMPV by real-time PCR. Real-time PCR primers and probes for hMPV F 100
gene were used for the diagnosis of hMPV infection and designed based on the sequence in 101
GenBank (accession number: DQ336144). Viral cDNA was amplified by using a real-time PCR with 102
the Premix Ex Taq Kit (Perfect Real Time, TaKaRa, China) in a LightCycler instrument (Roche 103
Diagnostics, American). Two microliters of cDNA was added to a 22uL real-time PCR master 104
mixture containing 200 uM deoxynucleoside triphosphates, 1.5 mM MgCl2, 1.5 U of Taq DNA 105
polymerase, and 50 pM each primer and probe. The thermocycling protocol was 94°C for 2 min, 106
followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final extension for 107
7 min at 72°C. The positive was defined at cycle threshold (Ct) of ≤36. Negative and positive control 108
was introduced in each real-time PCR. Positive control was the cDNA from a recombinant hMPV 109
infected Vero-E6 cells. The sequence of real-time PCR primers and probes for hMPV F gene was 110
as followed: hMPV-F(F): 5’-CGTTTCTAACATGCCGACATCTG-3, hMPV-F(R): 111
5’-GCTCCCGTAGACCCCTATCAG-3’, Probe 112
5’(FAM)-CCCTTTCTTCGCACCATCGCACGG(Eclipse)-3’, To verify the presence and sequence of 113
the virus, traditional RT-PCR was also used to amplify the hMPV F and G gene to ensure 114
reproducibility. All PCR assays were verified to be able to amplify F and G gene of the recombinant 115
hMPV positive control. 116
117
PCR amplification of full length G protein gene 118
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We selected 23 hMPV strains by using random digits table, including seven in the epidemic season 119
of 2008-2009, seven in 2009-2010 and nine in 2010-2011. Primers used to amplify the full length G 120
protein were hMPVGF 5'-GAGAACATTCGAGCAATAGACATG-3' and hMPVGR 121
5'-AGATAGACATTAACAGTGGATTCA-3' .DNA amplification was carried out by 35 cycles of 94 °C 122
for 1 min, 58 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 10 min. Five 123
microliters of PCR products were subjected to 1.5% agarose gel electrophoresis, stained with 124
GoldViewTM nucleic acid stain (SBS genetech) and visualized under UV light. 125
126
DNA sequencing 127
The PCR products were purified with a QIAquick gel extraction kit (QIAGEN, Germany) according to 128
the manufacturer’s instructions. The purified PCR products were sequenced in both directions using 129
the same PCR primers for amplification on an ABI PRISM 310 genetic analyzer (Applied 130
Biosystems) by using an ABI PRISM BigDye Terminator cycle sequencing ready reaction kit 131
(Applied Biosystems). 132
133
Phylogenetic analysis 134
Nucleotide and amino acid sequences of group A and B hMPV were compared independently with 135
the available hMPV sequences in the GenBank database using ClustalX version 1.81 and manually 136
edited with BioEdit version 7.0.1. Phylogenetic trees were constructed by the neighbor-joining 137
method in MEGA version 5. The statistical significance of the tree topology was tested by 138
bootstrapping (1,000 replicas). The phylogenetic analysis included the hMPV sequences in 139
GenBank belonging to four different subgroups: A1: CHN03-06( EF571502), FL-8-01( AY295989), 140
CAN99-81(AY574224) and NL1-00(AY296035); A2a: Arg-4-00(DQ362953), CAN97-83(AY297749); 141
A2b: CHN07-06(EF571506), BJ1819(DQ270215), BJ1887(DQ843659) and KOL-30-06(HQ599198); 142
BI: CHN12-06(EF571511), NL-11-00(AY296035), Arg-1-00(DQ362958) and NL-1-99(AY296034); 143
B2: CAN98-75(AY297748). 144
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Nucleotide sequence accession numbers：The GenBank accession numbers of the nucleotide 146
sequences obtained in the present study are JX082172-JX082194.147
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RESULTS 148
Epidemiology of hMPV 149
A total of 1410 specimens were collected from inpatients with ALRTI from April 2008 to March 2011, 150
and 144 (10.2%) were positive for hMPV by real-time RT-PCR. Positive rate of hMPV was 11.8% 151
during 2008-2009, 7.31% during 2009-2010 and 12.8% during 2010-2011. The prevalence of hMPV 152
in Chongqing was high in April, May and June of 2008-2009 and 2009-2010 while hMPV circulated 153
predominantly during the late winter and spring of 2010-2011 (Figure 1). 154
155
hMPV infected children were aged 1~71 months (median: 21 months). Most infected individuals 156
were infants aged 0~6 months, accounting for 45.9% of subjects positive for hMPV infection. Infants 157
aged 6-12 months and children aged 1~2 years accounted for 10.4% and 15.3%, respectively. 158
Children aged 2~5 years accounted for 20.1%. (Table 1） 159
160
All 144 hMPV-positive patients had clinical symptoms. The main clinical presentations included 161
fever (n=62; 41.5%), cough (n=142; 99.1%), wheezing (n=102; 71.1%) and cyanosis (n=110; 162
85.0%). The hMPV-infected children were diagnosed with bronchopneumonia (61.1%; 88/144), 163
