Implementation of a one-step real-time RT-PCR protocol for diagnosis of foot-and-mouth disease

Shaw, Andrew; Reid, Scott M.; Ebert, Katja; Hutchings, G.; Ferris, N.P.; King, Donald P.

doi:10.1016/j.jviromet.2007.02.009

Cited by 146 publications

(137 citation statements)

References 9 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%

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%

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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“…Methylation sequencing results were processed by the QUMA analysis software (http:// quma.cdb.riken.jp), and the real-time PCR results were processed using the 2 -ΔΔCt method (ΔCt = mean CXCR1 expression -mean β-ACTB expression) (Shaw et al, 2007). Data are reported as means ± SD.…”

Section: Data Processing and Analysismentioning

confidence: 99%

Methylation analysis of CXCR1 in mammary gland tissue of cows with mastitis induced by Staphylococcus aureus

Mao¹,

Zhu²,

Li³

et al. 2015

Genet. Mol. Res.

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ABSTRACT. Mastitis is the most important disease in the global dairy industry, and causes large economic losses. Staphylococcus aureus is one of most common pathogens that cause bovine mastitis. CXCR1 has been implicated as a prospective genetic marker for mastitis resistance in dairy cows; CXCR1 expression significantly increases when cows have mastitis. To investigate the mechanisms involved in its increased expression, bisulfite sequencing polymerase chain reaction (PCR) was used to detect the methylation status of CXCR1 CpG island, and quantitative fluorescence PCR was used to detect CXCR1 expression in bovine mammary tissue induced with S. aureus in three Chinese Holstein cows. No CpG island was found for bovine CXCR1 in the upstream 2-kb region, whereas one CpG island that contained 13 CpG sites was found in exon 1 of CXCR1. All of the CpG sites were under hypermethylation from 90 to 100% in the mammary tissues. When the 12607 CXCR1 methylation in cows with mastitis induced by S. aureus ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (4): 12606-12615 (2015) mammary gland mRNA expression of CXCR1 was 12.10-fold higher in infected cow quarters than in uninfected quarters, the methylation levels of the CpG site at position 519 were significantly lower in the infected quarters than in the uninfected quarters. Pearson correlation analysis showed that the methylation level at position 519 was significantly negatively correlated with the CXCR1 mRNA expression level (P < 0.05). These results indicate that the methylation of the CpG site at position 519 may regulate CXCR1 expression in cows with mastitis induced by S. aureus, but further studies are needed to elucidate the mechanisms involved.

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“…The 2 -ΔΔCt method was used to process the real-time PCR results (Shaw et al, 2007). Statistical analyses were carried out using the SPSS 15.0 software (SPSS Inc, USA), and the Student t-test was carried out to determine the significance of differences in the mRNA expression between different groups.…”

Section: Data Processing and Analysismentioning

confidence: 99%

Dynamic changes in TAP1 expression levels in newborn to weaning piglets, and its association with Escherichia coli F18 resistance

Wang¹,

Liu²,

Dong³

et al. 2014

Genet. Mol. Res.

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ABSTRACT. The transporter associated with antigen processing (TAP) transports peptides from the cytosol into the endoplasmic reticulum for subsequent loading onto the major histocompatibility complex (MHC) class I molecules. This study showed the dynamic changes in the TAP1 expression level in newborn to weaning piglets. Tissue expression profiles revealed that the TAP1 gene was expressed at low levels in all tissues, and the expression levels were relatively higher in the lung, spleen, lymph, and thymus; further, no significant difference was observed in the expression in each tissue among the 3 unweaned stages (8, 18, and 30 days). Nevertheless, the postweaning (35 days) expression levels in tissues, including the spleen, lung, lymph, duodenum, and jejunum were significantly higher than those in the unweaned stages. Furthermore, gene ontology and pathway analysis showed that TAP1 took part in 38 biological functions and 5 pathway processes, including ABC transporters and antigen processing and presentation. These analyses showed that the TAP1 gene, which was 3687 ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 13 (2): 3686-3692 (2014) TAP1 gene related to Escherichia coli F18 resistance related to MHC I immune regulation, had a stable and low expression level in unweaned stages; however, its expression increased in the postweaning stages. The high expression level of TAP1 indicated that the gene might play an important role in Escherichia coli F18 resistance.

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Implementation of a one-step real-time RT-PCR protocol for diagnosis of foot-and-mouth disease

Cited by 146 publications

References 9 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

Methylation analysis of CXCR1 in mammary gland tissue of cows with mastitis induced by Staphylococcus aureus

Dynamic changes in TAP1 expression levels in newborn to weaning piglets, and its association with Escherichia coli F18 resistance

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