The authors have declared that no competing interests exist.
Conceived and designed the experiments: DNT TMHP MTH LY MM HU. Performed the experiments: DNT. Analyzed the data: DNT TTLT TKHD. Contributed reagents/materials/analysis tools: SO SH MM HU. Wrote the paper: DNT HU.
Respiratory syncytial virus (RSV) is a major cause of acute respiratory infections (ARIs) in children worldwide and can cause high mortality, especially in developing countries. However, information on the clinical and molecular characteristics of RSV infection in developing countries is limited. From April 2010 to May 2011, 1,082 nasopharyngeal swabs were collected from children with ARI admitted to the Children's Hospital 2, Ho Chi Minh City, Vietnam. Samples were screened for RSV and genotyped by reverse transcription-PCR and sequencing. Demographic and clinical data was also recorded. RSV was found in 23.8% (257/1,082) of samples. RSV A was the dominant subgroup, accounting for 91.4% (235/257), followed by RSV B, 5.1% (13/257), and 9 cases (3.5%) were mixed infection of these subgroups. The phylogenetic analysis revealed that all group A strains belonged to the GA2 genotype. All group B strains belonged to the recently identified BA genotype, and further clustered into 2 recently described subgenotypes BA9 and BA10. One GA2 genotype strain had a premature stop codon which shortened the G protein length. RSV infection was significantly associated with younger age and higher severity score than those without. Co-infection with other viruses did not affect disease severity. RSV A caused more severe disease than RSV B. The results from this study will not only contribute to the growing database on the molecular diversity of RSV circulating worldwide but may be also useful in clinical management and vaccine development.
Respiratory syncytial virus (RSV) is the major cause of acute respiratory infections (ARIs) among infants and young children worldwide
Along with the F protein, the second variable region at the C-terminal of the G protein that contains much of the G gene variability is commonly used in molecular epidemiological studies
Although RSV has been recognized as an important pathogen in childhood, there is no published information regarding the molecular epidemiology and clinical characteristics of RSV infections in Vietnam. The aims of this study were to investigate the molecular epidemiology of RSV infections, as well as to compare the clinical characteristics of diseases caused by group A and B strains in hospitalized children in Ho Chi Minh City, Vietnam.
The study was conducted from April 2010 to May 2011 at the Respiratory Ward, the Children's Hospital 2, in Ho Chi Minh City, Vietnam. The Children's Hospital 2 is a 1,000-bed tertiary referral and university-affiliated hospital, receiving pediatric patients from most parts of the city as well as other provinces in the south of Vietnam. This area has a tropical climate with two distinct seasons: rainy season (May–October) and dry season (November–April). The daily temperature does not change much during the year conducting this study.
Under 15-year-old children admitted for an ARI condition with an onset of illness less than 7 days were enrolled. The study was approved by the Scientific and Ethical Committee of the Children's Hospital 2. The written consent was obtained from the parent or legal guardian of the participants. An ARI case was defined as any child presenting with cough and/or difficult breathing
Demographic and clinical data were recorded on a standardized questionnaire. The diagnosis was made on the basis of clinical findings and chest X-ray (CXR). ARI patients with the presence of an infiltrate on CXR were categorized as pneumonia. Bronchiolitis was defined as ARI patient under 2 years old presenting with wheezing and hyperaeration, atelectasis, or peribronchial thickening on CXR. Croup was characterized by hoarseness, cough, and stridor. URTI was defined as ARI with no abnormalities on CXR. The disease severity was assessed by using the previously published severity score
Nasopharyngeal flocked swabs (MicroRheologics, Brescia, Italy) were obtained by trained personnel on 2 fixed days each week from all enrolled children within 24 hr after admission. The specimens were immediately placed in tubes containing 2 ml sterile physiological saline fluid and stored at −20°C until further analysis at the laboratory.
Viral genomes were extracted directly from the specimens by using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and stored at −80°C.
All specimens were screened for RSV and other respiratory viruses such as influenza virus A and B, human metapneumovirus, parainfluenza virus types 1 to 4, human rhinoviruses (HRV), human coronaviruses (229E and OC43), adenovirus and human bocavirus by using multiplex hemi-nested (RT)-PCR as described previously
All samples positive for RSV by screening test were then subjected to grouping and genotyping by a hemi-nested PCR as described previously
The products of nested PCR were sequenced bi-directionally by the commercial company (Macrogen Japan Corp., Tokyo, Japan). The nucleotide sequences were analyzed and compared with the reference strains available in the GenBank database. The sequence data and the phylogenesis were analyzed using BioEdit v.7.0.5
The sequences of RSVs detected in this study have been submitted to GenBank and assigned accession numbers JX079948–JX079993.
