Validation of two real-time RT-PCR methods for foot-and-mouth disease diagnosis: RNA-extraction, matrix effect, uncertainty of measurement and precision

Goris, Nesya; Vandenbussche, Frank; Herr, Cécile; Villers, Jérôme; Stede, Yves Van der; Clercq, Kris De

doi:10.1016/j.jviromet.2009.05.005

Cited by 29 publications

(23 citation statements)

References 24 publications

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“…To confirm FMDV genome synthesis during virus replication, we quantified FMDV genome copies by using a previously described real-time reverse transcription-PCR (RT-PCR) assay (35,36). Briefly, total cellular RNA was extracted from FMDV-infected cells by using TRIzol (Invitrogen, Carlsbad, CA), and real-time PCR amplification was performed on a Stratagene Mx RealTime qPCR system (Agilent Technologies) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…For RNA synthesis, the 3 cell types were incubated with the mutants at an MOI of 0.1 for 1 h in 12-well plates, and the cells were subsequently washed 3 times by using phosphate-buffered saline (PBS) and supplemented with DMEM containing 2% FBS. The infected cell cultures were harvested at different time points (12,24,36, and 48 h) after 3 freeze-thaw cycles. Cell debris was removed by centrifugation, and total cellular RNA was extracted by using TRIzol (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

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Ribavirin-Resistant Variants of Foot-and-Mouth Disease Virus: the Effect of Restricted Quasispecies Diversity on Viral Virulence

Zeng

Wang

Xie

et al. 2014

J Virol

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Mutagenic nucleoside analogues can be used to isolate RNA virus high-fidelity RNA-dependent RNA polymerase (RdRp) variants, the majority of which are attenuated in vivo. However, attenuated foot-and-mouth disease virus (FMDV) high-fidelity RdRp variants have not been isolated, and the correlations between RdRp fidelity and virulence remain unclear. Here, the mutagen ribavirin was used to select a ribavirin-resistant population of FMDV, and 4 amino acid substitutions (D5N, A38V, M194I, and M296V) were identified in the RdRp-coding region of the population. Through single or combined mutagenesis using a reverse genetics system, we generated direct experimental evidence that the rescued D5N, A38V, and DAMM mutants but not the M194I and M296V mutants are high-fidelity RdRp variants. Mutagen resistance assays revealed that the higher replication fidelity was associated with higher-level resistance to ribavirin. In addition, significantly attenuated fitness and virulence phenotypes were observed for the D5N, A38V, and DAMM mutants. Based on a systematic quantitative analysis of fidelity and virulence, we concluded that higher replication fidelity is associated with a more attenuated virus. These data suggest that the resulting restricted quasispecies diversity compromises the adaptability and virulence of an RNA virus population. The modulation of replication fidelity to attenuate virulence may represent a general strategy for the rational design of new types of live, attenuated vaccine strains. IMPORTANCEThe ribavirin-isolated poliovirus (PV) RdRp G64S variant, the polymerases of which were of high replication fidelity, was attenuated in vivo. It has been proposed (M. Vignuzzi, E. Wendt, and R. Andino, Nat. Med. 14:154 -161, http://dx.doi.org/10.1038/nm 1726) that modulation of replication fidelity is a promising approach for engineering attenuated virus vaccines. The subsequently mutagen-isolated RdRp variants also expressed the high-fidelity polymerase, but not all of them were attenuated. Few studies have shown the exact correlation between fidelity and virulence. The present study investigates the effect of restricted quasispecies diversity on viral virulence via several attenuated FMDV high-fidelity RdRp variants. Our findings may aid in the rational design of a new type of vaccine strain.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Ribavirin-Resistant Variants of Foot-and-Mouth Disease Virus: the Effect of Restricted Quasispecies Diversity on Viral Virulence

Zeng

Wang

Xie

et al. 2014

J Virol

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“…A positive FMDV sample was included in each run, as well as a negative water sample. The cutoff value was set at a threshold cycle (Ct) value of 38 [21].…”

Section: Establishment Of Persistent Fmdv Infectionsmentioning

confidence: 99%

Establishment of persistent foot-and-mouth disease virus (FMDV) infection in MDBK cells

et al. 2015

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In addition to acute infection and disease, foot-and-mouth disease virus (FMDV) can cause persistent infection in ruminants. Such "carrier" animals represent a potential risk for FMDV transmission to susceptible animals. However, the mechanisms and the factors that determine FMDV persistence remain unknown. We describe here the establishment of FMDV type O persistent infection in a bovine epithelial cell line (Madin-Darby bovine kidney; MDBK). Preliminary experiments to assess the permissivity of MDBK cells to FMDV O infection revealed an unusual pattern of infection: after the initial phase of acute cell lysis, new monolayers formed within 48-72 h post-infection. We found that some cells survived cytolytic infection and subsequently regrew, thereby demonstrating that this bovine cell line can be persistently infected with FMDV type O. Further evidence that MDBK cells were persistently infected with FMDV includes: (i) detection of viral RNA in cells as well as in cell culture supernatants, (ii) detection of viral antigens in the cells by immunofluorescence analysis, and (iii) production of infectious viral particles for up to 36 cell passages. Furthermore, preliminary sequence analysis of persistent virus revealed a single nucleotide substitution within the VP1 coding region, resulting in the V50A amino acid substitution. This bovine model of FMDV persistence holds promise for the investigation of the viral and cellular molecular determinants that promote FMDV persistence.

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“…Primers and probes were designed to amplify FMDV 3D gene (Callahan et al, 2002), FMDV IRES sequence (Reid et al, 2002) and cellular β-actin gene (Toussaint et al, 2007). The final concentrations of primers and probes were as described by Goris and coworkers (Goris et al 2009) and are indicated in Table 2. In this test all probes were labelled by FAM fluorescent dye at the 5' end and TAMRA quencher dye at the 3' end.…”

Section: Two-step Simplex Rtrt-pcr Pan-fmdvmentioning

confidence: 99%

Establishment and validation of two duplex one-step real-time RT-PCR assays for diagnosis of foot-and-mouth disease

Górna

Relmy

Romey

et al. 2016

Journal of Virological Methods

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Validation of two real-time RT-PCR methods for foot-and-mouth disease diagnosis: RNA-extraction, matrix effect, uncertainty of measurement and precision

Cited by 29 publications

References 24 publications

Ribavirin-Resistant Variants of Foot-and-Mouth Disease Virus: the Effect of Restricted Quasispecies Diversity on Viral Virulence

Ribavirin-Resistant Variants of Foot-and-Mouth Disease Virus: the Effect of Restricted Quasispecies Diversity on Viral Virulence

Establishment of persistent foot-and-mouth disease virus (FMDV) infection in MDBK cells

Establishment and validation of two duplex one-step real-time RT-PCR assays for diagnosis of foot-and-mouth disease

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