"TGF-beta and CFS" and "Borna Disease Virus (BDV) and CFS"

INDEX

:Translator's Foreword
:Professor Kazuyoshi Ikuta
1: "TGF-beta and CFS"
2: Paper: "Borna Disease Virus and Chronic Fatigue Syndrome"
3: Result of BDV in CFS in the world report

Translator's Foreword

The following is revised summary of the study of correlation between BDV virus and CFS by Dr. Kazuyoshi Ikuta, at Osaka University, read at the third CFS conference in Tokyo, held in the 26 and the 27, June, 1998.
Translation from Japanese to English was done by Rika Kageyama, a CFS patient (having suffered from it for 22 years since 15 years old.).
If there is any misleading due to my misreading, responsibility lies with the translator, Rika Kageyama. If you find any problems in the following paper, please e-mail the translator directly.

Rika Kageyama

rkageyama@dol.hi-ho.ne.jp

I express, here, my heartfelt appreciation to Professor Kazuyoshi Ikuta and his student, Dr. Takaaki Nakaya who cooperated with me in their extremely busy time. I also want to express special appreciation to Dr. Yo Tomita at School of Music, Queen's University of Belfast who helped me to create this page sharing his very tight time. I also thank Ray Colliton at Co-Cure for giving me the opportunity to share this report on the Internet.

---- Revised Copyright, Rika Kageyama, 2006. Copying is not permitted without author's permission. ----

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"TGF-beta and CFS" "Correlation of Borna Disease Virus in CFS" and "Result of BDV in CFS in the world report"

By Professor Kazuyoshi Ikuta, Ph.D.

Section of Serology, Institute of Immunological Science

Osaka University,

Tel: 06-6879-8307
Fax: 06-6879-8310

URL: http://www.biken.osaka-u.ac.jp/biken/uirusumenneki/Aindex.htm

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1: "TGF-beta and CFS"

Dr. Kazuyoshi Ikuta, Professor at Section of Serology, Institute of Immunological Science, Osaka University, reported about the correlation between TGF-beta and its plasma level in CFS patients, at the third CFS conference in Tokyo, 26 June, 1998.
Based on the report by Nennett et al.,J. Clin. Immunol. 17, 160-166, 1997), his group studied TGF-beta plasma level in CFS patients. (25 CFS patients and 18 controls, comparing the averages of plasma level of TGF-beta)
The result was: In 25 CFS patients, only 6 showed lower peak of plasma TGF-beta level comparing to the highest peak of plasma TGF-beta level in control. Thus, they concluded that there is tendency to have high plasma TGF-beta level in CFS patients.

Dr. Ikuta also studied plasma TGF-beta level in two Japanese CFS family clusters which indicated strong relation with BDV infection. (Regarding the study of the correlation with the Japanese family cluster of CFS and BDV, I will show my translation of their paper, with author's permission, following)
Regarding TGF-beta in the two family members, the members also showed higher plasma TGF-beta level.

So, he pointed out the relationship between immuno-suprression in CFS, it is yet unclear which stared first, immuno-suprression or infection (egg or chicken problem). But, so far, any correlation between plasma level of TGF-beta and EBV antibody and/or BDV infection in CFS patients was not proved. Further study will be needed.

From the 3rd Conference of CFS, Tokyo, Japan: Reported by Rika Kageyama.

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2: Paper: "Borna Disease Virus and Chronic Fatigue Syndrome"

