A I131V Substitution in the Fusion Glycoprotein of Human Parainfluenza Virus Type 1 Enhances Syncytium Formation and Virus Growth.

Human parainfluenza virus type 1 (hPIV1) has two spike glycoproteins, the hemagglutinin-neuraminidase (HN) glycoprotein as a receptor-binding protein and the fusion (F) glycoprotein as a membrane-fusion protein. The F glycoprotein mediates both membrane fusion between the virus and cell and membrane fusion between cells, called syncytium formation. Wild-type C35 strain (WT) of hPIV1 shows little syncytium formation of infected cells during virus growth. In the present study, we isolated a variant virus (Vr) from the WT that showed enhanced syncytium formation of infected cells by using our previously established hPIV1 plaque formation assay. Vr formed a larger focus and showed increased virus growth compared with WT. Sequence analysis of the spike glycoprotein genes showed that the Vr had a single amino acid substitution of Ile to Val at position 131 in the fusion peptide region of the F glycoprotein without any substitutions of the HN glycoprotein. The Vr F glycoprotein showed enhanced syncytium formation in F and HN glycoprotein-expressing cells. Additionally, expression of the Vr F glycoprotein increased the focus area of the WT-infected cells. The single amino acid substitution at position 131 in the F glycoprotein of hPIV1 gives hPIV1 abilities to enhance syncytium formation and increase cell-to-cell spread. The present study supports the possibility that hPIV1 acquires increased virus growth in vitro from promotion of direct cell-to-cell transmission by syncytium formation.

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.

Rapid Fluorescent Detection Assay for Human Parainfluenza Viruses.

Human parainfluenza virus type 1 (hPIV1) does not form clear plaque by the conventional plaque formation assay because of slightly a cytopathic effects in many cell lines infected with hPIV1, thus making in virus titration, isolation and inhibitor evaluation difficult. We have succeeded in fluorescent histochemical visualization of sialidase activities of influenza A and B viruses, Newcastle disease virus and Sendai virus by using a novel fluorescent sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac). In this study, we applied the BTP3-Neu5Ac assay for rapid detection of hPIV1 and hPIV type 3. The BTP3-Neu5Ac assay could histochemically visualize dot-blotted hPIVs on a membrane and hPIV-infected cells as local fluorescence under UV irradiation. We succeeded in distinct fluorescent visualization of hPIV1-infected cells in only 3 d using the BTP3-Neu5Ac assay. Due to there being no fixation, hPIV1 was isolated directly from fluorescent stained focus cells by the BTP3-Neu5Ac assay. Establishment of a sensitive, easy, and rapid fluorescent focus detection assay for hPIV, hPIV1 in particular will contribute greatly to progress in hPIV studies.

A novel method for detection of Newcastle disease virus with a fluorescent sialidase substrate.

Newcastle disease virus (NDV), belonging to the family Paramixoviridae, causes respiratory and neuronal symptoms in almost all birds. NDV has haemagglutinin-neuraminidase (HN) glycoprotein possessing sialidase activity. HN glycoprotein is highly expressed on the surface of NDV-infected cells, resulting in much higher sialidase activity in NDV-infected cells than in non-infected cells. It was reported that mouse and human cancer cells up-regulating sialidase expression were histochemically stained with a fluorescent sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac), which deposits water-insoluble fluorescent compound BTP3 on locations of sialidase activity. By using the BTP3-Neu5Ac assay, we showed that NDV-infected cells and HN gene-expressing cells could be simply detected at room temperature after only 5min. Infection of the cells with the virus resulted in apparent green fluorescence, which disappeared with addition of a sialidase inhibitor. Cells that were stained in the BTP3-Neu5Ac assay were immunostained with an anti-NDV antibody. Moreover, BTP3-Neu5Ac staining was applied to a virus overlay binding assay with NDV particles. NDV-bound protein bands on guinea pig red blood cells were easily and rapidly detected by the BTP3-Neu5Ac assay after Western blotting. BTP3-Neu5Ac offers an easy and rapid protocol for fluorescent staining of NDV and virus-infected cells without antibodies.

Histochemical fluorescent staining of Sendai virus-infected cells with a novel sialidase substrate.

Sialidases, enzymes that remove terminal sialic acid residues, are pivotal in various biological processes such as malignancy and infection with pathogens. For histochemical staining of sialidase activity, we have developed a new synthetic sialidase substrate, sialic acid-conjugated fluorescent benzothiazolylphenol derivative (BTP3-Neu5Ac), for rapid, sensitive, and specific fluorescent staining of sialidase activity. Here, we showed the usefulness of BTP3-Neu5Ac for histochemical fluorescent staining of cells infected with Sendai virus (SV), which possesses sialidase activity. BTP3-Neu5Ac also visualised SV-infected regions of lung sections from SV-infected mice. We succeeded in histochemical fluorescent staining of SV both in vitro and in vivo. SV has been utilised in many virological and biotechnological studies such as developments of an oncolytic virus, a gene therapy vector, and a vaccine candidate. BTP3-Neu5Ac should contribute to rapid progress of such studies and researches on viral sialidase.

Imaging of influenza virus sialidase activity in living cells.

Influenza virus is rich in variation and mutations. It would be very convenient for virus detection and isolation to histochemically detect viral infection regardless of variation and mutations. Here, we established a histochemical imaging assay for influenza virus sialidase activity in living cells by using a new fluorescent sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac). The BTP3-Neu5Ac assay histochemically visualized influenza virus-infected cells regardless of viral hosts and subtypes. Influenza virus neuraminidase-expressed cells, viral focus formation, and virus-infected locations in mice lung tissues were easily, rapidly, and sensitively detected by the BTP3-Neu5Ac assay. Histochemical visualization with the BTP3-Neu5Ac assay is extremely useful for detection of influenza viruses without the need for fixation or a specific antibody. This novel assay should greatly improve the efficiency of detection, titration, and isolation of influenza viruses and might contribute to research on viral sialidase.