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

Fluorescent visualization of influenza A virus blotted on a PVDF membrane by using the BTP3-Neu5Ac assay.A PVDF membrane was dot-blotted with 2-fold dilutions (22 to 2−8 HAU) of avian influenza A virus strain A/duck/Hong Kong/313/4/1978 (H5N3). The membrane was incubated with 10 and 100 μM BTP3-Neu5Ac at 37°C for 10 min (a) or 1 hr (b) in the absence or presence of 1 μM zanamivir.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Fluorescent visualization of influenza A virus blotted on a PVDF membrane by using the BTP3-Neu5Ac assay.A PVDF membrane was dot-blotted with 2-fold dilutions (22 to 2−8 HAU) of avian influenza A virus strain A/duck/Hong Kong/313/4/1978 (H5N3). The membrane was incubated with 10 and 100 μM BTP3-Neu5Ac at 37°C for 10 min (a) or 1 hr (b) in the absence or presence of 1 μM zanamivir.

Mentions: To confirm the substrate reactivity of BTP3-Neu5Ac for sialidase activity of influenza A virus NA, we performed fluorescent visualization of influenza A virus blotted on polyvinylidene difluoride (PVDF) membranes with BTP3-Neu5Ac. Virus-blotted membranes were incubated with 10 or 100 μM BTP3-Neu5Ac at 37°C for 10 min or 1 hr. The use of 10 μM and 100 μM BTP3-Neu5Ac for 10 min enabled fluorescent detection up to 2−3 and 2−4 in viral hemagglutination unit (HAU) titer, respectively (Fig. 2a). Also, the use of 10 μM and 100 μM BTP3-Neu5Ac for 1 hr enabled fluorescent detection up to 2−5 and more than 2−8 in viral HAU titer, respectively (Fig. 2b). The fluorescent detection was incubation time- and viral dose-dependent and was completely inhibited in the presence of zanamivir, a specific sialidase inhibitor of influenza A and B viruses (Fig. 2a and 2b). These results indicated that BTP3-Neu5Ac was a suitable substrate for very rapid (within 10 min) and sensitive detection of influenza A virus sialidase activity.


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)

Fluorescent visualization of influenza A virus blotted on a PVDF membrane by using the BTP3-Neu5Ac assay.A PVDF membrane was dot-blotted with 2-fold dilutions (22 to 2−8 HAU) of avian influenza A virus strain A/duck/Hong Kong/313/4/1978 (H5N3). The membrane was incubated with 10 and 100 μM BTP3-Neu5Ac at 37°C for 10 min (a) or 1 hr (b) in the absence or presence of 1 μM zanamivir.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Fluorescent visualization of influenza A virus blotted on a PVDF membrane by using the BTP3-Neu5Ac assay.A PVDF membrane was dot-blotted with 2-fold dilutions (22 to 2−8 HAU) of avian influenza A virus strain A/duck/Hong Kong/313/4/1978 (H5N3). The membrane was incubated with 10 and 100 μM BTP3-Neu5Ac at 37°C for 10 min (a) or 1 hr (b) in the absence or presence of 1 μM zanamivir.
Mentions: To confirm the substrate reactivity of BTP3-Neu5Ac for sialidase activity of influenza A virus NA, we performed fluorescent visualization of influenza A virus blotted on polyvinylidene difluoride (PVDF) membranes with BTP3-Neu5Ac. Virus-blotted membranes were incubated with 10 or 100 μM BTP3-Neu5Ac at 37°C for 10 min or 1 hr. The use of 10 μM and 100 μM BTP3-Neu5Ac for 10 min enabled fluorescent detection up to 2−3 and 2−4 in viral hemagglutination unit (HAU) titer, respectively (Fig. 2a). Also, the use of 10 μM and 100 μM BTP3-Neu5Ac for 1 hr enabled fluorescent detection up to 2−5 and more than 2−8 in viral HAU titer, respectively (Fig. 2b). The fluorescent detection was incubation time- and viral dose-dependent and was completely inhibited in the presence of zanamivir, a specific sialidase inhibitor of influenza A and B viruses (Fig. 2a and 2b). These results indicated that BTP3-Neu5Ac was a suitable substrate for very rapid (within 10 min) and sensitive detection of influenza A virus sialidase activity.

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