Infection, Genetics and Evolution 27 (2014) 131–136
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Genetic variation of human respiratory syncytial virus among children with fever and respiratory symptoms in Shanghai, China, from 2009 to 2012 Jia Liu a,1, Yonglin Mu a,c,1, Wei Dong b, Fujia Yao a, Lili Wang a, Huajie Yan b, Ke Lan a,⇑, Chiyu Zhang a,⇑ a b c
Pathogen Diagnostic Center, Institut Pasteur of Shanghai, Chinese Academy of Science, Shanghai 200025, China Pediatric Department, Shanghai Nanxiang Hospital, Jiading District, Shanghai 201800, China College of Life and Environmental Science, Shanghai Normal University, Shanghai 200234, China
a r t i c l e
i n f o
Article history: Received 14 June 2014 Received in revised form 9 July 2014 Accepted 10 July 2014 Available online 19 July 2014 Keywords: Human respiratory syncytial virus (HRSV) New genotype Fever and respiratory symptoms Shanghai Co-circulation
a b s t r a c t Human respiratory syncytial virus (HRSV) of genus Pneumovirus is one of the most common pathogens causing severe acute lower respiratory tract infection in infants and children. No information on the genotype distribution of HRSV is available in East China (e.g. Shanghai). From August 2009 to December 2012, 2407 nasopharyngeal swabs were collected from outpatient children with fever and respiratory symptoms in Shanghai. HRSV infection was determined using a multiplex RT-PCR assay. The second hypervariable region (HVR2) of G protein gene of HRSV was amplified and sequenced from HRSV positive samples. Genotypes were characterized by phylogenetic analyses. Of 2407 nasopharyngeal samples, 184 (7.6%) were tested as HRSV positive. From 160 positive subjects with sufficient nasopharyngeal samples, 69 HVR2 sequences were obtained by RT-PCR and sequencing. Three HRSV epidemic seasons were observed from August 2009 to December 2012, and an extreme outbreak of HRSV occurred in the 2009–2010 epidemic season. A genotype shift of predominant HRSV strains from B group in the 2009– 2010 epidemic season to group A in the subsequent epidemic seasons was observed. Ten HRSV genotypes, including four group A genotypes NA1, NA3, NA4, and ON1, and six group B genotypes BA9, BA10, SAB4, CB1, BAc, and BA?, were detected in Shanghai. Seven genotypes (NA1, BA9–10, SAB4, CB1, BAc and BA?) were found in the 2009–2010 epidemic season. The co-circulation of multiple genotypes was associated with the extreme outbreak of HRSV among children with fever and respiratory symptoms in the 2009–2010 epidemic season. Ó 2014 Elsevier B.V. All rights reserved.
1. Background Human respiratory syncytial virus (HRSV) of genus Pneumovirus is one of the most common pathogens causing severe acute lower respiratory tract infection in infants and children, accounting for 15–40% pneumonia or bronchiolitis in children (Rudan et al., 2008). HRSV is a negative single-strand RNA virus with a genome of about 15 kilobases that encodes 11 proteins (Collins and Melero, 2011). The attachment glycoprotein (G protein) responsible for HRSV infection is the most variable protein (Teng et al., 2001). It contains two hypervariable regions (HVRs). The second HVR of G protein has the highest degree of divergence that can reflect overall gene variability, and is commonly used for genotyp⇑ Corresponding authors. Address: Pathogen Diagnostic Center, Institut Pasteur of Shanghai, Chinese Academy of Science, Hefei road 411, Shanghai, China. Tel.: +86 21 5492 3005. E-mail addresses: [email protected] (K. Lan), [email protected] (C. Zhang). 1 These authors contributed equally to this article. http://dx.doi.org/10.1016/j.meegid.2014.07.011 1567-1348/Ó 2014 Elsevier B.V. All rights reserved.
ing HRSV (Galiano et al., 2005). According to genetic variability of HRV2, two genetically distinct HRSV groups A and B have been divided (Mufson et al., 1985). Groups A and B were further subdivided into 11 (ON1, GA1–GA7, SAA1, NA1, and NA2) and 20 (GB1– GB4, BA1–BA10, SAB1–SAB4, URU1, and URU2) genotypes (Arnott et al., 2011; Eshaghi et al., 2012). One main feature of HRSV epidemic is that the predominant genotypes can shift among different epidemic seasons (Peret et al., 2000; Zhang et al., 2010b). The molecular epidemiological characteristics of HRSV were described previously in Southwestern and Northwestern China (Cui et al., 2013b; Qin et al., 2013; Zhang et al., 2010a,b). However, less information on the genotype distribution of HRSV is available in East China. HRSV infection is often associated with acute respiratory tract infections (ARTIs). There is less information available for HRSV infection among children with fever and respiratory symptoms. Here we report the molecular epidemiological characteristics of HRSV among children with fever and respiratory symptoms in Shanghai, China, from 2009 to 2012.
