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Volume 104, Issue 7

A highly discriminatory RNA strand-specific assay to facilitate analysis of the role of -acting elements in foot-and-mouth disease virus replication Open Access

Abstract

Foot-and-mouth-disease virus (FMDV), the aetiological agent responsible for foot-and-mouth disease (FMD), is a member of the genus within the family . In common with all picornaviruses, replication of the single-stranded positive-sense RNA genome involves synthesis of a negative-sense complementary strand that serves as a template for the synthesis of multiple positive-sense progeny strands. We have previously employed FMDV replicons to examine viral RNA and protein elements essential to replication, but the factors affecting differential strand production remain unknown. Replicon-based systems require transfection of high levels of RNA, which can overload sensitive techniques such as quantitative PCR, preventing discrimination of specific strands. Here, we describe a method in which replicating RNA is labelled with 5-ethynyl uridine. The modified base is then linked to a biotin tag using click chemistry, facilitating purification of newly synthesised viral genomes or anti-genomes from input RNA. This selected RNA can then be amplified by strand-specific quantitative PCR, thus enabling investigation of the consequences of defined mutations on the relative synthesis of negative-sense intermediate and positive-strand progeny RNAs. We apply this new approach to investigate the consequence of mutation of viral -acting replication elements and provide direct evidence for their roles in negative-strand synthesis.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): cre , FMDV , foot-and-mouth disease virus , negative sense , replication and strand-specific assay
Funding
This study was supported by the:
  • BBSRC (Award BB/T015748/1)
    • Principle Award Recipient: NicolaJ Stonehouse
  • BBSRC (Award BB/T015748/1)
    • Principle Award Recipient: DavidR Rowlands
  • bbsrc (Award BB/T015748/1)
    • Principle Award Recipient: MorganR Herod
  • BBSRC (Award BB/T015748/1)
    • Principle Award Recipient: SamuelJ Dobson
© 2023 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.

Erratum

An erratum has been published for this content:
Corrigendum: A highly discriminatory RNA strand-specific assay to facilitate analysis of the role of -acting elements in foot-and-mouth disease virus replication
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A highly discriminatory RNA strand-specific assay to facilitate analysis of the role of cis-acting elements in foot-and-mouth disease virus replication
J. Gen. Virol. 104, 001871 (2023); https://doi.org/10.1099/jgv.0.001871
/content/journal/jgv/10.1099/jgv.0.001871
/content/journal/jgv/10.1099/jgv.0.001871
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