Community acquired respiratory syncytial virus infections : detection by multiplex PCR and strain characterisation by partial G gene sequencing

Stockton, Joanne Dawn (2000). Community acquired respiratory syncytial virus infections : detection by multiplex PCR and strain characterisation by partial G gene sequencing. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000e2e1

Abstract

The aim of this project was to design an assay for the detection of respiratory syncytial virus (RSV) RNA extracted directly from clinical specimens. The assay was intended to address the question of whether RSV is a significant cause of respiratory illness in all age groups of the general community.

The amplification assay for the detection of RSV subtypes A and B was designed using primers located in the nucleocapsid gene. This RSV PCR was incorporated into a multiplex PCR together with primers specific to influenza A H1N1, H3N2 and influenza B, The multiplex assay was optimised and validated, and different amplicon detection methods were investigated with a view to develop a high throughput protocol.

The multiplex PCR assay was then applied to combined nose and throat swabs collected from members of the general community with influenza or influenza like illness, over a three year period (1995-1998). Analysis of these results revealed the co-circulation of RSV and influenza during the winters. RSV was shown to be an important contributor to respiratory illness in all age groups, being detectable in about 20% of patients with influenza like illness.

The RSV positive samples from the three winter seasons studied were processed to obtain sequence data suitable for molecular epidemiological analysis. A strategy to amplify and sequence the first variable region of the glycoprotein gene was developed. PCR amplification was successfully performed directly using stored clinical samples. Phylogenetic analyses of the amplicons revealed that different strain types circulated during each winter season.

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