Journal of Medical Virology 85:1639–1644 (2013)

Molecular Characterization of HumanRespiratory Syncytial Virus NA1 and GA5Genotypes Detected in Assam in Northeast India,2009–2012

Dipankar Biswas,* Kaushal Yadav, Biswajyoti Borkakoty, and Jagadish Mahanta

Regional Medical Research Centre, NE Region (Indian Council of Medical Research), Dibrugarh, Assam, India

A significant number of children die each yearfrom acute respiratory tract infections especial-ly in developing countries. Human respiratorysyncytial virus (RSV) is the most common virusidentified in such cases. Genetic characteriza-tion and the circulation pattern of RSV isimportant for future selection of appropriatevaccine strains. Limited information is avail-able on the circulation of RSV in developingcountries including India. The present studyaimed to provide baseline information on thegenetic variability of RSV in the Dibrugarhdistrict of Assam, northeast India. Clinicalspecimens collected from children aged�6 years for routine influenza surveillance inthe Dibrugarh district of Assam during theperiod 2009–2012, were screened for RSVby real-time reverse transcription-polymerasechain reaction. Genotyping was based onpartial sequencing of the RSV attachmentglycoprotein gene. RSV was detected in 7.9%(39/493) of cases. Only RSV group A viruseswere detected during the study period withpredominance of NA1 genotypes (89%). TwoRSV GA5 genotypes were found to be co-circulating during 2012. The specific aminoacid substitutions characteristics of the NA1genotypes were distinct from RSV strainsreported from the rest of India. It is concludedthat the circulating genotypes of RSV inAssam, northeast India are NA1 and GA5.To our knowledge this is the first reportof circulation of the NA1 genotype in India.J. Med. Virol. 85:1639–1644, 2013.# 2013 Wiley Periodicals, Inc.

KEY WORDS: human respiratory syncytialvirus; RSV; acute respiratorytract infection; NA1; northeastIndia

INTRODUCTION

Acute respiratory tract infection is the leadingcause of pediatric mortality worldwide with a signifi-cant number of deaths occurring in developing coun-tries [Williams et al., 2002]. One fourth of the totaldeaths among children under 5 years of age occur inIndia and approximately 20% (0.5 million) of thesedeaths are due to acute respiratory infections [Red-daiah and kapoor, 1988; Ahmad et al., 2000; Williamset al., 2002]. A viral cause is found in 20–40% ofchildren admitted to hospital with acute respiratoryinfections in India where the human respiratorysyncytial virus (RSV) is identified as the mostfrequent cause of infection [Misra et al., 1990; Johnet al., 1991; Maitreyi et al., 2000].RSV, classified in the Pneumovirus genus of the

Paramyxoviridae family is an enveloped virus with anegative-sense single-stranded RNA genome thatencodes 11 proteins [Tripp, 2005]. Based on reactionwith monoclonal antibodies against the G and Fglycoproteins and genetic differences, RSV has beenclassified into two major groups A and B [Andersonet al., 1985; Mufson et al., 1985; Cane, 2001].Genotyping of RSV-A and RSV-B viruses is based onthe sequence variability of the G protein gene [Peretet al., 1998]. Ten RSV-A genotypes have beendescribed from different geographical regions,

Grant sponsor: RMRC NE (ICMR) Dibrugarh, Assam, India

The authors declare no conflict of interest.

Ethics: A written informed consent was obtained from parents/guardian of the enrolled cases prior to the study. The study wasapproved by human ethics committee of RMRC NE (ICMR)Dibrugarh, Assam, India.

*Correspondence to: Dipankar Biswas, Regional MedicalResearch Centre, NE Region (Indian Council of Medical Re-search), P.O. Box 105, 786001 Dibrugarh, Assam, India.E-mail: biswas_dip@yahoo.com

Accepted 4 April 2013

DOI 10.1002/jmv.23636Published online 18 June 2013 in Wiley Online Library(wileyonlinelibrary.com).

