A capillary cytometer method to quantitate viable virus particles based on early detection of viral antigens and cellular events within single cells.
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.
Pheasey
Nigel
N
Department of Biochemical Engineering, University College London, Torrington Place, WC1E 7JE, UK.
John
Justin
J
Love
Colin
C
Coffin
Rob
R
Ward
John M
JM
Boushaba
Rihab
R
Hoare
Mike
M
Levy
M Susana
MS
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
D000914:Antibodies, Viral; D000956:Antigens, Viral