Development of a quantitative TaqMan RT-PCR for respiratory syncytial virus

Human respiratory syncytial virus (HRSV) is the main cause of acute lower respiratory infections in children under 2 years of age and causes repeated infections throughout life. We investigated the genetic variability of RSV-A circulating in Ontario during 2010–2011 winter season by sequencing and phylogenetic analysis of the G glycoprotein gene. Among the 201 consecutive RSV isolates studied, RSV-A (55.7%) was more commonly observed than RSV-B (42.3%). 59.8% and 90.1% of RSV-A infections were among children ≤12 months and ≤5 years old, respectively. On phylogenetic analysis of the second hypervariable region of the 112 RSV-A strains, 110 (98.2%) clustered within or adjacent to the NA1 genotype; two isolates were GA5 genotype. Eleven (10%) NA1-related isolates clustered together phylogenetically as a novel RSV-A genotype, named ON1, containing a 72 nucleotide duplication in the C-terminal region of the attachment (G) glycoprotein. The predicted polypeptide is lengthened by 24 amino acids and includes a23 amino acid duplication. Using RNA secondary structural software, a possible mechanism of duplication occurrence was derived. The 23 amino acid ON1 G gene duplication results in a repeat of 7 potential O-glycosylation sites including three O-linked sugar acceptors at residues 270, 275, and 283. Using Phylogenetic Analysis by Maximum Likelihood analysis, a total of 19 positively selected sites were observed among Ontario NA1 isolates; six were found to be codons which reverted to the previous state observed in the prototype RSV-A2 strain. The tendency of codon regression in the G-ectodomain may infer a decreased avidity of antibody to the current circulating strains. Further work is needed to document and further understand the emergence, virulence, pathogenicity and transmissibility of this novel RSV-A genotype with a72 nucleotide G gene duplication.

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“…Sub-grouping was undertaken targeting the nucleocapsid (N) gene using a modified duplex version of a previously published method [21].…”

Section: Methodsmentioning

confidence: 99%

Genetic Variability of Human Respiratory Syncytial Virus A Strains Circulating in Ontario: A Novel Genotype with a 72 Nucleotide G Gene Duplication

Eshaghi¹,

Duvvuri

Lai³

et al. 2012

PLoS ONE

301

417

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“…Different RSV genes have been targeted in these assays, such as the matrix, the polymerase (Kuypers et al, 2004), or the fusion protein genes (Mentel et al, 2003;van Elden et al, 2003), but the N gene is the most widely used due to its high conservation and the number of N sequences available in Genbank. Several assays were designed to subtype RSV, but most were validated more than 10 years ago (Dewhurst-Maridor et al, 2004;Do et al, 2012;Gueudin et al, 2003;Hu et al, 2003). In order to include Table 5 Validation of subtyping efficiency.…”

Section: Discussionmentioning

confidence: 99%

“…Real-time quantitative PCR (RT-qPCR) is the gold standard for both accurate RSV diagnosis and quantification and several assays have been developed during the last decades (Chi et al, 2007;Choudhary et al, 2013;Dewhurst-Maridor et al, 2004;Do et al, 2012;Gueudin et al, 2003;Hu et al, 2003;Kuypers, http://dx.doi.org/10.1016/j.jviromet.2016.05.004 0166-0934/© 2016 Elsevier B.V. All rights reserved. Mentel et al, 2003;van Elden et al, 2003), although these have to be adapted constantly to take into account genetic variations among circulating strains.…”

Section: Introductionmentioning

confidence: 99%

A new real-time RT-qPCR assay for the detection, subtyping and quantification of human respiratory syncytial viruses positive- and negative-sense RNAs

Essaidi-Laziosi

Lyon

Mamin

et al. 2016

Journal of Virological Methods

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a b s t r a c tHuman respiratory syncytial virus (RSV) is a major health problem and the main cause of hospitalization due to bronchiolitis. RSV is divided into two antigenic subgroups, RSV-A and -B that co-circulate worldwide. Rapid and sensitive detection is desirable for proper patient handling while assessment of viral load may help to evaluate disease severity and progression. Finally RSV subtyping is needed to determine the prevalence and pathogenicity of each RSV subgroup, as well as their sensitivity to treatment. In this study, we took into account the most recent circulating RSV variants and designed two quantitative TaqMan one-step RT-PCR assays to detect and quantify both RSV subgroups separately. Standard dilutions of transcripts of positive and negative polarities were included in the assay validation to assess potential differences in sensitivity on negative-sense genomes and positive-sense RNAs. In addition, RSV detection in respiratory specimens of different types and sampled in different populations was compared to commercially available RSV diagnostic tools. Altogether, the RSV-A and -B assays revealed sensitive and quantitative over a wide range of viral loads, with a slight improved sensitivity of the RSV-B assay on positive sense transcripts, and allowed accurate RSV subtyping. We thus provide a useful tool for both RSV diagnostics and research.

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“…RNA genomes in lung macerates were quantitated by the method of Dewhurst‐Maridor et al [] using the primers and probe designed by Maertzdorf et al [] and synthesized by Applied Biosystems, Carlsbad, California). Standard curves were constructed using RNA extracted from a HMPV174 infected cell lysate (infectivity titre 3.5 × 10 6 ffu/ml) arbitrary assumed to contain 10 6 genome units.…”

Section: Methodsmentioning

confidence: 99%

Immunogenicity in mice of human metapneumovirus with a truncated SH glycoprotein

Tedcastle

Fenwick

Robinson

et al. 2013

Journal of Medical Virology

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The SH glycoprotein of human metapneumovirus (HMPV) is twice the size of that of human respiratory syncytial virus and possesses a large, hydrophilic luminal domain. The glycoprotein is located on the surface of the virion and of virus infected cells and, if immunogenic, might be expected to play a role in anti-viral immunity. Initial attempts to study anti-SH antibody immunogenicity were thwarted by the instability of the SH gene on passage both in human bronchial epithelial cells and in mice. Repeated passage of virus isolates in human bronchial epithelial cells in culture resulted in the appearance and eventual predominance of HMPV mutants lacking all or most of the luminal domain of SH coincidental with the loss of productive infection in mouse lungs. Where infection was established in mice with an early cell culture passage, the virus recovered from mouse lung differed markedly from the inoculum, carrying 19 coding mutations in the SH luminal domain. Immunization of mice with a mutant virus variant expressing only 14 amino acids of the luminal domain of SH induced a cross-reactive antibody response to both the F glycoprotein and the SH glycoprotein but a largely sub-group specific response to the G glycoprotein. Similar patterns of response were achieved by immunization with individual HMPV glycoproteins expressed from recombinant vaccinia viruses. Recombinant truncated SH glycoprotein induced sub-group cross-reactive antibodies capable of neutralizing wild-type virus. Recombinant F glycoprotein also induced cross-reactive neutralizing antibodies whilst recombinant G glycoprotein induced largely strain-specific, non-neutralizing antibodies.

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Development of a quantitative TaqMan RT-PCR for respiratory syncytial virus

Cited by 40 publications

References 22 publications

Genetic Variability of Human Respiratory Syncytial Virus A Strains Circulating in Ontario: A Novel Genotype with a 72 Nucleotide G Gene Duplication

Genetic Variability of Human Respiratory Syncytial Virus A Strains Circulating in Ontario: A Novel Genotype with a 72 Nucleotide G Gene Duplication

A new real-time RT-qPCR assay for the detection, subtyping and quantification of human respiratory syncytial viruses positive- and negative-sense RNAs

Immunogenicity in mice of human metapneumovirus with a truncated SH glycoprotein

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