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2. Material and methods 2.1. Study subjects and sample collection From August 2009 to December 2012, 2407 nasopharyngeal swabs were collected from outpatient children with fever and respiratory symptoms in Shanghai Nanxiang Hospital, a district-level general hospital in Shanghai, China. Nanxiang Hospital is the biggest and best general hospital in Jiading district of Shanghai, and majority of patients in this hospital are from suburbs and rural area of Shanghai. Demographic information including symptom, age, gender and diagnosis were recorded for each patient. The study was approved by the Ethics committee of Shanghai Nanxiang Hospital and informed content was obtained from each child’s parent or guardian. These samples were transported to laboratory in virus transport media (hank’s buffer, BSA, HEPES and antibiotics) immediately and divided into three aliquot parts. One aliquot was used to extract RNA and the others were stored at 80 °C for future use. 2.2. RNA extraction and detection of HRSV infection by a multiplex RTPCR Total RNA was extracted from each sample using a QIAamp Viral RNA Mini Kit (Qiagen, Germany). Extracted RNA was tested for the viruses associated with ARTIs using a multiplex RT-PCR assay modified from ref (Wang et al., 2009), which can detect simultaneously 17 respiratory viruses (including HRSV) under five tubes. The detail protocol is available on request from the authors. For each HRSV positive sample determined by the multiplex RTPCR assay, another RT-PCR was performed to amplify the C terminus region (i.e. HVR2 region) of G gene of HRSV using previously described primers (Zhang et al., 2010b). All RT-PCR reactions were performed using the QIAGEN One Step RT-PCR Kit (Qiagen, Germany). Amplified products were subjected to direct sequencing. 2.3. HRSV genotyping and phylogenetic analysis Nucleotide sequences obtained in this study were aligned with HRSV genotype reference sequences reported previously using Muscle program implemented in MEGA 5.05. Genotype reference sequences were retrieved from GenBank according to strain name and/or GenBank accession numbers reported in previous studies. The phylogenetic trees were constructed using the neighbor-joining method implemented in MEGA 5.05. The evolutionary distances used to reconstruct phylogeny was calculated based on Kimura 2parameter model and the reliability of each branch was assessed with 1000 bootstrap replicates. HRSV genotyping of Shanghai sequences were determined according to the phylogenetic trees. When the Shanghai sequences are unable to closely cluster with any reference sequences, but form an independent cluster with a high bootstrap support (>85), they are defined as new genotypes. 2.4. Nucleotide sequence accession numbers The HVR2 sequences of HRSV reported in this article are available in GenBank under accession numbers of KJ658764–KJ658832. 3. Results and discussion 3.1. HRSV seasonal distribution and patient characteristic From 2407 samples from children with fever and respiratory symptoms, 184 (7.6%) samples were detected to HRSV positive. The HRSV prevalence among children with fever and respiratory symptoms was substantially lower than among children with
symptoms of acute respiratory tract infections (ARTIs) (Zhang et al., 2010a,b). Clinical manifestations of 154 HRSV infected cases with available information are shown in Table 1. The ratio of boys to girls was 1.2:1. Vast majority of patients (90.3%) were between one and 10 years old, and the most common symptoms observed were fever, coughing and runny nose (Table 1). HRSV epidemics exhibit distinct seasonal variation with high HRSV infection rates among children with fever and respiratory symptoms during the winter to spring months (December to February) (Supporting Fig. S1), well consistent with previous observations on children with ARTIS (Zhang et al., 2010a,b). The prevalence of HRSV among children with fever and respiratory symptoms in Shanghai was divided into three epidemic seasons by two threemonth intervals from May to July in 2010 and 2011, during which no HRSV infection was found. The sample numbers are almost similar for each month except January to June 2012. Half (50%) of HRSV cases appeared from August 2009 to April 2010 with a peak at January 2010, indicating that the 2009–2010 epidemic season experienced an extreme HRSV outbreak among children with fever and respiratory symptoms (Fig. 1A). 3.2. Genotype shift in HRSV prevalence Among 184 HRSV positive samples, only 160 samples had enough amounts to support further experiments for amplification of HVR2 fragment. From the 160 HRSV positive samples, 69 HVR2 sequences were obtained. The failure in the amplification of viral genomic fragments in other samples may be due to low viral load of these samples or the variations in primer binding sites. Phylogenetic analysis with the HVRs reference sequences retrieved from GenBank reveals that 23 were HRSV A group and 46 were B group (Fig. 2). HRSV B strains were predominant in the 2009– 2010 epidemic season, accounting for 91.5%. The predominant strains were changed from group B in the 2009–2010 epidemic season to group A (100% and 81.3%, respectively) in the 2010– 2011 and 2011–2012 epidemic seasons (Fig. 1B). The genotype shift trend of HRSV was consistent with that in other regions of China, where predominant strains were shifted from group A in 2006 to group B in 2009, and further to group A in 2011 (Qin et al., 2013; Zhang et al., 2010b). Of 23 HRSV A strains, vast majority (19/23, 82.6%) clustered with the reference sequences of NA1 genotype, indicating majority of A strains were NA1 genotype. One strain clustered with ON1 reference strains with high bootstrap value support (88%) (Fig. 2A), and carried the ON1-specific 24-amino acid repeat (Supporting Fig. S2A). Interestingly, one and two Shanghai strains clustered together with the NA3 and NA4 strains with high bootstrap support (97–100%). The NA3 and NA4 genotypes were two new genotypes recently identified in Beijing, the capital of China (Cui et al., 2013b). Their nomenclatures were because they clustered outside of NA2 strain and had distinct amino acid sequence characteristics from NA1 and NA2 (Fig. 2A). Compared with other NA and ON1 strains, NA3 strains have five genotype-specific amino acids at sites 255, 270, 280, 283 and 310, and NA4 strains have nine genotypespecific amino acids at sites 226, 239, 242, 244, 250, 262, 274, 316, and 321, as well as an additional residue (Tyr) at the C-terminal (Supporting Fig. S2A). Among 46 Shanghai HRSV B strains, 34 belonged to genotype BA that has a unique 20-amino acid repeat in the C-terminal region of G protein, accounting for 73.9%; 11 belonged to CB1 and one was SAB4 strain (Fig. 2B). The CB1 genotype was a new HRSV genotype identified in Beijing. The CB1 strains exhibit different amino acid sequence characteristics from GB2 stains (Supporting Fig. S2B). BA strains were further divided into ten genotypes, BA1–BA10. Of these BA strains, 21 were identified as BA9 and one was BA10 since they clustered within the clades of corresponding genotype
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J. Liu et al. / Infection, Genetics and Evolution 27 (2014) 131–136 Table 1 Clinical manifestations of HRSV infected cases.