Values were given as percentages for categorical variables, and as median with range for continuous variables. Categorical variables were compared by using χ2 test or Fisher's exact test, and continuous variables were compared by using the Mann-Whitney
From April 2010 to May 2011, 1,082 cases of ARI were enrolled in this study. The median age was 9 months (ranged from 0 to 161 months), 86% of patients were under 2 years old. The boy to girl ratio was 1.8∶1, indicating that boys were more commonly affected than girls.
One or more respiratory viruses were identified in 63.8% (690/1,082) of patients, including 23.8% (257/1,082) that were positive for RSV. RSV was the second most common virus following HRV. Additionally, 23% (59/257) of all RSV positive samples contained at least one other respiratory virus. Co-infection between RSV and HRV was the most frequent (51 cases). RSV A was the dominant subgroup with 91.4% (235/257). RSV subgroup B had 5.1% (13/257). Interestingly, 9 cases (3.5%) contained both subgroup A and B RSV. The RSV epidemic occurred during the rainy season, from May to October (
Twenty-seven RSV A and 19 RSV B samples were selected randomly for sequencing. Twenty-seven of the group A strains clustered into one genotype, GA2 (
Phylogenetic tree was constructed with MEGA 3.1 software using the neighbor-joining method. Bootstrap values of greater than 70% are shown at the branch nodes. The RSV strains in this study are marked with solid round. Prototype strain A2 (in bold face) for group A was also included. The genotype assignment is indicated by the brackets on the right.
All 19 of the group B strains belonged to the recently identified BA genotype, with a 60-nucleotide duplication in the second variable region of the G protein gene
Phylogenetic tree was constructed with MEGA 3.1 software using the neighbor-joining method. Bootstrap values of greater than 70% are shown at the branch nodes. The RSV strains in this study are marked with solid round. Prototype strain of BA genotype is marked with solid triangle. Prototype strain 18537 for group B was also included. The genotype assignment is indicated by the brackets on the right.
The deduced amino acid sequences of the group A and group B isolates were compared to those of the prototype A2 and BA strains, respectively (
Alignments are shown relative to the sequences of prototype strain A2 (GenBank accession number M11486) (A) and genotype BA strain BA4128/99B (GenBank accession number AY333364) (B). The amino acids shown correspond to strain A2 G protein positions 221 to 299 for the group A viruses or to strain BA4128/99B G protein positions 213 to 320 for the group B viruses. Identical residues are indicated by dashes. The two copies of the duplicated 20-amino-acid region in group B viruses are indicated by rectangles. Stop codons are indicated by asterisks. Potential N-glycosylation sites (NXT, where X is not proline) are underlined.
The G protein genes of the BA genotype were predicted to encode proteins of 2 different lengths, 312 (16/19 isolates) and 319 (3/19 isolates) amino acids. A serine (S) to proline (P) substitution was found within the 20-amino acid duplicated region at position 247 in all the Vietnamese BA strains. In addition, 14 of the 15 BA9 strains had a V271A substitution relative to the BA prototype strain, whereas all 4 BA10 strains had an E292G change. The majority (11/15) of the BA9 isolates also had amino acid substitutions at positions 270 (T270I) and 287 (H287Y).
The G protein is heavily glycosylated with both N-linked and O-linked sugars. Two putative N-glycosylation sites (NXT, where X is not proline) were identified in the second variable region of the G protein among the majority of the RSV A strains. The amino acid substitutions at some positions made these strains gain or lose the N-glycosylation sites. Similarly, two N-glycosylation sites were found to be conserved in all BA strains examined. Two of the BA10 isolates, 960-HCM/11.10-BA10 and 566-HCM/07.10-BA10, each acquired an additional N-glycosylation site as a result of amino acid substitutions at P231S and K258N, respectively.