Borna disease virus (BDV) is the causative agent of sporadic progressive encephalitis (Borna disease) in horses. Other hosts for natural BDV infection include cows, sheep, ostriches and cats. In laboratory experiments, BDV can infect a wide variety of animals from birds to mammals, and can be made to cause encephalomyelitis in rats. Rats, in fact, provide the best model for the laboratory study Borna disease. We want to emphasize that BDV infection in humans has been described and BDV antibody has been found in a high percentage of patients with psychiatric disorders, such as schizophrenia and depression. However, BDV antibody has also been found in healthy blood donors, though only in a small percentage. The relationship between BDV and psychiatric diseases is still unclear.
Chronic Fatigue Syndrome (CFS) is an illness which devastates the normal life style of previously healthy people, causing symptoms of prolonged general fatigue, fever, headaches, muscle pain, and various cognitive and memory deficits. The cause of the condition is unknown, and symptoms, unfortunately, generally last for often years. Because epidemics of the condition have been recognized, and because the disease is often heralded by flu-like symptoms (sore throat, fever, respiratory symptoms, etc.), researchers have long suspected an infectious etiology, possibly viral.
Up until now, Coxsackie B virus, Epstein-Barr virus (EBV), Human Herpes virus 6 and 7 (HHV-6,7), Human T-cell Lymphocytic Leukemia virus II (HTLV-II), Spuma virus and Hepatitis virus C have been considered as putative causes of CFS but no clear relationship between these viruses and the disease has yet been established.

We have studied BDV from a serological and molecular epidemiological point of view not only in patients suspected of viral infection but also healthy individuals. In our study, we confirm that a high percentage of patients with psychiatric disease have some relation with BDV. Furthermore, our study suggests a relationship between BDV and some of CFS patients.

I. BDV

Borna Disease Virus (BDV) is a neurotropic envelope virus containing non segmented, negative-, single-stranded RNA with approximately 8,900 bases. BDV codes for at least five proteins; nucleoprotein (p40), phosphoprotein (polymerase cofactor: p24), Matrix (gp18), Envelope protein (gp 56) and polymerase (p180) as shown in Figure 1. An additional open reading frame (ORF) encoding for p10 protein was recently identified.
Because of its similar genome structure, BDV had thought to be similar to the rabies virus or vesicular stomatitis virus in the rhabdovirus family.
However, the shape of BDV is spherical (diameter about 100 nm) and it replicates in the nucleus of the infected cell. Because of these differences from the rhabdovirus family, the International Committee on Taxonomy of Viruses (ICTV) has authorized a new family name, Bornaviridae, for this virus.

II. BDV Etiological study.

1. Serological diagnosis (ELISA and Immunoblotting)

Immunological study of BDV in humans was originally pursued using fluorescent antibodies obtained from BDV infected horse cells. These studies showed differences between patients with schizophrenia or depression and healthy individuals in Europe, America and Africa. The percentage of healthy individuals with positive anti-BDV antibody was measured to be between one to two percent, while in patients with schizophrenia and depression the percentage varied from four to seven percent and as high as 20%. Although the magnitude of the association between schizophrenia/depression and positive anti-BDV titers varies from study to study, it seems clear, nevertheless, that there is such an association. However, the typical antibody titer detected in these studies is quite low (between 1:10 to 1:40), inviting concerns about the specificity and sensitivity of the indirected fluorescent antibody assay.
To improve sensitivity and reproducibility, we have developed each ORF in BDV as fusion protein with glutathione-S-transferase (GST) in E. coli and used the purified GST-fusion proteins as antigens for ELISA and immunoblotting examination. Practical method is following.

Each protein, p40 (nucleoprotein), p24 (polymerase cofactor) and gp18 (matrix) in BDV, as fusion protein with GST, was expressed and purified as antigens for ELISA and immunoblotting examination.
ELISA was performed with following method. First, antigens (5 micro gram/ml) were absorbed on ELISA plate, then the sample (human plasma) with a 100-fold dilution was reacted. Next, as secondary antibody, HRP labelled anti-human IgG antibody (Jackson immuno Research Co) was responded to a 1,000-fold dilution. As a base, O-phenylen diamine was coloured and adsorbed value (OD 492 nm) was measured using micro plate radar U-2000 (Hitachi Co.,).

