Elsevier

Infection, Genetics and Evolution

Volume 27, October 2014, Pages 339-347
Infection, Genetics and Evolution

The genetic variability of glycoproteins among respiratory syncytial virus subtype A in China between 2009 and 2013

https://doi.org/10.1016/j.meegid.2014.07.030Get rights and content

Highlights

  • RSV-A NA1 genotype was predominant in China between 2009 and 2013.

  • ON1 strains emerged in November 2012.

  • ON1 genotype is under positive selection pressures.

  • ON1 genotype has rapidly disseminated across the world.

Abstract

Human respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in infants and children under 5 years of age. The novel genotype ON1 has a 72-nucleotide duplication, which is the largest duplicated genome portion of RSV. Whether the ON1 genotype will follow the pattern of the BA genotype, which has a 60-nucleotide duplication, and become the predominant RSV-A strain is a global concern. To obtain information regarding the prevalence of the ON1 genotype in Chongqing in Southwestern China, we examined the circulation pattern of RSV-A identified over four consecutive years (June 2009 to August 2013). In this study, 312 (12%) RSV-A strains were isolated from 2601 nasopharyngeal aspirates, and partial G gene was sequenced successfully in 250 isolates. Of the sequenced Chongqing RSV-A isolates, 237 (94.8%) strains were the NA1 genotype, 4 (1.6%) strains were the NA3 genotype, 4 (1.6%) strains were the NA4 genotype, 1 (0.4%) strain was the GA1 genotype, and 4 (1.6%) strains were identified as the ON1 genotype. Analysis of the distribution, phylogeny, and evolution of the ON1 strains that were collected globally until December 2013 revealed that the ON1 genotype has rapidly disseminated across the world under positive selection pressures. Future studies will determine whether this new genotype will continue to spread and become the dominant strain of RSV-A worldwide. These findings may contribute to the understanding of RSV evolution and to the potential development of a vaccine against RSV.

Introduction

Respiratory syncytial virus (RSV) was first isolated in 1956 (Blount et al., 1956) and is currently the leading cause of acute lower respiratory tract infections (ALRTI) in infants and young children under 5 years of age worldwide (Nair et al., 2010).

RSV is a single-stranded negative sense RNA virus in the family Paramyxoviridae and the subfamily Pneumovirinae with a genome of 15.2 kb in length that encodes 11 proteins in the following order: NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2, and L (Cane, 2001). G and F are the most important surface glycoproteins. The G protein is a type II glycoprotein that facilitates virus attachment. G has a cytoplasmic tail that extends from the N-terminus to amino acid (aa) 37, a membrane anchor spanning from aa 38 to aa 66, and an ectodomain from aa 67 to the C-terminus. There are two variable and heavily glycosylated domains of the ectodomain that are separated by a central region (aa 155–206). Another form of the G protein is secreted G, which begins at aa 48 of the G gene. Secreted G helps RSV to escape the antibody-dependent restriction of replication via effects as an antigen decoy that blocks interactions with the virus (Bukreyev et al., 2008).

Based on sequencing and monoclonal antibody reactions, RSV is divided into 2 subtypes, subtype A (RSV-A) and subtype B (RSV-B) (Mufson et al., 1985). Sequences of the G gene have been used for phylogenetic analyses because the G protein is most variable of the RSV proteins. Both RSV subtypes can be further categorized into genotypes. Currently, RSV-A includes 14 genotypes (GA1-GA7, SAA1, CB-A, NA1-4, and ON1) (Baek et al., 2012, Cui et al., 2013, Eshaghi et al., 2012, Peret et al., 1998, Peret et al., 2000, Shobugawa et al., 2009, Venter et al., 2001) and RSV-B includes 21 genotypes (GB1–GB4, SAB1–SAB4, URU1-2 and BA1-12) (Arnott et al., 2011, Baek et al., 2012, Blanc et al., 2005, Dapat et al., 2010, Kushibuchi et al., 2013, Peret et al., 1998, Peret et al., 2000, Trento et al., 2006, Yoshida et al., 2010). The G protein is the target of RSV-specific neutralizing antibodies and because G induces a protective immune response, it has been included in most RSV candidate vaccines and the G gene or protein has been included in some subunit vaccines. These phenomena suggest that the G protein is under immune pressure and that the different genotypes may be the result of immune pressure because positive selection of amino acid changes has been observed in the two hypervariable regions of the G protein ectodomain (Pretorius et al., 2013). The ON1 genotype has a 72-nucleotide duplication in the C terminal of the G gene, which is the largest duplication in RSV (Eshaghi et al., 2012). Prior to the discovery of this genotype, a 60-nucleotide duplication in the BA genotype was first detected in 1999 (Trento et al., 2010), and the BA genotype has been identified as the globally predominant strain of RSV-B. Based on the circulation pattern of the BA genotype, the ON1 genotype may become the predominant strain of RSV-A. The ON1 genotype provides a model for the investigation of the evolution of RSV-A because we can analyze the evolutionary path of RSV-A ON1 from time zero.

In this study, we evaluated the genetic variability of the G protein genes of RSV-A cultured isolates that were identified in Chongqing in Southwestern China from 2009 to 2013. Phylogenetic analyses were performed to establish the relationships of the Chinese ON1 isolates with the global ON1 strains and the previously described RSV-A genotypes that are deposited in GenBank. The molecular characterization and evolutionary rate of the ON1 genotype were also analyzed.

Section snippets

Specimen collection and viral isolation

All nasopharyngeal aspirates (NPAs) were collected from hospitalized infants or children with ALRTI from the Department of Respiratory Medicine, Chongqing Children’s Hospital in Southwestern China from June 2009 to August 2013. Following the study procedure, NPAs were collected upon admission to hospital and forwarded directly to the respiratory laboratory. NPAs were cultured in human laryngeal carcinoma HEp-2 cells for RSV isolation. The viral isolates were harvested by scraping when the

Clinical samples and isolates

In the present study, 423 (16.26%) RSV clinical strains were isolated from 2601 NPAs from June 2009 to August 2013. Three hundred and twelve (73.76%) and 110 (26%) isolates were identified as subtype A and subtype B, respectively, and 1 sample (0.24%) was detected as both RSV-A and RSV-B. All RSV-A isolates were selected for genotyping and further analysis. The age range of the patients from which the RSV-A samples were collected was 1–129 months (median: 5 months), and the majority of patients

Discussion

In this study, we isolated RSV from NPAs of hospitalized infants and children in Chongqing between 2009 and 2013. The phylogenetic analysis revealed that the RSV-A circulating in Chongqing could be clustered into 5 genotypes. The prevailing genotype was NA1, and the novel genotype ON1 with a 72-nucleotide duplication was also identified. The ON1 genotype has been disseminated globally in the course of its evolution.

The shifting patterns of the RSV subtypes in Chongqing were AAB from 2006 to

Acknowledgements

This project was supported by the China Special Grant for the Prevention and Control of Infection Diseases (2012zx10004212) and National Key Specialty [2011] 873. We thank all of the families for their enrollment in this study. We also thank the staff of the Department of Respiratory Medicine, Children’s Hospital of Chongqing Medical University for collecting the clinical information.

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