*
Age
Case
0–6 mon 6–12 mon 1–2 yr 2–5 yr 6–10 yr >10 yr Total
4 9 41 61 37 2 154
Gender*
Symptom incidence (no. [%])
Boys
Girls
Fever
Coughing
Runny nose
Sore throat
Expectoration
3 6 22 30 20 1 82
1 2 17 30 17 1 68
4(100.0) 9(100.0) 38(92.7) 56(91.8) 33(89.2) 1(50.0) 141
4(100.0) 9(100.0) 36(87.8) 54(88.5) 33(89.2) 2(100.0) 138
4(100.0) 6(66.6) 36(87.8) 50(81.9) 28(75.7) 1(50.0) 125
4(100.0) 4(44.4) 18(43.9) 32(52.5) 17(45.9) 0 75
3(75.0) 8(88.9) 18(43.9) 32(52.5) 17(45.9) 0 78
The gender information is not available for four cases.
defined as a new HRSV genotypes, recently (Cui et al., 2013b). Relative to other BA genotypes, BAc strains carries six genotype-specific amino acids at sites 221, 242, 259, 272, 285 and 315 (Supporting Fig. S2B). In addition, we found that one strain within the BA clade did not cluster with other BA strains with a high bootstrap value. It was defined as BA?, which means its genotype is undetermined (Fig. 2B). In 2013, four new HRSV genotypes, including NA3 and NA4 in group A and CB1 and BAc in group B, were identified in Beijing, China (Cui et al., 2013b). In this study, we also detected these new genotypes in Shanghai, about 1300 km away from Beijing, suggesting that these strains might be circulating in a larger geographic scope in China. Up to now, the strains of the four genotypes were only found in China, possibly implying that they originated in China. On the other hand, although CB1 and BAc were firstly detected in Beijing, more CB1 and BAc strains were found among children with fever and respiratory syndromes in Shanghai, both which accounted for about half (47.8%) of group B strains. Where is the geographic origin of these new HRSV genotypes in China needs to be determined. 3.3. HRSV outbreak and genotype diversity
Fig. 1. The epidemiological pattern of HRSV among children with fever and respiratory symptoms in Shanghai from August 2009 and December 2012. (A) Monthly distribution of total (right vertical axis) and HRSV positive (left vertical axis) sample numbers. (B) Monthly distribution of A and B group HRSV. (C) Monthly distribution of various HRSV genotypes.
references. Eleven strains clustered with the BAc clade with a support value of 99%. BAc genotype was also isolated in Beijing and
Analysis of the genotype distributions in different epidemic seasons showed that seven HRSV genotypes, including three novel genotypes (CB1, BAc and the undetermined genotype BA?), occurred among children with fever and respiratory symptoms in the 2009–2010 epidemic season, higher than two genotypes in the 2010–2011 epidemic season and four genotypes in the 2011– 2012 epidemic season (Fig. 1C). Because only 69 sequences were available and they were all from one district-level general hospital of Shanghai, the genotyping results in this study cannot completely represent the general situation of Shanghai. In contrast, it implies that HRSV genotypes circulating among children with fever and respiratory symptoms in Shanghai might be more than reported here (ten genotypes). On the other hand, because HRSV prevalence was lower among children with fever and respiratory symptoms than with ARTIs, the genotype characteristics of HRSV circulating among children with ARTIs might be also more complex. In despite of this, it is very rare that so many HRSV genotypes were co-circulating in same area in a short epidemic period (Fig. 1C). Among seven genotypes in the 2009–2010 epidemic season, three B group genotypes, BA9, CB1 and BAc were the top three most predominant strains, accounting for 38.3%, 23.4% and 23.4%, respectively, and only one group A genotype NA1 appeared in this epidemic season (from November 2009 to January 2010). Of importance is that there were four to five HRSV genotypes co-circulating in each month during the HRSV epidemic peak from November 2009 to February 2010 (Fig. 1C). These suggest that the extreme outbreak of HRSV among children with fever and respiratory symptoms in the 2009–2010 epidemic season was associated with co-circulation of multiple genotypes (especially two new
