Differences in viral load among human respiratory syncytial virus genotypes in hospitalized children with severe acute respiratory infections in the Philippines
© The Author(s). 2016
Received: 13 March 2016
Accepted: 17 June 2016
Published: 27 June 2016
Human respiratory syncytial virus (HRSV) is a leading viral etiologic agent of pediatric lower respiratory infections, including bronchiolitis and pneumonia. Two antigenic subgroups, HRSV-A and B, each contain several genotypes. While viral load may vary among HRSV genotypes and affect the clinical course of disease, data are scarce regarding the actual differences among genotypes. Therefore, this study estimated and compared viral load among NA1 and ON1 genotypes of HRSV-A and BA9 of HRSV-B. ON1 is a newly emerged genotype with a 72-nucleotide duplication in the G gene as observed previously with BA genotypes in HRSV-B.
Children <5 years of age with an initial diagnosis of severe or very severe pneumonia at a hospital in the Philippines from September 2012 to December 2013 were enrolled. HRSV genotypes were determined and the viral load measured from nasopharyngeal swabs (NPS). The viral load of HRSV genotype NA1 were significantly higher than those of ON1 and BA9. Regression analysis showed that both genotype NA1 and younger age were significantly associated with high HRSV viral load.
The viral load of NA1 was higher than that of ON1 and BA9 in NPS samples. HRSV genotypes may be associated with HRSV viral load. The reasons and clinical impacts of these differences in viral load among HRSV genotypes require further evaluation.
Human respiratory syncytial virus (HRSV) is a major etiologic agent of lower respiratory tract infections, including bronchiolitis and pneumonia, and a leading cause of hospitalizations among infants in both developing and developed countries . Two subgroups, HRSV-A and HRSV-B, have been described. A total of 11 HRSV-A genotypes have been identified, including GA1 to GA7 , SAA1 , NA1 and NA2 , and, most recently, ON1, which has a 72-nucleotide duplication in the G gene . A total of 19 HRSV-B genotypes have been reported, including GB1 to GB4 , SAB1 to SAB3 , and BA, with a 60-nucleotide duplication in the G gene, which is subdivided into BA1 to BA12 [6–9]. The newly-emerged BA genotypes have replaced the previous genotypes ; similarly, ON1, derived from NA1 of HRSV-A, has been recently reported in many regions worldwide [11–13]. Both BA and ON1 have nucleotide duplications in the hypervariable region of the G gene. However, the effect of these duplications on the spread of these genotypes remains unknown. Although, viral load is an important factor in HRSV disease and severity , to date, there are no data regarding differences in HRSV viral load among different genotypes. We therefore investigated HRSV viral load among three genotypes identified in hospitalized pediatric patients.
A case series study was conducted from September 2012 to August 2013 in the Biliran Provincial Hospital, a referral hospital on Biliran Island  in the Philippines. Children aged <5 years admitted with an initial diagnosis of severe or very severe pneumonia based on Integrated Management of Childhood Illness criteria (IMCI)  were asked participation into the study and written parental consent was obtained for each participant. Severe pneumonia were defined as cases with cough and/or difficulty of breathing accompanied by chest indrawing and/or one or more danger signs including inability to drink, convulsion or lethargic/asleep. Local medical staffs were trained with a standardized protocol for nasopharyngeal swab (NPS) samples through the study period. Samples were taken using EX-swab 002 (DENKA SEIKEN Tokyo, Japan), stirred in 3 ml viral transport medium, which consists of Hanks BSS (Balanced salt solution), 0.25 % gelatin, 0.035 % sodium bicarbonate, Streptomycin (500 μg/mL) and Penicillin (500 U/mL) and Amphotericin B (250 μg/mL) and kept at 0–4 °C. Viral RNA was extracted using the QIAamp MinElute Virus Spin Kit (QIAGEN, Hilden, Germany) and cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase and random primers (Thermo Scientific, Boston, MA, USA).
The first real-time polymerase chain reaction (PCR) for screening of HRSV was performed using an Applied Biosystems StepOnePlus system (Applied Biosystems, Foster City, CA, USA) as previously described, with some modifications  in Research Institute for Tropical Medicine (RITM), Metro Manila. The region of the N gene targeted by the real-time PCR is highly conserved among subgroups and genotypes. The detection limit of the assay was 100 copies for all genotypes.