�C 2013 WILEY PERIODICALS, INC.

designated as GA1 to GA7 [Peret et al., 1998, 2000],SAA1 (South Africa, A1) [Venter et al., 2001], andmost recently NA1 and NA2 genotypes [Shobugawaet al., 2009]. Genetic variability between RSV strainsis a signature characteristic that may alter thepathogenicity and contribute to the ability to causerepeated infections and outbreaks by immune systemevasion [Eshaghi et al., 2012]. There is limitedinformation available regarding the circulation pat-tern of RSV in developing countries including India.Presently, there are no data available regarding themolecular characteristics of RSV in northeasternregion of India. The present study aimed to providebaseline information on the circulating genotypes andsubtypes of RSV strains in Dibrugarh district ofAssam, northeast India.

MATERIALS AND METHODS

Patients

Patients were recruited from routine influenzasurveillance at three randomly selected health cen-ters representing both rural and urban areas ofDibrugarh, Assam during October 2009 to April 2012.The following inclusion criteria were adopted for thepurposes of the present study (1) Patients of both thesexes within 6 years of age, (2) Patients meeting thecase definition of influenza-like illness which wasdefined as fever >37.8˚C with a cough and/or sorethroat (Centre for Disease Control and Prevention,Atlanta, USA), (3) Parents who had given consent forparticipation of their children in the study.

Clinical Specimens

Clinical specimens of nasal, throat and/or nasopha-ryngeal swabs were collected in virus transportmedia [HiViral Transport Medium, HiMedia, Mum-bai, India] which was then transported to laboratoryunder cold chain conditions (þ4˚C). The patient’scharacteristics were recorded in a structured caseinvestigation form. A prior informed consent wastaken from parents/guardians of the children. Thestudy was approved by human ethical committee ofRegional Medical Research Centre, North East Re-gion (ICMR), Dibrugarh.

Viral RNA Extraction and RT-PCR

Viral RNA was isolated from the respiratory speci-mens using Viral RNA mini kit (Qiagen, Hilden,Germany) following manufacture’s instruction. Initialscreening was done by One step TaqMan RT-PCRusing SuperScript III Platinum One-Step Quantita-tive RT-PCR System with ROX (Invitrogen, Carlsbad,CA) in real time PCR system (LC 480, Roche,Rotkreuz, Switzerland) targeting the RSV F gene asdescribed earlier [Mentel et al., 2003]. RSV groupanalysis was done by a two step RT-PCR as follows.

First Strand c-DNA Synthesis

In the first step, c-DNA was synthesized in a 20 mlreaction mixture using Transcriptor c-DNA synthesiskit (Roche, Indianapolis, IN) following instruction inthe manual. Briefly, 5 ml of RNA was reverse tran-scribed using random hexamer primers, 200 mMconcentration of each deoxynucleotide triphosphate(Promega, Madison, WI), 20 U Protector RNase inhib-itor, 1�Transcriptor RT reaction buffer (50 mM Tris/HCl, 3 mM KCl, 8 mM MgCl2), and 10 U Transcrip-tor Reverse Transcriptase (Roche). The synthesis of c-DNA was carried out for 5 min at 25˚C, followed by60 min at 50˚C, and finally for 5 min at 85˚C.

RSV G Protein Gene Amplification

PCR was conducted in 50 ml reaction volumecontaining 1� PCR Master Mix (Promega), 5 ml of c-DNA, 0.5 mM of forward primer F1 (50-CAACTC-CATTGTTATTTGCC-30), and reverse primers GPA(50-GAAGTGTTCAACTTTGTACC-30) and GPB (50-AAGATGATTACCATTTTGAAGT-30) as described byPeret et al. [1998] for RSV group A and B identifica-tion. Amplification conditions consisted of 2 min at94˚C, followed by 30 cycles of PCR (948˚C for 1 min;50˚C for 1 min; 72˚C for 2 min), and final extensionfor 7 min at 72˚C. Amplified products were run on 2%Agarose gel stained with ethidium bromide.

DNA Sequencing

The PCR products were purified with High PurePCR Product purification kit (Roche, Mannheim,Germany) according to the manufacturer’s instruc-tions. Purified PCR products were cycle sequenced inthe forward and reverse direction with primer pairGPA/F1 using ABI Big Dye Terminator cycle se-quencing kit in an ABI 3130 genetic analyzer [Ap-plied Biosystems, Foster City, CA].

