Direct whole-genome deep-sequencing of human respiratory syncytial virus A and B from Vietnamese children identifies distinct patterns of inter- and intra-host evolution
Human respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in children < 2 years of age. Little is known about RSV intra-host genetic diversity over the course of infection or about the immune pressures that drive RSV molecular evolution. We performed whole-genome deep-sequencing on 53 RSV-positive samples (37 RSV subgroup A and 16 RSV subgroup B) collected from the upper airways of hospitalized children in southern Vietnam over two consecutive seasons. RSV A NA1 and RSV B BA9 were the predominant genotypes found in our samples, consistent with other reports on global RSV circulation during the same period. For both RSV A and B, the M gene was the most conserved, confirming its potential as a target for novel therapeutics. The G gene was the most variable and was the only gene under detectable positive selection. Further, positively selected sites in G were found in close proximity to and in some cases overlapped with predicted glycosylation motifs, suggesting that selection on amino acid glycosylation may drive viral genetic diversity. We further identified hotspots and coldspots of intra-host genetic diversity in the RSV genome, some of which may highlight previously unknown regions of functional importance.
ArnottA.,
VongS.,
MardyS.,
ChuS.,
NaughtinM.,
SovannL.,
BuecherC.,
BeautéJ.,
RithS.,
other authors.
2011; A study of the genetic variability of human respiratory syncytial virus (HRSV) in Cambodia reveals the existence of a new HRSV group B genotype. J Clin Microbiol 49:3504–3513 [View Article][PubMed]
BaekY. H.,
ChoiE. H.,
SongM. S.,
PascuaP. N.,
KwonH. I.,
ParkS. J.,
LeeJ. H.,
WooS. I.,
AhnB. H.,
other authors.
2012; Prevalence and genetic characterization of respiratory syncytial virus (RSV) in hospitalized children in Korea. Arch Virol 157:1039–1050 [View Article][PubMed]
BlancA.,
DelfraroA.,
FrabasileJ.,
ArbizaS.2005; Genotypes of respiratory syncytial virus group B identified in Uruguay. Arch Virol 150:603–609 [View Article][PubMed]
BotossoV. F.,
ZanottoP. M.,
UedaM.,
ArrudaE.,
GilioA. E.,
VieiraS. E.,
StewienK. E.,
PeretT. C.,
JamalL. F.,
other authors.
2009; Positive selection results in frequent reversible amino acid replacements in the G protein gene of human respiratory syncytial virus. PLoS Pathog 5:e1000254 [View Article][PubMed]
CaneP. A.1997; Analysis of linear epitopes recognised by the primary human antibody response to a variable region of the attachment (G) protein of respiratory syncytial virus. J Med Virol 51:297–304[PubMed][CrossRef]
CaneP. A.,
PringleC. R.1995; Evolution of subgroup A respiratory syncytial virus: evidence for progressive accumulation of amino acid changes in the attachment protein. J Virol 69:2918–2925[PubMed]
ChoudharyM. L.,
WadhwaB. S.,
JadhavS. M.,
ChadhaM. S.2013; Complete genome sequences of two human respiratory syncytial virus genotype A strains from India, RSV-A/NIV1114046/11 and RSV-A/NIV1114073/11. Genome Announc 1:e00165–e00113 [View Article][PubMed]
CoatesH. V.,
AllingD. W.,
ChanockR. M.1966; An antigenic analysis of respiratory syncytial virus isolates by a plaque reduction neutralization test. Am J Epidemiol 83:299–313[PubMed]
CollinsP. L.,
OlmstedR. A.,
SpriggsM. K.,
JohnsonP. R.,
Buckler-WhiteA. J.1987; Gene overlap and site-specific attenuation of transcription of the viral polymerase L gene of human respiratory syncytial virus. Proc Natl Acad Sci U S A 84:5134–5138 [View Article][PubMed]
CollinsP. L.,
MinkM. A.,
StecD. S.1991; Rescue of synthetic analogs of respiratory syncytial virus genomic RNA and effect of truncations and mutations on the expression of a foreign reporter gene. Proc Natl Acad Sci U S A 88:9663–9667 [View Article][PubMed]
ConnorsM.,
CroweJ. E.Jr,
FirestoneC. Y.,
MurphyB. R.,
CollinsP. L.1995; A cold-passaged, attenuated strain of human respiratory syncytial virus contains mutations in the F and L genes. Virology 208:478–484 [View Article][PubMed]
CroweJ. E.Jr,
FirestoneC. Y.,
WhiteheadS. S.,
CollinsP. L.,
MurphyB. R.1996; Acquisition of the ts phenotype by a chemically mutagenized cold-passaged human respiratory syncytial virus vaccine candidate results from the acquisition of a single mutation in the polymerase (L) gene. Virus Genes 13:269–273 [View Article][PubMed]
DapatI. C.,
ShobugawaY.,
SanoY.,
SaitoR.,
SasakiA.,
SuzukiY.,
KumakiA.,
ZaraketH.,
DapatC.,
other authors.
