A novel assay using 5-ethynyl uridine labeling and click chemistry successfully isolated nascent FMDV RNA, demonstrating that the viral cis-acting replication element is essential for negative-strand synthesis.
A novel strand-specific qPCR assay using 5-EU labeling successfully discriminates newly synthesized FMDV RNA from input RNA, revealing that the cis-acting replication element (cre) is essential for negative-strand synthesis.
A corrigendum of this article has been published full details can be found at https://doi.org/10.1099/jgv.0.001993 Foot-and-mouth-disease virus (FMDV), the aetiological agent responsible for foot-and-mouth disease (FMD), is a member of the genus Aphthovirus within the family Picornavirus . 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 in vivo 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 cis -acting replication elements and provide direct evidence for their roles in negative-strand synthesis.
Dobson et al. (Wed,) conducted a other in Foot-and-mouth disease virus (FMDV) replication. 5-ethynyl uridine (5-EU) labeling and click chemistry vs. Standard replicon transfection without nascent RNA selection was evaluated on Detection of nascent positive and negative strand viral RNA. A novel assay using 5-ethynyl uridine labeling and click chemistry successfully isolated nascent FMDV RNA, demonstrating that the viral cis-acting replication element is essential for negative-strand synthesis.