To examine the disease severity in the RSV positive and negative groups, clinical and demographic data were compared (
Characteristics (%) | RSV pos N = 257 | RSV neg N = 825 | p | RSV mono N = 198 | RSV co N = 59 | p | RSV A N = 177 | RSV B N = 12 | RSV A&B N = 9 | p |
Male | 63 | 65.2 | NS | 63.6 | 61 | NS | 64.4 | 50 | 66.7 | NS |
Age (m) |
7(3–14) | 11(4.5–19) |
|
7.5(3–14) | 6(2–13.5) | NS | 7 (3–14.5) | 7.5 (3–13.5) | 8 (2–11) | NS |
Age group | ||||||||||
<6 m | 42.8 | 29.9 |
|
41.4 | 47.5 | NS | 41.7 | 41.7 | 44.4 | NS |
6–<12 m | 26.5 | 23.2 | NS | 28.3 | 20.3 | NS | 27.8 | 16.7 | 44.4 | NS |
12–<24 m | 20.2 | 29.6 |
|
19.7 | 22.0 | NS | 18.9 | 41.7 | 11.1 | NS |
≥24 m | 10.5 | 17.3 |
|
10.6 | 10.2 | NS | 11.7 | 0 | 0 | NS |
Prematurity (<37 weeks) | 10.9 | 8.6 | NS | 10.7 | 11.9 | NS | 10.6 | 16.7 | 0 | NS |
Malnutrition | 6.2 | 10.5 |
|
7.1 | 3.4 | NS | 6.1 | 25 | 0 | RSV A v RSV B p = 0.046 |
Infection with other viruses | 23.0 | 52.5 |
|
NA | NA | NA | NA | NA | ||
Days before hos. |
2(2–4) | 3(2–5) |
|
2(2–4) | 3(2–4) | NS | 2 (2–4) | 2 (1–2.5) | 3 (3–5) | RSV B v RSV AB p = 0.023 |
Fever | 72.8 | 65.5 |
|
74.7 | 66.1 | NS | 73.3 | 83.3 | 100 | NS |
Cough | 93.8 | 89.9 | NS | 93.4 | 94.9 | NS | 92.8 | 100 | 100 | NS |
Runny nose | 82.5 | 70.5 |
|
84.3 | 76.3 | NS | 83.9 | 83.3 | 100 | NS |
SpO2≤92% | 5.8 | 9.3 | NS | 4.5 | 10.2 | NS | 4.4 | 8.3 | 0 | NS |
Tachypnea | 41.6 | 45.5 | NS | 42.9 | 37.3 | NS | 44.4 | 41.7 | 11.1 | NS |
Lower chest indrawing | 68.5 | 51.0 |
|
69.2 | 66.1 | NS | 70 | 50 | 77.8 | NS |
Wheezingd | 59.9 | 58.5 | NS | 59.6 | 61 | NS | 60.6 | 25 | 88.9 | RSV A v RSV B p = 0.03 |
Clinical Severity Score |
12(8–13) | 11(7–13) |
|
12(8–13) | 12(8–13) | NS | 12 (8–13) | 8 (7.5–12) | 13 (8–14) | RSV A v RSV B p = 0.031 |
Diagnosis | ||||||||||
URTIs | 16.0 | 23.4 |
|
17.7 | 10.2 | NS | 18.9 | 8.3 | 0 | NS |
Croup | 0.8 | 7.8 |
|
1 | 0 | NS | 1.1 | 0 | 0 | NS |
Bronchiolitis | 47.1 | 28.8 |
|
44.9 | 54.2 | NS | 42.8 | 66.7 | 66.7 | NS |
Pneumonia | 36.2 | 40.0 | NS | 36.4 | 35.6 | NS | 37.2 | 25 | 33.3 | NS |
Hos. duration |
6(4–8) | 5(4–8) | NS | 6(4–8) | 6(4–7.5) | NS | 6 (4–8) | 7 (5–8) | 6 (3–6) | NS |
Abbreviation: d, day; m, month; hos, hospitalization; URTI, upper respiratory infection; RSV, respiratory syncytial virus; NA, not applicable; NS, not significant; pos, positive; neg, negative; mono, mono-infection; co, co-infection.
Note: All results are expressed in percentages except for (a) in median with interquartile range between brackets.
Chi-squared test.