bronchiolitis (19.4%; 28/144), bronchial asthma exacerbation (15.9%; 23/144) and acute upper 164
respiratory infection (3.4%; 5/144). (Table 2）.Eighteen out of 144 inpatients had co-morbidities 165
including congenital heart disease (15/144), tracheomalacia (3/144) and congenital laryngeal stridor 166
(1/144). Eight patients developed respiratory failure and one required mechanical ventilation. 167
Among the eight patients with respiratory failure, congenital heart disease was identified in 3 168
patients and co-infection of other respiratory viruses was found in 4 patients, including 3 with RSV 169
and 1 with PIV3. 170
171
Other respiratory viruses co-infecting with hMPV 172
In addition, 29.2% (n=42) of hMPV positive patients were found to have concomitant infection of 173
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other respiratory viruses, including 19 with RSV, 11 with PIV3, 6 with ADV, 6 with InfB. Among them, 174
18 (42.9%) were younger than 1 year and diagnosed with ALRTI including pneumonia (n=12) and 175
bronchiolitis (n=3). 176
177
Phylogenetic analysis and genotype distribution patterns 178
Phylogenetic analysis of 23 hMPV strains in Chongqing confirmed two main genetic lineages A and 179
B. Interestingly, all the strains in Chongqing were clustered as A2b, A1 and B1, and subgroup B2 180
was not identified. During 2008 -2009, subgroup A2b，A1 and B1 co-circulated, with 82% (n=9) 181
belonging to A2b. Only one A1 and one B1 hMPV were detected in the same season. Interestingly, 182
only subgroup A2b was identified in the next two epidemic seasons (2009-2010 and 2010-2011). 183
The results of phylogenetic analysis of 23 hMPV strains were showed in Figure 2. 184
185
186
Sequencing of the full length of G protein gene revealed the homology ranged from 55.8- 58.4% at 187
nt level and 44.1-49.7% at the aa level between groups A and B isolates. Subgroup B1 strains 188
shared high homology with the prototype strain NL/1/99 (94.9% at nt.; 91.9% at aa), whereas 189
subgroup A2b strains revealed homology with the prototype strain CAN97-83 ranged from 190
89.6−99.8% at nt level and 82.3-84.9% at aa level. Subgroup A1 strains shared high homology with 191
the prototype strain CAN99-81 (100% in nt.). Compared with the prototype A2 strains circulating 192
from April 2006 to March 2008 in Chongqing, 21 A2 strains prevalent from April 2008 to March 2011 193
revealed homology ranging from 95.7-97.7% at nt level and 94.3-96.2% at aa level. The only one 194
subgroup B1 strains circulating in the present study shared high homology with the prototype B2 195
strains prevalent from April 2006 to March 2008 in Chongqing (92.8% at nt.; 90.7% at aa). 196
197
Amino acid analysis 198
The comparisons of deduced aa sequence of G protein gene of strains in Chongqing with their 199
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prototype strains revealed that intracellular and transmembrane regions were highly conserved 200
across the strains (supplemental data). Most of the changes in aa were observed in the extracellular 201
domain due to nt substitution. Changes in the stop codon were observed among strains of different 202
subgroups {nt 644 (UAG); nt 671 (UAG), nt 694 (UGA); 709(UAA)}, which corresponded to variable 203
lengths in polypeptides. Strains from subgroup A2b had two different stop codons resulting in G 204
proteins with 210aa (UAG) and 219aa (UAG), whereas subgroup B1 strains terminated at UGA stop 205
codon and exhibited protein with 231 aa. The subgroup A1 strains terminated at UAA stop codon 206
and exhibited protein with 236 aa. 207
208
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DISCUSSION 211
212
Respiratory tract infections are the major cause of hospitalization of young children in the winter 213
worldwide. Viruses most frequently associated with respiratory infections include rhinoviruses, 214
coronaviruses, influenza viruses, parainfluenza viruses, RSV and adenoviruses. In 2001, a new 215
member of the paramyxoviridae associated with respiratory infections, hMPV, was described in the 216
Netherlands. Serological studies indicated that nearly all children had been infected by age of 5 217
years (26). Some previous studies have suggested that hMPV is an important causative pathogen 218
for ALRTI in children in China (13,16), but the epidemiology of hMPV infection in countries with 219
large population remains largely unknown due to the limitations in these studies. 220
221
In the present study, 1410 specimens were collected from pediatric patients with ALRTI from April 222
2008 to March 2011 and tested for hMPV by real-time RT-PCR. The enrolled patients accounted for 223
a significant proportion of total inpatients with ALRTI in that period. Although we designed to enroll 224
all newly admitted patients in three fixed days in every week, the results were not necessarily free 225
