Journal of Medical Virology 87:1–9 (2015)
Molecular Characterization of Human Respiratory Syncytial Virus Subtype B: A Novel Genotype of Subtype B Circulating in China Luo Ren,1 Qiuyan Xiao,1 Lili Zhou,1 Qiuling Xia,1 and Enmei Liu2* 1
Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing, China 2 Department of Respiratory Medicine, Children’s Hospital of Chongqing Medical University, Chongqing, China
Human respiratory syncytial virus (HRSV) is major pathogen of lower respiratory tract infections in infants and young children worldwide. There have been many studies regarding HRSV subgroup A (HRSV-A) G protein genetic variability but little information about HRSV subtype B (HRSV-B) G protein genetic diversity and molecular evolution in China. Thus, a survey of the molecular epidemiology and evolution of the G protein in China is of high importance. In this study, the circulation and genetic diversity of HRSV in Chongqing, Southwestern China, from June 2009 to May 2013, were investigated. A total of 3,167 nasopharyngeal aspirates were obtained in this study, and it was found that HRSV-B predominated in the 2009–2010 and 2012–2013 epidemic seasons. This study identified the genetic variability of the glycoprotein G gene among 102 HRSV-B strains isolated by cell culture from Chongqing nasopharyngeal aspirates, and 68 Chinese HRSV-B sequences were deposited in GenBank. Genotyping and phylogenetic analysis revealed that the HRSV-B strains were clustered into three genotypes: BA (n ¼ 111, 65.29%), GB3 (n ¼ 5, 2.94%), and a new GB genotype (n ¼ 54, 31.77%) named GB5. The GB5 strains varied from other genotypes in the central conserved region and N-glycosylation sites. The estimated evolutionary rate of Chinese HRSV-B was 2.01 103 nucleotide substitutions/site/year, which is similar to the reports from Belgium and the Netherlands with 1.95 103 and 2.78 103 nucleotide substitutions/site/year, respectively. This study provides data on the circulating pattern and molecular characterization of HRSV-B genotypes in China during four consecutive years and may contribute to HRSV vaccine development. J. Med. Virol. 87:1–9, 2015. # 2014 Wiley Periodicals, Inc. C 2014 WILEY PERIODICALS, INC.
KEY WORDS:
human respiratory syncytial virus; subtype B; genotype; GB5; G glycoprotein; China
INTRODUCTION Human respiratory syncytial virus (HRSV) is a major pathogen of lower respiratory tract infections in infants and young children worldwide. It is estimated that HRSV causes over 30 million lower respiratory tract infections each year and results in 3 million hospitalizations, making it the most common cause of hospitalization in children under 5-years old [Nair et al., 2010]. As a species of the Paramyxoviridae family, HRSV has a nonsegmented, negative-sense RNA genome of approximately 15,200 nucleotides encoding 11 proteins [Cane, 2001]. Among these proteins, G protein, a type II glycoprotein, is N- and O-glycosylated [Johnson et al., 1987]. The variation in the G glycoprotein is the major difference among the major HRSV subtypes. HRSV has two antigenic subgroups, Abbreviations: HRSV, human respiratory syncytial virus; HRSV-A, human respiratory syncytial virus subgroup A; HRSVB, human respiratory syncytial virus subgroup B. Grant sponsor: China Special Grant for the Prevention and Control of Infection Diseases; Grant number: 2013ZX10004202002. The authors have no conflicts of interest to declare. Correspondence to: Enmei Liu, Department of Respiratory Medicine, Children’s Hospital of Chongqing Medical University, No. 136, the 2nd Zhongshan Road, Yuzhong District, Chongqing 400014, China. E-mail: [email protected] Accepted 25 March 2014 DOI 10.1002/jmv.23960 Published online 9 June 2014 in Wiley Online Library (wileyonlinelibrary.com).
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A and B, based on reactions with monoclonal antibodies against the G glycoprotein [Mufson et al., 1985] and genetic analysis. There has been no effective vaccine to prevent the spread of HRSV. Palivizumab, which recognizes the “A” antigenic site of the HRSV fusion protein, is the first and only approved humanized monoclonal antibody to prevent HRSV infection in infants and young children at high risk [The IMpact-RSV Study Group, 1998]. However, Palivizumab is not available in China. The G glycoprotein is the target of neutralizing antibodies, and intranasal administration of G protein elicits beneficial protective immunity and represents a promising vaccine against HRSV infection [Yu et al., 2008], suggesting that this protein may be under immune pressure [Woelk and Holmes, 2001]. Thus, a survey of the molecular epidemiology and evolution of the G protein in China is of high importance. A previous hospital-based study at Chongqing, Southwestern China, showed that the BA genotype of HRSV subgroup B (HRSV-B) with a 60-nucleotide insertion became the dominant genotype during the 2008–2009 epidemic season [Zhang et al., 2010a]. Notably, there have been more studies regarding HRSV subgroup A (HRSV-A) G protein genetic variability [Zhang et al., 2007, 2010a,b] but little information about HRSV-B G protein genetic diversity and molecular evolution in China. This study not only evaluated G protein genetic variability of HRSVB isolated clinically in Chongqing, China, but also analyzed the Chinese HRSV-B strains in GenBank from 2004 to 2013.
manufacturer’s instructions. Reverse transcription (RT-PCR) was performed to create cDNA using the SuperScript II Reverse Transcriptase System (Invitrogen, USA) with random primers. Subtypes were identified by PCR amplification as reported previously [Coiras et al., 2003].
MATERIALS AND METHODS
Phylogenetic Analysis
Sample Collection and Viral Isolation
The 270 and 330 nucleotides of HVR2 of the G gene found within non-BA and BA genotypes were compared, respectively, with reference strains available from GenBank. Multiple sequence alignments were made using MEGA 5.0 programs. Phylogenetic trees were constructed using the neighbor-joining algorithm, and the statistical significance of the tree topology tested by bootstrapping (1,000 replicates) was performed. Only bootstrap values >70% are shown in each tree. According to a previous study [Venter et al., 2001], sequences were arbitrarily considered a genotype if they clustered together with bootstrap values of 70–100% and the pairwise-distance (p-distance) was less than 0.07 to all other members in the same phylogenetic cluster. Besides the sequences of the Chinese HRSV-B strains, all reference sequences for HRSV-B included in the phylogenetic analysis are shown in Table I. Partial G protein gene sequences of newly obtained HRSV-B isolates in Chongqing have been deposited in GenBank with the accession numbers KC461262–KC461295.
All nasopharyngeal aspirates were collected from hospitalized infants or children with acute lower respiratory tract infections from the Department of Respiratory Medicine, Chongqing Children’s Hospital in Southwestern China from June 2009 to May 2013. This study was approved by the Ethics and Research Council of Chongqing Children’s Hospital, and signed consent was obtained from each child’s parent or foster parent. The nasopharyngeal aspirates were stored at 80˚C and cultured in human laryngeal carcinoma HEp-2 cells. The viral isolates were harvested by scraping when the cytopathic effect was more than 75%. The virus-cell suspensions were frozen and stored at 80˚C. RNA Extraction and HRSV Subtype Identification Before RNA extraction, the nasopharyngeal aspirates and frozen cultured samples were centrifuged at 3,000g and 4˚C for 5 min. RNA was extracted from 200 ml of nasopharyngeal aspirate supernatant and frozen cultured samples using a QIAamp Viral RNA Kit (Qiagen, Hilden, Germany) according to the J. Med. Virol. DOI 10.1002/jmv
PCR Amplification and Sequencing The cultured HRSV-B isolates were used to sequence the G gene. Partial HRSV-B G gene (0.8 kb), the ectodomain, including the first hypervariable region (HVR1, nt. 199 to nt. 489 of the G gene), the central conserved region (nt. 490 to nt. 548 of the G gene), and the secondary hypervariable region (HVR2, nt. 549 to the end of the G gene), was amplified according to a previous study [Zlateva et al., 2005]. The PCR products were sequenced using an ABI3730xl DNA Analyzer at BGI Tech Solutions Co., Ltd. (Beijing, China). Data Set Assembly Analysis of HRSV-B G genetic variability was conducted on a data set containing a combination of newly obtained HRSV-B isolates in Chongqing and strains from other provinces of China found in GenBank. The data sets were named as Chinese HRSV-B strains. The accession numbers were DQ270232 and DQ270233 [Deng et al., 2006], DQ289648–DQ289649 [Zhang et al., 2007], GU357503–GU357530 [Zhang et al., 2010b], GU550474–GU550503 [Zhang et al., 2010a], and JF713439–JF713446 (unpublished).
Amino Acid and N-Glycosylation Site Analysis The amino acid sequences within part of the ectodomain, from amino acid 101 to the C-terminal
A Novel Genotype of HRSV-B in China
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TABLE I. HRSV-B G Protein GenBank Reference Sequences Strain 8/60 9320/77 BA/1370/99 BA/1441/02 BA/4825/03 CB616/090 CB757/09 CH18537 CH93-9b NG-010-09 NG-022-06 NG-025-07 NG-040-07 NG-046-06
GenBank accession No.
Strain
GenBank accession No.
M73545 AY353550 DQ227364 DQ227381 DQ227401 HQ699300 HQ699304 M17213 AF065251 HM459857 HM459876 HM459889 HM459879 AB603478
NG-050-05 NG-056-05 NG-084-07 NG-087-07 NG-102-06 NG-119-07 NG-166-06 NG-182-03 NY01 SA0025 SA98V602 SA99V429 SA99V800 TX69208
AB603476 AB603474 HM459883 HM459870 AB603467 HM459878 AB603479 HM459859 AF233931 AF348825 AF348824 AF348813 AF348821 AF233933
test. The codon-based maximum likelihood IFEL method is subject to selective pressure at the population level. A P value < 0.1 was used to define sites as positively (dN > dS) or negatively (dN < dS) selected sites, respectively. RESULTS HRSV Distribution and Seasonal
end of newly obtained Chongqing HRSV-B isolates, were determined and compared to the reference strains CH18537 (non-BA genotype) and 1370/99 (BA genotype). Potential N-glycosylation sites (NXT, where X is not a proline) were predicted using Net NGlyc 1.0 [Julenius, 2007].
