Introduction

Acute respiratory tract infection (ARI) is the leading killer among children in the world (1.9 million per year), with the number of deaths occurring in developing countries ¹. One fourth (2.5 millions) of the total deaths among children less than 5 years of age occur in India and approximately 20% of these are due to ARTIs ^2-4. Other than that bacterial infection, influenza virus and Human respiratory syncytial virus (HRSV) have been identified as the predominant etiological agents for lower respiratory tract infections ^5-7.About 20-40 % of the children hospitalized due to RSV with ARI in India ^8-10. Human respiratory syncytial virus (RSV) is the second most frequent cause of respiratory infections, and the main cause of severe cases of bronchitis and pneumonia in infants and young children ¹¹.RSV has been considered as an important viral infection, there is no antiviral treatment and effective vaccine prophylaxis is currently available against to RSV infections^{12, 13}.

The RSV has been classified in the Pneumovirus genus of the family Paramyxoviridae, it is an enveloped virus with negative sense single stranded RNA genome that encodes for ten proteins ^22-24. Glycoprotein (G) and Fusion protein (F) are most immunogenic proteins that are expressed on the virion surface, responsible for inducing production of neutralizing antibodies ^{14, 18, 21-23}. Both within and between the major RSV subgroups, the G protein is the most changeable viral protein with a minimal conserved ectodomain that contains 2 hypervariable regions (HVRs). The second HVR (HVR2) brings the C terminus of the protein and is commonly sequenced to examine the genetic variability of Human respiratory syncytial virus strains within a given population ^25-27.

The RSV has been classified into two antigenic subgroups RSV-A and RSV-B respectively, initially on the basis of the reactivity of the virus with monoclonal antibodies directed against the attachment G protein ^{14-16,22,24,28-30} and currently through genetic analyses ^{17,19,23,26,31-33}. The subtypes have been further subdivided into various genotypes. To date, 11 RSV-A (ON1, GA1–GA7, SAA1, NA1& NA2) and 17 RSV-B (GB1–GB4, SAB1-SAB3& BA1–BA10) genotypes have been identified ²⁰.

HRSV has been recognized as an important respiratory virus, there is a dearth of information on genetic diversity of HRSV strains among children in India, with few reports from North India ^34-39,52. In south India RSV molecular epidemiology is narrow. Therefore, this is the first study was to determine the prevalence and molecular epidemiology of RSV A and B in Chennai, Tamilnadu, south India.

Materials and Methods

Sampling site, collection and transport of clinical specimens

During the study period from January 2011 to December 2014, 850  Influenza virus negative clinical samples (throat/Nasal swab)  are subjected to detection of  other respiratory viruses  like, Respiratory Syncytial Virus ,Para influenza viruses, Human Metapneumo viruses, samples are collected from out patient of  Chennai Government Hospitals (Saidapet Government Hospital, Stanly Government Hospital, Periyar Nagar peripheral Government  Hospital, Institute of Child Health).In the present study we are included only RSV data. Throat and nasal swab were collected following standard protocols (WHO criteria), Patient demographic details, clinical symptoms were recorded. Samples were transported to the laboratory  with virus transport medium (Hanks’ balanced salt solution with 200 IU penicillin ml^-1, 200 mg streptomycin ml^-1and 2% BSA)  The present study was approved by the Institutional Ethical Committee of the King Institute of Preventive Medicine and Research, Gundy, Chennai. The present study was carried in the Dept of Virology King Institute of Preventive Medicine and Research, Guindy, Chennai.

Table 1: Yearly Distibution of RSV infections in Chennai. Click here to View Table

Viral RNA Extraction

Viral RNA was isolated from the respiratory specimens using Viral RNA mini kit (Qiagen) followed by manufacture’s directions. The RNA was stored in aliquots at -80⁰C until use.

RT-PCR for detection of HRSV strains

Nucleic acid amplification was performed on GeneAmp PCR system 9600 thermal cycler (ABI- Applied Bio systems) using with Invitrogen Superscript III platinum one step RT-PCR kit for detection and sub typing of the RSV as described earlier ⁴⁰. The amplified products were analyzed on a 1.5 % Agarose gel electrophoresis.

RSV Genotyping

RSV-A and RSV-B specific oligonucleotide primers (second hypervariable region of the G-protein) were used for genotyping and sequencing of the G protein. PCR protocols were described earlier ⁴².

