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Original Paper
Nephron 1995;70:449-454
Takako Yamamotoac
Yutaka Nakajimab
Masahiko Yamamotob
Takashi Hironakac
Kanji Hiraic
Yoshiko Nakamuraa
Epstein-Barr Virus Activity in
Patients on Chronic
Hemodialysis
Departments of
a Clinical Pathology and
b Internal Medicine, Showa University
Fujigaoka Hospital, Yokohama, Kanagawa,
c Department of Cell Regulation, Division of
Virology and Immunology, Medical
Research Institute, Tokyo Medical and
Dental University, Tokyo, Japan
Key Words
Abstract
Epstein-Barr virus
Hemodialysis
EBNA-1
EBV DNA
Patients with uremia are susceptible to viral infections, especially to EpsteinBarr virus (EBV). Sixty-one patients with end-stage renal diseases on chronic
hemodialysis (HD), 14 patients with impaired renal function (CRF), and 27
healthy controls were studied with regard to EBV infection. Uremic patients
(HD and CRF) had a significantly higher incidence of EBV infection and high­
er titers of anti-EBV VCA-IgG antibody than healthy controls. The antiEBNA-1 titer was significantly higher in patients whose dialysis period was
more than 3 months than in whom the dialysis period was 3 months or less.
Immunoblotting analysis also showed stronger EBNA-1 signals in hemodialy­
sis patients than EBNA-2, which was strongly detected in the CRF group and
in healthy controls. EBV DNA was detected by Southern blot hybridization
after PCR amplification of peripheral leukocytes, and occurred at a greater
incidence in hemodialysis patients than in the other groups. Taken together,
these results demonstrated that hemodialysis patients had persistent EBV
infection.
Patients in end-stage renal failure are highly suscepti­
ble to infectious diseases and these present profound
problems which are complicated by the immune deficit
found in uremia [1]. Myobacterial [2] and viral infections
[3-5] have an especially high incidence in these patients.
Judging by the incidence of viral infections, patients
usually do not respond adequately to vaccinations [4,6,7]
and the titers of antibodies to some viruses are inversely
correlated to impaired renal function [8], So once uremic
Presented in part at the 40th Japanese Conference on
Virology, Kobe. October 1992.
Accepted:
August 11. 1994
patients are exposed to viral agents, the risk of chronic
and latent infection of resident viruses is increased [5, 9]
and then resulting complications can develop [9],
Epstein-Barr virus (EBV) and cytomegalovirus (CMV)
are members of the Herpesviridae and cause a variety of
diseases, including infection and neoplasms, in humans.
EBV infects lymphocytes causing infectious mononucleo­
sis as a primary infection [10]. The latent state in lympho­
cytes results in chronic infection [11, 12] and EBV is reac­
tivated under immunosuppressive conditions [13, 14].
The patients with chronic EBV infection have VCA-IgM-
Takako Yamamoto. MD
Department of Clinical Pathology
Showa University Fujigaoka Hospital
1-30, Fujigaoka, Aoba-ku
Yokahama 227 (Japan)
© 1995 S. Kargcr AG. Basel
0028-2766/95/0704-0449
S8.00/0
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Introduction
negative, IgG-positive and EBNA-positive sera [ 11,13], It
is reported that EBV is possibly related to collagen dis­
eases including rheumatoid arthritis (RA) [15] and sys­
temic lupus erythematosus (SLE) [16], as there are signifi­
cantly high titers of VCA-IgG and EBNA in some of these
patients. On the other hand, CMV can infect various
types of cells causing systemic infection in an immunodeficient host [17]. CMV also latently infects lymphocytes
and is reactivated under immunosuppressive conditions
[18].
Chronic renal dysfunction results in various complica­
tions as systemic infection. One of these complications,
fo-microglobulin amyloidosis, is not infrequently seen in
such patients especially in those on hemodialysis. It is
known that acute hemodialysis patients have an immunosuppressed condition. In these patients, EBV and CMV
could cause systemic infection. To our knowledge, no
detailed studies of EBV infection in uremic patients have
been reported. We therefore evaluated EBV-related anti­
body and its mode of activity in uremic patients in com­
parison with CMV activity.
sample buffer (60 m.V/ Tris-HCl pH 6.8. 2% SDS. 40% glycerol, 5%
2-mercaptoethanol, and 0.05% bromophenol blue). The proteins
were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel
as described by Laemmli [23]. Electrophoretic transfer of proteins
from gels to a membrane filter (Clear Blot Membrane-P; Atto Co.