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genotypes CB1 and BAc, both which accounted for 46.8% of all strains in this season) since these new emerging genotypes might evade easily pre-existing immunity induced by past infection with hetero-genotypes (Collins and Melero, 2011). Some previous studies showed that HRSV group A and/or certain genotype (e.g. GA2) of group A were associated with disease severity, while other studies suggested that there was no significant difference in disease severity between groups A and B (Collins and Melero, 2011; Gilca et al., 2006). In this study, we found that 66.7% of HRSV strains in Shanghai belonged to group B, and half of group B strains were new group B genotypes (i.e. CB1, BAc, and BA?) recently identified in China. Because of small sample size, whether there was an association between these new HRSV genotypes and disease severity needs to be further determined. ON1 genotype was characterized by a unique 24-amino acid repeat in the C-terminal region of G protein and firstly identified between November 2010 and January 2011 in Ontario, Canada
(Eshaghi et al., 2012). Since then, it was detected sporadically in other regions (e.g. German, Japan, South Africa) of the world (Lee et al., 2012; Valley-Omar et al., 2013), and Beijing (Cui et al., 2013a,b). We found one ON1 strain in Shanghai (Fig. 2A). The Shanghai strain appeared in February 2011, a time very close to the time that the initial ON1 strains appeared in Canada, and had a closer genetic relationship and shared high sequence homology with the Canada strains in G protein (Fig. 2A and Supporting Fig. S2A). Whether the Shanghai ON1 strain was transmitted from Canada or had an independent origin in Shanghai remains to be further determined. As a new HRSV genotype identified in China, CB1 has three genotype-specific amino acids at sites 245, 274, and 295 compared with other genotypes (BA, GB and SAB) (Supporting Fig. S2B). Interestingly, we found that the early CB1 strains (in 2009) had a threeamino acid insertion (232TGK235) in the G protein compared with other group B genotypes, but the late strains lost this insertion
Fig. 2. Phylogenetic trees of HRSV A (A) and B (B) group strains from Shanghai. The trees were constructed using MEGA 5.0. The reliability of each branch was assessed with 1000 bootstrap replicates. The prototype strain A2 (AF035006) and strain B1 (AF013254) were used as outgroup in the phylogenic analyses of HRSV groups A and B, respectively. Only bootstrap values P70% were shown. The sequences from Shanghai and Beijing are highlighted by solid circles and triangles, respectively. The new HRSV genotypes recently identified in Beijing are highlighted by the red boxes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2 (continued)
(Supporting Fig. S2B). How this insertion originated is unclear. Because the sampling times of three CB1 strains from Beijing were unavailable, we did not sure whether the similar observation also occurred in Beijing.
4. Conclusions We report a high prevalence of HRSV occurred among children with fever and respiratory symptoms in the 2009–2010 epidemic
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season in Shanghai from 2009 to 2012. We found that seven genotypes (NA1, BA9–10, SAB4, CB1, BAc and BA?) occurred in the 2009–2010 epidemic season. The co-circulation of multiple genotypes was associated with the extreme outbreak of HRSV among children with fever and respiratory symptoms in the 2009–2010 epidemic season in Shanghai. Funding This work was supported by grants from the China National Mega-projects for Infectious Diseases (2012ZX10004211-002 and 2013ZX10004101-005), the Public Health Key Disciplines in Shanghai-Health Microbiology (12GWZX0801), and Health Bureau Key Disciplines in Jiading District of Shanghai-Pediatric Respiratory Specialty (ZD03). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2014.07. 011. References 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. Collins, P.L., Melero, J.A., 2011. Progress in understanding and controlling respiratory syncytial virus: still crazy after all these years. Virus Res. 162, 80– 99. Cui, G., Qian, Y., Zhu, R., Deng, J., Zhao, L., Sun, Y., Wang, F., 2013a. Emerging human respiratory syncytial virus genotype ON1 found in infants with pneumonia in Beijing, China. Emerg. Microbes Infect. 2, e22. Cui, G., Zhu, R., Qian, Y., Deng, J., Zhao, L., Sun, Y., Wang, F., 2013b. 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.
Eshaghi, A., Duvvuri, V.R., Lai, R., Nadarajah, J.T., Li, A., Patel, S.N., Low, D.E., Gubbay, J.B., 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. Galiano, M.C., Luchsinger, V., Videla, C.M., De Souza, L., Puch, S.S., Palomo, C., Ricarte, C., Ebekian, B., Avendano, L., Carballal, G., 2005. Intragroup antigenic diversity of human respiratory syncytial virus (group A) isolated in Argentina and Chile. J. Med. Virol. 77, 311–316. Gilca, R., De Serres, G., Tremblay, M., Vachon, M.L., Leblanc, E., Bergeron, M.G., Dery, P., Boivin, G., 2006. Distribution and clinical impact of human respiratory syncytial virus genotypes in hospitalized children over 2 winter seasons. J. Infect. Dis. 193, 54–58. Lee, W.-J., Kim, Y.-J., Kim, D.-W., Lee, H.S., Lee, H.Y., Kim, K., 2012. Complete genome sequence of human respiratory syncytial virus genotype A with a 72-nucleotide duplication in the attachment protein G gene. J. Virol. 86, 13810–13811. Mufson, M.A., Orvell, C., Rafnar, B., Norrby, E., 1985. Two distinct subtypes of human respiratory syncytial virus. J. Gen. Virol. 66, 2111–2124. Peret, T.C., Hall, C.B., Hammond, G.W., Piedra, P.A., Storch, G.A., Sullender, W.M., Tsou, C., Anderson, L.J., 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. Qin, X., Zhang, C., Zhao, Y., Zhao, X., 2013. Genetic variability of subgroup A and B respiratory syncytial virus strains circulating in southwestern China from 2009 to 2011. Arch. Virol. 158, 1487–1495. Rudan, I., Boschi-Pinto, C., Biloglav, Z., Mulholland, K., Campbell, H., 2008. Epidemiology and etiology of childhood pneumonia. Bull. World Health Organ. 86, 408–416. Teng, M.N., Whitehead, S.S., Collins, P.L., 2001. Contribution of the respiratory syncytial virus G glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo. Virology 289, 283–296. Valley-Omar, Z., Muloiwa, R., Hu, N.C., Eley, B., Hsiao, N.Y., 2013. Novel respiratory syncytial virus subtype ON1 among children, Cape Town, South Africa, 2012. Emerg. Infect. Dis. 19, 668–670. Wang, W., Ren, P., Sheng, J., Mardy, S., Yan, H., Zhang, J., Hou, L., Vabret, A., Buchy, P., Freymuth, F., Deubel, V., 2009. Simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-PCR assays. J. Virol. Methods 162, 40–45. Zhang, R.F., Jin, Y., Xie, Z.P., Liu, N., Yan, K.L., Gao, H.C., Song, J.R., Yuan, X.H., Xiao, N.G., Guo, M.W., Zhou, Q.H., Hou, Y.D., Duan, Z., 2010a. Human respiratory syncytial virus in children with acute respiratory tract infections in China. J. Clin. Microbiol. 48, 4193–4199. Zhang, Z.Y., Du, L.N., Chen, X., Zhao, Y., Liu, E.M., Yang, X.Q., Zhao, X.D., 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.
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Genetic variation of human respiratory syncytial virus among children with fever and respiratory symptoms in Shanghai, China, from 2009 to 2012 Jia Liu a,1, Yonglin Mu a,c,1, Wei Dong b, Fujia Yao a, Lili Wang a, Huajie Yan b, Ke Lan a,⇑, Chiyu Zhang a,⇑ a b c
Pathogen Diagnostic Center, Institut Pasteur of Shanghai, Chinese Academy of Science, Shanghai 200025, China Pediatric Department, Shanghai Nanxiang Hospital, Jiading District, Shanghai 201800, China College of Life and Environmental Science, Shanghai Normal University, Shanghai 200234, China
a r t i c l e
i n f o
Article history: Received 14 June 2014 Received in revised form 9 July 2014 Accepted 10 July 2014 Available online 19 July 2014 Keywords: Human respiratory syncytial virus (HRSV) New genotype Fever and respiratory symptoms Shanghai Co-circulation
a b s t r a c t Human respiratory syncytial virus (HRSV) of genus Pneumovirus is one of the most common pathogens causing severe acute lower respiratory tract infection in infants and children. No information on the genotype distribution of HRSV is available in East China (e.g. Shanghai). From August 2009 to December 2012, 2407 nasopharyngeal swabs were collected from outpatient children with fever and respiratory symptoms in Shanghai. HRSV infection was determined using a multiplex RT-PCR assay. The second hypervariable region (HVR2) of G protein gene of HRSV was amplified and sequenced from HRSV positive samples. Genotypes were characterized by phylogenetic analyses. Of 2407 nasopharyngeal samples, 184 (7.6%) were tested as HRSV positive. From 160 positive subjects with sufficient nasopharyngeal samples, 69 HVR2 sequences were obtained by RT-PCR and sequencing. Three HRSV epidemic seasons were observed from August 2009 to December 2012, and an extreme outbreak of HRSV occurred in the 2009–2010 epidemic season. A genotype shift of predominant HRSV strains from B group in the 2009– 2010 epidemic season to group A in the subsequent epidemic seasons was observed. Ten HRSV genotypes, including four group A genotypes NA1, NA3, NA4, and ON1, and six group B genotypes BA9, BA10, SAB4, CB1, BAc, and BA?, were detected in Shanghai. Seven genotypes (NA1, BA9–10, SAB4, CB1, BAc and BA?) were found in the 2009–2010 epidemic season. The co-circulation of multiple genotypes was associated with the extreme outbreak of HRSV among children with fever and respiratory symptoms in the 2009–2010 epidemic season. Ó 2014 Elsevier B.V. All rights reserved.
1. Background Human respiratory syncytial virus (HRSV) of genus Pneumovirus is one of the most common pathogens causing severe acute lower respiratory tract infection in infants and children, accounting for 15–40% pneumonia or bronchiolitis in children (Rudan et al., 2008). HRSV is a negative single-strand RNA virus with a genome of about 15 kilobases that encodes 11 proteins (Collins and Melero, 2011). The attachment glycoprotein (G protein) responsible for HRSV infection is the most variable protein (Teng et al., 2001). It contains two hypervariable regions (HVRs). The second HVR of G protein has the highest degree of divergence that can reflect overall gene variability, and is commonly used for genotyp⇑ Corresponding authors. Address: Pathogen Diagnostic Center, Institut Pasteur of Shanghai, Chinese Academy of Science, Hefei road 411, Shanghai, China. Tel.: +86 21 5492 3005. E-mail addresses: [email protected] (K. Lan), [email protected] (C. Zhang). 1 These authors contributed equally to this article. http://dx.doi.org/10.1016/j.meegid.2014.07.011 1567-1348/Ó 2014 Elsevier B.V. All rights reserved.
ing HRSV (Galiano et al., 2005). According to genetic variability of HRV2, two genetically distinct HRSV groups A and B have been divided (Mufson et al., 1985). Groups A and B were further subdivided into 11 (ON1, GA1–GA7, SAA1, NA1, and NA2) and 20 (GB1– GB4, BA1–BA10, SAB1–SAB4, URU1, and URU2) genotypes (Arnott et al., 2011; Eshaghi et al., 2012). One main feature of HRSV epidemic is that the predominant genotypes can shift among different epidemic seasons (Peret et al., 2000; Zhang et al., 2010b). The molecular epidemiological characteristics of HRSV were described previously in Southwestern and Northwestern China (Cui et al., 2013b; Qin et al., 2013; Zhang et al., 2010a,b). However, less information on the genotype distribution of HRSV is available in East China. HRSV infection is often associated with acute respiratory tract infections (ARTIs). There is less information available for HRSV infection among children with fever and respiratory symptoms. Here we report the molecular epidemiological characteristics of HRSV among children with fever and respiratory symptoms in Shanghai, China, from 2009 to 2012.
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2. Material and methods 2.1. Study subjects and sample collection From August 2009 to December 2012, 2407 nasopharyngeal swabs were collected from outpatient children with fever and respiratory symptoms in Shanghai Nanxiang Hospital, a district-level general hospital in Shanghai, China. Nanxiang Hospital is the biggest and best general hospital in Jiading district of Shanghai, and majority of patients in this hospital are from suburbs and rural area of Shanghai. Demographic information including symptom, age, gender and diagnosis were recorded for each patient. The study was approved by the Ethics committee of Shanghai Nanxiang Hospital and informed content was obtained from each child’s parent or guardian. These samples were transported to laboratory in virus transport media (hank’s buffer, BSA, HEPES and antibiotics) immediately and divided into three aliquot parts. One aliquot was used to extract RNA and the others were stored at 80 °C for future use. 2.2. RNA extraction and detection of HRSV infection by a multiplex RTPCR Total RNA was extracted from each sample using a QIAamp Viral RNA Mini Kit (Qiagen, Germany). Extracted RNA was tested for the viruses associated with ARTIs using a multiplex RT-PCR assay modified from ref (Wang et al., 2009), which can detect simultaneously 17 respiratory viruses (including HRSV) under five tubes. The detail protocol is available on request from the authors. For each HRSV positive sample determined by the multiplex RTPCR assay, another RT-PCR was performed to amplify the C terminus region (i.e. HVR2 region) of G gene of HRSV using previously described primers (Zhang et al., 2010b). All RT-PCR reactions were performed using the QIAGEN One Step RT-PCR Kit (Qiagen, Germany). Amplified products were subjected to direct sequencing. 2.3. HRSV genotyping and phylogenetic analysis Nucleotide sequences obtained in this study were aligned with HRSV genotype reference sequences reported previously using Muscle program implemented in MEGA 5.05. Genotype reference sequences were retrieved from GenBank according to strain name and/or GenBank accession numbers reported in previous studies. The phylogenetic trees were constructed using the neighbor-joining method implemented in MEGA 5.05. The evolutionary distances used to reconstruct phylogeny was calculated based on Kimura 2parameter model and the reliability of each branch was assessed with 1000 bootstrap replicates. HRSV genotyping of Shanghai sequences were determined according to the phylogenetic trees. When the Shanghai sequences are unable to closely cluster with any reference sequences, but form an independent cluster with a high bootstrap support (>85), they are defined as new genotypes. 2.4. Nucleotide sequence accession numbers The HVR2 sequences of HRSV reported in this article are available in GenBank under accession numbers of KJ658764–KJ658832. 3. Results and discussion 3.1. HRSV seasonal distribution and patient characteristic From 2407 samples from children with fever and respiratory symptoms, 184 (7.6%) samples were detected to HRSV positive. The HRSV prevalence among children with fever and respiratory symptoms was substantially lower than among children with
symptoms of acute respiratory tract infections (ARTIs) (Zhang et al., 2010a,b). Clinical manifestations of 154 HRSV infected cases with available information are shown in Table 1. The ratio of boys to girls was 1.2:1. Vast majority of patients (90.3%) were between one and 10 years old, and the most common symptoms observed were fever, coughing and runny nose (Table 1). HRSV epidemics exhibit distinct seasonal variation with high HRSV infection rates among children with fever and respiratory symptoms during the winter to spring months (December to February) (Supporting Fig. S1), well consistent with previous observations on children with ARTIS (Zhang et al., 2010a,b). The prevalence of HRSV among children with fever and respiratory symptoms in Shanghai was divided into three epidemic seasons by two threemonth intervals from May to July in 2010 and 2011, during which no HRSV infection was found. The sample numbers are almost similar for each month except January to June 2012. Half (50%) of HRSV cases appeared from August 2009 to April 2010 with a peak at January 2010, indicating that the 2009–2010 epidemic season experienced an extreme HRSV outbreak among children with fever and respiratory symptoms (Fig. 1A). 