Immunoblot was performed using the following method. Fusion protein with GST was blotted onto PVDF membrane (Immobilon: Millipore Co.,) after its electrophoresis in 12 and 15 percent of SDS-PAGE, using semi-dry method.
The result of primary and secondary antibody reaction was corresponded with ELISA examination. HRP-1000 (Konica co,.) was used for colouring. ELISA examination has advantage in shortening the time to examine many samples, comparing to immunoblotting examination. However, ELISA has a disadvantage in distinction of specificity. Particularly, exclusion of nonspecific reaction is very important.So, we are trying to develop an indirect sandwich ELISA which is thought to be increased a specificity. This method uses antibody serum (a 100-fold dilution) after adsorbed monoclonal antibody on plates as primary antibody. Indirect sandwich ELISA can also differentiate human sera which equally reacts to both of GST and GST-BDV (nonspecific reaction) or reacts only to GST-BDV (specific reaction). In our preliminary study, the indirect sandwich ELISA has higher advantage on accuracy of excluding these confusing antibodies. As we anticipated, the diagnostic result in the indirect sandwich ELISA method and immunoblotting were almost corresponding.

2. Molecular biological diagnoses (RT-PCR)

Up to the present, in the various kinds of tissues of naturally infected animals by BDV, the high percentage of BDV gene has been found in mainly brain tissues. BDV genes were also found, using RT-PCR examination, in peripheral blood mononuclear cell (PBMC) in rats infected by BDV in laboratory experiment. Additionally, BDV antigen-positive-cells were found in a part of PBMC in human. This provided us to detect BDV gene using RT-PCR examination from blood cells. To improve the percentage of BDV gene, we developed a new method, RT-nested-PCR. As a target gene, we focused on p24 coding region of BDV in PBMC from patients with psychiatric diseases.
(Figure 2).

RT-nested-PCR method has already proven the reliability with its high sensitivity as it can detect single BDV infected cell (MDCK/BDV) among 1,000,000 cells. In the same period (1995), German research group, Dr. Bode et al., has established their finding of BDV gene, using RT-nested-PCR method, focusing on p40 coding region in PBMC from patients with psychiatric diseases. Following will be the practical method of RT-PCR which we performed.

PBMC was separated, using Ficoll-paque (1.077g/ml: Pharmacia Biotech Co.) from obtained whole blood, about 5 ml. We extracted RNA from obtained PBMC, using Isogen (Nippon Gene Co.). For our RT-PCR performance, two sets of methods were used for 1st PCR: cDNA synthesis and 1st PCR reaction were performed using (1) EZ rTth RNA PCR kit (Perkin Elmer Co.) and (2) reverse transcription kit (SuperScript II: Gibco-BRL Co.) and AmpliTaq DNA polymerase kit. For 2nd PCR, AmpliTaq DNA polymerase kit was used in both methods.

Two sets of primers were used for 1st PCR at p24 coding region:
5'-TGACCCAACCAGTAGACCA-3' (1387-1405) as (+) sense primer and
5'-GTCCCATTCATCCGTTGTC-3'(1865-1847) as (-) sense primer. Another two sets of primers were used for 2nd PCR at p24 coding region:
5'-TCAGACCCAGACCAGCGAA-3' (1443-1461) as (+) sense primer and
5'-AGCTGGGGATAAATGCGCG-3' (1834-1816) as (-) sense primer. We followed the manufacture's protocol of PCR reacting conditions. The final PCR products were separated on 1.5 percent agarose gel electrophoresis then blotted onto a Hybond-N+membrane (Amersham Co.).
In Southern hybridisation, four kinds of 32P-labeled synthetic oligonucleotides were used as probes (BDV p24 region: sense nucleotides; (1462-1485, 1485-1507 and 1637-1658) with the antisense nucleotide; (1811-1791)). Reactionary pictures were analysed by BAS1000 (Fuji Film Co.). Obtained PCR product was cloned onto the pCR-TM II vector (Invitrogen Corp., Sand Diego, CA) and was sequenced. As a control, we performed RT-PCR of glyceraldehyde-3-phosphate dehydroegnase (GAPDH; house keeping gene) to check the quality of extracted RNA : 5'-GATGCTGGCGCTGAGTACGTCG-3' (325-346) as (+) sense primer and 5'-GTGGTGCAGGAGGCATTGCTGA-3' (521-500) as (-) sense primer. Finally, we performed hybridisation, using two probes: sense oligonucleotide (371-390) and antisense oligonucleotide (435-416).