from selection bias because we only collected NPAs in the daytime and some of admitted patients 226
refused to provide NPAs. The age and sex distributions and diseases between the enrolled 1410 227
patients in this study and the whole 9231 inpatients in the same period were not statistically different, 228
reflecting that the selected subjects may be relatively representative. To detect hMPV, F protein 229
gene was chosen becasue it is highly conserved and has been used in previous studies (16,17). 230
The frequency of hMPV infection varied from 5% to 30% among hospitalized children with ALRTI in 231
most studies (1, 18, 22, 25,) and 7.31% to 12.8% annually in this study by using real-time PCR to 232
detect the hMPV F protein gene. The prevalence of 46% hMPV infection varied from year to year, 233
with the lowest in the epidemic season from 2009 to 2010. The general prevalence of hMPV 234
infection was relatively high as compared with that in some other regions, probably due to a higher 235
population density in this city. Thus, hMPV appeared to bring a significant disease burden in 236
Chongqing and was a common prevalent virus for ALRTI. Several seroprevalence studies have 237
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previously shown that 90-100% of children are infected by the ages of 5-10 years. Comparable to 238
other reports (4, 5, 20), the present study showed 71.4% of children with hMPV infection were aged 239
<2 years. The high prevalence of hMPV infection in little children (<6 months) might be attributed to 240
weak immune function. The symptoms of hMPV infection among children were similar to those 241
described previously in respiratory syncytial viral infection (11, 21, 27). We found the most common 242
clinical manifestations of hMPV-associated ALRTI were fever, cough, wheezing and cyanosis. The 243
main diseases ranged from acute laryngotracheobronchitis, asthmatic bronchitis, bronchitis, to 244
bronchiolitis and pneumonia. We found hMPV infection was related to wheezing, asthma 245
exacerbations or bronchiolitis, as previous studies described (7, 8, 13). Consistent with our findings, 246
other studies have shown that hMPV infection may lead to severe respratory distress requiring 247
prolonged hospitalization, mechanical ventilation, and admission to an intensive care unit (2, 11, 14). 248
Thus hMPV infection is associated with a substantial clinical and likely economic impact. 249
250
Although the winter-spring season is traditionally regarded as the typical epidemic season of 251
hMPV infection in the temperate regions, our results showed that the hMPV infection peaked in the 252
spring-summer season (April-June) of 2008-2009 and 2009-2010 and winter-spring season 253
(November-February) of 2010-2011. The hMPV seasonality observed in this study was comparable 254
to that reported in Japan, which showed a biennial hMPV infection, with a peak between November 255
and March in odd years and between March and June in the successive even years (19).. The 256
difference in the hMPV seasonality between our study and other studies is unclear. Previous 257
studies have shown that climate can affect the seasonality of hMPV infection (14, 18, 22). However, 258
the climate records for Chongqing fail to indicate significant variability in the same months of 259
different years. Apparently, more studies with long period are needed to elucidate the real pattern of 260
hMPV circulation in this region. 261
262
Because of the similar seasonal distribution of hMPV and other respiratory viruses, the potential 263
co-infection likely existed. Some studies have found a co-infection rate of 10%-75% in hMPV and 264
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other respiratory virus (10, 15, 23). In this study, co-infection of hMPV with other respiratory viruses 265
was detected in 29.2% of hMPV-infected patients, and the co-infected viruses included respiratory 266
syncytial virus, influenza virus, parainfluenza virus and adenovirus. Of 25 patients identified as a 267
co-infection with RSV, 3 developed respiratory failure, indicating that hMPV and RSV co-infection 268
may lead to a more severe disease than infection alone. 269
270
Phylogenetic analysis of G protein gene sequences in the present study showed both the group A 271
and B hMPV circulated in Chongqing. Generally, group A hMPV is reported more common than 272
group B (9, 14, 16). In the present study, among 23 hMPV strains, 22 were grouped as A and 1 as B 273
(subgroup B1). HMPV group A was a likely predominant virus circulating in Chongqing, which was 274
in consistent with other studies (8, 17, 20). Genotype A2b, A1 and B1 viruses were detected in the 275
epidemic season of 2008-2009, of which A2b were more common. In 2009-2010 and 2010-2011, 276
only genotype A2b viruses were identified. Genotype B2 viruses were not identified in the present 277
