Herpes B virus DNA was specifically amplified by PCR, targeting the regions that did not cross-react with herpes simplex virus (HSV). The amplified products, which were shown to be highly genetic polymorphisms among herpes B virus isolates, were identified by microplate hybridization with probes generated by PCR. The products immobilized in microplate wells were hybridized with the biotin-labeled probes derived from the SMHV strain of herpes B virus. The amplified products derived from the SMHV and E2490 strains of herpes B virus were identified by microplate hybridization. PCR products amplified from the trigeminal ganglia of seropositive cynomolgus macaques were identified as herpes B virus DNA. The utility of the PCR-microplate hybridization assay for genetic detection and identification of the polymorphic region of herpes B virus was determined.Herpes B virus infection, caused by herpes B virus harbored in its natural host, Asian macaques, is a fatal encephalomyelitis in humans (6,23,26,37). The primary means of transmission of herpes B virus to humans has been suggested to occur by direct and/or indirect contact with virus-contaminated secretion from the natural host (38). Forty-three cases of herpes B virus infection have been reported since the first reports by Sabin (7,26). The infection has a high mortality if not treated by antiviral therapy in the early stages of infection. Thus, development of an assay is essential for prevention and early diagnosis of herpes B virus infection.Herpes B virus is an alphaherpesvirus that shares some characteristics with the herpes simplex virus (HSV) (9). Both of the neurotropic viruses establish latency in the sensory nerve ganglia of their natural hosts (2, 34). Stress induces reactivation of the viruses from the latent state, resulting in shedding of infectious viruses from mucosal tissue (14,35). In addition, herpes B virus shows strong serological cross-reactivity with HSV (5, 18). The genetic arrangement is almost identical between herpes B virus and HSV (8,21,22,25), and the nucleotide sequences of the herpes B virus genes have been reported to show high homology with the corresponding HSV genes (1,21,22,25). These similarities suggest that a sample from a patient with herpes B virus infection also contains HSV and that misidentification occurs in a diagnosis of the infection. In addition, HSV type 1 (HSV-1) was reported to be isolated and detected from a pet monkey and from white-faced monkeys with fatal infection, suggesting that a macaque, a natural host of herpes B virus, is infected with HSV (13, 30). Thus, accurate diagnosis of herpes B virus infection in both human and the natural herpes B virus host requires a specific assay to distinguish herpes B virus from the closely related HSV.PCR was considered suitable for specific detection of herpes B virus. PCR was designed to amplify the herpes B virus target sequence, which is the most divergent in the genome sequence among HSV-1, HSV-2, and herpes B virus (4, 25). Furthermore, the amplified region was shown...