Phylogenetic Analysis

Sequences were edited manually in Bioedit soft-ware v 7.0.9.0 [Hall, 1999]. The reference sequenceswere downloaded from NCBI GenBank. The list ofreference sequences used in the study is given inTable I. All the Phylogenetic analysis and multiplesequence alignment were conducted with MEGAsoftware v 5 [Tamura et al., 2011]. The nucleotidesequence spanning bases 673 to 912 (270 bp) ofprototype strain A2 (GenBank Accession No.M11486) [Anderson et al., 1985] was used for compar-ing RSV group A strains.

Analysis of N- and O-glycosylation Sites

Potential N-glycosylation (Asn-Xaa-Ser/Thr; whereXaa is any amino acid other than Proline) andO-glycosylation sites were predicted using NetNGlyc1.0 [NetNglyC 1.0] and NetOGlyc 3.1 [Julenius et al.,2005]. The deduced amino acid sequences of the

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second hyper-variable region of RSV-A strains werecompared to those of prototype A2 (GenBank Acces-sion No. M11486) and NA1 strains (GenBank Acces-sion No. AB470478).

Nucleotide Sequence Accession Numbers

The GenBank accession numbers of the nucleotidesequences obtained in the present study areJF907049-55 and KC117365-75.

RESULTS

Patient Characteristics

A total of 493 cases comprising of 271 male and222 female children within the age ranging from4 months to 6 years were tested during the periodwhere the RSV was detected in 7.9% of cases (39/493). All the cases had history of respiratory illnesssuch as fever, cough, and nasal discharge for 2–3days. The prevalence of RSV was 10.9% (37/339)within the first three years of life and out of the RSVpositive cases about 95% (37/39) belonged to this agegroup. Among the RSV cases there was predominanceof male (24/39) over females (15/39) (Odds ratio 1.34,95% CI: 0.7 to 2.6, P ¼ 0.4). Most of the isolates(72%) were obtained during the month of January toMarch.

Circulation of RSV Genotypes

Partial sequences of the RSV Glycoprotein (G) genewere obtained from 18 of 39 RSV-positive samples.Only the RSV group A viruses were detectedthroughout the period (2009–2012) with predomi-

nance of NA1 genotypes (89%). RSV subtype GA5was detected in two cases during 2012 (Fig. 1).

Intra and Intergenotype Diversityof RSV-A Strains

The overall nucleotide and amino acid divergenceamong RSV-A strains were 5% and 10% respectively.When only NA1 strains were compared, the averageintragenotype nucleotide and amino acid divergencewas 3% and 5% respectively. The rate of divergencebetween prototype A2 strain and the strains ofnortheast India ranged from 9% to 14% at thenucleotide level and 19–26% at the amino acid level.The northeastern NA1 sequences diverged from theprototype NA1 strain (NG-16-04) [Shobugawa et al.,2009] by 1–5% at the nucleotide level and 2–9% atamino acid level. The NA1 strains diverged from GA5strains by 13% and 26% at the nucleotide and aminoacid level, respectively.

Amino Acid Sequence Analysis of RSV-A Strainsfrom Dibrugarh (Assam), Northeast India

The predicted amino acid lengths of northeastIndia NA1 and GA5 strains were 297 and 298respectively. Of four previously reported NA1 geno-type-specific amino acid substitutions [Shobugawaet al., 2009], in the present study the amino acidsubstitutions Asp237 was present in 93.8% of isolates,Ser292 and a premature stop codon at position 298was present in all the NA1 isolates whereas Leu274

was absent among NA1 strains (Fig. 2).The RSV NA1 strains from northeast India were

distinct from NA1 strains of Japan [Shobugawa

TABLE I. RSV (group A) G Protein Gene Sequences Used in the Study

Strains GenBank accession no. Country of isolation Year of isolation Refs.

