Limits...
Imaging of influenza virus sialidase activity in living cells.

Kurebayashi Y, Takahashi T, Otsubo T, Ikeda K, Takahashi S, Takano M, Agarikuchi T, Sato T, Matsuda Y, Minami A, Kanazawa H, Uchida Y, Saito T, Kawaoka Y, Yamada T, Kawamori F, Thomson R, von Itzstein M, Suzuki T - Sci Rep (2014)

Bottom Line: It would be very convenient for virus detection and isolation to histochemically detect viral infection regardless of variation and mutations.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka 4228526, Japan [2].

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

Histochemical visualization of virus-infected cells by using the BTP3-Neu5Ac assay.(a) MDCK cells were infected with avian influenza A virus strain A/duck/Hong Kong/313/4/1978 (H5N3) at a multiplicity of infection (MOI) of 0.01 to 1. After culture for 12 hr, the infected cells were incubated with 10 μM BTP3-Neu5Ac at 37°C for 10 min. (b) MDCK cells were infected with A/duck/Hong Kong/313/4/1978 (H5N3) and cultured for 12 hr. The infected cells were incubated with 10 μM BTP3-Neu5Ac in the absence or presence of zanamivir at 37°C for 10 min. (c) MDCK cells were infected with A/duck/Hong Kong/313/4/1978 (H5N3) and cultured for 10 hr. The infected cells were fixed with 4% paraformaldehyde-PBS and immunostained with mouse anti-NA monoclonal antibody (red). Then the immunostained cells were incubated with 10 μM BTP3-Neu5Ac at 37°C for 3 min (green). (d) MDCK cells were infected with influenza A virus strains [A/PR/8/1934 (H1N1), A/Memphis/1/1971 (H3N2), and A/Shizuoka/833/2009 (H1N1pdm)], and influenza B virus strain (B/Lee/1940). After culture for 12 hr, the infected cells were incubated with 10 μM BTP3-Neu5Ac at 37°C for 10 min. Scale bars indicate 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4007088&req=5

f3: Histochemical visualization of virus-infected cells by using the BTP3-Neu5Ac assay.(a) MDCK cells were infected with avian influenza A virus strain A/duck/Hong Kong/313/4/1978 (H5N3) at a multiplicity of infection (MOI) of 0.01 to 1. After culture for 12 hr, the infected cells were incubated with 10 μM BTP3-Neu5Ac at 37°C for 10 min. (b) MDCK cells were infected with A/duck/Hong Kong/313/4/1978 (H5N3) and cultured for 12 hr. The infected cells were incubated with 10 μM BTP3-Neu5Ac in the absence or presence of zanamivir at 37°C for 10 min. (c) MDCK cells were infected with A/duck/Hong Kong/313/4/1978 (H5N3) and cultured for 10 hr. The infected cells were fixed with 4% paraformaldehyde-PBS and immunostained with mouse anti-NA monoclonal antibody (red). Then the immunostained cells were incubated with 10 μM BTP3-Neu5Ac at 37°C for 3 min (green). (d) MDCK cells were infected with influenza A virus strains [A/PR/8/1934 (H1N1), A/Memphis/1/1971 (H3N2), and A/Shizuoka/833/2009 (H1N1pdm)], and influenza B virus strain (B/Lee/1940). After culture for 12 hr, the infected cells were incubated with 10 μM BTP3-Neu5Ac at 37°C for 10 min. Scale bars indicate 200 μm.

Mentions: We examined the usefulness of BTP3-Neu5Ac for histochemical visualization of influenza virus-infected cells. Madin-Darby canine kidney (MDCK) cells were infected with avian influenza A virus strain A/duck/Hong Kong/313/4/1978 (H5N3) for 12 hr and incubated with BTP3-Neu5Ac at 37°C for 10 min. The infected cells were fluorescently and histochemically visualized in a virus dose-dependent manner (Fig. 3a). The fluorescence was completely inhibited in the presence of zanamivir (Fig. 3b), indicating that the fluorescence with BTP3-Neu5Ac was dependent on the viral sialidase activity but not on endogenous mammalian sialidase activity. Moreover, a fluorescence image with BTP3-Neu5Ac coincided with an immunohistochemical image with anti-influenza A virus NA monoclonal antibody (Fig. 3c). Sialidase activity of the fixed cells expressing NA remained approximately 66% of that of the non-fixed cells expressing NA (see Supplementary Fig. S1 online). Cells infected with old human influenza A virus strains [A/PR/8/1934 (H1N1) and A/Memphis/1/1971 (H3N2)], a recent clinical strain [A/Shizuoka/833/2009 (H1N1pdm)], and influenza B virus strain (B/Lee/1940) were clearly detected by the BTP3-Neu5Ac assay (Fig. 3d). The results indicate that the BTP3-Neu5Ac assay is applicable to sialidase activities of all influenza viruses, regardless of the host, surface antigens (subtypes), and internal antigens (A and B types). We also obtained high magnification images of intracellular staining of fixed infected cells with BTP3-Neu5Ac by using confocal laser microscopy. In infected cells at 8 hr postinfection, distribution of viral NA proteins coincided with fluorescence of BTP3 (Fig. 4).