RT-PCR and Nested PCR for RSV A Genotyping

Genotyping of RSV A, amplification of the second hypervariable region of the Glycoprotein gene by either external or nested PCR. The external PCR was carried out with 10 pmol of forward primer RSVA-G513-F(5′ AGTGTTCAACTTTGTACCCTGC3′) and reverse primer RSVA-F131-R (5’CTGCACTGCATGTTGATTGAT3′) ⁴¹ with Superscript III platinum one step RT-PCR kit (Invitrogen). Thermal cycling conditions are 50°C for 30 min 94°C for 5 min, followed by 40 cycles of PCR, 1 cycle consisting of 30 s at 94°C, 30 s at 58°C, and 1 min at 72°C, and a final extension is 10 min at 72°C. The 583 bp amplified products were analyzed by electrophoresis. In the case of external PCR results negative, 5 μl of the external PCR product was used as a template for nested PCR with 10 pmol of primers RSVA-G606-F (5’AACCACCACCAAGCCCACAA3′) and RSV-F22-R (5’CAACTCCATTGTTATTTGCC3′) ⁴¹, with Phusion DNA PCR kit (New England Bio labs).The cycling conditions were the same as for the external PCR (absence of RT step), except annealing temperature, which was 53°C. The nested amplicons of 391 bp were visualized by agarose gel electrophoresis.

RT-PCR for RSV-B Genotyping

For RSV B, RT-PCR assay was performed by using Superscript III platinum one step RT-PCR kit (Invitrogen) in a 25μl volume containing 20 pmol each of forward primer BGF (5’GCAGCCATAATATTCATCATCTCT3′) and reverse primer BGR (5’TGCCCCAGRTTTAATTTCGTTC3′) ⁴¹, and 5 μl of the extracted RNA. PCR reactions were conducted on a GeneAmp PCR system 9600 thermal cycler (ABI) thermal cyclic conditions are   50°C for 30 min for reverse transcription and 94°C for 5 min, 40 cycles of 94°C for 30 s, 63°C for 1 min, 72°C for 1 min, and the final extension is, at 72°C for 10 min. Amplified products were subjected to 1.5% agarose gel electrophoresis.

Sequencing and Phylogenetic Analysis

The PCR products were purified with a Purification kit (Qiagen), according to the manufacturer’s directions. The purified products were sequenced in both directions on an ABI Prism 3130 genetic analyzer (ABI) by using an ABI Prism BigDye Terminator cycle sequencing kit 3.1 (ABI). Sequences were edited manually in Bioedit software v 7.0.9.0 ⁴³. The reference global sequences were downloaded from NCBI GenBank. All the Phylogenetic analysis and multiple sequence alignment were conducted with MEGA software v 5 ⁴⁴.

Statistical analysis

Statistical analyses were carried out with SPSS16.0 software. Significance of differences in frequencies of age groups was tested using a chi – square test. P value was found to be statistically significant (P<0.05).

Results

Yearly Distibution of RSV infections in chennai

Out of 850 samples 124 (14.5%) samples were positive for RSV, in which 84(9.8%) were RSV A, 40 (4.7%) were RSV B, (Table I). There was an increase in the percentage positivity in 2012when compared to reaming years. During the study period from 2011-2014,

Prevalence of RSV infection in different age groups

Among  patients  with RSV  infection,  47.5%  were   less  than 1 year age group, 29.8 %  were between  1-5 year age group, 14.5% were between 6-14 years age group and  8 % were above 14 years age (Figure I).

Figure 1: Proportion of suspected and confirmed cases of RSV infection by age group, during the study period in Chennai. Click here to View figure

Relation of meteorological factors with RSV infection

During the study period January 2011-December 2014 their positivity and number of samples correlated with meteorological data (Rain fall and Temperature) obtained from the Meteorological Department, Chennai, Government of India. The percentage of RSV positivity was found to be higher during the months of October to December in all the four years (Figure II).

Figure 2: The correlation between meteorological variables and the number of RSV positive samples obtained during the study. Click here to View figure

Phylogenetic analysis

During the study period, out of 124 positive samples 27 samples (20 RSV A, 7RSV B )  are subjected to sequencing. Phylogenetic tree constructed with RSV global reference sequences and Indian sequences (Dibrugarh, Delhi and Pune, Kolkata, Lucknow ). Phylogenetic analysis detected that all RSV-A glycoprotein sequences obtained in this study clustered in ON1 group of NA1 subtypes. A newly designated genotype ON1, within the NA1 genotype was first documented from Ontario, Canada and those samples were collected in the period Dec 2010 ⁴⁷. Phylogenetic analysis revealed that all RSV-B glycoprotein sequences obtained in this study clustered in genotype BA, in which 4 sequences clustered in genotype BA9, 3 sequences clustered in newly designated BA12 genotype first observed in Malaysia ⁴⁸ (Figure III).