The positive cDNA samples were shipped from RITM to laboratory at Tohoku University and viral load was defined as viral RNA copy number estimated from mean threshold cycle (Ct) values in duplicated samples obtained from the second real-time PCR with synthesized viral genomic RNA standard samples for each genotype (N gene, 658 nucleotides). For subgrouping and genotyping, the C-terminus region of the G gene was amplified and sequenced as previously described .
Patient characteristics, clinical symptoms, elapsed time between illness onset and specimen collection, viral coinfection (influenza virus, human rhinovirus/enterovirus, human metapneumovirus, and human parainfluenza virus) and viral load (Log10 RNA copies/μL) were compared among HRSV genotypes using chi-square and Kruskal-Wallis tests and one-way analysis of variance (ANOVA). The beta coefficients for associations between viral load and genotypes or other factors were adjusted by generalized linear regression. Statistical analysis was performed using IBM SPSS Statistics for Windows, version 20 (IBM Corp, Armonk, NY, USA) and a P value of < 0.05 was considered statistically significant.
Characteristics of 139 hospitalized patients due to severe acute respiratory infection by HRSV genotypes
n = 17
n = 51
n = 71
Age in months, median [IQR]
Days between illness onset and specimen collection, median [IQR]
Difficulty of breathing
Inability of drink
Peripheral oxygen saturation < 90 % (%)
Viral coinfection (%)
Viral load (Log10 RNA copies/μL), median [IQR]
< 6 months
(n = 6)
(n = 27)
(n = 21)
Distribution and adjusted coefficients of viral and patient factors for HRSV viral load
β (95 % CI)
−0.21 (−0.57, 0.15)
−0.55 (−0.92, −0.18)
0.28 (−0.02, 0.58)
Days between illness onset and specimen collection
0.15 (−0.16, 0.46)
−1.13 (−1.53, −0.73)
−0.92 (−1.27, −0.56)
−0.19 (−0.82, 0.44)
Genetic differences among genotypes may explain the differences in viral load. ON1 has a 72-nucleotide insertion in the G gene . This insertion is absent in the NA1 genotype and is the largest insertion described to date in this genus, which may affect the viral structure and pathogenesis. BA9, also characterized by an insertion in the G gene, also showed significantly lower viral load compared to NA1. The G protein is one of the major HRSV protective antigens and has significant roles in cell attachment , cell fusion , and immune response . Insertions and duplications in the G gene may have affected the efficiency of HRSV replication in the newly emerged ON1 and BA genotypes. Detailed molecular analysis of the G protein gene sequence of several HRSV strains commonly reveal multiple short sequence repeats. The earlier report in HRSV-B of the 60-nucleotide duplication (the BA genotypes) had shown an exceptional example of repeated sequence in the G protein, which emerged and replaced previous genotypes within HRSV-B . Further studies are needed to better understand why viral load differs among genotypes as well as the impact of new insertions in the G gene on HRSV pathogenesis.
Limitation of our observation is the small number of patients in NA1 group and NPS sampling by different study personnel, which may have had inter- sampling variation. Also viral load in NPS may not enhance the severity of lower respiratory infection. Others factors that may influence HRSV viral load such as, prematurity, breast feeding status and use of steroids or other immunosuppressive medication were not assessed in this study.
In the present study, age and viral genotypes were host and viral factors that independently predicted HRSV viral load. Age is a well-known predictor of HRSV disease severity  and is also associated with HRSV viral load. Although other studies using molecular techniques did not observe differences in viral load between HRSV subgroups [23, 24], differences in sample size, sample collection, or circulating genotypes during their study periods could explain this difference in findings. Our findings suggest the critical need for further studies to investigate potential differences in viral load among genotypes and their association with disease severity.
Ct, cycle threshold; HRSV, human respiratory syncytial virus; NPS, nasopharyngeal swab; PCR, polymerase chain reaction
We are grateful to the staff of Biliran Provincial Hospital and Tohoku-RITM Collaborating Research Center on Emerging and Reemerging Diseases. We also thank R. Kojima, S. Abe, M. Kishi, and M. Estanislao for their technical assistance. This work was supported by a grant-in-aid from the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) from Japan Agency for Medical Research and Development (AMED), Japan, Science and Technology Research Partnership for Sustainable Development (SATREPS) from Japan International Cooperation Agency (JICA) and AMED.
FK Study protocol development, performed experiments and manuscript drafting. MO Laboratory diagnosis and data quality assurance. YF Performed experiments and contributed to reviewing the manuscript. AS Study protocol development and hospital site operation. RT Hospital site operation and data analysis. IL Laboratory diagnosis and data analysis. CD Manuscript review. RM Molecular genetic studies. MSO Study coordination and laboratory data analysis. GR Hospital site coordination and clinical data management. VT Study planning, protocol development and data quality assurance. SL Study planning, protocol development, and study supervision. MS Data analysis and manuscript revision. HO Study planning, protocol development, and critical review of the revised manuscript. All authors read and approved the final manuscript.
None of the authors have any competing interests in the manuscript.
Ethics approval and consent to participate
This study was approved by the Ethics Committee of Tohoku University Graduate School of Medicine and Institutional Review Board of RITM.
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- Borchers AT, Chang C, Gershwin ME, Gershwin LJ. Respiratory syncytial virus--a comprehensive review. Clin Rev Allergy Immunol. 2013;45:331–79.View ArticlePubMedGoogle Scholar
- Peret TC, Hall CB, Schnabel KC, Golub JA, Anderson LJ. Circulation patterns of genetically distinct group A and B strains of human respiratory syncytial virus in a community. J Gen Virol. 1998;79(Pt 9):2221–9.View ArticlePubMedGoogle Scholar
- Venter M, Madhi SA, Tiemessen CT, Schoub BD. Genetic diversity and molecular epidemiology of respiratory syncytial virus over four consecutive seasons in South Africa: identification of new subgroup A and B genotypes. J Gen Virol. 2001;82:2117–24.View ArticlePubMedGoogle Scholar
- Shobugawa Y, Saito R, Sano Y, Zaraket H, Suzuki Y, Kumaki A, Dapat I, Oguma T, Yamaguchi M, Suzuki H. Emerging genotypes of human respiratory syncytial virus subgroup A among patients in Japan. J Clin Microbiol. 2009;47:2475–82.View ArticlePubMedPubMed CentralGoogle Scholar
- Eshaghi A, Duvvuri VR, Lai R, Nadarajah JT, Li A, Patel SN, Low DE, Gubbay JB. Genetic variability of human respiratory syncytial virus A strains circulating in Ontario: a novel genotype with a 72 nucleotide G gene duplication. PLoS One. 2012;7:e32807.View ArticlePubMedPubMed CentralGoogle Scholar
- Khor CS, Sam IC, Hooi PS, Chan YF. Displacement of predominant respiratory syncytial virus genotypes in Malaysia between 1989 and 2011. Infect Genet Evol. 2013;14:357–60.View ArticlePubMedGoogle Scholar
- Trento A, Casas I, Calderón A, Garcia-Garcia ML, Calvo C, Perez-Breña P, Melero JA. Ten years of global evolution of the human respiratory syncytial virus BA genotype with a 60-nucleotide duplication in the G protein gene. J Virol. 2010;84:7500–12.View ArticlePubMedPubMed CentralGoogle Scholar
- Trento A, Viegas M, Galiano M, Videla C, Carballal G, Mistchenko AS, Melero JA. Natural history of human respiratory syncytial virus inferred from phylogenetic analysis of the attachment (G) glycoprotein with a 60-nucleotide duplication. J Virol. 2006;80:975–84.View ArticlePubMedPubMed CentralGoogle Scholar
- Tran DN, Pham TM, Ha MT, Tran TT, Dang TK, Yoshida LM, Okitsu S, Hayakawa S, Mizuguchi M, Ushijima H. Molecular epidemiology and disease severity of human respiratory syncytial virus in Vietnam. PLoS One. 2013;8:e45436.View ArticlePubMedPubMed CentralGoogle Scholar
