A SYBR-green I quantitative real-time reverse transcription-PCR assay for rabies viruses with different virulence

Wang, Lindong; Liu, Ye; Zhang, Shoufeng; Wang, Ying; Zhao, Junqing; Fu-chun, Miao; Hu, Rongliang

doi:10.1007/s12250-014-3378-1

Cited by 5 publications

(2 citation statements)

References 10 publications

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“…A number of real-time RT-PCRs have been designed for specifically detecting individual strains or lineages of RABV 2 and also to detect lyssaviruses across the genus 3,4,5,6,7,8,9 . All assays will have a limit of detection dependent on how conserved the primer (and if necessary, the probe) sequences are across the genus.…”

Section: Introductionmentioning

confidence: 99%

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Marston

Jennings

MacLaren

et al. 2019

JoVE

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Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to confirm rabies diagnosis but have only recently been accepted by the OIE (World Organisation for Animal Health) as a primary method to detect rabies infection. Real-time RT-PCR assays provide real-time data, and are closed-tube systems, minimizing the risk of contamination during setup. DNA intercalating fluorochrome real-time RT-PCR assays do not require expensive probes, minimizing the cost per sample, and when the primers are designed in conserved regions, assays that are specific across virus genera rather than specific to just one virus species are possible. Here we describe a pan-lyssavirus SYBR real-time RT-PCR assay that detects lyssaviruses across the Lyssavirus genus, including the most divergent viruses IKOV, WCBV and LLEBV. In conjunction with dissociation curve analysis, this assay is sensitive and specific, with the advantage of detecting all lyssavirus species. The assay has been adopted in many diagnostic laboratories with quality assured environments, enabling robust, rapid, sensitive diagnosis of animal and human rabies cases.

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Section: Introductionmentioning

confidence: 99%

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Marston

Jennings

MacLaren

et al. 2019

JoVE

View full text Add to dashboard Cite

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“…All reactions were performed in triplicate and analyzed using the 2 −ΔΔ C t method. The sequences of the qRT-PCR primers used were as follows (Table 1) and have been described previously [35,46,47].…”

Section: Methodsmentioning

confidence: 99%

Exosome-Mediated Delivery of Inducible miR-423-5p Enhances Resistance of MRC-5 Cells to Rabies Virus Infection

Wang

Teng

Zhao

et al. 2019

IJMS

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The human diploid cell line Medical Research Council -5 (MRC-5) is commonly utilized for vaccine development. Although a rabies vaccine developed in cultured MRC-5 cells exists, the poor susceptibility of MRC-5 cells to the rabies virus (RABV) infection limits the potential yield of this vaccine. The underlying mechanism of MRC-5 cell resistance to RABV infection remains unknown. In this study, we demonstrate that viral infection increased exosomal release from MRC-5 cells; conversely, blocking exosome release promoted RABV infection in MRC-5 cells. Additionally, RABV infection up-regulated microRNA (miR)-423-5p expression in exosomes, resulting in feedback inhibition of RABV replication by abrogating the inhibitory effect of suppressor of cytokine signaling 3 (SOCS3) on type I interferon (IFN) signaling. Furthermore, intercellular delivery of miR-423-5p by exosomes inhibited RABV replication in MRC-5 cells. We also show that RABV infection increased IFN-β production in MRC-5 cells and that blocking the type I IFN receptor promoted RABV infection. In conclusion, MRC-5 cells were protected from RABV infection by the intercellular delivery of exosomal miR-423-5p and the up-regulation of IFN-β. These findings reveal novel antiviral mechanisms in MRC-5 cells against RABV infection. miR-423-5p, exosomes, and IFN signaling pathways may therefore be potential targets for improving MRC-5 cell-based rabies vaccine production.

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