Figure 3: Phylogenetic Analysis of RSVA and RSV B Click here to View figure

Discussion

RSV is one of the most regular viral pathogens causing lower respiratory tract infections among infants, young children and elderly individuals worldwide. Different studies discovered RSV positivity in the range from 15% to 35% in pediatric population ^{45, 46.}  In India, Kolkata (Eastern India) 10.3% ³⁹, Assam (North East)  7.9% ⁵², Delhi (Northern India) 17% ³⁵, Pune (Northern India) 18.61 % ³⁴ were detected positive, in the present study during the study period from 2011-2014 positivity rate was 14.5%.  Previous studies show that RSV group A is more predominant than group B infections in globally ^{33, 49-51}. RSV-A was the predominantly circulating group in 2011, RSV-B was the predominantly circulating in 2005–2006 while RSV-A in 2007–2008 in Kolkata ³⁹ and RSV-A during the 2001–2005 in Delhi ³⁵. RSV-A was the predominant circulating strain in Assam during 2009–2012 ⁵². Our Present study shows that, 9.8% were RSV A, 4.7% were RSV B, suggesting that RSV-A was the predominant subtype circulating in Chennai, south India. Little is known regarding the seasonality of RSV in tropical and subtropical countries like India.

Most infected individuals were children less than 2 years of age. Globally, the rate of RSV infection has been reported to decrease with increasing age ^{46, 53, 54}. We observed an increased prevalence of RSV in the less than one year (47.5%) and 1-5 years (29.8%) age groups compared with greater than 5-14 (14.5%) and above 14 years old children, which is similar to previous studies done in Kolkata, India ³⁹. We observed that RSV infection revealed seasonal variations which peaked during the monsoon and winter months. Similar finding were reported in Pune reported a peak in monsoon and winter ³⁴, while Kolkata and Delhi reported peaks in the winter months ^{36, 39}. Similar findings were reported from Cambodia ⁴⁶, Germany ⁵³ and Belgium ⁵⁴.

In recent years, GA2 and GA5 were the most familiar genotypes among RSV-A worldwide. GA2 was the most common genotype of RSV-A found around the world and has persisted for many years ^{36, 46, 53, 54}. In earlier studies from Delhi, subgroups GA2 and GA5 were prevalent among the circulating RSV-A strains during the 2002 to 2003 season and genotype GA5 was predominant in the 2003 to 2005 seasons. Sequences from Pune area in 2006 clustered in genotype GA2, GA5 and the recently designated group NA2. Sequences previously documented from different parts of India clustered together in the GA2, GA3, GA5 and NA2 genotypes. Sequences from Dibrugarh, eastern India from 2009 to 2012 mainly clustered in NA1 genotype and two sequences in GA5 genotype⁵². In the present study not described about Amino acid analysis, this is the drawback of our study, further studies will be performed and rectify the hitch. In our study, phylogenetic analysis revealed that all RSV-A strains are clustered in newly designated ON1, within the NA1 genotype.

RSV group B genotype BA, first detected in Argentina in 1999 and has since become the predominant worldwide ^{36, 39, 46, 54}. BA genotype is further classified from BA1 to BA12. BA genotype has been the most reported genotype in previous studies from India ^{35, 39}. Our BA sequences clustered along with the BA9 and BA12 genotypes. Four sequences from the present study clustered in cluster BA within BA9 genotype, and three sequences in genotype BA12. However, it required long term surveillance throughout the country.

Conclusion

This is the first report contributing preliminary data on the RSV molecular characterization in Chennai, south India. In the present study concludes that RSV group A viruses predominated than group B infections during the study period in Chennai. RSV infection is pediment in the age group between less than one to five years children. In Chennai RSV infection exhibited seasonal variations which peaked during the monsoon and winter months. Phylogenetic analysis of RSV-A belonged to the ON1 within NA1 genotype, RSV-B belonged to genotype BA of BA9 and BA12. In the present study not described about Amino acid analysis,  next studies will be carried out and fulfilled the hitch.  However, further studies will be required from the rest of South India, which shares understanding the molecular epidemiology of RSV in south India.. RSV Genotyping data will helpful for future vaccine candidates.

Acknowledgement

The authors thank to Indian Council of Medical Research (ICMR), New Delhi for financially supported in this study. The authors acknowledge the Director, National Institute of Virology, Pune, for providing support in this influenza surveillance project and VDL staff for support in laboratory work and collection of samples.

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