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A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection
Published on: August 14, 2019
Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells
Tadanobu Takahashi1, Takashi Agarikuchi1, Yuuki Kurebayashi1
1Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka, Japan.
Abstract:
Mumps viruses show diverse cytopathic effects (CPEs) of infected cells and viral plaque formation (no CPE or no plaque formation in some cases) depending on the viral strain, highlighting the difficulty in mumps laboratory studies. In our previous study, a new sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac), was developed for visualization of sialidase activity. BTP3-Neu5Ac can easily and rapidly perform histochemical fluorescent visualization of influenza viruses and virus-infected cells without an antiviral antibody and cell fixation. In the present study, the potential utility of BTP3-Neu5Ac for rapid detection of mumps virus was demonstrated. BTP3-Neu5Ac could visualize dot-blotted mumps virus, virus-infected cells, and plaques (plaques should be called focuses due to staining of infected cells in this study), even if a CPE was not observed. Furthermore, virus cultivation was possible by direct pick-up from a fluorescent focus. In conventional methods, visible appearance of the CPE and focuses often requires more than 6 days after infection, but the new method with BTP3-Neu5Ac clearly visualized infected cells after 2 days and focuses after 4 days. The BTP3-Neu5Ac assay is a precise, easy, and rapid assay for confirmation and titration of mumps virus.
Insights
A new sialidase substrate, BTP3-Neu5Ac, enables rapid fluorescent detection of mumps virus, overcoming challenges posed by variable cytopathic effects. This method significantly shortens detection times compared to conventional techniques.
Area of Science:
- Virology
- Biochemistry
Background:
- Mumps virus detection is challenging due to strain-dependent variations in cytopathic effects (CPE) and plaque formation.
- Conventional methods for mumps virus visualization and titration can be time-consuming, often requiring over 6 days post-infection.
Purpose of the Study:
- To evaluate the utility of a novel sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac), for the rapid detection and titration of mumps virus.
- To demonstrate the effectiveness of BTP3-Neu5Ac in visualizing mumps virus and infected cells, even in the absence of observable CPE.
Main Methods:
- Development and application of the BTP3-Neu5Ac substrate for histochemical fluorescent visualization.
- Testing BTP3-Neu5Ac on dot-blotted mumps virus, infected cells, and viral focuses.
- Assessing the feasibility of virus cultivation from fluorescent focuses detected by BTP3-Neu5Ac.
Main Results:
- BTP3-Neu5Ac successfully visualized mumps virus, infected cells, and focuses, irrespective of CPE presence.
- Fluorescent visualization of infected cells was achieved within 2 days and focuses within 4 days, significantly faster than conventional methods.
- Virus cultivation was successfully performed directly from fluorescent focuses.
Conclusions:
- The BTP3-Neu5Ac assay provides a precise, rapid, and easy method for the confirmation and titration of mumps virus.
- This novel substrate overcomes limitations of traditional mumps virus detection methods, offering a significant advancement in laboratory diagnostics.