Ltd, Tokyo, Japan) and Western blot analysis with 1:100 diluted sera
and horseradish-peroxidase coupled anti-human IgG were per­
formed [24].
Detection o f EBVD N A and CMVDNA by PCR
Genomic DNA was extracted from the heparinized peripheral
blood leukocytes (PBL) [25]. One microliter (500 ng) of sample DNA
was subjected to the PCR reaction, performed on 30 cycles using
0.4 \iM of each primer. For detection of EBV DNA, a set of primers
and a probe were chosen from the 134-bp sequences in the EBV BamHI-W fragment [26]. MIE primers and a probe [27] were also used to
detect CMV DNA sequences. Southern blot hybridization was done
with an alkaline phosphatase-linked oligonucleotide probe as de­
scribed previously [27],
Statistical Analysis
Distribution of anti-EBNA-1 and anti-EBNA-2 antibodies in the
different groups of patients was evaluated by the x2 test. The frequen­
cy of detection of DNA and the antibody titers against the different
antigens in the different groups of patients were compared by Wilcoxon test, p values < 0 .0 5 w ere considered statistically significant.
Materials and Methods
Results
Measurement o f Serum Antibody Titers
Antibody titers to VCA-IgM. -IgG, -IgA (Kayaku-VCA slide;
Wako Pure Chemical Industries, Ltd, Osaka, Japan) [19], EA-IgG,
-IgA (Kayaku-EA slide; Wako) [20] and EBNA-1 (Capy kit TdT; Cos­
mo Bio Co. Ltd, Tokyo, Japan) [21] were determined by indirect
immunofluorescence.
Immunoblot Analysis o f Anti-EBNA-1 and Anli-EBNA-2 IgG
Antibodies in Sera
Nuclear lysates of BJAB. Raji, P3HRI, and B95-8 cells were pre­
pared, essentially as described by Wen et al. [22]. After final centrifu­
gation, the nuclei were lyophilized and suspended in electrophoresis
450
Antibody Titers to EBV Antigens
As shown in table 2, patients with uremia (HD-2,
HD-1, CRF) had increased frequencies of elevated antiVCA-IgG titers (^1:640) compared to healthy controls.
The geometric mean titers (GMT) of anti-VCA-IgG in
these groups were also significantly higher than those in
the healthy control group. The incidence of elevated
anti-VCA-IgA titers (i=l:20) in the HD-2 group was sig­
nificantly higher than that in the healthy control, 32%
compared with 0%, p < 0.01. No anti-VCA-IgM anti­
bodies were detected in any sera (data not shown). The
frequency of elevated anti-EA-IgG antibodies was in­
creased in the uremic groups (HD-1, HD-2 and CRF)
and the HD-1 group especially had a statistically higher
incidence of positive anti-EA-IgG sera (1=1:20) than the
healthy control. No anti-EA-IgA antibody was detected
in any sera, except for 2 in the HD-2 group (data not
shown). The frequency of elevated anti-EBNA-1 anti­
body differed between the hemodialysis groups (HD-1
and HD-2) and the nonhemodialysis groups (CRF and
N). Regarding EBNA-1 titers, GMT in the HD-2 group
was significantly higher than in the other groups. GMT
in the HD-1 group was 2-fold higher than in the CRF
and the N groups.
Yamamoto/Nakajima/Yamamoto/
Hironaka/Hirai/Nakamura
EBV in Chronic Hemodialysis
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Patients
Sixty-one patients with end-stage renal diseases on chronic hemo­
dialysis (HD), 14 patients with impaired renal function (CRF), and
27 healthy controls (N) were studied. The HD group consisted of 31
patients whose dialysis periods exceeded 3 months (HD-2) and 30
patients whose dialysis periods were less than 3 months (HD-1) (ta­
ble 1). Patients were excluded from this study if they had diabetes
mellitus, collagen diseases or infectious conditions, or if they were
receiving corticosteroids.
All hemodialysis patients had arteriovenous fistula; dialysis was
performed 3 times weekly in the HD-2 group and twice weekly in the
HD-1 group. Two types of dialyzer membrane were used; cellulosis
membranes (AM-PC; Asahi Medical Co. Ltd., Tokyo, Japan. TF-a;
Teijin Co. Ltd.. Tokyo. Japan) in the 20 patients in the HD-1 group
and the 16 patients in the HD-2 group, and polymethylmethacrylate
membranes (Toray Co. Ltd., Tokyo, Japan) in the others. None of
them has experience of blood transfusion.
Table 1. Characterization of patients
Group
Number of patients (male)
Age (mean ± SD)
Hemodialysis period, months
(mean ± SD)
Serum creatinine, mg/dl
(mean ± SD)
Underlying diseases
CGN
NSc
Others
HD-2
HD-1
CRF
N
31(19)
52.4 ±18.3
14(6)
30(16)
56.1 ±23.6 42.4 ±10.1
78.5 ±62.7
0.9 ±0.8
13.9 ± 3.8
10.6 ± 2.0
3.5 ±3.6
22
6
3
18
10
2
9
5
0
27(12)
52.8 ±20.6
I.0±0.1
Groups HD-2, HD-1. CRF, and N are defined in the text. CGN = Chronic glomerulo­
nephritis; NSc = nephrosclerosis.
Table 2. Frequencies and levels of anti­
body titers to EBV-specific antigens in sera
from patients
Group
HD-2
HD-1
CRF
N
Patients Antibodies to
31
30
14
27
VCA-IgG
VCA-lgA
EA-lgG
EBNA-1
5640. % GMT
520, %
520,%
5320, % GMT
32*
40**
50**
8
32**
10
14
0
26
47**
29
11
71***
30**.*
8*»
H**
461*
394**'1►
405*
251
786***
237*-***
132***
110***
GMT = Geometric mean titers.
* p < 0.0 1 vs. healthy controls: ** p < 0.01 vs. healthy controls; *** p < 0.001 vs. healthy
controls. ’‘ p < 0.05 vs. HD-2; ** p < 0.02 vs. HD-2; *** p < 0.001 vs. HD-2.
showed no differences in the signal intensity of EBNA-1
and EBNA-2 (fig. 1). No EBNAs were detected in 2 sera of
each nonhemodialysis group (CRF and N).
Detection o f EBV and CMV DNA
EBV-specific DNA sequences were detected at a great­
er incidence in hemodialysis patients (HD-1 and HD-2)
than in nonhemodialysis patients (CRF) and healthy con­
trols (N) by Southern blot hybridization after PCR am­
plification (table 3). For standard assay, EBV DNA could
detect one clone 9 cell containing a single copy of EBV
genome per cell in a background of 1 x 106 EBV-negative
BJAB cells. On the other hand, a high frequency of detec­
tion of CMV DNA was observed only in the HD-1 group.
There were no differences of frequency among the HD-2
group, the CRF group, and healthy controls (N).
451
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Immunoblot Analysis
To analyze the pattern of antibodies to EBNAs in the
sera from hemodialysis patients, Western blot was per­
formed. One major band corresponding to EBNA-1 was
detected in all sera in the hemodialysis groups (HD-1 and
HD-2) which were tested on Raji, B95-8, and P3HRI
cells. These antigens, however, could not be detected on
EBV-negative BJAB cells (fig. 1). The antibody to 90 kD
antigen corresponding to EBNA-2 was detected as a faint
band, only in a few cases in sera from the hemodialysis
patients (HD-2: 1 of 16; HD-1: 3 of 11), but the band for
EBNA-1 was strongly found in all the hemodialysis pa­
tients. In contrast, the anti-EBNA-2 antibody was fre­
quently detected in the sera from the CRF group and the
N group and the antibody for EBNA-1 was detected in 3
and 9 sera from the CRF group and the N group, respec­
tively (fig. 1). The CRF group and the N group also
EBNA-1
EBNA-1
and
EBNA-2
EBNA-2
Samples, n
{-)
HD-2
16
0
16
r*
r*
HD-1
11
0
11
3*
3*
CRF
6
2
3
4
3
N
11
2
9
e
8
*p < 0.05 vs healthy controls (N)
**p < 0.01 vs healthy controls (N)
« CC
«
Standards
:= . in I
m o> m
CL CD a .
