A study of bovine virus diarrhea mucosal disease virus by plaque technique

Abstract

Bovine virus diarrhea-mucosal disease (BVD-MD), NADL, strain formed 3-4 mm plaques on monolayers of bovine embryo kidney (BEK), lung and testicular (BET) cell cultures on post inoculation day four. Plaques were 1.5 mm on the post inoculation day five in lamb testicular cell cultures. Neutral red incorporated in first overlay had inhibitory effect on plaque formation in these cell-virus systems. The study of effects of environmental variables on plaquing efficiency indicated that virus adsorption rate was temperature dependent and approximately 80% virus was adsorbed onto BET monolayers in two hours. Rate of adsorption was slightly superior in BEK monolayers than the ones recorded in BET cell cultures. Virus diluent should contain calcium and magnesium ions for maximum plaquing efficiency. Cultures maintained under lamb serum should be washed for the development of maximum number of plaques. Virus particles could diffuse through agar overlay to initiate infection and form delayed plaques. Size of the plaques was proportional to the concentration of agar in overlay medium. Plaquing efficiency was also dependent upon pH of the overlay and optimum pH for maximum efficiency was 7.3 - 7.7.NADL strain of BVD-MD virus was sensitive to trypsin but resistant to 5'-Bromodeoxyuridine. Thermostability studies showed that 0.5% virus survived when incubated at 37 degrees C for 48 hours. The virus was sensitive to freezing and thawing. Comparative titers of virus determined and expressed as PFU and TCID(50) were almost similar.