3.2. Genotype shift in HRSV prevalence Among 184 HRSV positive samples, only 160 samples had enough amounts to support further experiments for amplification of HVR2 fragment. From the 160 HRSV positive samples, 69 HVR2 sequences were obtained. The failure in the amplification of viral genomic fragments in other samples may be due to low viral load of these samples or the variations in primer binding sites. Phylogenetic analysis with the HVRs reference sequences retrieved from GenBank reveals that 23 were HRSV A group and 46 were B group (Fig. 2). HRSV B strains were predominant in the 2009– 2010 epidemic season, accounting for 91.5%. The predominant strains were changed from group B in the 2009–2010 epidemic season to group A (100% and 81.3%, respectively) in the 2010– 2011 and 2011–2012 epidemic seasons (Fig. 1B). The genotype shift trend of HRSV was consistent with that in other regions of China, where predominant strains were shifted from group A in 2006 to group B in 2009, and further to group A in 2011 (Qin et al., 2013; Zhang et al., 2010b). Of 23 HRSV A strains, vast majority (19/23, 82.6%) clustered with the reference sequences of NA1 genotype, indicating majority of A strains were NA1 genotype. One strain clustered with ON1 reference strains with high bootstrap value support (88%) (Fig. 2A), and carried the ON1-specific 24-amino acid repeat (Supporting Fig. S2A). Interestingly, one and two Shanghai strains clustered together with the NA3 and NA4 strains with high bootstrap support (97–100%). The NA3 and NA4 genotypes were two new genotypes recently identified in Beijing, the capital of China (Cui et al., 2013b). Their nomenclatures were because they clustered outside of NA2 strain and had distinct amino acid sequence characteristics from NA1 and NA2 (Fig. 2A). Compared with other NA and ON1 strains, NA3 strains have five genotype-specific amino acids at sites 255, 270, 280, 283 and 310, and NA4 strains have nine genotypespecific amino acids at sites 226, 239, 242, 244, 250, 262, 274, 316, and 321, as well as an additional residue (Tyr) at the C-terminal (Supporting Fig. S2A). Among 46 Shanghai HRSV B strains, 34 belonged to genotype BA that has a unique 20-amino acid repeat in the C-terminal region of G protein, accounting for 73.9%; 11 belonged to CB1 and one was SAB4 strain (Fig. 2B). The CB1 genotype was a new HRSV genotype identified in Beijing. The CB1 strains exhibit different amino acid sequence characteristics from GB2 stains (Supporting Fig. S2B). BA strains were further divided into ten genotypes, BA1–BA10. Of these BA strains, 21 were identified as BA9 and one was BA10 since they clustered within the clades of corresponding genotype
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J. Liu et al. / Infection, Genetics and Evolution 27 (2014) 131–136 Table 1 Clinical manifestations of HRSV infected cases.
*
Age
Case
0–6 mon 6–12 mon 1–2 yr 2–5 yr 6–10 yr >10 yr Total
4 9 41 61 37 2 154
Gender*
Symptom incidence (no. [%])
Boys
Girls
Fever
Coughing
Runny nose
Sore throat
Expectoration
3 6 22 30 20 1 82
1 2 17 30 17 1 68
4(100.0) 9(100.0) 38(92.7) 56(91.8) 33(89.2) 1(50.0) 141
4(100.0) 9(100.0) 36(87.8) 54(88.5) 33(89.2) 2(100.0) 138
4(100.0) 6(66.6) 36(87.8) 50(81.9) 28(75.7) 1(50.0) 125
4(100.0) 4(44.4) 18(43.9) 32(52.5) 17(45.9) 0 75
3(75.0) 8(88.9) 18(43.9) 32(52.5) 17(45.9) 0 78
The gender information is not available for four cases.
defined as a new HRSV genotypes, recently (Cui et al., 2013b). Relative to other BA genotypes, BAc strains carries six genotype-specific amino acids at sites 221, 242, 259, 272, 285 and 315 (Supporting Fig. S2B). In addition, we found that one strain within the BA clade did not cluster with other BA strains with a high bootstrap value. It was defined as BA?, which means its genotype is undetermined (Fig. 2B). In 2013, four new HRSV genotypes, including NA3 and NA4 in group A and CB1 and BAc in group B, were identified in Beijing, China (Cui et al., 2013b). In this study, we also detected these new genotypes in Shanghai, about 1300 km away from Beijing, suggesting that these strains might be circulating in a larger geographic scope in China. Up to now, the strains of the four genotypes were only found in China, possibly implying that they originated in China. On the other hand, although CB1 and BAc were firstly detected in Beijing, more CB1 and BAc strains were found among children with fever and respiratory syndromes in Shanghai, both which accounted for about half (47.8%) of group B strains. Where is the geographic origin of these new HRSV genotypes in China needs to be determined. 3.3. HRSV outbreak and genotype diversity
Fig. 1. The epidemiological pattern of HRSV among children with fever and respiratory symptoms in Shanghai from August 2009 and December 2012. (A) Monthly distribution of total (right vertical axis) and HRSV positive (left vertical axis) sample numbers. (B) Monthly distribution of A and B group HRSV. (C) Monthly distribution of various HRSV genotypes.
