Reverse plaque formation method for titration of non-cytopathogenic bovine viral diarrhea-mucosal disease virus

The reverse plaque formation (RPF) method with a semi-micro plate was applied to the titration of a non-cytopathogenic (non-CP) strain of bovine viral diarrhea-mucosal disease (BVD-MD) virus. All the five non-CP strains used in this experiment formed reverse plaques (RP) on bovine testicle cell culture under methyl cellulose overlay. The RPF was inhibited by the pretreatment of a non-CP virus strain with immune rabbit serum to a reference strain. The specificity of the RPF method was demonstrated by the linear test and Poisson distribution test. Comparative titration of commercial BVD-MD vaccines was carried out by the semi-micro RPF method and the tube method based on the exaltation on Newcastle disease virus. The virus titer obtained by the former was slightly higher than that obtained by the latter. The former was proved to be a method of high sensitivity for determining non-CP virus.