2010; New genotypes within respiratory syncytial virus group B genotype BA in Niigata, Japan. J Clin Microbiol 48:3423–3427 [View Article][PubMed]
DoA. H.,
van DoornH. R.,
NghiemM. N.,
BryantJ. E.,
HoangT. H.,
DoQ. H.,
VanT. L.,
TranT. T.,
WillsB.,
other authors.
2011; Viral etiologies of acute respiratory infections among hospitalized Vietnamese children in Ho Chi Minh City, 2004-2008. PLoS One 6:e18176 [View Article][PubMed]
DoL. A.,
van DoornH. R.,
BryantJ. E.,
NghiemM. N.,
Nguyen VanV. C.,
VoC. K.,
NguyenM. D.,
TranT. H.,
FarrarJ.,
de JongM. D.2012; A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial virus. J Virol Methods 179:250–255 [View Article][PubMed]
EmpeyK. M.,
PeeblesR. S.Jr,
KollsJ. K.2010; Pharmacologic advances in the treatment and prevention of respiratory syncytial virus. Clin Infect Dis 50:1258–1267 [View Article][PubMed]
Escribano-RomeroE.,
RawlingJ.,
García-BarrenoB.,
MeleroJ. A.2004; The soluble form of human respiratory syncytial virus attachment protein differs from the membrane-bound form in its oligomeric state but is still capable of binding to cell surface proteoglycans. J Virol 78:3524–3532 [View Article][PubMed]
EshaghiA.,
DuvvuriV. R.,
LaiR.,
NadarajahJ. T.,
LiA.,
PatelS. N.,
LowD. E.,
GubbayJ. B.2012; Genetic variability of human respiratory syncytial virus A strains circulating in Ontario: a novel genotype with a 72 nucleotide G gene duplication. PLoS One 7:e32807 [View Article][PubMed]
FearnsR.,
CollinsP. L.,
PeeplesM. E.2000; Functional analysis of the genomic and antigenomic promoters of human respiratory syncytial virus. J Virol 74:6006–6014 [View Article][PubMed]
GarcíaO.,
MartínM.,
DopazoJ.,
ArbizaJ.,
FrabasileS.,
RussiJ.,
HortalM.,
Perez-BreñaP.,
MartínezI.,
other authors.
1994; Evolutionary pattern of human respiratory syncytial virus (subgroup A): cocirculating lineages and correlation of genetic and antigenic changes in the G glycoprotein. J Virol 68:5448–5459[PubMed]
GauntE. R.,
JansenR. R.,
PoovorawanY.,
TempletonK. E.,
TomsG. L.,
SimmondsP.2011; Molecular epidemiology and evolution of human respiratory syncytial virus and human metapneumovirus. PLoS One 6:e17427 [View Article][PubMed]
GradY. H.,
NewmanR.,
ZodyM.,
YangX.,
MurphyR.,
QuJ.,
MalboeufC. M.,
LevinJ. Z.,
LipsitchM.,
DeVincenzoJ.2014; Within-host whole-genome deep sequencing and diversity analysis of human respiratory syncytial virus infection reveals dynamics of genomic diversity in the absence and presence of immune pressure. J Virol 88:7286–7293 [View Article][PubMed]
JohnsonP. R.Jr,
OlmstedR. A.,
PrinceG. A.,
MurphyB. R.,
AllingD. W.,
WalshE. E.,
CollinsP. L.1987a; Antigenic relatedness between glycoproteins of human respiratory syncytial virus subgroups A and B: evaluation of the contributions of F and G glycoproteins to immunity. J Virol 61:3163–3166[PubMed]
JohnsonP. R.,
SpriggsM. K.,
OlmstedR. A.,
CollinsP. L.1987b; The G glycoprotein of human respiratory syncytial viruses of subgroups A and B: extensive sequence divergence between antigenically related proteins. Proc Natl Acad Sci U S A 84:5625–5629 [View Article][PubMed]
KarronR. A.,
BuonagurioD. A.,
GeorgiuA. F.,
WhiteheadS. S.,
AdamusJ. E.,
Clements-MannM. L.,
HarrisD. O.,
RandolphV. B.,
UdemS. A.,
other authors.