- Dapat IC, Shobugawa Y, Sano Y, Saito R, Sasaki A, Suzuki Y, Kumaki A, Zaraket H, Dapat C, Oguma T, et al. New genotypes within respiratory syncytial virus group B genotype BA in Niigata, Japan. J Clin Microbiol. 2010;48:3423–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Ren L, Xia Q, Xiao Q, Zhou L, Zang N, Long X, Xie X, Deng Y, Wang L, Fu Z, et al. The genetic variability of glycoproteins among respiratory syncytial virus subtype A in China between 2009 and 2013. Infect Genet Evol. 2014;27:339–47.View ArticlePubMedGoogle Scholar
- Pierangeli A, Trotta D, Scagnolari C, Ferreri ML, Nicolai A, Midulla F, Marinelli K, Antonelli G, Bagnarelli P: Rapid spread of the novel respiratory syncytial virus A ON1 genotype, central Italy, 2011 to 2013. Euro Surveill 2014;19:1–10.Google Scholar
- Agoti CN, Otieno JR, Gitahi CW, Cane PA, Nokes DJ. Rapid spread and diversification of respiratory syncytial virus genotype ON1, Kenya. Emerg Infect Dis. 2014;20:950–9.View ArticlePubMedPubMed CentralGoogle Scholar
- DeVincenzo JP, El Saleeby CM, Bush AJ. Respiratory syncytial virus load predicts disease severity in previously healthy infants. J Infect Dis. 2005;191:1861–8.View ArticlePubMedGoogle Scholar
- Kosai H, Tamaki R, Saito M, Tohma K, Alday PP, Tan AG, Inobaya MT, Suzuki A, Kamigaki T, Lupisan S, et al. Incidence and risk factors of childhood pneumonia-like episodes in biliran island, Philippines-a community-based study. PLoS One. 2015;10:e0125009.View ArticlePubMedPubMed CentralGoogle Scholar
- UNICEF. WHODoCaAHaD: Handbook IMCI : integrated management of childhood illness / World Health Organization. Geneva: World Health Organization, Dept. of Child and Adolescent Health and Development; UNICEF.: World Health Organization, Dept. of Child and Adolescent Health and Development; UNICEF; 2005.Google Scholar
- Bonroy C, Vankeerberghen A, Boel A, De Beenhouwer H. Use of a multiplex real-time PCR to study the incidence of human metapneumovirus and human respiratory syncytial virus infections during two winter seasons in a Belgian paediatric hospital. Clin Microbiol Infect. 2007;13:504–9.View ArticlePubMedGoogle Scholar
- Ohno A, Suzuki A, Lupisan S, Galang H, Sombrero L, Aniceto R, Okamoto M, Saito M, Fuji N, Otomaru H, et al. Genetic characterization of human respiratory syncytial virus detected in hospitalized children in the Philippines from 2008 to 2012. J Clin Virol. 2013;57:59–65.View ArticlePubMedGoogle Scholar
- Wright PF, Gruber WC, Peters M, Reed G, Zhu Y, Robinson F, Coleman-Dockery S, Graham BS. Illness severity, viral shedding, and antibody responses in infants hospitalized with bronchiolitis caused by respiratory syncytial virus. J Infect Dis. 2002;185:1011–8.View ArticlePubMedGoogle Scholar
- Hotard AL, Laikhter E, Brooks K, Hartert TV, Moore ML. Functional analysis of the 60-nucleotide duplication in the respiratory syncytial Virus Buenos Aires strain attachment glycoprotein. J Virol. 2015;89:8258–66.View ArticlePubMedPubMed CentralGoogle Scholar
- Heminway BR, Yu Y, Tanaka Y, Perrine KG, Gustafson E, Bernstein JM, Galinski MS. Analysis of respiratory syncytial virus F, G, and SH proteins in cell fusion. Virology. 1994;200:801–5.View ArticlePubMedGoogle Scholar
- Tripp RA, Moore D, Jones L, Sullender W, Winter J, Anderson LJ. Respiratory syncytial virus G and/or SH protein alters Th1 cytokines, natural killer cells, and neutrophils responding to pulmonary infection in BALB/c mice. J Virol. 1999;73:7099–107.PubMedPubMed CentralGoogle Scholar
- Do LA, van Doorn HR, Bryant JE, Nghiem MN, Van Nguyen VC, Vo CK, Nguyen MD, Tran TH, Farrar J, de Jong MD. A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial virus. J Virol Methods. 2012;179:250–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Kuypers J, Wright N, Morrow R. Evaluation of quantitative and type-specific real-time RT-PCR assays for detection of respiratory syncytial virus in respiratory specimens from children. J Clin Virol. 2004;31:123–9.View ArticlePubMedGoogle Scholar