Imaging of influenza virus sialidase activity in living cells.

Kurebayashi Y, Takahashi T, Otsubo T, Ikeda K, Takahashi S, Takano M, Agarikuchi T, Sato T, Matsuda Y, Minami A, Kanazawa H, Uchida Y, Saito T, Kawaoka Y, Yamada T, Kawamori F, Thomson R, von Itzstein M, Suzuki T - Sci Rep (2014)

Histochemical visualization of virus-infected cells by using the BTP3-Neu5Ac assay.(a) MDCK cells were infected with avian influenza A virus strain A/duck/Hong Kong/313/4/1978 (H5N3) at a multiplicity of infection (MOI) of 0.01 to 1. After culture for 12 hr, the infected cells were incubated with 10 μM BTP3-Neu5Ac at 37°C for 10 min. (b) MDCK cells were infected with A/duck/Hong Kong/313/4/1978 (H5N3) and cultured for 12 hr. The infected cells were incubated with 10 μM BTP3-Neu5Ac in the absence or presence of zanamivir at 37°C for 10 min. (c) MDCK cells were infected with A/duck/Hong Kong/313/4/1978 (H5N3) and cultured for 10 hr. The infected cells were fixed with 4% paraformaldehyde-PBS and immunostained with mouse anti-NA monoclonal antibody (red). Then the immunostained cells were incubated with 10 μM BTP3-Neu5Ac at 37°C for 3 min (green). (d) MDCK cells were infected with influenza A virus strains [A/PR/8/1934 (H1N1), A/Memphis/1/1971 (H3N2), and A/Shizuoka/833/2009 (H1N1pdm)], and influenza B virus strain (B/Lee/1940). After culture for 12 hr, the infected cells were incubated with 10 μM BTP3-Neu5Ac at 37°C for 10 min. Scale bars indicate 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4007088&req=5

f3: Histochemical visualization of virus-infected cells by using the BTP3-Neu5Ac assay.(a) MDCK cells were infected with avian influenza A virus strain A/duck/Hong Kong/313/4/1978 (H5N3) at a multiplicity of infection (MOI) of 0.01 to 1. After culture for 12 hr, the infected cells were incubated with 10 μM BTP3-Neu5Ac at 37°C for 10 min. (b) MDCK cells were infected with A/duck/Hong Kong/313/4/1978 (H5N3) and cultured for 12 hr. The infected cells were incubated with 10 μM BTP3-Neu5Ac in the absence or presence of zanamivir at 37°C for 10 min. (c) MDCK cells were infected with A/duck/Hong Kong/313/4/1978 (H5N3) and cultured for 10 hr. The infected cells were fixed with 4% paraformaldehyde-PBS and immunostained with mouse anti-NA monoclonal antibody (red). Then the immunostained cells were incubated with 10 μM BTP3-Neu5Ac at 37°C for 3 min (green). (d) MDCK cells were infected with influenza A virus strains [A/PR/8/1934 (H1N1), A/Memphis/1/1971 (H3N2), and A/Shizuoka/833/2009 (H1N1pdm)], and influenza B virus strain (B/Lee/1940). After culture for 12 hr, the infected cells were incubated with 10 μM BTP3-Neu5Ac at 37°C for 10 min. Scale bars indicate 200 μm.
Mentions: We examined the usefulness of BTP3-Neu5Ac for histochemical visualization of influenza virus-infected cells. Madin-Darby canine kidney (MDCK) cells were infected with avian influenza A virus strain A/duck/Hong Kong/313/4/1978 (H5N3) for 12 hr and incubated with BTP3-Neu5Ac at 37°C for 10 min. The infected cells were fluorescently and histochemically visualized in a virus dose-dependent manner (Fig. 3a). The fluorescence was completely inhibited in the presence of zanamivir (Fig. 3b), indicating that the fluorescence with BTP3-Neu5Ac was dependent on the viral sialidase activity but not on endogenous mammalian sialidase activity. Moreover, a fluorescence image with BTP3-Neu5Ac coincided with an immunohistochemical image with anti-influenza A virus NA monoclonal antibody (Fig. 3c). Sialidase activity of the fixed cells expressing NA remained approximately 66% of that of the non-fixed cells expressing NA (see Supplementary Fig. S1 online). Cells infected with old human influenza A virus strains [A/PR/8/1934 (H1N1) and A/Memphis/1/1971 (H3N2)], a recent clinical strain [A/Shizuoka/833/2009 (H1N1pdm)], and influenza B virus strain (B/Lee/1940) were clearly detected by the BTP3-Neu5Ac assay (Fig. 3d). The results indicate that the BTP3-Neu5Ac assay is applicable to sialidase activities of all influenza viruses, regardless of the host, surface antigens (subtypes), and internal antigens (A and B types). We also obtained high magnification images of intracellular staining of fixed infected cells with BTP3-Neu5Ac by using confocal laser microscopy. In infected cells at 8 hr postinfection, distribution of viral NA proteins coincided with fluorescence of BTP3 (Fig. 4).

Bottom Line: It would be very convenient for virus detection and isolation to histochemically detect viral infection regardless of variation and mutations.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka 4228526, Japan [2].

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus