title: A capillary cytometer method to quantitate viable virus particles based on early detection of viral antigens and cellular events within single cells.
abstract: The traditional plaque forming and TCID(50) methods to determine replication competent virus titres rely on several cycles of replication and infection to generate a plaque with an incubation period of 24-72 h post-infection typically required. We developed a method to quantify infective viral particles based on early detection of cellular events by capillary cytometry. The method uses a capillary cytometer as a precise cell counter that can discriminate infected from non-infected cells. The general protocol was developed using a Guava PCA, genetically modified HSV-1 virus and polyclonal antibodies against antigens expressed on the cell membrane. Infection was detected after 1 h incubation and a plateau in the number of infected cells was observed between 7 and 9 h. A good correlation between titres obtained by the plaque forming method and the proposed method was observed for a ratio of infected to total cells between 0.5 and 0.05. The rapid and automated analysis (10 s/1000 events acquired per sample) makes the method particularly useful for high-throughput applications. The proposed method can be extended easily to determine the titre of other viruses providing a powerful tool for virology and antiviral screening.
authors.affiliation: Department of Biochemical Engineering, University College London, Torrington Place, WC1E 7JE, UK.
authors.forename: Nigel
authors.initials: N
authors.lastname: Pheasey
authors.forename: Justin
authors.initials: J
authors.lastname: John
authors.forename: Colin
authors.initials: C
authors.lastname: Love
authors.forename: Rob
authors.initials: R
authors.lastname: Coffin
authors.forename: John M
authors.initials: JM
authors.lastname: Ward
authors.forename: Rihab
authors.initials: R
authors.lastname: Boushaba
authors.forename: Mike
authors.initials: M
authors.lastname: Hoare
authors.forename: M Susana
authors.initials: MS
authors.lastname: Levy
mesh_terms: D000818:Animals; D000914:Antibodies, Viral; D000956:Antigens, Viral / Q000032:analysis*; D002460:Cell Line; D006224:Cricetinae; D005434:Flow Cytometry / Q000379:methods*; D018259:Herpesvirus 1, Human / Q000254:growth & development* / Q000276:immunology / Q000302:isolation & purification; D013223:Statistics as Topic; D010948:Viral Plaque Assay; D014780:Viruses / Q000254:growth & development* / Q000302:isolation & purification
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral