Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells.

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

Fluorescent visualization of dot-blotted mumps virus with BTP3-Neu5Ac.

<p>Mumps virus was dot-blotted onto a PVDF membrane at the indicated HAU. The membrane was incubated with 10 μM BTP3-Neu5Ac in PBS at 37°C for 15 min. Fluorescent bands of dot-blotted viruses on the membrane were visualized under UV irradiation. The membrane was also incubated with 100 μM X-Neu5Ac in PBS at 37°C for 15 min or 24 hr. “Con” indicates no blot of viruses (PBS only).</p

Fluorescent visualization of mumps virus focuses with BTP3-Neu5Ac.

<p>(A) Fluorescent visualization of mumps virus focuses. Vero cells were inoculated with 1 ml/well of mumps virus at the indicated ffu/ml. Cells were overlaid by an overlay medium containing 0.5% agarose. After culture at 37°C for 96 hr, 1 ml/well of 800 μM BTP3-Neu5Ac was dropped onto the overlay medium and incubated at 37°C for 6 hr. Fluorescent focuses were visualized under UV irradiation at 365 nm. (B) Immunostaining with rabbit anti-mumps virus antibody in (A). The cells were fixed with ethanol/acetic acid (v/v = 5: 1) at 4°C overnight, followed by additional fixation with methanol for 30 sec. Populations of infected cells (viral focuses) were immunostained with rabbit anti-mumps virus antibody and HRP-labeled Protein A. No infection was used as a negative control. (C) Virus cultivation by direct pick-up from a fluorescent focus. Two fluorescent focuses in (A) were picked up. A new monolayer of Vero cells was inoculated with respective fluorescent focus suspensions in SFM and cultured at 37°C for 48 hr. Virus replication from each focus was confirmed by immunostaining of the infected cells with rabbit anti-mumps virus antibody.</p