title: A novel method for detection of Newcastle disease virus with a fluorescent sialidase substrate.
abstract: 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.
authors.affiliation: Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka 4228526, Japan.
authors.forename: Tadanobu
authors.initials: T
authors.lastname: Takahashi
authors.affiliation: Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka 4228526, Japan.
authors.forename: Maiko
authors.initials: M
authors.lastname: Takano
authors.affiliation: Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka 4228526, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Agarikuchi
authors.affiliation: Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka 4228526, Japan.
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Kurebayashi
authors.affiliation: Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka 4228526, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Minami
authors.affiliation: Department of Organic Chemistry, School of Pharmaceutical Sciences, Hiroshima International University, Kure-shi, Hiroshima 7370112, Japan.
authors.forename: Tadamune
authors.initials: T
authors.lastname: Otsubo
authors.affiliation: Department of Organic Chemistry, School of Pharmaceutical Sciences, Hiroshima International University, Kure-shi, Hiroshima 7370112, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ikeda
authors.affiliation: Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka 4228526, Japan. Electronic address: suzukit@u-shizuoka-ken.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D001717:Birds; D005456:Fluorescent Dyes / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods*; D006168:Guinea Pigs; D009439:Neuraminidase / Q000378:metabolism*; D009521:Newcastle Disease / Q000821:virology*; D009522:Newcastle disease virus / Q000201:enzymology* / Q000302:isolation & purification*; D014773:Virology / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D009439:Neuraminidase
keywords: BTP3-Neu5Ac; Fluorescent detection; Haemagglutinin-neuraminidase; Newcastle disease virus; Sialidase activity; Sialidase synthetic substrate