1997; Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant. Proc Natl Acad Sci U S A 94:13961–13966 [View Article][PubMed]
KorberB.2000; HIV signature and sequence variation analysis. In Computational Analysis of HIV Molecular Sequences pp. 55–72 Edited by
RodrigoA. G.,
LearnG. H.
Dordrecht: Kluwer;
KrzywinskiM.,
ScheinJ.,
BirolI.,
ConnorsJ.,
GascoyneR.,
HorsmanD.,
JonesS. J.,
MarraM. A.2009; Circos: an information aesthetic for comparative genomics. Genome Res 19:1639–45[CrossRef]
KuoL.,
GrosfeldH.,
CristinaJ.,
HillM. G.,
CollinsP. L.1996; Effects of mutations in the gene-start and gene-end sequence motifs on transcription of monocistronic and dicistronic minigenomes of respiratory syncytial virus. J Virol 70:6892–6901[PubMed]
KuoL.,
FearnsR.,
CollinsP. L.1997; Analysis of the gene start and gene end signals of human respiratory syncytial virus: quasi-templated initiation at position 1 of the encoded mRNA. J Virol 71:4944–4953[PubMed]
LambR. A.,
CollinsP. L.,
KolakofskyD.,
MeleroJ. A.,
NagaiY.,
OldstoneM. B. A.,
PringleC. R.,
RimaB. K.2005; Paramyxoviridae. In Eighth Report of the International Committee on Taxonomy of Viruses pp. 655–668 Edited by
FauquetC. M.,
MayoM. A.,
ManiloffJ.,
DesselbergerU.,
BallL. A.
London: Elsevier Academic Press;
LeeW. J.,
KimY. J.,
KimD. W.,
LeeH. S.,
LeeH. Y.,
KimK.2012; Complete genome sequence of human respiratory syncytial virus genotype A with a 72-nucleotide duplication in the attachment protein G gene. J Virol 86:13810–13811 [View Article][PubMed]
LiJ.,
RahmehA.,
MorelliM.,
WhelanS. P.2008; A conserved motif in region V of the large polymerase proteins of nonsegmented negative-sense RNA viruses that is essential for mRNA capping. J Virol 82:775–784 [View Article][PubMed]
LlorenteM. T.,
García-BarrenoB.,
CaleroM.,
CamafeitaE.,
LópezJ. A.,
LonghiS.,
FerrónF.,
VarelaP. F.,
MeleroJ. A.2006; Structural analysis of the human respiratory syncytial virus phosphoprotein: characterization of an alpha-helical domain involved in oligomerization. J Gen Virol 87:159–169 [View Article][PubMed]
LlorenteM. T.,
TaylorI. A.,
López-ViñasE.,
Gomez-PuertasP.,
CalderL. J.,
García-BarrenoB.,
MeleroJ. A.2008; Structural properties of the human respiratory syncytial virus P protein: evidence for an elongated homotetrameric molecule that is the smallest orthologue within the family of paramyxovirus polymerase cofactors. Proteins 72:946–958 [View Article][PubMed]
MartínezI.,
DopazoJ.,
MeleroJ. A.1997; Antigenic structure of the human respiratory syncytial virus G glycoprotein and relevance of hypermutation events for the generation of antigenic variants. J Gen Virol 78:2419–2429 [View Article][PubMed]
MartínezI.,
ValdésO.,
DelfraroA.,
ArbizaJ.,
RussiJ.,
MeleroJ. A.1999; Evolutionary pattern of the G glycoprotein of human respiratory syncytial viruses from antigenic group B: the use of alternative termination codons and lineage diversification. J Gen Virol 80:125–130 [View Article][PubMed]
MathesonJ. W.,
RichF. J.,
CohetC.,
GrimwoodK.,
HuangQ. S.,
PennyD.,
HendyM. D.,
KirmanJ. R.2006; Distinct patterns of evolution between respiratory syncytial virus subgroups A and B from New Zealand isolates collected over thirty-seven years. J Med Virol 78:1354–1364 [View Article][PubMed]
McKennaA.,
HannaM.,
BanksE.,
SivachenkoA.,
CibulskisK.,
KernytskyA.,
GarimellaK.,
AltshulerD.,
GabrielS.,
other authors.
2010; The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297–1303 [View Article][PubMed]
MeleroJ. A.,
García-BarrenoB.,
MartínezI.,
PringleC. R.,
CaneP. A.1997; Antigenic structure, evolution and immunobiology of human respiratory syncytial virus attachment (G) protein. J Gen Virol 78:2411–2418 [View Article][PubMed]
MinkM. A.,
StecD. S.,
CollinsP. L.1991; Nucleotide sequences of the 3′ leader and 5′ trailer regions of human respiratory syncytial virus genomic RNA. Virology 185:615–624 [View Article][PubMed]
NeiM.,
GojoboriT.1986; Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol 3:418–426[PubMed]
NielsenR.,
YangZ.1998; Likelihood models for detecting positively selected amino acid sites and applications to the HIV-1 envelope gene. Genetics 148:929–936[PubMed]
OlmstedR. A.,
MurphyB. R.,
LawrenceL. A.,
ElangoN.,
MossB.,
CollinsP. L.1989; Processing, surface expression, and immunogenicity of carboxy-terminally truncated mutants of G protein of human respiratory syncytial virus. J Virol 63:411–420[PubMed]
OttoT. D.,
SandersM.,
BerrimanM.,
NewboldC.2010; Iterative Correction of Reference Nucleotides (iCORN) using second generation sequencing technology. Bioinformatics 26:1704–1707 [View Article][PubMed]
PalomoC.,
García-BarrenoB.,
PeñasC.,
MeleroJ. A.1991; The G protein of human respiratory syncytial virus: significance of carbohydrate side-chains and the C-terminal end to its antigenicity. J Gen Virol 72:669–675 [View Article][PubMed]
PalomoC.,
CaneP. A.,
MeleroJ. A.2000; Evaluation of the antibody specificities of human convalescent-phase sera against the attachment (G) protein of human respiratory syncytial virus: influence of strain variation and carbohydrate side chains. J Med Virol 60:468–474[PubMed][CrossRef]
ParveenS.,
SullenderW. M.,
FowlerK.,
LefkowitzE. J.,
KapoorS. K.,
BroorS.2006; Genetic variability in the G protein gene of group A and B respiratory syncytial viruses from India. J Clin Microbiol 44:3055–3064 [View Article][PubMed]
PeretT. C.,
HallC. B.,
SchnabelK. C.,
GolubJ. A.,
AndersonL. J.1998; Circulation patterns of genetically distinct group A and B strains of human respiratory syncytial virus in a community. J Gen Virol 79:2221–2229 [View Article][PubMed]
PeretT. C.,
HallC. B.,
HammondG. W.,
PiedraP. A.,
StorchG. A.,
SullenderW. M.,
TsouC.,
AndersonL. J.2000; Circulation patterns of group A and B human respiratory syncytial virus genotypes in 5 communities in North America. J Infect Dis 181:1891–1896 [View Article][PubMed]
Rebuffo-ScheerC.,
BoseM.,
HeJ.,
KhajaS.,
UlatowskiM.,
BeckE. T.,
FanJ.,
KumarS.,
NelsonM. I.,
HenricksonK. J.2011; Whole genome sequencing and evolutionary analysis of human respiratory syncytial virus A and B from Milwaukee, WI 1998-2010. PLoS One 6:e25468 [View Article][PubMed]
RocaA.,
LoscertalesM. P.,
QuintóL.,
Pérez-BreñaP.,
VazN.,
AlonsoP. L.,
SaizJ. C.2001; Genetic variability among group A and B respiratory syncytial viruses in Mozambique: identification of a new cluster of group B isolates. J Gen Virol 82:103–111 [View Article][PubMed]
RuedaP.,
DelgadoT.,
PortelaA.,
MeleroJ. A.,
García-BarrenoB.1991; Premature stop codons in the G glycoprotein of human respiratory syncytial viruses resistant to neutralization by monoclonal antibodies. J Virol 65:3374–3378[PubMed]
RuedaP.,
PalomoC.,
García-BarrenoB.,
