: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
The following is revised summary of the study of correlation between BDV virus
and CFS by Dr. Kazuyoshi Ikuta, at
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
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
---- Revised Copyright, Rika Kageyama, 2006. Copying is not permitted without author's permission. ----
Currently Professor Emeritus
Section of
Serology, Institute of Immunological Science
Tel: 06-6879-8307
Fax: 06-6879-8310
URL: http://virology.biken.osaka-u.ac.jp/
Dr. Kazuyoshi Ikuta, Professor at Section of
Serology,
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,
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.
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.
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,
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 (
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.
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).
Exploiting the above diagnostic method, we performed
molecular biological and serological diagnosis using PBMC and plasma of 25 CFS
patients in
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.
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.
Many publications have suggested that BDV can infect
humans. In 1996, research groups in U.S.A and
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.
Figure 1 BDV genome structure.
Figure 2 RT-nested-PCR amplification region.
<picture>
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.
<picture>
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.
<picture>
Figure 3: Detection of BDV RNA fragment in peripheral blood mononuclear cells from the family cluster of patients with CFS by RT-nested-PCR.
<picture>
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.
1) de la Torre J C: Molecular biology of Borna disease virus: prototype of a new group of animal viruses. J Virol 68: 7669-7675, 1994.
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.
3) Rott R et al: Detection of serum antibodies to Borna disease virus in patients with psychiatric disorders. Science 228: 755-756, 1985.
4) Bode L et al: Borna disease virus-specific antibodies in patients with HIV infection and with mental disorders. Lancet ii: 689, 1988.
5) Buchwald D, Komaroff A L: Review of laboratory findings in patients with chronic fatigue syndrome. Rev Inf Dis 13: S12-8,1991.
6) Komaroff A L, Buchwald D:Symptoms and signs in chronic fatigue syndrome. Rev Inf Dis 13: S8-11, 1991.
7) Levy J A: Viral studies of chronic fatigue syndrome (Review). Clin Inf Dis 18 (Suppl 1): S117, 1994.
8) Kishi M et al: Demonstration of human Borna disease virus RNA in human peripheral blood mononuclear cells. FEBS Lett 364: 293-297, 1995.
10) Bode L et al: Borna disease virus genome transcribed and expressed in psychiatric patients. Nature Med 11: 232-236,1995.
11) Holmes G P et al:Chronic fatigue syndrome: a working case definition. Ann Int Med 108: 387-389, 1988.
12) Fukuda K et al: The Chronic Fatigue Syndrome: a comprehensive approach to its definition and study. Ann Int Med 121: 953-959, 1994.
13) Kitani T et al: Possible correlation between Borna disease virus infection and Japanese patients with chronic fatigue syndrome. Microbiol Immunol 40: 459- 462, 1996.
14) Bode L et al: First isolates of infectious Borna disease virus from patients with mood disorders. Mol Psychiatry 1: 200-212,1996.
15) de la Torre J C et al: Sequence characterization of human Borna disease virus. Virus Res 44: 33-44, 1996.
16) Bode L et al: No serologic evidence of Borna disease virus in patients with chronic fatigue syndrome. Clin Inf Dis 15:1049, 1992.
17) Nakaya T et al: Demonstration of Borna disease virus RNA in peripheral blood mononuclear cells derived from Japanese patients with chronic fatigue syndrome. FEBS Lett 378:145-149,1996.
18)Milner I B et al: Is there a Gulf War syndrome? JAMA 271: 661,1994.
19) Auwanit W et al: Unusually high seroprevalence of
Borna disease virus in clade E human immunodeficiency virus type 1-infected
patients with sexually transmitted disease in
20) Nakaya T et al: Expression of Borna disease virus
in clinical samples from patients with brain malignant tumors. Proc
21) Landay A L et al: Chronic fatigue syndrome: Clinical condition associated with immune activation. Lancet 338: 707-712, 1991.