In this study, among 3,167 nasopharyngeal aspirates collected between June 2009 and May 2013, 1,035 (33%) were tested to be positive for HRSV. HRSV-A and HRSV-B were identified in 671 (65%) and 343 (33%) specimens, respectively, and both HRSV-A and HRSV-B were identified in 21 (2%) specimens. HRSV-B strains accounted for 59–79% among the cases in the summer of 2009 to the spring of 2010, whereas HRSV-A strains accounted for 73– 96% among the HRSV infections in the summer of 2010 to the spring of 2012, respectively (Fig. 1). In the summer of 2012 to the spring of 2013, group B strains predominated, accounting for 71–84% among all the HRSV cases (the variation was stated between seasons, Fig. 1).
Evolutionary Rate Estimation
Assembly of G Genetic Variability Data
To estimate the evolutionary rate of Chinese HRSV-B, an exploratory root-to-tip linear regression analysis was performed by Path-O-Gen (available at http://tree.bio.ed.ac.uk/software/pathogen/). A maximum likelihood (ML) tree was constructed by MEGA 5.0 with a general time-reversible (GTR) substitution model and a discretized gamma distribution to model rate variation among sites. This program takes the ML tree as the input and performs a linear regression between the genetic distance from the root and the sampling date for each strain. The exploratory analysis was performed with the sampling year for the Chinese HRSV-B strains.
In the present study, 2,415 nasopharyngeal aspirates were randomly selected by random numbers to inoculate into HEp-2 cells. A total of 374 (15.49%) clinical isolates were identified (265 for HRSV-A, 108 for HRSV-B, and 1 isolate detected HRSV-A and HRSV-B). For HRSV-B, the partial G gene was sequenced successfully in 102 HRSV-B cultured isolates. Among the 102 isolates in Chongqing, there were 72 different sequences, of which each of 60 isolates had a unique sequence in the G gene, and the remaining 42 isolates shared 10 different sequences. The central conserved region (amino acids 164– 186, according to strain CH18537) was recognized among the 102 HRSV-B G protein sequences. Additionally, 68 HRSV-B G protein sequences detected in other provinces of China [Deng et al., 2006; Zhang et al., 2007, 2010a,b] were analyzed. The nucleotide sequences from the Chinese strains were compared to 28 HRSV-B sequences available in GenBank (Table I).
Selective Pressures Analysis Positively and negatively selected sites among the present strains were calculated to derive the synonymous (dS) and nonsynonymous (dN) rates at every codon in the alignment using Datamonkey (http:// www.datamonkey.org/). An excess of nonsynonymous substitutions (dN > dS) were interpreted as positive selection and increased fitness by replacement substitutions, while a paucity of replacement changes (dN < dS) indicated that negative selection removes such substitutions from the gene pool. Single likelihood ancestor counting (SLAC), fixed effects likelihood (FEL), and internal fixed effects likelihood (IFEL) were used to identify the diversifying selection. SLAC bases its inference on the expected and inferred numbers of synonymous and nonsynonymous substitutions and FEL directly estimates dN and dS based on a codon-substitution model to derive the p-value for the test dN6¼dS using a likelihood ratio
Phylogenetic Analysis of HRSV-B The Chinese HRSV-B strains were clustered into three genotypes: BA (n ¼ 111, 65.29%), GB3 (n ¼ 5, 2.94%), and a new GB genotype (n ¼ 54, 31.77%) named GB5 with a bootstrap value of 99% (Fig. 2). The p-distance within the GB5 genotype was 0.016. Although the GB5 genotype was close genetically to the GB1 and GB2 genotypes, the p-distance between GB5 to GB1 and GB5 to GB2 were 0.138 0.002 and 0.068 0.003, respectively, suggesting that GB5 was a new genotype [Venter et al., 2001; Arnott et al., 2011] (Table II). J. Med. Virol. DOI 10.1002/jmv
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Fig. 1. Seasonal distribution of HRSV-positive samples. Data are represented as percentages between June 2009 and May 2013. SP, spring, including March, April, and May; SU, summer, including June, July, and August; FA, fall, including September, October, and November; WI, winter, including December, January, and February.
The BA genotype with duplication of 60 nucleotides, first reported in 1999 [Trento et al., 2003], became the dominant strain of HRSV-B in Niigata, Japan [Sato et al., 2005] and Belgium [Zlateva et al., 2005, 2007] in 2003, in New Delhi in 2004 [Parveen et al., 2006], in Brazil in 2005 [Botosso et al., 2009], in South Africa in 2006 [Visser et al., 2008], and in Madrid in 2007 [Trento et al., 2010]. However, the BA genotypes predominated in 2009– 2010, whereas the GB5 genotype outbreak was prominent in 2010–2011 in Chongqing (Fig. 3). The BA9 genotype replaced the non-BA genotype to become dominant from 2011 to 2013 in Chongqing (Fig. 3), suggesting that the BA genotypes expanded with fluctuation. Amino Acid and N-Glycosylation Site Analysis Due to the usage of alternative termination codons, deletions, insertions, one frameshift mutations, premature stop codons, the lengths of the deduced G protein sequences among the HRSV-B strains were significantly different. The predicted complete G proteins of the Chinese HRSV-B strains had four different amino acid lengths (290, 293, 296, and 297 Fig. 2. Phylogenetic trees for HRSV-B nucleotide sequences based on the second variable region of the G protein. Details for the reference GenBank sequences used for phylogenetic analysis are given in Table I. The newly identified Chongqing (CQ) and reported Chinese (B, Beijing; Chongqing, LZY) HRSV-B strains were compared with reference GenBank sequences throughout the world, including strains in Sweden (8/60), Massachusetts (9320/77), Niigata (NG), Rochester (CH), Houston (TX), Rochester (NY), Soweto (South Africa, SA), Madrid (Spain, BA genotype), and Chungbuk (Korea, CB). The phylogenetic tree was constructed with a neighbor-joining algorithm using MEGA 5 software. Only bootstrap values greater than 70% are displayed at the branch nodes. The genotypes are indicated at the right by brackets. The BA1-4, BA8, BA10, and CB-B genotypes were compressed as solid triangles.
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TABLE II. p-Distance Between Chinese HRSV-B Genotypes Genotype
BA1
BA10
BA2
BA3
BA4
BA8
BA9
CB-B
GB5
GB1
GB2
GB3
GB4
SAB1 SAB2 SAB3
BA1 BA10 BA2 BA3 BA4 BA8 BA9 CB-B GB5 GB1 GB2 GB3 GB4 SAB1 SAB2 SAB3
0.05 0.048 0.032 0.033 0.037 0.051 0.061 0.09 0.119 0.05 0.04 0.106 0.065 0.061 0.045
0.066 0.053 0.046 0.038 0.049 0.061 0.111 0.134 0.076 0.063 0.136 0.095 0.084 0.062
0.049 0.044 0.052 0.064 0.083 0.105 0.142 0.077 0.073 0.129 0.093 0.089 0.076
0.027 0.04 0.052 0.066 0.102 0.126 0.058 0.047 0.11 0.076 0.069 0.046
0.036 0.045 0.059 0.106 0.128 0.063 0.05 0.119 0.082 0.074 0.056
0.039 0.059 0.099 0.13 0.062 0.054 0.125 0.084 0.076 0.05
0.069 0.111 0.129 0.077 0.066 0.135 0.087 0.088 0.062
0.102 0.121 0.058 0.053 0.111 0.078 0.07 0.058
0.13 0.068 0.093 0.138 0.101 0.12 0.097
0.091 0.11 0.156 0.117 0.136 0.125
0.053 0.086 0.048 0.067 0.059
0.105 0.059 0.104 0.062 0.104 0.082 0.04 0.112 0.071 0.048
Bolds indicate the p-distance between GB5 and other genotypes.
amino acids) for non-BA genotypes and five different amino acid lengths (280, 310, 312, 313, and 317 amino acids) for BA genotypes, respectively (Fig. 4). The reported alternative stop codons [Zlateva et al., 2005] were observed in both the GB5 and BA genotypes (at amino acid positions 291, 294, and 298 for the GB5 genotype, referring to strain CH18537; at amino acid positions 311, 314, and 318 for the BA genotype, referring to strain BA/1370/99, Fig. 4). The previously reported six-nucleotide deletion (30 -AAAACC50 ) [Zlateva et al., 2005], causing amino acid deletions at positions 159 and 160, was observed in all of the newly obtained 102 HRSV-B strains in Chongqing. As a result of a 3-amino acid insertion at position 226 (QKT, referring to strain CH18537), three strains of the GB5 genotype (Chongqing/B/09/15, CQ911Sep2010 and CQ2607-Dec2011) had a protein with 296 amino acids. There was a premature stop codon at amino acid position 283 (referring to strain BA/ 1370/99) of the CQ2795-Jan2012 strain, leading to a BA genotype strain with a protein of 280 amino acids. In addition to the difference in the number of amino acids, a nonsynonymous amino acid substitution was identified in the central conserved region of the GB5 genotype (Q-R, at amino acid position 180). No N-glycosylation site was identified after amino acid 100 in reference to the HRSV-B sequence of strain CH18537 [Johnson et al., 1987]. However, three N-glycosylation sites were identified in genotype GB5 strains at amino acids 258, 276, and 290 (referring to strain CH18537, Fig. 4A). The N-glycosylation sites at amino acids 296 and 310 (referring to strain BA/1370/99, Fig. 4B) were common for the BA genotype.
tendency to develop respiratory failure (32.4% vs. 16.4%, P ¼ 0.067, Table III). Evolutionary Rate In order to estimate the rate of nucleotide substitutions and the time of the most recent common ancestor (TMRCA) of the Chinese HRSV-B strains throughout the 10 years of circulation, an exploratory root-to-tip linear regression analysis using 170 Chinese HRSV-B strains was performed. The correlation between phylogenetic root-to-tip divergence and time of sampling of HRSV-B strains is displayed in a regression plot (Fig. 5). The rate of substitution as estimated from this linear regression analysis was 2.01 103 nucleotide substitutions/site/year. The TMRCA of Chinese HSRV-B was estimated to date back to 1972.
Clinical Manifestations There were no significant differences observed in the clinical manifestations between children infected with the BA9 and GB5 genotypes (Table III). However, the patients infected with GB5 strains had a
Fig. 3. Yearly distribution of HRSV-B genotypes in Chongqing between 2009 and 2013. Data are represented as number of HRSV-B samples. SP, spring, including March, April, and May; SU, summer, including June, July, and August.
