Full text of DOI:10.1111/j.1348-0421.2002.tb02683.x

Microbiol. Immunol., 46(3), 177―180, 2002
Detection of Herpesvirus DNA in the Serum of
Immunocompetent Children
,
1
1
1
1
2
Shinya Hara* , Hiroshi Kimura , Yo Hoshino , Naoko Tanaka , Kazuo Nishikawa ,
3
4
5
Masaru Ihira , Tetsushi Yoshikawa , and Tsuneo Morishima
1
5
Department of Pediatrics, Department of Health Science, Nagoya University School of Medicine, Nagoya, Aichi 461
―
8673,
4
Japan, Laboratory of Virology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medi-
cine, Nagoya, Aichi 466―8550, Japan, Department of Pediatrics, Ekisaikai General Hospital, Nagoya, Aichi 454―8052, Japan,
and Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi 470―1192, Japan
2
3
Received November 13, 2001. Accepted December 25, 2001
Abstract: The DNA of herpesviruses such as Epstein-Barr virus (EBV), cytomegalovirus (CMV), human her-
pesvirus-6 (HHV6), and human herpesvirus-7 (HHV7) has been detected in the serum of patients with pri-
mary infection or with immunosuppression. However, it is unknown how frequently herpesvirus DNA can
be detected in the serum of immunocompetent children, or whether the detection of herpesvirus DNA indi-
cates an active infection or virus-related diseases. Using a real-time polymerase chain reaction assay, attempts
were made to detect herpesvirus DNA in the serum of 176 ambulatory children who visited a hospital for
various reasons. EBV was detected in 4 (2.2%), HHV6 in 4 (2.2%), and HHV7 in 2 (1.1%) of 176 children,
but CMV was not detected. Of the 10 positive patients, only 4 were considered, by virtue of clinical and sero-
logical characteristics, to have primary infections. The other 4 positive patients had other infections,
such as mycoplasma and salmonella. Although herpesvirus DNA could be detected in the serum of
immunocompetent children, there was not always a relationship between clinical manifestations and the
detection of virus DNA. When herpesvirus DNA is detected in the serum, a careful interpretation is necessary
to diagnose a primary infection or a virus-associated disease.
Key words: Herpesviruses, Serum, Real-time polymerase chain reaction assay
Presently, 8 herpesviruses are known to be associated
with human disease. They are classified into three sub-
families: herpes simplex virus type 1, herpes simplex
virus type 2 and varicella-zoster virus are α her-
pesviruses. Cytomegalovirus (CMV), human her-
pesvirus-6 (HHV6), and human herpesvirus-7 (HHV7)
are β herpesviruses. Epstein-Barr virus (EBV) and
human herpesvirus-8 are γ herpesviruses. Primary infec-
tion with these viruses occurs mostly in childhood, and
latent infection persists in the host for life. It has been
reported that some herpesvirus can be detected in the
serum of patients who either have an active virus infec-
tion or are affected by an immunosuppressive condi-
tion, such as AIDS or the sequelae of organ or stem
cell transplantation (2, 3, 5, 7). Specifically, latent β and
γ herpesviruses in blood cells are detectable in blood
from patients with these conditions (15, 18). It has also
been reported that the quantification of these viral DNA
is useful in the diagnosis of virus-associated diseases and
in monitoring virus activity (2, 9, 14, 20). However, it is
unknown how frequently herpesvirus DNA can be detect-
ed in the serum of immunocompetent children or whether
the detection of herpesvirus DNA indicates an active
infection or virus-related disease. In this study, sera
were collected from immunocompetent children, and a
real-time polymerase chain reaction (PCR) was used to
detect EBV, CMV, HHV6, and HHV7. The relative
importance of viral detection in these children is dis-
cussed, along with clinical and serological data.
Materials and Methods
Patients and samples. The patients enrolled in this
study were 176 ambulatory children (96 boys and 80
girls, mean age of 4.9 years old, range from 0
―
15 years),
Abbreviations: CMV, cytomegalovirus; EBNA, EB nuclear
antigen; EBV, Epstein-Barr virus; ELISA, enzyme-linked
immunosorbent assay; HHV6, human herpesvirus-6; HHV7,
human herpesvirus-7; PCR, polymerase chain reaction; VCA,
viral capsid antigen.
