A capillary cytometer method to quantitate viable virus particles based on early detection of viral antigens and cellular events within single cells

• Published in: Journal of virological methods 2006-11, Vol.137 (2), p.213-218
• Main Authors: Pheasey, Nigel, John, Justin, Love, Colin, Coffin, Rob, Ward, John M., Boushaba, Rihab, Hoare, Mike, Levy, M. Susana
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Viral; Antigens, Viral - analysis; Biological and medical sciences; Capillary cytometer; Cell Line; Cricetinae; Flow Cytometry - methods; Fundamental and applied biological sciences. Psychology; Herpes simplex virus 1; Herpesvirus 1, Human - growth & development; Herpesvirus 1, Human - immunology; Herpesvirus 1, Human - isolation & purification; Infectivity; Microbiology; Plaque forming units; Statistics as Topic; Techniques used in virology; Viable virus quantitation; Viral Plaque Assay; Virology; Viruses - growth & development; Viruses - isolation & purification
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2006.06.013

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.