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Fig. 4. Deduced amino acid alignments of the central conserved and HVR2 of the G protein from 102 newly identified Chongqing HRSV-B strains. Alignments are shown relative to the sequences of prototyped strains CH18537 and BA/1370/99. Identical residues are indicated by dots. Stop codons are
indicated by asterisks. Potential N-glycosylation sites (NXT, where X is not a proline) are indicated by dotted rectangles. The solid rectangles show the nonsynonymous amino acid substitution Q180R of GB5 strains and the 60 nucleotide duplication of the BA genotype, respectively.
Analysis of Selective Pressure
genotype of HRSV-B, was found to be circulated in China. It has been reported that the subtype circulation pattern of HRSV-B became predominant after two consecutive seasons with a high prevalence of subgroup A [Coggins et al., 1998; Scott et al., 2004; Arbiza et al., 2005]. However, in an earlier hospitalbased study of Chongqing, China, HRSV-B predominated in the epidemic season during 2008–2009 [Zhang et al., 2010a], indicating that there were two switches of HRSV subgroups in the past 7 years in Chongqing. In accordance with the previous study [Zhang et al., 2010a] and the recent study in Beijing, China [Cui et al., 2013], HRSV-B predominated over the HRSV circulation pattern within 2 years after two epidemic seasons of HRSV-A in Chongqing. A similar circulation pattern was observed in Cambodia [Arnott et al., 2011]. The shift in the predominant group might be correlated in part with variability in the G-protein gene and the immune response of the population.
We used SLAC, FEL, and IFEL methods to identify the diversified selection. In HRV2, five positively selected sites were identified (amino acid positions 223, 271, 278, 305, and 315, referring to strain BA/ 1370/99, Table IV). While 15 negatively selected sites were identified at amino acid positions 216, 220, 235, 252, 260, 272, 279, 285, 288, 294, 295, 297, 302, 210, and 311 (referring to strain BA/1370/99, Table IV). DISCUSSION The present study analyzed the G gene of HRSV-B isolates from nasopharyngeal aspirates from June 2009 to May 2013 in Chongqing, Southwestern China. Various bioinformatics methods were used to better understand the genotype variability, molecular epidemiology, and evolutionary rate of circulating strains. The results demonstrated that HRSV-B was predominant in the epidemic seasons of 2009–2010 and 2012–2013 and that the GB5 genotype, a novel
TABLE III. Demographic Characteristics of the Genotyped Population
Group
No. of cases
Gender (Male, %)
Age (month, median, range)
Fever (N, %)
Cough (N, %)
Wheezing (N, %)
Days of Hospitalization (median, range)
Respiratory failure (N, %)
BA9 GB5 Total
67 34 101
44 (65.7) 25 (73.5) 7 (5.5)
5 (1–40) 5.5 (1–42) 5 (1–42)
16 (23.9) 10 (29.4) 26 (25.7)
67 (100) 34 (100) 101 (100)
45 (67.2) 25 (73.5) 70 (69.3)
7 (3–17) 6.5 (3–14) 7 (3–17)
11 (16.4) 11 (32.4) 22 (21.8)
A strain which was identified as GB3 genotype was not included in this table for only one case was reported.
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Fig. 5. Linear root-to-tip regression plot presenting the correlation between the branch lengths and the sampling dates of the HRSV-B isolates included in this study.
In addition to the reported genotypes of GA1 to GA7 [Peret et al., 1998, 2000] and SAA1 [Venter et al., 2001], four novel genotypes of HRSV-A, NA1, NA2 [Shobugawa et al., 2009], CB-A [Baek et al., 2012], and ON1 [Eshaghi et al., 2012] have been reported in the past 5 years. HRSV-B genotypes include GB1 to GB4, SAB1 to SAB3, URU1, URU2, BA1 to BA6 [Peret et al., 1998, 2000; Blanc et al., 2005; Trento et al., 2006; Yoshida et al., 2010], and new genotypes of SAB4 [Arnott et al., 2011], BA7 to BA10 [Dapat et al., 2010], BA11 and CB-B [Baek et al., 2012] and BA12 [Kushibuchi et al., 2013]. Another novel HRSV-B genotype named GB5 was observed in this study. Fifty-four GB5 strains were identified in different provinces of China from 2005 TABLE IV. Positively and Negatively Selected Sites of Chinese HRSV-B Codon
IFEL
Positively selected sites 223 271 278 305 315 Negatively selected sites 216 220 235 252 260 272 279 285 288 294 295 297 302 310 311
FEL
SLAC
SLAC, single likelihood ancestor counting; FEL, fixed effects likelihood; IFEL, internal fixed effects likelihood. P < 0.1.
to 2013. During the preparation of this manuscript, the GB5 genotype was reported as CB1 [Cui et al., 2013] and THB [Auksornkitti et al., 2014] genotypes in Beijing and Thailand, respectively, indicating that the GB5 strains also can be identified in other countries. Among the GB5 genotypes, a nonsynonymous amino acid substitution (Q180R) in the central conserved region of amino acids 164–186 that contains a highly conserved region with 13 amino acids, cysteine residues, and a CX3C motif [Collins and Crowe, 2007], inhibits innate immunity elicited by HRSV and endotoxins [Polack et al., 2005]. Three N-glycosylation sites at amino acids 258, 276, and 290 (referring to strain CH18537) emerged from the GB5 strains. N-glycosylated sites have an impact on the antigenic structure of the G protein and virus infectivity [Lambert, 1988; Garcia-Beato et al., 1996]. After first being detected in 1999, the BA genotype replaced the non-BA genotypes to become the worldwide dominant HRSV-B strain in 2003–2007 [Sato et al., 2005; Zlateva et al., 2005, 2007; Parveen et al., 2006; Visser et al., 2008; Botosso et al., 2009; Trento et al., 2010], indicating the great adaptation of this genotype. However, during the expansion of the BA genotype, there was an outbreak of GB5 in 2010– 2011 in Chongqing, suggesting that BA genotypes expanded with fluctuation. This was consistent with the pattern of BA genotype expansion worldwide [Trento et al., 2010]. On the other hand, a recent study reported that new non-BA strains related to the BA genotype reemerged in Kenya, where BA strains were predominant for years [Agoti et al., 2013]. This observation provides a mechanism for the replacement of BA strains if the BA selective advantage diminishes. Thus, it is important to monitor whether the GB5 strains reappear in China. The estimated evolutionary rate of the G protein was greater than that of the whole genome [Tan et al., 2013]. The estimated evolutionary rate of Chinese HRSV-B was 2.01 103 nucleotide substitutions/site/year, which is very similar to the reports from Belgium and the Netherlands with 1.95 103 and 2.78 103 nucleotide substitutions/site/year, respectively [Zlateva et al., 2005; Tan et al., 2013]. While the evolutionary rates of the HRSV-A G protein gene were 1.83 103 and 2.22 103 nucleotide substitutions/site/year [Zlateva et al., 2004; Tan et al., 2013], indicating that HRSV-B had an equally high potential for accumulation of genetic diversity of the intra- and intersubtypes. As a target for the neutralizing antibody, the G glycoprotein is under immune pressure. In the present study, five positive-selected sites at amino acid positions 223, 271, 278, 305, and 315 were identified, referring to strain BA/1370/99. The 223 site was also identified as a positive-selected site in previous studies [Woelk and Holmes, 2001; Zlateva et al., 2005], suggesting that this site may be correlated with the immune response to HRSV-B infection. J. Med. Virol. DOI 10.1002/jmv
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In conclusion, a novel genotype of HRSV-B, GB5 circulating in China, was identified and molecular analysis of the G protein demonstrated that GB5 strains had variations in the central conserved region and N-glycosylation sites in this study. These findings may contribute to the development of a potential vaccine based on the HRSV-B G protein in China. Further molecular and epidemiological surveillance of HRSV is important to understand HRSV transmission and select appropriate vaccine strains. ACKNOWLEDGMENTS We thank all the families for their enrollment in this study. We also thank the staff in the Department of Respiratory Medicine, Children’s Hospital of Chongqing Medical University for clinical information collection. REFERENCES Agoti CN, Gitahi CW, Medley GF, Cane PA, Nokes DJ. 2013. Identification of group B respiratory syncytial viruses that lack the 60-nucleotide duplication after six consecutive epidemics of total BA dominance at coastal Kenya. Influenza Other Respir Viruses 7:1008–1012. Arbiza J, Delfraro A, Frabasile S. 2005. Molecular epidemiology of human respiratory syncytial virus in Uruguay: 1985–2001—A review. Mem Inst Oswaldo Cruz 100:221–230. Arnott A, Vong S, Mardy S, Chu S, Naughtin M, Sovann L, Buecher C, Beaute J, Rith S, Borand L, Asgari N, Frutos R, Guillard B, Touch S, Deubel V, Buchy P. 2011. A study of the genetic variability of human respiratory syncytial virus (HRSV) in Cambodia reveals the existence of a new HRSV group B genotype. J Clin Microbiol 49:3504–3513. Auksornkitti V, Kamprasert N, Thongkomplew S, Suwannakarn K, Theamboonlers A, Samransamruajkij R, Poovorawan Y. 2014. Molecular characterization of human respiratory syncytial virus, 2010–2011: Identification of genotype ON1 and a new subgroup B genotype in Thailand. Arch Virol 159:499–507. Baek YH, Choi EH, Song MS, Pascua PN, Kwon HI, Park SJ, Lee JH, Woo SI, Ahn BH, Han HS, Hahn YS, Shin KS, Jang HL, Kim SY, Choi YK. 2012. Prevalence and genetic characterization of respiratory syncytial virus (RSV) in hospitalized children in Korea. Arch Virol 157:1039–1050. Blanc A, Delfraro A, Frabasile S, Arbiza J. 2005. Genotypes of respiratory syncytial virus group B identified in Uruguay. Arch Virol 150:603–609. Botosso VF, Zanotto PM, Ueda M, Arruda E, Gilio AE, Vieira SE, Stewien KE, Peret TC, Jamal LF, Pardini MI, Pinho JR, Massad E, Sant’anna OA, Holmes EC, Durigon EL, Consortium V. 2009. Positive selection results in frequent reversible amino acid replacements in the G protein gene of human respiratory syncytial virus. PLoS Pathog 5:e1000254. Cane PA. 2001. Molecular epidemiology of respiratory syncytial virus. Rev Med Virol 11:103–116. Coggins WB, Lefkowitz EJ, Sullender WM. 1998. Genetic variability among group A and group B respiratory syncytial viruses in a children’s hospital. J Clin Microbiol 36:3552–3557. Coiras MT, Perez-Brena P, Garcia ML, Casas I. 2003. Simultaneous detection of influenza A, B, and C viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-PCR assay. J Med Virol 69:132–144. Collins PL, Crowe JE, Jr. 2007. Fields BN, Knipe DM, Howley PM, editors. Fields virology. Respiratory syncytial virus and metapneumovirus. Philadelphia, PA: Wolters Kluwer Health/Lippincott Williams & Wilkins, pp 1601–1635. Cui G, Zhu R, Qian Y, Deng J, Zhao L, Sun Y, Wang F. 2013. Genetic variation in attachment glycoprotein genes of human respiratory syncytial virus subgroups a and B in children in recent five consecutive years. PLoS ONE 8:e75020.
J. Med. Virol. DOI 10.1002/jmv
Dapat IC, Shobugawa Y, Sano Y, Saito R, Sasaki A, Suzuki Y, Kumaki A, Zaraket H, Dapat C, Oguma T, Yamaguchi M, Suzuki H. 2010. New genotypes within respiratory syncytial virus group B genotype BA in Niigata, Japan. J Clin Microbiol 48:3423–3427. Deng J, Zhu R, Qian Y, Zhao L, Wang F. 2006. Sequence analysis of G glycoprotein of human respiratory syncytiai virus subtype B strains isolated from children with acute respiratory infections in Beijing, China in years 2000–2004. Chin J Microbiol Immunol 26:1–5. Eshaghi A, Duvvuri VR, Lai R, Nadarajah JT, Li A, Patel SN, Low DE, Gubbay JB. 2012. Genetic variability of human respiratory syncytial virus A strains circulating in Ontario: A novel genotype with a 72 nucleotide G gene duplication. PLoS ONE 7: e32807. Garcia-Beato R, Martinez I, Franci C, Real FX, Garcia-Barreno B, Melero JA. 1996. Host cell effect upon glycosylation and antigenicity of human respiratory syncytial virus G glycoprotein. Virology 221:301–309. Johnson PR, Spriggs MK, Olmsted RA, Collins PL. 1987. The G glycoprotein of human respiratory syncytial viruses of subgroups A and B: Extensive sequence divergence between antigenically related proteins. Proc Natl Acad Sci USA 84:5625–5629. Julenius K. 2007. NetCGlyc 1.0: Prediction of mammalian Cmannosylation sites. Glycobiology 17:868–876. Kushibuchi I, Kobayashi M, Kusaka T, Tsukagoshi H, Ryo A, Yoshida A, Ishii H, Saraya T, Kurai D, Yamamoto N, Kanou K, Saitoh M, Noda M, Kuroda M, Morita Y, Kozawa K, Oishi K, Tashiro M, Kimura H. 2013. Molecular evolution of attachment glycoprotein (G) gene in human respiratory syncytial virus detected in Japan 2008–2011. Infect Genet Evol 18:168–173. Lambert DM. 1988. Role of oligosaccharides in the structure and function of respiratory syncytial virus glycoproteins. Virology 164:458–466. Mufson MA, Orvell C, Rafnar B, Norrby E. 1985. Two distinct subtypes of human respiratory syncytial virus. J Gen Virol 66:2111–2124. Nair H, Nokes DJ, Gessner BD, Dherani M, Madhi SA, Singleton RJ, O’Brien KL, Roca A, Wright PF, Bruce N, Chandran A, Theodoratou E, Sutanto A, Sedyaningsih ER, Ngama M, Munywoki PK, Kartasasmita C, Simoes EA, Rudan I, Weber MW, Campbell H. 2010. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: A systematic review and meta-analysis. Lancet 375:1545–1555. Parveen S, Broor S, Kapoor SK, Fowler K, Sullender WM. 2006. Genetic diversity among respiratory syncytial viruses that have caused repeated infections in children from rural India. J Med Virol 78:659–665. Peret TC, Hall CB, Schnabel KC, Golub JA, Anderson LJ. 1998. Circulation patterns of genetically distinct group A and B strains of human respiratory syncytial virus in a community. J Gen Virol 79:2221–2229. Peret TC, Hall CB, Hammond GW, Piedra PA, Storch GA, Sullender WM, Tsou C, Anderson LJ. 2000. Circulation patterns of group A and B human respiratory syncytial virus genotypes in 5 communities in North America. J Infect Dis 181:1891–1896. Polack FP, Irusta PM, Hoffman SJ, Schiatti MP, Melendi GA, Delgado MF, Laham FR, Thumar B, Hendry RM, Melero JA, Karron RA, Collins PL, Kleeberger SR. 2005. The cysteine-rich region of respiratory syncytial virus attachment protein inhibits innate immunity elicited by the virus and endotoxin. Proc Natl Acad Sci USA 102:8996–9001. Sato M, Saito R, Sakai T, Sano Y, Nishikawa M, Sasaki A, Shobugawa Y, Gejyo F, Suzuki H. 2005. Molecular epidemiology of respiratory syncytial virus infections among children with acute respiratory symptoms in a community over three seasons. J Clin Microbiol 43:36–40. Scott PD, Ochola R, Ngama M, Okiro EA, Nokes DJ, Medley GF, Cane PA. 2004. Molecular epidemiology of respiratory syncytial virus in Kilifi district, Kenya. J Med Virol 74:344–354. Shobugawa Y, Saito R, Sano Y, Zaraket H, Suzuki Y, Kumaki A, Dapat I, Oguma T, Yamaguchi M, Suzuki H. 2009. Emerging genotypes of human respiratory syncytial virus subgroup A among patients in Japan. J Clin Microbiol 47:2475–2482. Tan L, Coenjaerts FE, Houspie L, Viveen MC, van Bleek GM, Wiertz EJ, Martin DP, Lemey P. 2013. The comparative genomics of human respiratory syncytial virus subgroups A and
A Novel Genotype of HRSV-B in China B: Genetic variability and molecular evolutionary dynamics. J Virol 87:8213–8226. The IMpact-RSV Study Group. 1998. Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. Pediatrics 102:531–537. Trento A, Galiano M, Videla C, Carballal G, Garcia-Barreno B, Melero JA, Palomo C. 2003. Major changes in the G protein of human respiratory syncytial virus isolates introduced by a duplication of 60 nucleotides. J Gen Virol 84:3115–3120. Trento A, Viegas M, Galiano M, Videla C, Carballal G, Mistchenko AS, Melero JA. 2006. Natural history of human respiratory syncytial virus inferred from phylogenetic analysis of the attachment (G) glycoprotein with a 60-nucleotide duplication. J Virol 80:975–984. Trento A, Casas I, Calderon A, Garcia-Garcia ML, Calvo C, PerezBrena P, Melero JA. 2010. Ten years of global evolution of the human respiratory syncytial virus BA genotype with a 60nucleotide duplication in the G protein gene. J Virol 84:7500– 7512. Venter M, Madhi SA, Tiemessen CT, Schoub BD. 2001. Genetic diversity and molecular epidemiology of respiratory syncytial virus over four consecutive seasons in South Africa: Identification of new subgroup A and B genotypes. J Gen Virol 82:2117–2124. Visser A, Delport S, Venter M. 2008. Molecular epidemiological analysis of a nosocomial outbreak of respiratory syncytial virus associated pneumonia in a kangaroo mother care unit in South Africa. J Med Virol 80:724–732. Woelk CH, Holmes EC. 2001. Variable immune-driven natural selection in the attachment (G) glycoprotein of respiratory syncytial virus (RSV). J Mol Evol 52:182–192. Yoshida LM, Suzuki M, Yamamoto T, Nguyen HA, Nguyen CD, Nguyen AT, Oishi K, Vu TD, Le TH, Le MQ, Yanai H, Kilgore
9 PE, Dang DA, Ariyoshi K. 2010. Viral pathogens associated with acute respiratory infections in central vietnamese children. Pediatr Infect Dis J 29:75–77. Yu JR, Kim S, Lee JB, Chang J. 2008. Single intranasal immunization with recombinant adenovirus-based vaccine induces protective immunity against respiratory syncytial virus infection. J Virol 82:2350–2357. Zhang Y, Xu W, Shen K, Xie Z, Sun L, Lu Q, Liu C, Liang G, Beeler JA, Anderson LJ. 2007. Genetic variability of group A and B human respiratory syncytial viruses isolated from 3 provinces in China. Arch Virol 152:1425–1434. Zhang RF, Jin Y, Xie ZP, Liu N, Yan KL, Gao HC, Song JR, Yuan XH, Xiao NG, Guo MW, Zhou QH, Hou YD, Duan Z. 2010a. Human respiratory syncytial virus in children with acute respiratory tract infections in China. J Clin Microbiol 48:4193– 4199. Zhang ZY, Du LN, Chen X, Zhao Y, Liu EM, Yang XQ, Zhao XD. 2010b. Genetic variability of respiratory syncytial viruses (RSV) prevalent in Southwestern China from 2006 to 2009: emergence of subgroup B and A RSV as dominant strains. J Clin Microbiol 48:1201–1207. Zlateva KT, Lemey P, Vandamme AM, Van Ranst M. 2004. Molecular evolution and circulation patterns of human respiratory syncytial virus subgroup a: Positively selected sites in the attachment G glycoprotein. J Virol 78:4675–4683. Zlateva KT, Lemey P, Moes E, Vandamme AM, Van Ranst M. 2005. Genetic variability and molecular evolution of the human respiratory syncytial virus subgroup B attachment G protein. J Virol 79:9157–9167. Zlateva KT, Vijgen L, Dekeersmaeker N, Naranjo C, Van Ranst M. 2007. Subgroup prevalence and genotype circulation patterns of human respiratory syncytial virus in Belgium during ten successive epidemic seasons. J Clin Microbiol 45:3022–3030.
J. Med. Virol. DOI 10.1002/jmv
Molecular Characterization of Human Respiratory Syncytial Virus Subtype B: A Novel Genotype of Subtype B Circulating in China Luo Ren,1 Qiuyan Xiao,1 Lili Zhou,1 Qiuling Xia,1 and Enmei Liu2* 1
Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing, China 2 Department of Respiratory Medicine, Children’s Hospital of Chongqing Medical University, Chongqing, China
Human respiratory syncytial virus (HRSV) is major pathogen of lower respiratory tract infections in infants and young children worldwide. There have been many studies regarding HRSV subgroup A (HRSV-A) G protein genetic variability but little information about HRSV subtype B (HRSV-B) G protein genetic diversity and molecular evolution in China. Thus, a survey of the molecular epidemiology and evolution of the G protein in China is of high importance. In this study, the circulation and genetic diversity of HRSV in Chongqing, Southwestern China, from June 2009 to May 2013, were investigated. A total of 3,167 nasopharyngeal aspirates were obtained in this study, and it was found that HRSV-B predominated in the 2009–2010 and 2012–2013 epidemic seasons. This study identified the genetic variability of the glycoprotein G gene among 102 HRSV-B strains isolated by cell culture from Chongqing nasopharyngeal aspirates, and 68 Chinese HRSV-B sequences were deposited in GenBank. Genotyping and phylogenetic analysis revealed that the HRSV-B strains were clustered into three genotypes: BA (n ¼ 111, 65.29%), GB3 (n ¼ 5, 2.94%), and a new GB genotype (n ¼ 54, 31.77%) named GB5. The GB5 strains varied from other genotypes in the central conserved region and N-glycosylation sites. The estimated evolutionary rate of Chinese HRSV-B was 2.01 103 nucleotide substitutions/site/year, which is similar to the reports from Belgium and the Netherlands with 1.95 103 and 2.78 103 nucleotide substitutions/site/year, respectively. This study provides data on the circulating pattern and molecular characterization of HRSV-B genotypes in China during four consecutive years and may contribute to HRSV vaccine development. J. Med. Virol. 87:1–9, 2015. # 2014 Wiley Periodicals, Inc. C 2014 WILEY PERIODICALS, INC.
KEY WORDS:
human respiratory syncytial virus; subtype B; genotype; GB5; G glycoprotein; China
INTRODUCTION Human respiratory syncytial virus (HRSV) is a major pathogen of lower respiratory tract infections in infants and young children worldwide. It is estimated that HRSV causes over 30 million lower respiratory tract infections each year and results in 3 million hospitalizations, making it the most common cause of hospitalization in children under 5-years old [Nair et al., 2010]. As a species of the Paramyxoviridae family, HRSV has a nonsegmented, negative-sense RNA genome of approximately 15,200 nucleotides encoding 11 proteins [Cane, 2001]. Among these proteins, G protein, a type II glycoprotein, is N- and O-glycosylated [Johnson et al., 1987]. The variation in the G glycoprotein is the major difference among the major HRSV subtypes. HRSV has two antigenic subgroups, Abbreviations: HRSV, human respiratory syncytial virus; HRSV-A, human respiratory syncytial virus subgroup A; HRSVB, human respiratory syncytial virus subgroup B. Grant sponsor: China Special Grant for the Prevention and Control of Infection Diseases; Grant number: 2013ZX10004202002. The authors have no conflicts of interest to declare. Correspondence to: Enmei Liu, Department of Respiratory Medicine, Children’s Hospital of Chongqing Medical University, No. 136, the 2nd Zhongshan Road, Yuzhong District, Chongqing 400014, China. E-mail: [email protected] Accepted 25 March 2014 DOI 10.1002/jmv.23960 Published online 9 June 2014 in Wiley Online Library (wileyonlinelibrary.com).
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A and B, based on reactions with monoclonal antibodies against the G glycoprotein [Mufson et al., 1985] and genetic analysis. There has been no effective vaccine to prevent the spread of HRSV. Palivizumab, which recognizes the “A” antigenic site of the HRSV fusion protein, is the first and only approved humanized monoclonal antibody to prevent HRSV infection in infants and young children at high risk [The IMpact-RSV Study Group, 1998]. However, Palivizumab is not available in China. The G glycoprotein is the target of neutralizing antibodies, and intranasal administration of G protein elicits beneficial protective immunity and represents a promising vaccine against HRSV infection [Yu et al., 2008], suggesting that this protein may be under immune pressure [Woelk and Holmes, 2001]. Thus, a survey of the molecular epidemiology and evolution of the G protein in China is of high importance. A previous hospital-based study at Chongqing, Southwestern China, showed that the BA genotype of HRSV subgroup B (HRSV-B) with a 60-nucleotide insertion became the dominant genotype during the 2008–2009 epidemic season [Zhang et al., 2010a]. Notably, there have been more studies regarding HRSV subgroup A (HRSV-A) G protein genetic variability [Zhang et al., 2007, 2010a,b] but little information about HRSV-B G protein genetic diversity and molecular evolution in China. This study not only evaluated G protein genetic variability of HRSVB isolated clinically in Chongqing, China, but also analyzed the Chinese HRSV-B strains in GenBank from 2004 to 2013.
manufacturer’s instructions. Reverse transcription (RT-PCR) was performed to create cDNA using the SuperScript II Reverse Transcriptase System (Invitrogen, USA) with random primers. Subtypes were identified by PCR amplification as reported previously [Coiras et al., 2003].
MATERIALS AND METHODS
Phylogenetic Analysis
Sample Collection and Viral Isolation
The 270 and 330 nucleotides of HVR2 of the G gene found within non-BA and BA genotypes were compared, respectively, with reference strains available from GenBank. Multiple sequence alignments were made using MEGA 5.0 programs. Phylogenetic trees were constructed using the neighbor-joining algorithm, and the statistical significance of the tree topology tested by bootstrapping (1,000 replicates) was performed. Only bootstrap values >70% are shown in each tree. According to a previous study [Venter et al., 2001], sequences were arbitrarily considered a genotype if they clustered together with bootstrap values of 70–100% and the pairwise-distance (p-distance) was less than 0.07 to all other members in the same phylogenetic cluster. Besides the sequences of the Chinese HRSV-B strains, all reference sequences for HRSV-B included in the phylogenetic analysis are shown in Table I. Partial G protein gene sequences of newly obtained HRSV-B isolates in Chongqing have been deposited in GenBank with the accession numbers KC461262–KC461295.
All nasopharyngeal aspirates were collected from hospitalized infants or children with acute lower respiratory tract infections from the Department of Respiratory Medicine, Chongqing Children’s Hospital in Southwestern China from June 2009 to May 2013. This study was approved by the Ethics and Research Council of Chongqing Children’s Hospital, and signed consent was obtained from each child’s parent or foster parent. The nasopharyngeal aspirates were stored at 80˚C and cultured in human laryngeal carcinoma HEp-2 cells. The viral isolates were harvested by scraping when the cytopathic effect was more than 75%. The virus-cell suspensions were frozen and stored at 80˚C. RNA Extraction and HRSV Subtype Identification Before RNA extraction, the nasopharyngeal aspirates and frozen cultured samples were centrifuged at 3,000g and 4˚C for 5 min. RNA was extracted from 200 ml of nasopharyngeal aspirate supernatant and frozen cultured samples using a QIAamp Viral RNA Kit (Qiagen, Hilden, Germany) according to the J. Med. Virol. DOI 10.1002/jmv
PCR Amplification and Sequencing The cultured HRSV-B isolates were used to sequence the G gene. Partial HRSV-B G gene (0.8 kb), the ectodomain, including the first hypervariable region (HVR1, nt. 199 to nt. 489 of the G gene), the central conserved region (nt. 490 to nt. 548 of the G gene), and the secondary hypervariable region (HVR2, nt. 549 to the end of the G gene), was amplified according to a previous study [Zlateva et al., 2005]. The PCR products were sequenced using an ABI3730xl DNA Analyzer at BGI Tech Solutions Co., Ltd. (Beijing, China). Data Set Assembly Analysis of HRSV-B G genetic variability was conducted on a data set containing a combination of newly obtained HRSV-B isolates in Chongqing and strains from other provinces of China found in GenBank. The data sets were named as Chinese HRSV-B strains. The accession numbers were DQ270232 and DQ270233 [Deng et al., 2006], DQ289648–DQ289649 [Zhang et al., 2007], GU357503–GU357530 [Zhang et al., 2010b], GU550474–GU550503 [Zhang et al., 2010a], and JF713439–JF713446 (unpublished).
Amino Acid and N-Glycosylation Site Analysis The amino acid sequences within part of the ectodomain, from amino acid 101 to the C-terminal
A Novel Genotype of HRSV-B in China
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TABLE I. HRSV-B G Protein GenBank Reference Sequences Strain 8/60 9320/77 BA/1370/99 BA/1441/02 BA/4825/03 CB616/090 CB757/09 CH18537 CH93-9b NG-010-09 NG-022-06 NG-025-07 NG-040-07 NG-046-06
GenBank accession No.
Strain
GenBank accession No.
M73545 AY353550 DQ227364 DQ227381 DQ227401 HQ699300 HQ699304 M17213 AF065251 HM459857 HM459876 HM459889 HM459879 AB603478
NG-050-05 NG-056-05 NG-084-07 NG-087-07 NG-102-06 NG-119-07 NG-166-06 NG-182-03 NY01 SA0025 SA98V602 SA99V429 SA99V800 TX69208
AB603476 AB603474 HM459883 HM459870 AB603467 HM459878 AB603479 HM459859 AF233931 AF348825 AF348824 AF348813 AF348821 AF233933
test. The codon-based maximum likelihood IFEL method is subject to selective pressure at the population level. A P value < 0.1 was used to define sites as positively (dN > dS) or negatively (dN < dS) selected sites, respectively. RESULTS HRSV Distribution and Seasonal
end of newly obtained Chongqing HRSV-B isolates, were determined and compared to the reference strains CH18537 (non-BA genotype) and 1370/99 (BA genotype). Potential N-glycosylation sites (NXT, where X is not a proline) were predicted using Net NGlyc 1.0 [Julenius, 2007].
