Development of a DNA probe for identification of bovine herpesvirus 4

Abstract

Summary A sensitive and specific dna probe for detection and identification of bovine herpesvirus 4 (bhv-4) was developed. Cloned fragments from a library of HindIII fragments of the bhv-4 (DN-599) genome were labeled with 32P or digoxigenin and were tested for sentitivity and specificity in detecting viral dna by dot-blot hybridization. Two probes were identified that detected 10 pg of purified viral dna, and detected viral dna in 0.001 μg of total dna extracted from bhv-4-infected cells. Both probes labeled with 32P and 1 labeled with digoxigenin detected viral dna in samples prepared from cells infected with 2 prototype strains (DN-599 and Movar 33/63) and 4 field isolates of bhv-4. The dna probes did not hybridize to total dna prepared from uninfected bovine cells or from cells infected with bhv-1, bhv-2, alcelaphine herpesvirus 1, pseudorabies virus, or equine herpesvirus 1. One probe, labeled with digoxigenin, was tested further by dot-blot hybridization with infected cell lysates that were simply treated with sodium dodecyl sulfate and proteinase K prior to application to the membrane, avoiding extensive dna purification procedures. This simplified procedure also resulted in specific detection of field isolates of bhv-4 and prototype strains of bhv-4.