Borna disease virus (BDV) is the prototype of a new virus family, Bornaviridae, within the Mononegavirales order, which has a nonsegmented, negative-sense, single-stranded RNA genome (12,42). BDV is a noncytolytic neurotropic virus that infects a wide variety of animal species from birds to primates (7,17,26,36,51,52) and causes Borna disease (BD), which is characterized by central nervous system dysfunction with variable manifestations ranging from fatal neuronal damage to almost asymptomatic viral persistence (19,25,28,29). The wide host range of the virus and behavioral disturbances in animals with BD have suggested that BDV infection may be associated with human psychiatric disorders (5,8,9,15,23,33,40,48,49,53). Furthermore, seroepidemiological data and the detection of BDV RNA in peripheral blood mononuclear cells (PBMCs) by nested reverse transcriptase PCR (RT-PCR) have also suggested a possible involvement of BDV in human psychiatric disorders. However, there is controversy over the prevalence of BDV antibodies and BDV RNA in the PBMCs of patients with psychiatric disorders.In previous serological studies, the prevalence of anti-BDV antibodies in psychiatric patients varied from 0 to 30% in different laboratories. The differences in prevalence could be due to the use of different assay systems (immunofluorescence [IF] assay [4,5,37,38], Western blot [WB] analysis [8,14,15,21,23,24,40,47,50], enzyme-linked immunosorbent assay [14,20,24], and electrochemiluminescence immunoassay [ECLIA] [53]) with different sensitivities and specificities. Particularly, in most of the previous reports that examined BDV antibodies by WB analysis, the specificity for BDV has not been considered, giving rise to the possibility of false-positive results. One of the aims of the present study was to establish a WB analysis with specificity for the detection of BDV antibodies in human sera and to reevaluate the prevalence of BDV antibodies. Furthermore, we also tried to detect anti-BDV antibodies by ECLIA and IF assay to make elaborate inquires into the prevalence of anti-BDV antibodies. To our knowledge, this is the first report on the evaluation of the prevalence of anti-BDV antibodies examined by three different methods.Experimental animal models of BD demonstrated that BD is caused by T-cell-mediated immunopathology in the brain (1a, 2, 3, 16, 28, 31, 32, 35, 41, 43-45). The evidence has suggested that BDV-specific CD4 ϩ and CD8 ϩ T cells in the brain play an