study. Indeed, circulation of hMPV of different genotypes has been reported to vary annually, with 278
replacement of predominant genotypes every 1-3 years in a given population. Such genotype 279
replacement is believed to result from adaptive immunity of a population to the predominant 280
circulating genotype. However, genotype A2b viruses were predominant in the consecutive three 281
epidemic seasons in Chongqing, which was consistent with the findings in epidemiological study 282
from April 2006 to March 2008 (6). We found that genotype A2b viruses prevalent in the study 283
period revealed high homology with those circulated from April 2006 to March 2008 which may be 284
attributed to the low G gene diversity. 285
286
Although a report described that group A hMPV may be more pathogenic than group B hMPV (22), 287
no significant difference in the severity of ALRTI between groups or subgroups because only one 288
group B hMPV strain was detected. Further studies are required to better understand the clinical 289
significance, seasonality, and molecular epidemiology of hMPV. 290
291
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The predicted G protein of hMPV strains had different lengths which may be attributed to amino acid 292
substitution, insertion, deletion and/or change in the stop codon. G proteins with 4 different lengths 293
were identified in this study and this was the first report showing hMPV strain with 210 amino acids 294
circulating in China. 295
296
In summary, hMPV subgroups are observed among inpatients in Chongqing China in the past three 297
consecutive epidemic seasons. Genotype A2b has become a dominant genotype in the three 298
epidemic seasons. This study thus contributes to a better knowledge on the molecular epidemiology 299
of hMPV in China mainland. Comprehensive information on the prevalence of hMPV is important for 300
the selection of appropriate vaccine strains and thus may contribute to vaccine development. 301
302
Acknowledgements 303
This work was supported by the National Natural Science Foundation of China (No. 30330590) and 304
in part by Program for Chongqing Excellent Young Scientists Fund (CSCT, 2008BA5040). 305
306
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25. Sumino KC，Asapov E，Pierce RA , et a1. 2005. Detection of severe human metapneumovirus 364
infection by real-time polymerase chain reaction and histopathological assessment. J Infect 365
Dis. 192(6):1052-1060. 366
26. van den Hoogen BG, Bestebroer TM, Osterhaus ADME, et al. 2002. Analysis of the genomic 367
sequence of a human metapneumovirus.Virology. 295:119-132. 368
27.Van den Hoogen BG, de Jong JC, Groen J, et al. 2001. A newly discovered human pneumovirus 369
isolated from young children with respiratory tract disease. Nat Med. 7: 719–724. 370
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Fig. 1 Season distribution of hMPV infection in infants and children with ALRTI in Chongqing 389
between April 2008 and March 2011. 390
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Fig. 2. Phylogenetic trees of nucleotide sequences of G protein gene of Chinese hMPV group A 403
(panel A) and B (panel B). The numbers at the branch nodes represent the number of bootstrap 404
probabilities. Reference sequences for each genotype (A1, A2a, A2b, B1,B2) were obtained from 405
GenBank. Additional sequences were included for the comparison by selecting representatives of 406
distinct clusters in previous studies and selecting isolates from GenBank that gave the best hits in 407
blast searches with each of the Chinese clusters. The genotype assignment is indicated at the right 408
by brackets. 409
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415 Table 1. Age distribution of 144 patients with hMPV infection 416
417 Age Number of patients Percentage(%) 0-6 mo 66 45.9
6-12mo 15 10.4 1-2yr 22 15.3 2-5yr 29 20.1 >5yr 12 8.3 Total 144 100
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Table 2. Clinical features of 144 patients with hMPV infection 421 422
Clinical presentation/diagnostic disease
Number of patients
Percentage(%)
fever 62 41.5 cough 142 99.1
wheezing 102 77.1 cyanosis 110 85
bronchopneumonia 88 61.1 bronchiolitis 28 19.4
bronchial asthma exacerbation 23 15.9 acute upper respiratory infection 5 3.4
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Table 1. Age distribution of 144 patients with hMPV infection
Age Number of patients Percentage(%) 0-6 mo 66 45.9
6-12mo 15 10.4 1-2yr 22 15.3 2-5yr 29 20.1 >5yr 12 8.3 Total 144 100
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Table 2. Clinical features of 144 patients with hMPV infection
Clinical presentation/diagnostic disease
Number of patients
Percentage(%)
fever 62 41.5 cough 142 99.1
wheezing 102 77.1 cyanosis 110 85
bronchopneumonia 88 61.1 bronchiolitis 28 19.4
bronchial asthma exacerbation 23 15.9 acute upper respiratory infection 5 3.4
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