Fig 1.
Fluorescent visualization scheme of mumps virus sialidase activity using BTP3-Neu5Ac.
Sialidase activity of mumps virus releases BTP3 from BTP3-Neu5Ac by hydrolysis of chemical bonding between BTP3 and Neu5Ac. BTP3 is a water-insoluble crystalline fluorescent compound (excitation/emission = 372/526 nm) and deposits on locations of sialidase activity of the virus and infected cells.
Fig 2.
Fluorescent visualization of dot-blotted mumps virus with BTP3-Neu5Ac.
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).
Fig 3.
Fluorescent visualization of mumps virus-infected cells with BTP3-Neu5Ac.
(A) Fluorescent visualization of mumps virus-infected cells. Vero cells were inoculated with mumps virus and then incubated at 37°C for 48 hr. The infected cells were stained with 10 μM BTP3-Neu5Ac at 37°C for 15 min in the absence (left panel) or presence (center panel) of 1 mM DANA, a pan-sialidase inhibitor. (B) Fluorescent visualization of HN-expressing cells. COS-7 cells were transfected with an expression vector containing the HN gene of mumps virus and then incubated at 37°C for 48 hr. The HN-expressing cells were stained with 10 μM BTP3-Neu5Ac at 37°C for 15 min in the absence (left panel) or presence (center panel) of 1 mM DANA. (C) Fluorescent double staining of the infected cells with BTP3-Neu5Ac and anti-mumps virus antibody. Vero cells were inoculated with mumps virus at 37°C for 48 hr. Cells were fixed with 4% paraformaldehyde at room temperature for 10 min. The infected cells were immunostained with rabbit anti-mumps virus antibody and Hilyte PLUS 555-labeled goat anti-rabbit IgG secondary antibody. Then the infected cells were stained with 10 μM BTP3-Neu5Ac at 37°C for 15 min. Scale bars indicate 200 μm.
Fig 4.
Fluorescent visualization of mumps virus focuses with BTP3-Neu5Ac.
(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.