CL
°? CL
CL
:= . Î
I
Ic
°9 tr
I
_ _ CL
:= . m
Is
03
05
CO
CC CO C l
—
-E B N A -2
^EBNA-1
*
12
33
19
200 116 97 4 '
66 2 -
Fig. 1. Western immunoblotting detec­
P3HRI
R aji
B JA B
N
Standards
kD
Œ
_
X
CO
•
CL
is
CL CD CL
_____ _
E8NA-2
EBNA-1
CO
£
=. m X
03
05
CL CD
CO CL
I
CO
Cl
-I
-*
S
— __ - EBNA-2
^E B N A -l
_
4 5 .0 -
14
17
19
HD-1
17
19
HD-2
Discussion
EBV
CMV
26/31 ( 8 4 % r
15/15 (100%)++
7/14(50%)
13/26(50%)
15/31 (48%)
14/15(93%)*
3/14(21%)
14/26(54%)
DNAs were extracted from PBL and 1-pl samples o f DNA were
subjected to PCR amplification.
N/N = Number of patients with positive signals/number of
patients examined by Southern blot hybridization using PCR-amplified products.
+ p < 0.05 vs. healthy controls; *+ p < 0.01 vs. healthy controls.
This study demonstrated that most sera from hemodi­
alysis patients showed elevated titers of EBV-specific
antibodies, especially EBNA-1. Elevated anti-VCA-IgG
and anti-EA-IgG titers have been reported in RA [15], in
SLE [16] and in a variety of immunosuppressive condi­
tions [13, 14]. In the case of significantly EBV-reactivated
conditions such as Burkitt lymphoma or nasopharyngeal
carcinoma (NPC), elevated anti-VCA-IgG and anti-EAIgG titers have been shown to be more pronounced and
EBNA titers are also elevated [28]. Anti-VCA-IgA and
anti-EA-IgA can be detected only in patients with NPC
[29, 30] and EBNA titers are low or even undetectable in
Yamamoto/Nakajima/Yamamoto/
H ironaka/ H irai/Naka mura
EBV ir. Chronic Hemodialysis
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Table 3. Detection of EBV and CMV DNA
452
“ cI c
05
•
tion of EBNA-l and EBNA-2 proteins with
sera. 100 jig nuclear lysate of Raji cells and
50 ug nuclear lysate of B95-8 and P3HRI
cells were clectrophoresed on a 10% sodium
dodecyl sulfate-polyacrylamide gel. The electroblotted proteins were detected with 1:100
dilution of human serum. Groups N. CRF,
H D -1, and HD-2 are defined in the text.
HD-2
HD-1
CRF
N
03
13
CRF
the case of EBV reactivation due to congenital or acquired
immunodeficiency [31, 32]. Hemodialysis patients are in
an immunosuppressive condition for long periods. We
found that the pattern of EBV-specific antibodies was dif­
ferent between hemodialysis patients and the others; ele­
vated EBNA-1 titers and VCA-IgA antibody positivity
were observed, particularly in the HD-2 group. The immunoblot analysis also showed the detection of EBNA-1
was more frequent and stronger than that of EBNA-2,
compared to the other groups. In contrast to hemodialysis
patients, it has been reported that high levels of antiEBNA-2 antibody were detected in patients with RA and
SLE [33]. Anti-EBNA-1 antibody usually appeared at a
later phase of EBV infection than anti-EBNA-2 antibody.
It is known that patients with persistent EBV [34] infec­
tion have higher titers of EBNA-1 than of EBNA-2.
Therefore, we considered that hemodialysis patients had
persistent EBV infection. As indicated that control of
EBV activity is dependent on cytotoxic T cells [35], we
suggest that an increase of EBV activity is due to reduced
activated T cells and production of IL-2, in the patients
with high titers of EBNA-1 (unpubl. data).
EBV DNA was frequently detected in ethidium bro­
mide staining and Southern blot hybridization of the PCR
products of peripheral blood DNA in hemodialysis pa­
tients. The frequency of detection of EBV DNA was not
significantly different between the HD-1 and HD-2
groups. With regard to CMV, which is known to be reacti­
vated in immunosuppressive conditions, the frequency of
detection of CMV DNA was increased in the H D-1 group,
but the incidence of CMV DNA detection in the HD-2
group was not greater than that in the N group. It has been
reported that immunological conditions are most sup­
pressive [36] in the HD-1 group, compared with the other
groups (HD-2, CRF, and N). These results suggested that
the activity of EBV is different from that of CMV under
the conditions of hemodialysis treatment and chronic
renal failure and further, that factors other than immuno­
suppression also promote EBV activity.
These findings indicated enhanced activity of EBV in
uremic patients; it is possible that activated EBV might be
related to a variety of complications in patients on long­
term hemodialysis. Further studies are required to exam­
ine whether EBV is involved in immunosuppression or
whether the reactivation of EBV due to immunosuppres­
sion induces a variety of complications.
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