In this study, among 3,167 nasopharyngeal aspirates collected between June 2009 and May 2013, 1,035 (33%) were tested to be positive for HRSV. HRSV-A and HRSV-B were identified in 671 (65%) and 343 (33%) specimens, respectively, and both HRSV-A and HRSV-B were identified in 21 (2%) specimens. HRSV-B strains accounted for 59–79% among the cases in the summer of 2009 to the spring of 2010, whereas HRSV-A strains accounted for 73– 96% among the HRSV infections in the summer of 2010 to the spring of 2012, respectively (Fig. 1). In the summer of 2012 to the spring of 2013, group B strains predominated, accounting for 71–84% among all the HRSV cases (the variation was stated between seasons, Fig. 1).
Evolutionary Rate Estimation
Assembly of G Genetic Variability Data
To estimate the evolutionary rate of Chinese HRSV-B, an exploratory root-to-tip linear regression analysis was performed by Path-O-Gen (available at http://tree.bio.ed.ac.uk/software/pathogen/). A maximum likelihood (ML) tree was constructed by MEGA 5.0 with a general time-reversible (GTR) substitution model and a discretized gamma distribution to model rate variation among sites. This program takes the ML tree as the input and performs a linear regression between the genetic distance from the root and the sampling date for each strain. The exploratory analysis was performed with the sampling year for the Chinese HRSV-B strains.
In the present study, 2,415 nasopharyngeal aspirates were randomly selected by random numbers to inoculate into HEp-2 cells. A total of 374 (15.49%) clinical isolates were identified (265 for HRSV-A, 108 for HRSV-B, and 1 isolate detected HRSV-A and HRSV-B). For HRSV-B, the partial G gene was sequenced successfully in 102 HRSV-B cultured isolates. Among the 102 isolates in Chongqing, there were 72 different sequences, of which each of 60 isolates had a unique sequence in the G gene, and the remaining 42 isolates shared 10 different sequences. The central conserved region (amino acids 164– 186, according to strain CH18537) was recognized among the 102 HRSV-B G protein sequences. Additionally, 68 HRSV-B G protein sequences detected in other provinces of China [Deng et al., 2006; Zhang et al., 2007, 2010a,b] were analyzed. The nucleotide sequences from the Chinese strains were compared to 28 HRSV-B sequences available in GenBank (Table I).
Selective Pressures Analysis Positively and negatively selected sites among the present strains were calculated to derive the synonymous (dS) and nonsynonymous (dN) rates at every codon in the alignment using Datamonkey (http:// www.datamonkey.org/). An excess of nonsynonymous substitutions (dN > dS) were interpreted as positive selection and increased fitness by replacement substitutions, while a paucity of replacement changes (dN < dS) indicated that negative selection removes such substitutions from the gene pool. Single likelihood ancestor counting (SLAC), fixed effects likelihood (FEL), and internal fixed effects likelihood (IFEL) were used to identify the diversifying selection. SLAC bases its inference on the expected and inferred numbers of synonymous and nonsynonymous substitutions and FEL directly estimates dN and dS based on a codon-substitution model to derive the p-value for the test dN6¼dS using a likelihood ratio
Phylogenetic Analysis of HRSV-B The Chinese HRSV-B strains were clustered into three genotypes: BA (n ¼ 111, 65.29%), GB3 (n ¼ 5, 2.94%), and a new GB genotype (n ¼ 54, 31.77%) named GB5 with a bootstrap value of 99% (Fig. 2). The p-distance within the GB5 genotype was 0.016. Although the GB5 genotype was close genetically to the GB1 and GB2 genotypes, the p-distance between GB5 to GB1 and GB5 to GB2 were 0.138 0.002 and 0.068 0.003, respectively, suggesting that GB5 was a new genotype [Venter et al., 2001; Arnott et al., 2011] (Table II). J. Med. Virol. DOI 10.1002/jmv
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Fig. 1. Seasonal distribution of HRSV-positive samples. Data are represented as percentages between June 2009 and May 2013. SP, spring, including March, April, and May; SU, summer, including June, July, and August; FA, fall, including September, October, and November; WI, winter, including December, January, and February.
The BA genotype with duplication of 60 nucleotides, first reported in 1999 [Trento et al., 2003], became the dominant strain of HRSV-B in Niigata, Japan [Sato et al., 2005] and Belgium [Zlateva et al., 2005, 2007] in 2003, in New Delhi in 2004 [Parveen et al., 2006], in Brazil in 2005 [Botosso et al., 2009], in South Africa in 2006 [Visser et al., 2008], and in Madrid in 2007 [Trento et al., 2010]. However, the BA genotypes predominated in 2009– 2010, whereas the GB5 genotype outbreak was prominent in 2010–2011 in Chongqing (Fig. 3). The BA9 genotype replaced the non-BA genotype to become dominant from 2011 to 2013 in Chongqing (Fig. 3), suggesting that the BA genotypes expanded with fluctuation. Amino Acid and N-Glycosylation Site Analysis Due to the usage of alternative termination codons, deletions, insertions, one frameshift mutations, premature stop codons, the lengths of the deduced G protein sequences among the HRSV-B strains were significantly different. The predicted complete G proteins of the Chinese HRSV-B strains had four different amino acid lengths (290, 293, 296, and 297 Fig. 2. Phylogenetic trees for HRSV-B nucleotide sequences based on the second variable region of the G protein. Details for the reference GenBank sequences used for phylogenetic analysis are given in Table I. The newly identified Chongqing (CQ) and reported Chinese (B, Beijing; Chongqing, LZY) HRSV-B strains were compared with reference GenBank sequences throughout the world, including strains in Sweden (8/60), Massachusetts (9320/77), Niigata (NG), Rochester (CH), Houston (TX), Rochester (NY), Soweto (South Africa, SA), Madrid (Spain, BA genotype), and Chungbuk (Korea, CB). The phylogenetic tree was constructed with a neighbor-joining algorithm using MEGA 5 software. Only bootstrap values greater than 70% are displayed at the branch nodes. The genotypes are indicated at the right by brackets. The BA1-4, BA8, BA10, and CB-B genotypes were compressed as solid triangles.
J. Med. Virol. DOI 10.1002/jmv
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A Novel Genotype of HRSV-B in China
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TABLE II. p-Distance Between Chinese HRSV-B Genotypes Genotype
BA1
BA10
BA2
BA3
BA4
BA8
BA9
CB-B
GB5
GB1
GB2
GB3
GB4
SAB1 SAB2 SAB3
BA1 BA10 BA2 BA3 BA4 BA8 BA9 CB-B GB5 GB1 GB2 GB3 GB4 SAB1 SAB2 SAB3
0.05 0.048 0.032 0.033 0.037 0.051 0.061 0.09 0.119 0.05 0.04 0.106 0.065 0.061 0.045
0.066 0.053 0.046 0.038 0.049 0.061 0.111 0.134 0.076 0.063 0.136 0.095 0.084 0.062
0.049 0.044 0.052 0.064 0.083 0.105 0.142 0.077 0.073 0.129 0.093 0.089 0.076
0.027 0.04 0.052 0.066 0.102 0.126 0.058 0.047 0.11 0.076 0.069 0.046
0.036 0.045 0.059 0.106 0.128 0.063 0.05 0.119 0.082 0.074 0.056
0.039 0.059 0.099 0.13 0.062 0.054 0.125 0.084 0.076 0.05
0.069 0.111 0.129 0.077 0.066 0.135 0.087 0.088 0.062
0.102 0.121 0.058 0.053 0.111 0.078 0.07 0.058
0.13 0.068 0.093 0.138 0.101 0.12 0.097
0.091 0.11 0.156 0.117 0.136 0.125
0.053 0.086 0.048 0.067 0.059
0.105 0.059 0.104 0.062 0.104 0.082 0.04 0.112 0.071 0.048
Bolds indicate the p-distance between GB5 and other genotypes.
amino acids) for non-BA genotypes and five different amino acid lengths (280, 310, 312, 313, and 317 amino acids) for BA genotypes, respectively (Fig. 4). The reported alternative stop codons [Zlateva et al., 2005] were observed in both the GB5 and BA genotypes (at amino acid positions 291, 294, and 298 for the GB5 genotype, referring to strain CH18537; at amino acid positions 311, 314, and 318 for the BA genotype, referring to strain BA/1370/99, Fig. 4). The previously reported six-nucleotide deletion (30 -AAAACC50 ) [Zlateva et al., 2005], causing amino acid deletions at positions 159 and 160, was observed in all of the newly obtained 102 HRSV-B strains in Chongqing. As a result of a 3-amino acid insertion at position 226 (QKT, referring to strain CH18537), three strains of the GB5 genotype (Chongqing/B/09/15, CQ911Sep2010 and CQ2607-Dec2011) had a protein with 296 amino acids. There was a premature stop codon at amino acid position 283 (referring to strain BA/ 1370/99) of the CQ2795-Jan2012 strain, leading to a BA genotype strain with a protein of 280 amino acids. In addition to the difference in the number of amino acids, a nonsynonymous amino acid substitution was identified in the central conserved region of the GB5 genotype (Q-R, at amino acid position 180). No N-glycosylation site was identified after amino acid 100 in reference to the HRSV-B sequence of strain CH18537 [Johnson et al., 1987]. However, three N-glycosylation sites were identified in genotype GB5 strains at amino acids 258, 276, and 290 (referring to strain CH18537, Fig. 4A). The N-glycosylation sites at amino acids 296 and 310 (referring to strain BA/1370/99, Fig. 4B) were common for the BA genotype.
tendency to develop respiratory failure (32.4% vs. 16.4%, P ¼ 0.067, Table III). Evolutionary Rate In order to estimate the rate of nucleotide substitutions and the time of the most recent common ancestor (TMRCA) of the Chinese HRSV-B strains throughout the 10 years of circulation, an exploratory root-to-tip linear regression analysis using 170 Chinese HRSV-B strains was performed. The correlation between phylogenetic root-to-tip divergence and time of sampling of HRSV-B strains is displayed in a regression plot (Fig. 5). The rate of substitution as estimated from this linear regression analysis was 2.01 103 nucleotide substitutions/site/year. The TMRCA of Chinese HSRV-B was estimated to date back to 1972.
Clinical Manifestations There were no significant differences observed in the clinical manifestations between children infected with the BA9 and GB5 genotypes (Table III). However, the patients infected with GB5 strains had a
Fig. 3. Yearly distribution of HRSV-B genotypes in Chongqing between 2009 and 2013. Data are represented as number of HRSV-B samples. SP, spring, including March, April, and May; SU, summer, including June, July, and August.