MeleroJ. A.1995; The three C-terminal residues of human respiratory syncytial virus G glycoprotein (Long strain) are essential for integrity of multiple epitopes distinguishable by antiidiotypic antibodies. Viral Immunol 8:37–46 [View Article][PubMed]
SalterA.,
LaoiB. N.,
CrowleyB.2011; Molecular epidemiology of human respiratory syncytial virus subgroups A and B identified in adults with hematological malignancy attending an Irish hospital between 2004 and 2009. J Med Virol 83:337–347 [View Article][PubMed]
SpannK. M.,
TranK. C.,
ChiB.,
RabinR. L.,
CollinsP. L.2004; Suppression of the induction of alpha, beta, and lambda interferons by the NS1 and NS2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages [corrected]. J Virol 78:4363–4369 [View Article][PubMed]
StamatakisA.2006; RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690 [View Article][PubMed]
StecD. S.,
HillM. G.III,
CollinsP. L.1991; Sequence analysis of the polymerase L gene of human respiratory syncytial virus and predicted phylogeny of nonsegmented negative-strand viruses. Virology 183:273–287 [View Article][PubMed]
SwansonK. A.,
SettembreE. C.,
ShawC. A.,
DeyA. K.,
RappuoliR.,
MandlC. W.,
DormitzerP. R.,
CarfiA.2011; Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. Proc Natl Acad Sci U S A 108:9619–9624 [View Article][PubMed]
TanL.,
LemeyP.,
HouspieL.,
ViveenM. C.,
JansenN. J.,
van LoonA. M.,
WiertzE.,
van BleekG. M.,
MartinD. P.,
CoenjaertsF. E.2012; Genetic variability among complete human respiratory syncytial virus subgroup A genomes: bridging molecular evolutionary dynamics and epidemiology. PLoS One 7:e51439 [View Article][PubMed]
TolleyK. P.,
MarriottA. C.,
SimpsonA.,
PlowsD. J.,
MatthewsD. A.,
LonghurstS. J.,
EvansJ. E.,
JohnsonJ. L.,
CaneP. A.,
other authors.
1996; Identification of mutations contributing to the reduced virulence of a modified strain of respiratory syncytial virus. Vaccine 14:1637–1646 [View Article][PubMed]
TrentoA.,
GalianoM.,
VidelaC.,
CarballalG.,
García-BarrenoB.,
MeleroJ. A.,
PalomoC.2003; Major changes in the G protein of human respiratory syncytial virus isolates introduced by a duplication of 60 nucleotides. J Gen Virol 84:3115–3120 [View Article][PubMed]
TrentoA.,
CasasI.,
CalderónA.,
García-GarcíaM. L.,
CalvoC.,
Perez-BreñaP.,
MeleroJ. A.2010; 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 84:7500–7512 [View Article][PubMed]
VenterM.,
MadhiS. A.,
TiemessenC. T.,
SchoubB. D.2001; 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 82:2117–2124 [View Article][PubMed]
VignuzziM.,
StoneJ. K.,
ArnoldJ. J.,
CameronC. E.,
AndinoR.2006; Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature 439:344–348 [View Article][PubMed]
WeeseD.,
EmdeA. K.,
RauschT.,
DöringA.,
ReinertK.2009; RazerS - fast read mapping with sensitivity control. Genome Res 19:1646–1654 [View Article][PubMed]
WhiteheadS. S.,
JuhaszK.,
FirestoneC. Y.,
CollinsP. L.,
MurphyB. R.1998; Recombinant respiratory syncytial virus (RSV) bearing a set of mutations from cold-passaged RSV is attenuated in chimpanzees. J Virol 72:4467–4471[PubMed]
YoshidaL. M.,
SuzukiM.,
YamamotoT.,
NguyenH. A.,
NguyenC. D.,
NguyenA. T.,
OishiK.,
VuT. D.,
LeT. H.,
other authors.
2010; Viral pathogens associated with acute respiratory infections in central Vietnamese children. Pediatr Infect Dis J 29:75–77 [View Article][PubMed]
Direct whole-genome deep-sequencing of human respiratory syncytial virus A and B from Vietnamese children identifies distinct patterns of inter- and intra-host evolution