Immunological and PCR analyses for Borna disease virus in psychiatric patients and blood donors in Japan

Abstract

The involvement of Borna disease virus (BDV) in psychiatric diseases in humans remains controversial. T-cell memory response and seroprevalence of BDV in patients with psychiatric disorders and blood donors in Japan were evaluated collectively by Western blot (WB) analysis with inhibition test, electrochemiluminescence immunoassay, immunofluorescence assay, and T-cell proliferative response as well as detection of BDV p24 RNA in peripheral blood mononuclear cells (PBMCs). Positive proliferative responses to both BDV p40 and p24 proteins were detected in 9% of patients with mood disorders (4 of 45), 4% of schizophrenic patients (2 of 45), and 2% of blood donors (1 of 45). By WB analysis, the antibody to BDV p40 was detected only in 2% of patients with mood disorders (1 of 45). The BDV p24 antibody was detected in 2% of patients with mood disorders (1 of 45) and 9% of schizophrenic patients. (4 of 45) No plasma reacted with both BDV proteins. The finding of a lower seroprevalence than previously reported suggests the presence of false-positive cases in the previous report. BDV RNA was detected only in 2% of patients with mood disorders (1 of 45). In these three serological assays, T-cell responses, and PCR analysis, there was no significant difference in the prevalence among the three groups. However, we found three psychiatric patients who were positive for both BDV antibodies and T-cell proliferative responses and one patient who was positive for BDV RNA in PBMCs. These findings suggest the usefulness of the proliferative T-cell response and that certain individuals are infected with BDV or a BDV-related virus.

FIG. 1

Purification of BDV p40 and p24 proteins. BDV proteins expressed as GST fusion proteins were purified by cleavage from GST by factor Xa and Mono Q column chromatography. (A) SDS-PAGE of purified BDV proteins. BDV p40 protein was electrophoresed with the expected mobility, 40 kDa (lane 1). Purified BDV p24 protein exhibited three major bands (22, 20, and 17.5 kDa), and two minor bands (21 and 19 kDa) (lane 2). (B) Specificity of BDV proteins. Lanes 1 and 2, immunoreactivities of monospecific rabbit anti-BDV p40 serum and normal rabbit serum (1:16,000 and 1:50, respectively) to BDV p40 protein. Lanes 3 and 4, immunoreactivities of monospecific rabbit anti-BDV p24 serum and normal rabbit serum (1:4,000 and 1:50, respectively) to BDV p24 protein. The electrophoretic mobilities and size (in kilodaltons) of molecular markers are indicated on the left. The positions of p40 and p24 are indicated by arrowheads.

FIG. 2

Sensitivity and specificity of Western blot analysis. (A) Titration of rabbit anti-BDV p40 (a) and p24 (b) antibodies. (a) Rabbit anti-BDV p40 antibody was serially diluted twofold from 1:2,000 to 1:64,000 (lanes 1 to 6). (b) Rabbit anti-BDV p24 antibody was serially diluted twofold from 1:500 to 1:16,000 (lanes 1 to 6). Lanes 7 illustrate immunoreactivity of normal rabbit serum (1:50). (B) Specificity of rabbit anti-BDV p40 (a) and p24 (b) antibodies. (a) Lane 1, immunoreactivity of monospecific rabbit anti-BDV p40 antibody to BDV p40 protein. Specificity of the immunoreactivity of rabbit anti-BDV p40 antibodies was demonstrated by inhibiting the reaction with 20 μg of BDV p40 (lane 2) and GST (lane 3) protein per ml. (b) Lane 1, immunoreactivity of monospecific rabbit anti-BDV p24 antibody to BDV p24 protein. Specificity of the immunoreactivity of rabbit anti-BDV p24 antibodies was demonstrated by inhibiting the reaction with 20 μg of BDV p24 (lane 2) and GST (lane 3) protein per ml. The electrophoretic mobilities and sizes (in Kilodaltons) of molecular markers are indicated on the left. Arrowheads indicate the positions of BDV p40 and p24.

FIG. 3

Detection of antibodies to BDV proteins in human plasma. Recombinant BDV p40 and p24 proteins were separated by SDS-PAGE and analyzed by Western blot analysis. (A) Screening test for antibodies to BDV p40 (a) and p24 (b) in human sera. Lanes 1 to 20, immunoreactivities of 18 representative human plasma samples (lanes 1 to 9 and 11 to 19), rabbit anti-p40 antibody (lane 10), and rabbit anti-p24 antibody (lane 20). (B) Specificity of human plasma to BDV p40 (left panel) and p24 (right panel) proteins was demonstrated by inhibition tests. M10 and M43 show the reactions of two positive plasma samples, and M2 and C10 show the reactions of two negative plasma samples. Lanes a, immunoreactivity of human plasma without preincubation with p40 or p24 protein. Lanes b and c, immunoreactivity of human plasma preincubated with 20 μg of p40 or p24 (lanes b) and GST (lanes c) proteins, per ml. The electrophoretic mobilities and sizes (in kilodaltons) of molecular markers are indicated on the left. Arrowheads indicate the positions of p40 and p24.

FIG. 4

Detection of antibodies to BDV in patient M10 with mood disorders by IF. The patient plasma at a dilution of 1:160 was applied on acetone-fixed BVD-infected MDCK cells (A) and MDCK cells (B). Note the dot-like staining in the nuclei-of BDV-infected MDCK cells but not in MDCK cells.