III. Epidemiological Examination of BDV in Japanese Patients with Chronic Fatigue Syndrome.

Exploiting the above diagnostic method, we performed molecular biological and serological diagnosis using PBMC and plasma of 25 CFS patients in Japan (15 males and 10 females: age 19-57 years old, with average age of 37.9 years). The patients were diagnosed with CFS in accordance with guidelines established by the Centres for Disease Control and Prevention in the United State of America, established in 1988 and 1994. Result of the diagnosis was shown in table 1. Positive signal of BDV gene was detected in 3 of 25 patients (12%) and anti-BDV antibody was detected in 6 of 25 patients (24%) with serological diagnosis using ELISA and immunoblot. In these patients with positive results, only single patient had positive signals in both gene and antibody. Therefore, we concluded that 8 patients of 25 patients (32 percent) had BDV gene and/or BDV antibody. Retrospectively, considering the low percentage of positive signals in our past study using 100 healthy blood donors, only five percentage of BDV gene and one percentage of BDV antibody, the high percentage of positive BDV in CFS patients in our current study was remarkable. Furthermore, these results were also confirmed in 89 CFS patients (average age of 33 years) who were diagnosed in accordance with the above guidelines during in March 1991 and in April 1995. Generally CFS patients have some of the elevated antibody titers to EBV, Herpes Simplex Virus, Cytomegalo virus, etc, and this is thought to be caused by a nonspecific polyclonal B-lymphocytic response. This report invited us to measure antibody titers of HHV-6 and EBV in our samples.
However, any correlation between the antibody titers between EBV and HHV-6 in CFS patients were not found. Therefore, we concluded that anti-BDV antibody was specifically elevated among these patients with CFS.

IV. BDV Infection in a Family Cluster of Patients with Chronic Fatigue Syndrome.

Here, we would like to introduce a family cluster of patients with CFS infected with BDV. Among 5 family members (father, mother, two sons and one daughter), 4 members except for the elder son developed CFS almost at the same time. It suggested the possibility of this family cluster with some viral infection. Hence, we examined antibody response in each BDV antigen and analysed gene and genotype of BDV in PBMC of the family members by follow up study.
The family structure in our study was following: Father (46 years old), mother (46 years old), elder son (19 years old), younger son (17 years old) and daughter (14 years old). All are current (1997) ages. The father and daughter were diagnosed with CFS according to the Holmes guideline (Centre of Disease Control: CDC 1988). The mother and younger son were diagnosed as lacking several minor criteria, but their symptoms fulfilled the new CDC guidelines in 1994. Only elder son did not have any symptoms of CFS and kept healthy condition. We summarised the condition and its changes in (Figure 2) .

We examined BDV in this family members twice, in April 1995 and in September 1996, using donated blood. Table 3 was the summary of the result.
BDV gene was detected from four family members, except for the elder son (Figure 3). In our first study in 1995, we got the following results: the father and daughter had antibody against three antigens (p40, p24 and gp18). However, the mother and younger son had only anti-p40 and anti-p24 antibody, respectively. The elder son had no antibody against BDV. The result of RT-PCR analysis among this family was following: BDV gene was detected from the father, younger son and daughter in both time points, while the gene was detected in 1995 (first time point) but not in 1996 (second time point) from the mother. PCR products detected in 1995 and 1996 were cloned and analysed their sequence. Based on the sequence, we analysed it using a phylogenetic tree. This analysis showed that the genotype between the mother and father were slightly different and that two children had a part of the genotype from both father's and mother's. The father and daughter, who had elevated titers of three kinds of antibodies (anti p24, p40 and gp18 antibody), were more serious condition at the onset period and their severity has lasted afterward. In contrast, in the mother and younger son, who had only one antibody of p40 and p24, respectively, symptom was less serious and their conditions improved afterward. Therefore, we assumed that elevated anti-BDV antibody titers in father and daughter may be the result of activated replication of virus particle. In addition, mRNA of BDV p24 in PBMC in the two patients having serious condition was detected twice in our two separate studies, in 1995 and 1996. It even proved our above assumption. On the contrary, though BDV gene was detected from PBMC in the mother in our first study, the gene was not found in our second study. This suggested the correlation with severity of CFS condition and BDV: less serious condition in the mother and younger son was thought to be caused by decreased virus antibodies or virus itself in PBMC in these patients.