references. Eleven strains clustered with the BAc clade with a support value of 99%. BAc genotype was also isolated in Beijing and
Analysis of the genotype distributions in different epidemic seasons showed that seven HRSV genotypes, including three novel genotypes (CB1, BAc and the undetermined genotype BA?), occurred among children with fever and respiratory symptoms in the 2009–2010 epidemic season, higher than two genotypes in the 2010–2011 epidemic season and four genotypes in the 2011– 2012 epidemic season (Fig. 1C). Because only 69 sequences were available and they were all from one district-level general hospital of Shanghai, the genotyping results in this study cannot completely represent the general situation of Shanghai. In contrast, it implies that HRSV genotypes circulating among children with fever and respiratory symptoms in Shanghai might be more than reported here (ten genotypes). On the other hand, because HRSV prevalence was lower among children with fever and respiratory symptoms than with ARTIs, the genotype characteristics of HRSV circulating among children with ARTIs might be also more complex. In despite of this, it is very rare that so many HRSV genotypes were co-circulating in same area in a short epidemic period (Fig. 1C). Among seven genotypes in the 2009–2010 epidemic season, three B group genotypes, BA9, CB1 and BAc were the top three most predominant strains, accounting for 38.3%, 23.4% and 23.4%, respectively, and only one group A genotype NA1 appeared in this epidemic season (from November 2009 to January 2010). Of importance is that there were four to five HRSV genotypes co-circulating in each month during the HRSV epidemic peak from November 2009 to February 2010 (Fig. 1C). These suggest that the extreme outbreak of HRSV among children with fever and respiratory symptoms in the 2009–2010 epidemic season was associated with co-circulation of multiple genotypes (especially two new
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genotypes CB1 and BAc, both which accounted for 46.8% of all strains in this season) since these new emerging genotypes might evade easily pre-existing immunity induced by past infection with hetero-genotypes (Collins and Melero, 2011). Some previous studies showed that HRSV group A and/or certain genotype (e.g. GA2) of group A were associated with disease severity, while other studies suggested that there was no significant difference in disease severity between groups A and B (Collins and Melero, 2011; Gilca et al., 2006). In this study, we found that 66.7% of HRSV strains in Shanghai belonged to group B, and half of group B strains were new group B genotypes (i.e. CB1, BAc, and BA?) recently identified in China. Because of small sample size, whether there was an association between these new HRSV genotypes and disease severity needs to be further determined. ON1 genotype was characterized by a unique 24-amino acid repeat in the C-terminal region of G protein and firstly identified between November 2010 and January 2011 in Ontario, Canada
(Eshaghi et al., 2012). Since then, it was detected sporadically in other regions (e.g. German, Japan, South Africa) of the world (Lee et al., 2012; Valley-Omar et al., 2013), and Beijing (Cui et al., 2013a,b). We found one ON1 strain in Shanghai (Fig. 2A). The Shanghai strain appeared in February 2011, a time very close to the time that the initial ON1 strains appeared in Canada, and had a closer genetic relationship and shared high sequence homology with the Canada strains in G protein (Fig. 2A and Supporting Fig. S2A). Whether the Shanghai ON1 strain was transmitted from Canada or had an independent origin in Shanghai remains to be further determined. As a new HRSV genotype identified in China, CB1 has three genotype-specific amino acids at sites 245, 274, and 295 compared with other genotypes (BA, GB and SAB) (Supporting Fig. S2B). Interestingly, we found that the early CB1 strains (in 2009) had a threeamino acid insertion (232TGK235) in the G protein compared with other group B genotypes, but the late strains lost this insertion
Fig. 2. Phylogenetic trees of HRSV A (A) and B (B) group strains from Shanghai. The trees were constructed using MEGA 5.0. The reliability of each branch was assessed with 1000 bootstrap replicates. The prototype strain A2 (AF035006) and strain B1 (AF013254) were used as outgroup in the phylogenic analyses of HRSV groups A and B, respectively. Only bootstrap values P70% were shown. The sequences from Shanghai and Beijing are highlighted by solid circles and triangles, respectively. The new HRSV genotypes recently identified in Beijing are highlighted by the red boxes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2 (continued)
(Supporting Fig. S2B). How this insertion originated is unclear. Because the sampling times of three CB1 strains from Beijing were unavailable, we did not sure whether the similar observation also occurred in Beijing.
4. Conclusions We report a high prevalence of HRSV occurred among children with fever and respiratory symptoms in the 2009–2010 epidemic
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season in Shanghai from 2009 to 2012. We found that seven genotypes (NA1, BA9–10, SAB4, CB1, BAc and BA?) occurred in the 2009–2010 epidemic season. The co-circulation of multiple genotypes was associated with the extreme outbreak of HRSV among children with fever and respiratory symptoms in the 2009–2010 epidemic season in Shanghai. Funding This work was supported by grants from the China National Mega-projects for Infectious Diseases (2012ZX10004211-002 and 2013ZX10004101-005), the Public Health Key Disciplines in Shanghai-Health Microbiology (12GWZX0801), and Health Bureau Key Disciplines in Jiading District of Shanghai-Pediatric Respiratory Specialty (ZD03). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2014.07. 011. References 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. Collins, P.L., Melero, J.A., 2011. Progress in understanding and controlling respiratory syncytial virus: still crazy after all these years. Virus Res. 162, 80– 99. Cui, G., Qian, Y., Zhu, R., Deng, J., Zhao, L., Sun, Y., Wang, F., 2013a. Emerging human respiratory syncytial virus genotype ON1 found in infants with pneumonia in Beijing, China. Emerg. Microbes Infect. 2, e22. Cui, G., Zhu, R., Qian, Y., Deng, J., Zhao, L., Sun, Y., Wang, F., 2013b. 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.
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