AN IMMUNOPLAQUE ASSAY DISTINGUISHING BETWEEN CYTOPATHOGENIC AND NONCYTOPATHOGENIC BIOTYPES OF BOVINE VIRAL DIARRHEA VIRUS

Cytopathogenic (cp) and noncytopathogenic (ncp) biotypes of bovine virus diarrhoea virus (BVDV) could be clearly distinguished by hollow plaques with peripheral immunostaining in infected fetal calf kidney (FCK) monolayers cultures or the appearance of homogenously stained immunoplaques (IPs), respectively. Of 70 BVDV isolates formerly classified as cytopathogenic according to microscopic observations in FCK cell cultures, 46 produced IPs of both types while the remaining 24 isolates induced only the IP type characteristic of the ncp BVDV biotype. The same was true for 24 additional isolates classified by light microscopy as ncp after ten blind passages in FCK cell cultures. Thus it appeared that in 1/3 of the cases changes in FCK cell cultures had been formerly misinterpreted as BVDV induced cytopathic effects (CPE). Of the 46 isolates which produced both ncp and cp immunoplaques 5 BVDV isolates were selected and successfully subjected to cloning. Clones of the cp biotype were developed by picking native plaques from unfixed FCK cell monolayers. In case of ncp IP, cloning was attempted by picking randomly in the spaces between visible native plaques. Pairs of clones from each of the field isolates proved to consist of pure plaque populations of cp BVDV and ncp BVDV biotypes, respectively, at the dilution of viral suspension which allowed the classification of a single IP according to morphology. Analysis of the clones using 15 monoclonal antibodies against the major viral glycoprotein revealed far-reaching antigenic homogeneity for each pair with only few but distinct exceptions.