Mann-Whitney-
To address the question of whether children with RSV co-infection have different characteristics to those with RSV mono-infection, attempts were made to compare these two groups (
To assess the relationship between clinical severity and RSV subgroup, subjects that were co-infected with other viruses were excluded. The 177 cases with RSV A, 12 cases with RSV B, and 9 cases with mixed infection of both RSV A and B were compared (
In multivariate analysis adjusted for age, sex, prematurity, malnutrition and infection with other viruses, the difference on days before hospitalization and on the clinical severity score remained significant between the RSV positive and negative group (
RSV is one of the most important respiratory pathogens among infants worldwide. The identification of RSV in about one fourth of cases in this study confirmed that this virus is a dominant agent of respiratory disease in children. This finding is in line with previous reports from Vietnam and other countries
Regarding the seasonality in this study, the RSV epidemic peaked during the rainy season. Interestingly, RSV activity was completely absent for the 3 months of the dry season (February to April, 2011). The presence of RSV infection seasonality in relation to rainfall has been observed in India
RSV is the most common viral pathogen causing lower respiratory tract infections (LRTIs) among infants and young children. However, there has been no information about the molecular epidemiology of RSV in Vietnam. This report provides data on the molecular characteristics of RSV from hospitalized children in Ho Chi Minh City and represents the first such study in Vietnam. Information from this study will contribute to the growing database on the molecular diversity of RSV circulating worldwide. The results of this study indicated that RSV subgroup A and B co-existed in one epidemic and cases of RSV subgroup A infection predominated over those of subgroup B. These findings are in agreement with majority of studies in many countries around the world including Germany
The identification of the GA2 as the predominant genotype in this study was consistent with the results of other reports. GA2 was the most common genotype of RSV group A found around the world and has persisted for many years
In this study, 26/27 of GA2 genotype strains were predicted to encode a G protein of 297 amino acids in length, while one strain was shorter, of 286 amino acids. This mutant strain was sequenced directly from the clinical sample. To our knowledge, there has been one previous report of such a mutant strain, by Cane and Pringle
Surprisingly, all Vietnamese subgroup B viruses fell into a new genotype BA, which was first detected in Buenos Aires, Argentina during 1999
The Vietnamese BA strains had two different G protein lengths, 312 and 319 amino acids, which were reported in previous studies
Potential N-glycosylation sites are thought to play an important role in helping viruses escape from the host immune response
The demographic and clinical information is important to put the results into a practical context and allow them to be applied in clinical practice. In the current study, statistical significance was achieved for the detection of RSV and its association with clinical severity. The RSV-positive patients tended to have more severe symptoms and a higher severity score that probably led them to seek hospital care earlier than the RSV-negative patients.
The fact that nearly half of the RSV infections (42.8%) occurred in children under 6 months showed that patients in this age group are the most vulnerable to RSV infections, despite the presence of maternal antibodies. Maternal antibodies are able to protect against severe RSV in children, however relatively high tilters are required
The detection of co-infection has led to the speculation that the presence of several types of virus in one respiratory specimen may affect the clinical presentation of ARIs. Viruses might interact indirectly or directly, and the effect may depend upon which viruses are co-infecting. The combination of hMPV and RSV has been associated with increased disease severity requiring the use of mechanical ventilation
Regarding clinical manifestation of each group, by using a composite severity score, we found that infections involving RSV group A (alone or mixed with RSV group B) were associated with more severe disease than infection with RSV group B. Besides virus factors, the clinical severity of RSV infection is also associated with epidemiological and host factors, which include socioeconomic status, age, prematurity, and underlying heart and/or lung disease. To exclude these confounding factors, patients with underlying diseases or having co-infection with other viruses were not included.
Subgroup B infected children were admitted to the hospital less frequently than subgroup A infected children. Either subgroup B strains cause such a mild illness that there is no need to hospitalize the patient or the prevalence of subgroup B is truly low. However, in two studies, the proportions of subgroup A and subgroup B infected children in the hospital and in the community were similar
Several studies have examined the relationship between clinical severity and RSV subgroups. In approximately half of these studies, group A seemed to be associated with more severe clinical disease
The current study was limited to only one epidemic season. Because the predominant subgroups shift from year to year, the immunity against the previous circulating groups could have altered the severity of disease caused by specific subgroups. Therefore, continued observation and analysis of additional seasons is required to determine the association between subgroups and severity of disease.
In summary, RSV was found to be the viral pathogen most commonly and frequently associated with severe acute respiratory diseases in infants and children. Co-infections between RSV and other respiratory viruses did not lead to increased disease severity. The molecular characteristics of RSV were determined for the first time in Vietnam, and carried characteristics both similar to strains from other parts of the world and specific to Vietnam. Subgroup A and B of RSV were co-circulating and subgroup A caused more severe disease than subgroup B. Similar surveillance should be continued to follow the epidemiology of this virus. A better understanding of RSV epidemiology is essential to enable prediction of outbreaks and for planning preventive and therapeutic control measures.
We deeply thank Dr. Nguyen Tran Quynh Nhu and Ms. Pham Thi Ngoc Hue, Cardiovascular Department, Children's Hospital 2, Ho Chi Minh City for their help in patient counseling and sample collection. DNT is a recipient of the scholarship from the Honjo International Scholarship Foundation.