A2 GA1 M11486 Australia 1961 Wertz et al. [1985]AL19471-5 GA1 AF233902 United States 1994–1995 Peret et al. [2000]AL19376-1.GA2 AF233900 United States 1994–1995 Peret et al. [2000]MO55.GA2 AF233915 United States 1994–1995 Peret et al. [2000]TX69564.GA2 AF233923 United States 1994–1995 Peret et al. [2000]LLC235-267.GA2 AY114149 Singapore 2000–2001 Lim et al. [2003]Ken/7/00 GA2 AY524663 Kenya 2000 Scott et al. [2004]MO16 GA3 AF233913 United States 1994–1995 Peret et al. [2000]TX68481 GA3 AF233920 United States 1994–1995 Peret et al. [2000]NY/CH09/93 GA4 AF065254 United States 1993 Peret et al. [1998]AL19556-3 GA5 AF233903 United States 1994–1995 Peret et al. [2000]MO01 GA5 AF233909 United States 1994–1995 Peret et al. [2000]Sal/173/99 GA5 AY472094 Brazil 1999 Moura et al. [2004]AL19452-2 GA6 AF233901 United States 1994–1995 Peret et al. [2000]NY20 GA6 AF233918 United States 1994–1995 Peret et al. [2000]MO02 GA7 AF233910 United States 1994–1995 Peret et al. [2000]SA98V603 SAA1 AF348807 South Africa 1998 Venter et al. [2001]SA99V1239 SAA1 AF348808 South Africa 1999 Venter et al. [2001]Cam2008-7262 NA1 JN119952 Cambodia 2008 Arnott et al. [2011]Cam2006-2093 GA2 JN119884 Cambodia 2006 Arnott et al. [2011]Cam2007-1176 GA5 JN119905 Cambodia 2007 Arnott et al. [2011]NG-016-04 NA1 AB470478 Japan 2004 Shobugawa et al. [2009]DEL/3W/04 GA2 DQ248923 India 2004 Parveen et al. [2006]DEL/138/02 GA5 DQ248894 India 2002 Parveen et al. [2006]DEL/192/02 GA2 DQ248924 India 2002 Parveen et al. [2006]

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RSV Genotypes in Dibrugarh (Assam), Northeast India 1641

et al., 2009] in having amino acid substitutionsThr269 and Ser289 (Fig. 2). Along with previouslydescribed GA5 genotype-specific amino acid substitu-tions [Reiche and Schweiger, 2009] Asn250, Ser251,Thr274, Ile279, Ile295, and Asp297, the GA5 strainsfrom northeast India had additional three amino acidsubstitutions, Ile238, Pro241, and Leu256. The aminoacid substitutions noted in this study were analogousto recently reported Cambodian NA1 and GA5 strains[Arnott et al., 2011]. The N-glycosylation site locatedat amino acid position 237 as reported in previousstudies [Reiche and Schweiger, 2009] including Cam-bodian GA5 strains [Arnot et al., 2011] was absent inGA5 strains from northeast India due to amino acidsubstitutution Tyr237.The program NetOglyc predicted 22 and 34 � 2

serine and threonine residues to be potentially O-glycosylated with score predictors (G scores) of be-tween 0.5 and 0.8 in the deduced 85 to 86-amino-acidsequences of group A strains NA1 and GA5 fromnortheast India respectively. The G-score is the scorefrom the best general predictor; a residue is predictedas glycosylated when the G-score is >0.5, the higherthe score the more confident the prediction. Threesites were observed with extensive O-glycosylationamong the RSV-A strains from northeast India. TwoTTKP motifs located at amino acid positions 219 and227 were conserved among all the strains fromnortheast India. A potential O-glycosylation site, aKPT motif located at amino acid position 233, waspresent in all isolates of 2009, one strain eachisolated in 2011 and 2012 (Fig. 2).

