Elsevier

Infection, Genetics and Evolution

Volume 47, January 2017, Pages 125-131
Infection, Genetics and Evolution

Research paper
Molecular detection and characterization of respiratory syncytial virus B genotypes circulating in Pakistani children

https://doi.org/10.1016/j.meegid.2016.11.024Get rights and content

Highlights

  • The high incidence of respiratory infections in Pakistani children is complicated by dearth of epidemiological data.

  • This study highlights the clinical impact of RSV as a dominant cause of pediatric respiratory infections in Pakistan.

  • Our findings call for continued surveillance to understand epidemiology of RSV infections in Pakistan.

  • Our data will benefit to improve public health interventions for better control and prevention of respiratory infections.

Abstract

Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory tract infections in young children, but very little is known about its epidemiology and circulating genotypes in Pakistan. This study analyzed the epidemiological and molecular characteristics of RSV B genotypes in Pakistani children below 5 years with acute respiratory tract infections (ARIs) during three consecutive winter seasons from 2010 to 2013. A total of 1941 samples were analyzed for RSV infection by real time PCR and 24% (472/1941) samples were found positive out of which 22.3% (105/472) were sub-typed as RSV-B. The frequency of outpatient cases was higher (62.5%; 295/472) as compared to hospitalized patients (37.5%; 177/472). Patient ages ranged from 2 month to 5 years with a mean age of 1.48 ± 1.2 (years) and a median age of 1 year. Children below one year made up the highest percentage of enrolled subjects and male to female ratio of RSVB positive cases was nearly equivalent (1:1.1). The most common clinical symptoms were cough (96%), fever (80%) and sore throat (50%). All Pak RSVB strains ascribed to the BA genotype showing 91.9–97.1% and 86.2–95.3% homology at the nucleotide and amino acid levels respectively in comparison to BA prototype strain. On phylogenetic analysis, three genotypes of Pakistan RSV B viruses were observed; BA-9 and BA-10 which have been reported previously from other regions, and a third novel genotype assigned as BA-13 which formed a distinct cluster with protein length of 319 AA and showed 9–11 unique AA substitutions. All the RSV B isolates had two potential N-glycosylation sites in HVR2 of G protein and with heavy O-glycosylation of serine and threonine residues (G scores of 0.5–0.7). This study highlights the diversity of RSVB viruses and the significance of RSV as a dominant viral etiologic agent of pediatric ARI. It also emphasizes the need for continued molecular surveillance for early detection of prevalent and newly emerging genotypes to understand epidemiology of RSV infections in various regions of Pakistan.

Introduction

Human respiratory syncytial virus (RSV) is a single stranded negative sense RNA virus that belongs to genus Orthopneumovirus of the Pneumoviridae family. It has a non-segmented genome comprising of 10 genes that encode for 11 proteins (http://www.ictvonline.org/proposals/2015.011a-iM.A.v2.Pneumoviridae.pdf) (Collins and Crowe, 2007). It is an important causative agent of acute lower respiratory tract infections (ALRI) particularly in infants and young children, elderly and individuals with compromised immune status in developed as well as developing countries (Selwyn, 1990, Englund et al., 1991, Falsey et al., 2005, Fry et al., 2010, Hall et al., 2009, Nair et al., 2010). Primary RSV infections occur early during the first year of life in over half of infants and nearly every child has been infected at least once by 2 years of age (Viegas et al., 2004). The clinical presentation may range from mild upper respiratory tract infections to life threatening bronchiolitis and pneumonia and reinfections are very common throughout life. Children infected with one antigenic group are likely to be re-infected with the heterologous group (Parveen et al., 2006, Yamaguchi et al., 2011, Sullender et al., 1998).

