IMMUNOLOGICAL OVERLAP AND IDENTIFICATION OF GROUP A ARBOVIRUSES BY A GRADIENT PLATE PLAQUE TECHNIQUE

A semi-quantitative, gradient plate, plaque-neutralization technique was developed for studying immunological overlap and identification of Group A arboviruses. Two principles of the technique are 1 a concentration gradient of antibody is provided that continues into a control area without antibody, and 2 neutralization is measured by inhibition of plaque formation in agarcell suspension. The virus sample is streaked over an agar surface with an inoculating loop so that the line of plaques extends from control area into antibody-gradient area to provide an estimate of plaque neutralization overlap. Venezuelan VEE, eastern EEE, western WEE equine encephalitis and Sindbis viruses were studied. Results showed that reciprocal cross-neutralization occurred between EEE, WEE, and Sindbis virus with one exception of a one-way cross. VEE did not cross with any virus except possibly EEE using anti-VEE serum. In a comparison with two quantitative plaque-neutralization methods, the gradient method gave qualitative results similar to the quantitative method in which antiserum was incorporated into the agar. However, it showed cross- neutralization among more viruses than the quantitative method, which depends upon incubation of virus and antibody prior to plating for reduction of plaques.