Assay of Chikungunya virus in cell monolayers by immunofluorescence

Chikungunya virus was quantitatively assayed by counting immunofluorescent foci after infection of BHK21/C13 cell monolayers. The speed and efficiency of virus attachment to cells were markedly enhanced when augmented by centrifugal force. By this procedure, a proportionality was obtained between the number of immunofluorescent foci and the volume of inoculum. Virus penetration into cells was linear and complete within 15 min at 35 C. From observations on the sequential development of viral antigen within cells and immunofluorescent focus counts, foci of infected cells may be enumerated as early as 16 hr after inoculation of cell monolayers. A linear function was demonstrated between immunofluorescent focus counts and relative virus concentration. The immunofluorescent assay was comparable in sensitivity but more precise and rapid than virus assays based on the intracerebral inoculation of suckling mice or on plaque counting. By the immunofluorescent procedure, the 50% neutralizing end point of antiviral serum was rapidly and quantitatively determined.