Respiratory syncytial virus has been classified into two major antigenic types, A and B, (Mufson et al., 1988, Anderson et al., 1985) based on viral reactivity against monoclonal antibodies for the ‘G’ glycoprotein; this is a type II trans-membrane protein with two variable regions flanking a conserved central region (Anderson et al., 1985, Johnson et al., 1987, Mufson et al., 1985). More recently genotype classification of RSV A and B has been based on genetic analysis of the G gene sequences (Dapat et al., 2010, Van Niekerk and Venter, 2011, Shobugawa et al., 2009, Trento et al., 2006). Within the RSV A and B types, subgroups or genotypes have been demonstrated on the basis of antigenic differences on account of variations in the second hyper-variable region [HVR2] located at the C-terminal end of the G glycoprotein (Melero et al., 1997). Unlike other respiratory viruses such as influenza, RSV has multifarious circulation patterns, with multiple genotypes co-circulating within the same community and changeover of the prevalent genotypes with others or new ones over successive epidemic seasons (Dapat et al., 2010, Van Niekerk and Venter, 2011, Shobugawa et al., 2009, Cane and Pringle, 1995, Sullender, 2000, Arnott et al., 2011, Coggins et al., 1998, Goto-Sugai et al., 2010, Hall et al., 1990, Khor et al., 2013, Reiche and Schweiger, 2009, Trento et al., 2006, Zhang et al., 2010, Zlateva et al., 2007).

Several genotypes of both RSV-A and B have been reported by various research groups, based on molecular characterization of G protein. For RSV-A, GA1–GA7 (Peret et al., 1998), SAA1 (Venter et al., 2001), NA1 and NA2 within GA2 (Shobugawa et al., 2009), and the recently reported ON1genotypes have been described over the years (Tabatabai et al., 2014).Similarly for RSV-B, the described genotypes include GB1–GB4 (Peret et al., 1998), SAB1–SAB3 (Venter et al., 2001), SAB4 (Arnott et al., 2011), BA1–BA6 (Trento et al., 2006), BA7–BA10 (Dapat et al., 2010), BA11 (Baek et al., 2012), and BA12 (Khor et al., 2013). The BA (named after first detection in Buenos Aires) genotype was first reported in 1999 and has a 60 nucleotide insertion in the second hyper-variable region of the G gene. This genotype has now practically effaced the other RSV genotypes and has been in circulation globally.

There is limited information available regarding the molecular epidemiology of RSV from Pakistan (Ghafoor et al., 1990, Meropol et al., 2013, Aamir et al., 2013). The aim of the study was to investigate RSV A and RSV B prevalence and genotype circulation patterns among pediatric patients with influenza like illness (ILI) and severe acute respiratory illness (SARI) in Pakistan from 2010 to 2013. As only one RSV A genotype GA2/NA1 was detected through all the three seasons, the present study focuses only on evolution of RSV B genotypes.

Section snippets

Clinical specimens

Nasal, nasopharyngeal or throat swabs were collected from patients who fulfilled the WHO case definition for ILI and SARI and referred to National Institute of Health from tertiary care hospitals across Pakistan between October and April for three successive years from 2010 to 2013. Informed written consent was taken from the parents or guardians of the selected patients. The study was approved by the Internal Review Board of the National Institute of Health, Islamabad, Pakistan.

RNA extraction and real time PCR

RNA extraction

Results

From October to April of three successive years (2010  2013), a total of 1941 samples from children under five years were analyzed for RSV infection by real time PCR and 24% (472/1941) samples were found positive. Among these 77.7% (367/472) and 22.3% (105/472) were sub-typed as RSV-A and RSV-B respectively. The frequency of ILI cases was higher (62.5%; 295/472) as compared to SARI cases (37.5%; 177/472). The mean age (in years) of ILI cases was 1.67 ± 1.4 and 0.97 ± 1.11 for SARI cases.

Discussion

Respiratory syncytial virus (RSV) is a foremost causative agent responsible for bronchiolitis and pneumonia in children, the elderly and individuals who are immune-compromised, a common trend seen in both developed and developing countries. As the G protein of RSV shows a high level of genetic variability, repeated infections with various genotypes are common. This is demonstrated by the emergence of the novel BA genotype with a 60-nucleotide duplication that has dominated the subtype B

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