Unfortunately, because our BDV gene study in patients with CFS was confined on particularly PBMC, it is necessary to pursue further molecule biological study of BDV in other cells and tissues, besides PBMC. More investigation about the location of BDV virus and about the correlation between an amount of BDV (viral load) and a severity of CFS condition are needed.

Conclusion

Many publications have suggested that BDV can infect humans. In 1996, research groups in U.S.A and Germany reported the success of isolation of BDV from PBMC in patients with psychiatric diseases and the entire nucleotide sequence of BDV originated from humans was determined. Moreover, American research group has described the success of detection of BDV gene in autopsied brain tissues from patients with hippocampus sclerosis. On the other hand, though German team has published in 1992 that no correlation was found in their serological examination between CFS and BDV, our molecular biological and serological examinations showed remarkably high positive percentage of BDV in CFS patients. The discrepancy in the results between German group and our study has yet been cleared at this moment and the difference may be caused by sensitivity of methods or by varieties of individual condition among the patients diagnosed with CFS. However, the personal communication from Dr. Dobbin at CDC, Atlanta, in the US, mentioned that almost the same percentage of BDV infection, comparing to our study, was found in CFS patients in Sweden.

Onset of CFS has usually been thought sporadic but outbreaks have also been reported, such as, high respiratory epidemic associated with severe prolonged general fatigue has occurred among teachers in a high school (California) in 1989 and mass onset of CFS-like symptoms in American participants in the gulf war in 1990. In addition, we note that BDV prevalence was higher in patients infected with human immunodeficiency virus (HIV) compared with healthy controls.
Also, BDV RNA was detectable in malignant brain tumors (glioblastoma multiforme) from patients at immunosuppressive state. Considering that some of CFS patients often associates with various immunosuppression and abnormal immune functions, further study is needed to clarify the correlation of BDV and immunosuppression.

Figures and Charts

Figure 1 BDV genome structure.

Figure 2 RT-nested-PCR amplification region.

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p24 gene (phosphorylated protein) was amplified by RT-nested-PCR then hybridized using four 32P-labelled oligonucleotide probes.

Table 2: Summary of signs and symptoms in the family cluster with CFS in this study.

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Seriousness of symptoms is divided into four groups. (-), non; (+), moderate; (++), severe; (+++), very severe. The symptoms in the most severe period were shown on left side, and the severity of symptoms at the second blood collection (September 1996) were shown on right side.

Table 3: Summary of BDV examination in the family cluster of patients with CFS.

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Figure 3: Detection of BDV RNA fragment in peripheral blood mononuclear cells from the family cluster of patients with CFS by RT-nested-PCR.

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A: The PCR products were separated on agarose gel electrophoresis, then stained with ethidium bromide. The results are shown on upper panel. Lower panel shows the results of the Southern blot hybridisation. Extracted RNA from BDV infected cell (MDCK/BDV) and uninfected cell (MDCK) were used as positive and negative control, respectively. B: The internal control, GAPDH mRNA was also amplified in the same RNA samples by RT-PCR. Ethidium bromide (EtBr) staining (upper column) and corresponding Southern blot hybridization profiles are shown. M.W. indicates size markers.

Reference

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2) Narayan O et al: Pathogenesis of Borna disease in rats: immune mediated viral ophtalmoencephalopathy causing blindness and behavioral abnormalities. J Inf Dis 48: 305-315, 1983.

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14) Bode L et al: First isolates of infectious Borna disease virus from patients with mood disorders. Mol Psychiatry 1: 200-212,1996.

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18)Milner I B et al: Is there a Gulf War syndrome? JAMA 271: 661,1994.

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3: Result of BDV in CFS in the world report

Success of virus isolation from PBMC

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