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Fig. 4. Deduced amino acid alignments of the central conserved and HVR2 of the G protein from 102 newly identified Chongqing HRSV-B strains. Alignments are shown relative to the sequences of prototyped strains CH18537 and BA/1370/99. Identical residues are indicated by dots. Stop codons are
indicated by asterisks. Potential N-glycosylation sites (NXT, where X is not a proline) are indicated by dotted rectangles. The solid rectangles show the nonsynonymous amino acid substitution Q180R of GB5 strains and the 60 nucleotide duplication of the BA genotype, respectively.
Analysis of Selective Pressure
genotype of HRSV-B, was found to be circulated in China. It has been reported that the subtype circulation pattern of HRSV-B became predominant after two consecutive seasons with a high prevalence of subgroup A [Coggins et al., 1998; Scott et al., 2004; Arbiza et al., 2005]. However, in an earlier hospitalbased study of Chongqing, China, HRSV-B predominated in the epidemic season during 2008–2009 [Zhang et al., 2010a], indicating that there were two switches of HRSV subgroups in the past 7 years in Chongqing. In accordance with the previous study [Zhang et al., 2010a] and the recent study in Beijing, China [Cui et al., 2013], HRSV-B predominated over the HRSV circulation pattern within 2 years after two epidemic seasons of HRSV-A in Chongqing. A similar circulation pattern was observed in Cambodia [Arnott et al., 2011]. The shift in the predominant group might be correlated in part with variability in the G-protein gene and the immune response of the population.
We used SLAC, FEL, and IFEL methods to identify the diversified selection. In HRV2, five positively selected sites were identified (amino acid positions 223, 271, 278, 305, and 315, referring to strain BA/ 1370/99, Table IV). While 15 negatively selected sites were identified at amino acid positions 216, 220, 235, 252, 260, 272, 279, 285, 288, 294, 295, 297, 302, 210, and 311 (referring to strain BA/1370/99, Table IV). DISCUSSION The present study analyzed the G gene of HRSV-B isolates from nasopharyngeal aspirates from June 2009 to May 2013 in Chongqing, Southwestern China. Various bioinformatics methods were used to better understand the genotype variability, molecular epidemiology, and evolutionary rate of circulating strains. The results demonstrated that HRSV-B was predominant in the epidemic seasons of 2009–2010 and 2012–2013 and that the GB5 genotype, a novel
TABLE III. Demographic Characteristics of the Genotyped Population
Group
No. of cases
Gender (Male, %)
Age (month, median, range)
Fever (N, %)
Cough (N, %)
Wheezing (N, %)
Days of Hospitalization (median, range)
Respiratory failure (N, %)
BA9 GB5 Total
67 34 101
44 (65.7) 25 (73.5) 7 (5.5)
5 (1–40) 5.5 (1–42) 5 (1–42)
16 (23.9) 10 (29.4) 26 (25.7)
67 (100) 34 (100) 101 (100)
45 (67.2) 25 (73.5) 70 (69.3)
7 (3–17) 6.5 (3–14) 7 (3–17)
11 (16.4) 11 (32.4) 22 (21.8)
A strain which was identified as GB3 genotype was not included in this table for only one case was reported.
J. Med. Virol. DOI 10.1002/jmv
A Novel Genotype of HRSV-B in China
7
Fig. 5. Linear root-to-tip regression plot presenting the correlation between the branch lengths and the sampling dates of the HRSV-B isolates included in this study.
In addition to the reported genotypes of GA1 to GA7 [Peret et al., 1998, 2000] and SAA1 [Venter et al., 2001], four novel genotypes of HRSV-A, NA1, NA2 [Shobugawa et al., 2009], CB-A [Baek et al., 2012], and ON1 [Eshaghi et al., 2012] have been reported in the past 5 years. HRSV-B genotypes include GB1 to GB4, SAB1 to SAB3, URU1, URU2, BA1 to BA6 [Peret et al., 1998, 2000; Blanc et al., 2005; Trento et al., 2006; Yoshida et al., 2010], and new genotypes of SAB4 [Arnott et al., 2011], BA7 to BA10 [Dapat et al., 2010], BA11 and CB-B [Baek et al., 2012] and BA12 [Kushibuchi et al., 2013]. Another novel HRSV-B genotype named GB5 was observed in this study. Fifty-four GB5 strains were identified in different provinces of China from 2005 TABLE IV. Positively and Negatively Selected Sites of Chinese HRSV-B Codon
IFEL
Positively selected sites 223 271 278 305 315 Negatively selected sites 216 220 235 252 260 272 279 285 288 294 295 297 302 310 311
FEL
SLAC
SLAC, single likelihood ancestor counting; FEL, fixed effects likelihood; IFEL, internal fixed effects likelihood. P < 0.1.
to 2013. During the preparation of this manuscript, the GB5 genotype was reported as CB1 [Cui et al., 2013] and THB [Auksornkitti et al., 2014] genotypes in Beijing and Thailand, respectively, indicating that the GB5 strains also can be identified in other countries. Among the GB5 genotypes, a nonsynonymous amino acid substitution (Q180R) in the central conserved region of amino acids 164–186 that contains a highly conserved region with 13 amino acids, cysteine residues, and a CX3C motif [Collins and Crowe, 2007], inhibits innate immunity elicited by HRSV and endotoxins [Polack et al., 2005]. Three N-glycosylation sites at amino acids 258, 276, and 290 (referring to strain CH18537) emerged from the GB5 strains. N-glycosylated sites have an impact on the antigenic structure of the G protein and virus infectivity [Lambert, 1988; Garcia-Beato et al., 1996]. After first being detected in 1999, the BA genotype replaced the non-BA genotypes to become the worldwide dominant HRSV-B strain in 2003–2007 [Sato et al., 2005; Zlateva et al., 2005, 2007; Parveen et al., 2006; Visser et al., 2008; Botosso et al., 2009; Trento et al., 2010], indicating the great adaptation of this genotype. However, during the expansion of the BA genotype, there was an outbreak of GB5 in 2010– 2011 in Chongqing, suggesting that BA genotypes expanded with fluctuation. This was consistent with the pattern of BA genotype expansion worldwide [Trento et al., 2010]. On the other hand, a recent study reported that new non-BA strains related to the BA genotype reemerged in Kenya, where BA strains were predominant for years [Agoti et al., 2013]. This observation provides a mechanism for the replacement of BA strains if the BA selective advantage diminishes. Thus, it is important to monitor whether the GB5 strains reappear in China. The estimated evolutionary rate of the G protein was greater than that of the whole genome [Tan et al., 2013]. The estimated evolutionary rate of Chinese HRSV-B was 2.01 103 nucleotide substitutions/site/year, which is very similar to the reports from Belgium and the Netherlands with 1.95 103 and 2.78 103 nucleotide substitutions/site/year, respectively [Zlateva et al., 2005; Tan et al., 2013]. While the evolutionary rates of the HRSV-A G protein gene were 1.83 103 and 2.22 103 nucleotide substitutions/site/year [Zlateva et al., 2004; Tan et al., 2013], indicating that HRSV-B had an equally high potential for accumulation of genetic diversity of the intra- and intersubtypes. As a target for the neutralizing antibody, the G glycoprotein is under immune pressure. In the present study, five positive-selected sites at amino acid positions 223, 271, 278, 305, and 315 were identified, referring to strain BA/1370/99. The 223 site was also identified as a positive-selected site in previous studies [Woelk and Holmes, 2001; Zlateva et al., 2005], suggesting that this site may be correlated with the immune response to HRSV-B infection. J. Med. Virol. DOI 10.1002/jmv
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In conclusion, a novel genotype of HRSV-B, GB5 circulating in China, was identified and molecular analysis of the G protein demonstrated that GB5 strains had variations in the central conserved region and N-glycosylation sites in this study. These findings may contribute to the development of a potential vaccine based on the HRSV-B G protein in China. Further molecular and epidemiological surveillance of HRSV is important to understand HRSV transmission and select appropriate vaccine strains. ACKNOWLEDGMENTS We thank all the families for their enrollment in this study. We also thank the staff in the Department of Respiratory Medicine, Children’s Hospital of Chongqing Medical University for clinical information collection. REFERENCES Agoti CN, Gitahi CW, Medley GF, Cane PA, Nokes DJ. 2013. Identification of group B respiratory syncytial viruses that lack the 60-nucleotide duplication after six consecutive epidemics of total BA dominance at coastal Kenya. Influenza Other Respir Viruses 7:1008–1012. Arbiza J, Delfraro A, Frabasile S. 2005. Molecular epidemiology of human respiratory syncytial virus in Uruguay: 1985–2001—A review. Mem Inst Oswaldo Cruz 100:221–230. Arnott A, Vong S, Mardy S, Chu S, Naughtin M, Sovann L, Buecher C, Beaute J, Rith S, Borand L, Asgari N, Frutos R, Guillard B, Touch S, Deubel V, Buchy P. 2011. A study of the genetic variability of human respiratory syncytial virus (HRSV) in Cambodia reveals the existence of a new HRSV group B genotype. J Clin Microbiol 49:3504–3513. Auksornkitti V, Kamprasert N, Thongkomplew S, Suwannakarn K, Theamboonlers A, Samransamruajkij R, Poovorawan Y. 2014. Molecular characterization of human respiratory syncytial virus, 2010–2011: Identification of genotype ON1 and a new subgroup B genotype in Thailand. Arch Virol 159:499–507. Baek YH, Choi EH, Song MS, Pascua PN, Kwon HI, Park SJ, Lee JH, Woo SI, Ahn BH, Han HS, Hahn YS, Shin KS, Jang HL, Kim SY, Choi YK. 2012. Prevalence and genetic characterization of respiratory syncytial virus (RSV) in hospitalized children in Korea. Arch Virol 157:1039–1050. Blanc A, Delfraro A, Frabasile S, Arbiza J. 2005. Genotypes of respiratory syncytial virus group B identified in Uruguay. Arch Virol 150:603–609. Botosso VF, Zanotto PM, Ueda M, Arruda E, Gilio AE, Vieira SE, Stewien KE, Peret TC, Jamal LF, Pardini MI, Pinho JR, Massad E, Sant’anna OA, Holmes EC, Durigon EL, Consortium V. 2009. Positive selection results in frequent reversible amino acid replacements in the G protein gene of human respiratory syncytial virus. PLoS Pathog 5:e1000254. Cane PA. 2001. Molecular epidemiology of respiratory syncytial virus. Rev Med Virol 11:103–116. Coggins WB, Lefkowitz EJ, Sullender WM. 1998. Genetic variability among group A and group B respiratory syncytial viruses in a children’s hospital. J Clin Microbiol 36:3552–3557. Coiras MT, Perez-Brena P, Garcia ML, Casas I. 2003. Simultaneous detection of influenza A, B, and C viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-PCR assay. J Med Virol 69:132–144. Collins PL, Crowe JE, Jr. 2007. Fields BN, Knipe DM, Howley PM, editors. Fields virology. Respiratory syncytial virus and metapneumovirus. Philadelphia, PA: Wolters Kluwer Health/Lippincott Williams & Wilkins, pp 1601–1635. Cui G, Zhu R, Qian Y, Deng J, Zhao L, Sun Y, Wang F. 2013. Genetic variation in attachment glycoprotein genes of human respiratory syncytial virus subgroups a and B in children in recent five consecutive years. PLoS ONE 8:e75020.
J. Med. Virol. DOI 10.1002/jmv
Dapat IC, Shobugawa Y, Sano Y, Saito R, Sasaki A, Suzuki Y, Kumaki A, Zaraket H, Dapat C, Oguma T, Yamaguchi M, Suzuki H. 2010. New genotypes within respiratory syncytial virus group B genotype BA in Niigata, Japan. J Clin Microbiol 48:3423–3427. Deng J, Zhu R, Qian Y, Zhao L, Wang F. 2006. Sequence analysis of G glycoprotein of human respiratory syncytiai virus subtype B strains isolated from children with acute respiratory infections in Beijing, China in years 2000–2004. Chin J Microbiol Immunol 26:1–5. Eshaghi A, Duvvuri VR, Lai R, Nadarajah JT, Li A, Patel SN, Low DE, Gubbay JB. 2012. Genetic variability of human respiratory syncytial virus A strains circulating in Ontario: A novel genotype with a 72 nucleotide G gene duplication. PLoS ONE 7: e32807. Garcia-Beato R, Martinez I, Franci C, Real FX, Garcia-Barreno B, Melero JA. 1996. Host cell effect upon glycosylation and antigenicity of human respiratory syncytial virus G glycoprotein. Virology 221:301–309. Johnson PR, Spriggs MK, Olmsted RA, Collins PL. 1987. The G glycoprotein of human respiratory syncytial viruses of subgroups A and B: Extensive sequence divergence between antigenically related proteins. Proc Natl Acad Sci USA 84:5625–5629. Julenius K. 2007. NetCGlyc 1.0: Prediction of mammalian Cmannosylation sites. Glycobiology 17:868–876. Kushibuchi I, Kobayashi M, Kusaka T, Tsukagoshi H, Ryo A, Yoshida A, Ishii H, Saraya T, Kurai D, Yamamoto N, Kanou K, Saitoh M, Noda M, Kuroda M, Morita Y, Kozawa K, Oishi K, Tashiro M, Kimura H. 2013. Molecular evolution of attachment glycoprotein (G) gene in human respiratory syncytial virus detected in Japan 2008–2011. Infect Genet Evol 18:168–173. Lambert DM. 1988. Role of oligosaccharides in the structure and function of respiratory syncytial virus glycoproteins. Virology 164:458–466. Mufson MA, Orvell C, Rafnar B, Norrby E. 1985. Two distinct subtypes of human respiratory syncytial virus. J Gen Virol 66:2111–2124. Nair H, Nokes DJ, Gessner BD, Dherani M, Madhi SA, Singleton RJ, O’Brien KL, Roca A, Wright PF, Bruce N, Chandran A, Theodoratou E, Sutanto A, Sedyaningsih ER, Ngama M, Munywoki PK, Kartasasmita C, Simoes EA, Rudan I, Weber MW, Campbell H. 2010. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: A systematic review and meta-analysis. Lancet 375:1545–1555. Parveen S, Broor S, Kapoor SK, Fowler K, Sullender WM. 2006. Genetic diversity among respiratory syncytial viruses that have caused repeated infections in children from rural India. J Med Virol 78:659–665. Peret TC, Hall CB, Schnabel KC, Golub JA, Anderson LJ. 1998. Circulation patterns of genetically distinct group A and B strains of human respiratory syncytial virus in a community. J Gen Virol 79:2221–2229. Peret TC, Hall CB, Hammond GW, Piedra PA, Storch GA, Sullender WM, Tsou C, Anderson LJ. 2000. Circulation patterns of group A and B human respiratory syncytial virus genotypes in 5 communities in North America. J Infect Dis 181:1891–1896. Polack FP, Irusta PM, Hoffman SJ, Schiatti MP, Melendi GA, Delgado MF, Laham FR, Thumar B, Hendry RM, Melero JA, Karron RA, Collins PL, Kleeberger SR. 2005. The cysteine-rich region of respiratory syncytial virus attachment protein inhibits innate immunity elicited by the virus and endotoxin. Proc Natl Acad Sci USA 102:8996–9001. Sato M, Saito R, Sakai T, Sano Y, Nishikawa M, Sasaki A, Shobugawa Y, Gejyo F, Suzuki H. 2005. Molecular epidemiology of respiratory syncytial virus infections among children with acute respiratory symptoms in a community over three seasons. J Clin Microbiol 43:36–40. Scott PD, Ochola R, Ngama M, Okiro EA, Nokes DJ, Medley GF, Cane PA. 2004. Molecular epidemiology of respiratory syncytial virus in Kilifi district, Kenya. J Med Virol 74:344–354. Shobugawa Y, Saito R, Sano Y, Zaraket H, Suzuki Y, Kumaki A, Dapat I, Oguma T, Yamaguchi M, Suzuki H. 2009. Emerging genotypes of human respiratory syncytial virus subgroup A among patients in Japan. J Clin Microbiol 47:2475–2482. Tan L, Coenjaerts FE, Houspie L, Viveen MC, van Bleek GM, Wiertz EJ, Martin DP, Lemey P. 2013. The comparative genomics of human respiratory syncytial virus subgroups A and
A Novel Genotype of HRSV-B in China B: Genetic variability and molecular evolutionary dynamics. J Virol 87:8213–8226. The IMpact-RSV Study Group. 1998. Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. Pediatrics 102:531–537. Trento A, Galiano M, Videla C, Carballal G, Garcia-Barreno B, Melero JA, Palomo C. 2003. Major changes in the G protein of human respiratory syncytial virus isolates introduced by a duplication of 60 nucleotides. J Gen Virol 84:3115–3120. Trento A, Viegas M, Galiano M, Videla C, Carballal G, Mistchenko AS, Melero JA. 2006. Natural history of human respiratory syncytial virus inferred from phylogenetic analysis of the attachment (G) glycoprotein with a 60-nucleotide duplication. J Virol 80:975–984. Trento A, Casas I, Calderon A, Garcia-Garcia ML, Calvo C, PerezBrena P, Melero JA. 2010. Ten years of global evolution of the human respiratory syncytial virus BA genotype with a 60nucleotide duplication in the G protein gene. J Virol 84:7500– 7512. Venter M, Madhi SA, Tiemessen CT, Schoub BD. 2001. Genetic diversity and molecular epidemiology of respiratory syncytial virus over four consecutive seasons in South Africa: Identification of new subgroup A and B genotypes. J Gen Virol 82:2117–2124. Visser A, Delport S, Venter M. 2008. Molecular epidemiological analysis of a nosocomial outbreak of respiratory syncytial virus associated pneumonia in a kangaroo mother care unit in South Africa. J Med Virol 80:724–732. Woelk CH, Holmes EC. 2001. Variable immune-driven natural selection in the attachment (G) glycoprotein of respiratory syncytial virus (RSV). J Mol Evol 52:182–192. Yoshida LM, Suzuki M, Yamamoto T, Nguyen HA, Nguyen CD, Nguyen AT, Oishi K, Vu TD, Le TH, Le MQ, Yanai H, Kilgore
9 PE, Dang DA, Ariyoshi K. 2010. Viral pathogens associated with acute respiratory infections in central vietnamese children. Pediatr Infect Dis J 29:75–77. Yu JR, Kim S, Lee JB, Chang J. 2008. Single intranasal immunization with recombinant adenovirus-based vaccine induces protective immunity against respiratory syncytial virus infection. J Virol 82:2350–2357. Zhang Y, Xu W, Shen K, Xie Z, Sun L, Lu Q, Liu C, Liang G, Beeler JA, Anderson LJ. 2007. Genetic variability of group A and B human respiratory syncytial viruses isolated from 3 provinces in China. Arch Virol 152:1425–1434. Zhang RF, Jin Y, Xie ZP, Liu N, Yan KL, Gao HC, Song JR, Yuan XH, Xiao NG, Guo MW, Zhou QH, Hou YD, Duan Z. 2010a. Human respiratory syncytial virus in children with acute respiratory tract infections in China. J Clin Microbiol 48:4193– 4199. Zhang ZY, Du LN, Chen X, Zhao Y, Liu EM, Yang XQ, Zhao XD. 2010b. Genetic variability of respiratory syncytial viruses (RSV) prevalent in Southwestern China from 2006 to 2009: emergence of subgroup B and A RSV as dominant strains. J Clin Microbiol 48:1201–1207. Zlateva KT, Lemey P, Vandamme AM, Van Ranst M. 2004. Molecular evolution and circulation patterns of human respiratory syncytial virus subgroup a: Positively selected sites in the attachment G glycoprotein. J Virol 78:4675–4683. Zlateva KT, Lemey P, Moes E, Vandamme AM, Van Ranst M. 2005. Genetic variability and molecular evolution of the human respiratory syncytial virus subgroup B attachment G protein. J Virol 79:9157–9167. Zlateva KT, Vijgen L, Dekeersmaeker N, Naranjo C, Van Ranst M. 2007. Subgroup prevalence and genotype circulation patterns of human respiratory syncytial virus in Belgium during ten successive epidemic seasons. J Clin Microbiol 45:3022–3030.
J. Med. Virol. DOI 10.1002/jmv
