Borna disease virus (BDV) infection in psychiatric patients and healthy controls in Iran

Three hundred and fourteen Iranian subjects, including 114 psychiatric patients, 131 sex and age matched healthy controls, and 69 blood donors were included in this study. The association between BDV infection markers in blood plasma and five DSM IV- categorized psychiatric diseases, as well as gender and age of the individuals were analyzed.

Basic data are given in Table 1. One hundred and fourteen acute psychiatric patients, who had been admitted to local departments of psychiatry in Tehran, were included. All patients met the Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV) -criteria on the basis of interviews and medical records. They could be divided into five main groups and different DSM IV codes, including 64 bipolar disorder (BD)-, 12 major depressive disorder (MDD)-, 18 schizophrenia-, 15 schizoaffective- and 5 obsessive compulsive disorder (OCD) patients (Table 2). Additionally, 69 blood donors and 131 sex- and age matched, mentally healthy subjects (based on the supervision of the psychiatrists) were included and regarded as controls. All individuals were negative for Hepatitis B- and C-viruses, as well as HIV. The study was approved by the Ethic Committee of the Neuroscience Research Center at Shahid Beheshti University of Medical Sciences, and all patients -or an authorized representative- gave their written informed consent for participation. Blood samples of all individuals were collected prior to any medical treatment and plasma or sera were kept at -20°C.

The BDV infection markers, circulating immune complexes (CICs), virus antigens (N- and P- protein, N/P-complexes; abbreviated Ag), and antibodies (Ab), were assayed using the triple enzyme immune assay (EIA) system, as described [25]. According to the double- sandwich format, two BDV-specific monoclonal antibodies (mAbs), anti-N mAb (W₁) and anti-P mAb (Kfu₂), were used to bind any BDV-N- and P-protein or N/P -heterodimers in plasma, either circulating antigen bound to virus- specific host antibodies (CIC- EIA) or free antigen (pAg-EIA). CICs were visualized through alkaline phosphatase (AP)-coupled anti-human IgG and substrate, whereas the Ag-EIA needs a BDV-specific detecting antibody (rabbit hyper-immune serum) followed by AP phosphatase coupled anti-rabbit IgG and substrate. The specificity and sensitivity of the BDV mAbs have been further characterized [34]. In particular, epitope mapping has revealed that both these mAbs are binding to powerful conformational epitopes on either protein, which are formed through 5 binding sites in case of the anti-N mAb (W₁) and 3 binding sites in case of the anti-P mAb (Kfu₂). None of the W1 binding sites are overlapping with P-protein binding domains on the N-protein, confirming that commonly occurring N/P heterodimers are recognized by W_1, as well. In addition, none of either W₁- and Kfu₂- binding sites are overlapping with functionally important sites on N and P-protein, like NLS (nuclear localization signal) and NES (nuclear export signal). The extraordinarily high binding capacities of these mAbs to native N and P proteins have been determined through affinity-chromatography methods using N and P protein from the brain of a horse with Borna disease, resulting in dissociation constants (K_D) of 2.31 × 10^-9 for W1 and 3.33 × 10^-9 for Kfu2. Like for other antigen assays, recombinant proteins have been used to determine sensitivity and further confirm specificity. The detection limit of 1.5-3 ng/ml of purified recombinant N-protein (rN) has been determined for W₁ mAb. Diluting of rN in CIC-, Ag- and Ab- EIA-negative serum did not make any difference, confirming specificity. Additionally, N-protein could be demonstrated in the immune precipitate (IP using W₁) of a strongly antigen positive patient’s plasma by western blot, whereas the IP of an antigen-negative plasma showed nothing but the heavy and light chain of the mAb [34]. Furthermore, using recombinant P-protein, either the non-phosphorylated or phosphorylated form, revealed that mAb Kfu₂ only detects the activated phosphorylated form. Regarding the antibody assay we followed the exact protocol given earlier [25]. All three assays use the basic coating of antibody-stabilized monoclonal antibodies (W₁ and Kfu₂) as a standard immune module [34].

According to the primary experimental setting, a standardized cut off value has been specified as a mean of negative values plus 2 standard deviations, regularly reaching an extinction of < = 0.1 which separates negative and positive scores. The initial dilutions of the samples were 1:20, 1:2, and 1:100 to allow the same cut off value for testing CICs, free Ag, and Ab, respectively [25]. Results were visualized through alkaline phosphatase conjugated antibodies and a colorimetric substrate, absorbance measured in a multichannel photometer (405 nm), and values imported to statistical software [25].

Repetition of one third of the sample collection was performed and essentially gave the same results.

All data of the patients and controls were submitted to parametric and non-parametric statistical analyses. A comparison of the groups was carried out using independent T-Tests, ANOVA and Chi square tests. The prevalence of BDV infection markers was calculated as based on the cut off value of 0.1. Subjects were classified according to clinical diagnostic, gender and age as independent variables, as based on the CIC data measured.

The detailed evaluation of CIC tests were based on standardized scoring of the OD-values of >0.100- 0.300 to be +, >0.300- 0.600 to be ++, > 0.600 - 1.000 to be +++, and > 1.000 to be ++++ [25]. Prevalence and odds ratios (OR) were calculated. Chi square tests were used for an estimation of statistical differences between the groups. Furthermore, binary logistic regression for an estimation of an individual influence of three basic variables, namely age, gender and clinical diagnosis, on CIC titers was applied.

As shown in Table 1, efforts have been made to include gender and age matched control subjects, but comparability could finally not be achieved. The large disparity in both the female-to-male ratios and age of blood donors compared to patients considerably accounted for this limitation (gender: chi square = 7.758, p = 005; age: by t test, p = 0.015). As shown in Table 2, the majority of bipolar patients (BD) were either manic (59.4%) or in a mixed episode (25%), whereas only 15.6% experienced a recent depression. Of all patients, only 19.3% (10 BMD, 12 MDD patients out of 114) presented with a recent depressive episode.

Based on CICs we found a mean prevalence of subclinical infection of 29.5% in the healthy Iranian controls, displaying a slightly higher prevalence in blood donors (33.3%) as compared to the healthy subject cohort (27.5%) for whom any mental illness has been excluded.

Gender and age had no significant influence on CIC prevalence, but psychiatric patients showed significant differences compared to the control group (p = 0.036), presenting with a mean CIC prevalence of 40.4%. Particularly, the patients with bipolar disorder were statistically significantly different with reference to CIC prevalence, OR and OR estimate (OR Est.) (p = 0.014). It is noteworthy that the CIC prevalence found in patients with mood disorders (BD, MDD, and schizoaffective disorders; N = 91) was doubling that of schizophrenia patients (44% vs. 22%), a difference which turned out to be statistically significant (p = 0.026), as well. The statistical evaluations are given in Table 3.

Based on the cut-off value of 0.1 [25] valid for all tests of the triple-EIA system to differentiate the negative from positive results, free Abs were measured in 7.8% and 16.7% of the bipolar (BMD) and schizophrenia patients, respectively, whereas the controls presented with 5.3%. Free Ag was present in 5.6% of the schizophrenic patients (1 out of 18), vs. 1 % in the controls (2 out of 200). Other patient groups were negative in both tests (for details see Table 4). The dynamic balance between CIC formation, antigens, and antibodies accounts for their relative amounts simultaneously present in a sample. The cross-sectional design of the study provides an infection profile only valid at a given time point, thereby limiting the explanatory power of triple-EIA results.

According to the cut off values, as shown in Table 5, dividing all data into a negative and positive group and performing only non-parametric analyses resulted in many data unavailable for statistical inference. Instead, we used quantitative CIC data from the EIA-reading (after subtraction of the OD values for blanks) for parametric statistical analyses.A noticeable increase in CIC levels of both, the bipolar disorder (0.147) and the major depressive disorder (0.163) groups became obvious, being statistically significant when compared to control subjects. The values for 95% CI of CICs are illustrated in Figure 1. The CIC levels within the total population tend to be elevated among females when compared to males (p = 0.089). Therefore, the influence of sex on CIC extinction values was also analyzed in these patient groups (Figure 2). A significant increase in CIC levels in female patients was recognized when compared to males (p = 0.031).