DISCUSSION

In the Present Study, RSV was Detected in 7.9%of Patients by Real-Time RT-PCR

The nucleotide sequence analysis of the RSVstrains from northeast India showed existence of theRSV group A viruses with predominance of NA1subtype.There is little information available regarding the

seasonality, genetic diversity and the circulation ofRSV in developing countries where the diseaseburden is the greatest [Olsen et al., 2010; Nair et al.,2011]. Genetic variability of RSV in India has beenobserved in a few recent studies where circulation ofgenotypes GA2, GA5 and RSV group B viruses wasdocumented [Parveen et al., 2006; Agrawal et al.,2009; Raghuram et al., 2011]. However, the circula-tion of NA1 genotype has not yet been reported fromIndia. Thus the genetic diversity of RSV in this partof India is distinct from other Indian RSV strains.Most of the cases were detected in the month ofJanuary to March, though sporadic cases weredetected throughout the year. The NCBI BLASTsearch of the sequences obtained in the present studyshowed homology with Canadian NA1 strain (ON160-0111A) isolated recently [Eshaghi et al., 2012], withGA2 strain WI/629-Q0198/10 isolated in Wisconsin,

Fig. 1. Phylogenetic analysis of the C-terminal secondhypervariable region of the G protein gene of Dibrugarh(Assam), northeast India strains and reference HRSV group Aisolates. A few sequences corresponding to Cambodian NA1 andIndian GA2, GA5 were also included. Phylogeny was con-structed using the Maximum-likelihood method with 1,000bootstrap replicates in MEGA v 5. The tree is drawn to scale,with branch lengths in the same units as those of the evolution-ary distances used to infer the phylogenetic tree. The evolution-ary distances were computed using the Tamura-Nei model. Thestrains from Dibrugarh, northeast India were named in theorder-organism name (abbreviated) > place of isolation > iso-late number > year of isolation. The following reference sequen-ces were downloaded from GenBank to construct the trees:AL19471-5 and A2 (GA1); Ken/7/00, LLC235-267, AL19376-1,MO55, TX69564, Cam2006-2093, DEL/192/02 and DEL/3W/04(GA2); MO16 and TX68481 (GA3); NY/CH09/93 (GA4); MO01,Sal/173/99,AL19556-3,Cam2007-1176 and DEL/138/02 (GA5);AL19452-2 and NY20 (GA6); MO02 (GA7); SA98V603 (SAA1),NG-016-04 and Cam2008-7262 (NA1). The list of nucleotidesequences used in the construction of phylogenetic tree alongwith their GenBank Accession number, year, and country ofisolation are given in Table I.

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USA in 2010 [Rebuffo-Scheer et al., 2011] and strainNL20752390/07-08 isolated in Netherland during2007–2008 [Gaunt et al., 2011]. The sequence varia-tions of the RSV NA1 strains in the present studywere analogous to the Cambodian NA1 strains re-ported recently [Arnott et al., 2011].At present, information regarding specific disease

pattern such as disease severity associated with NA1strains are lacking, however in a recent study it wasreported that NA1 genotypes were associated withreinfection along with GA5 and NA2 [Yamaguchiet al., 2011]. The limitation of the study is that onlyoutdoor patients were evaluated. Thus further stud-ies including both outdoor and indoor patients (hospi-talized) would be required to firmly establish thedisease pattern associated with NA1 genotypes.In the present study molecular characterization of

RSV was based on sequence analysis directly fromclinical specimens which also eliminate any changesdue to adaptation in cell culture. Also samples werescreened first by real-time RT-PCR which provides ahighly sensitive method for the detection of RSV.In conclusion, the present study describes the

molecular characteristics of the RSV genotypes inAssam, northeast India in children with influenza-like illness. To the best of our knowledge, this is thefirst report of the detection of RSV NA1 genotypes inIndia. The occurrence of only RSV group A genotypesover the study period reinforces that RSV group Astrains are globally more frequent, potentially due tohigher genetic variability in comparison to RSVgroup B strains [Roca et al., 2001; Parveen et al.,2006] which may pose challenge to future vaccinedevelopment. The identification and characterizationof the virus indicates that RSV is also a diseaseburden in this part of India, however further studies

will be required from the northeastern part of Indiawhich shares international boundaries with China,Myanmar, Bangladesh, Bhutan and thus may con-tribute to understanding the global epidemiology ofRSV.

ACKNOWLEDGMENTS

The authors thank Miss Moitreye Lahan, MissSonali Dey, and Dr. Pranjal Jyoti Baruah for theirtechnical help in the study.

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