*
Address correspondence to Dr. Shinya Hara, Department of
Pediatrics, Nagoya University School of Medicine, 65 Tsurumai-
cho, Showa-ku, Nagoya, Aichi 466 8550, Japan. Fax: 052 744
974. E-mail: shinyah@med.nagoya-u.ac.jp
―
―
―
2
1
77

1
78
S. HARA ET AL
who visited Nagoya Ekisaikai General Hospital. They
consisted of patients with a variety of health complaints,
including upper respiratory tract infection, pneumoniae,
bronchitis, bronchial asthma, gastroenteritis, fever of
unknown origin, and treated epilepsy. None had any
underlying immunodeficiency or was receiving any
immunosuppressive therapy. Blood specimens were drawn
for clinical purposes. Informed consent was obtained
from the parents of all children enrolled in this study.
Real-time PCR assay. Viral DNA was detected by the
use of a real-time quantitative PCR assay. DNA was
extracted from 200 µl of serum with a QIAamp Blood
Kit (Qiagen, Hilden, Germany), eluted in 50 µl of dis-
tilled water, and stored at －20 C until use. Five micro-
liters of solution were used for the real-time PCR assay.
The primers and probe conditions for EBV, CMV, and
HHV6 were as described in previous papers (8, 18, 19).
The PCR primers and probe for HHV7 were derived
from a DNA sequence of the U37 gene. The sequence
data were obtained from GenBank. The upstream primer
was 5'-CGGAAGTCACTGGAGTAATGACAA-3', and
the downstream primer was 5'-CCAATCCTTCC-
GAAACCGAT-3'. A fluorogenic probe (5'-CTCGCA-
GATTGCTTGTTGGCCATG-3') was located between
the primers. The PCR reaction was performed with a
TaqMan PCR kit and a Model 7700 Sequence Detector
expressed as copies per ml. The detection limits of
each virus DNA were approximately 50
―
100 copies/ml.
To exclude contamination from our laboratory, adequate
care was taken in the experimental design (11). For
positive cases, DNA was extracted from the original
samples, and the PCR was repeated to confirm the
results.
Antibodies to 4 viruses. Antibodies to EBV and
CMV were tested by enzyme-linked immunosorbent
assay (ELISA). The DiaSorin VCA IgG, VCA IgM, and
EBNA IgG ELISA kits (DiaSorin, Stillwater, U.S.A.)
were used to measure EBV-specific antibodies, such as
anti-viral capsid antigen (VCA) IgG, IgM, and anti-EB
nuclear antigen (EBNA) antibody. Specific anti-CMV
IgG antibody was determined by using Enzygnost Anti-
CMV/IgG (Behring Diagnostics GmbH, Deerfield,
U.S.A.). Antibodies to HHV6 and HHV7 were tested by
an indirect immunofluorescence assay (IFA), as previ-
ously described (17, 21).
Results
Frequency of Viral DNA Detection
We found EBV-DNA in 4 (2.2%) of the 176 sera,
HHV6-DNA in 4 (2.2%), and HHV7-DNA in 2 (1.1%).
No CMV-DNA was detected. All samples were assessed
for antibodies to EBV and CMV. Ninety-six samples
were positive for anti-CMV IgG antibody, which indi-
cated previous or recent infection by CMV, and 81 sam-
ples were negative, suggesting that these children had not
been infected. A total of 116 samples were positive for
anti-VCA IgG antibody, indicating that these patients
were previously or recently infected with EBV. EBV-
DNA was detected in 4 of these 116 samples (3.4%), and
all 4 were positive for anti-VCA IgG antibody. EBV-
DNA was not detected in samples that were negative for
anti-VCA IgG antibody.
(PE Applied Biosystems, Foster, U.S.A.), as previously
described (8). Real-time fluorescence measurements
were made and a threshold cycle value for each sample
was calculated by determining the point at which the flu-
orescence exceeded a threshold limit (10 times the stan-
dard deviation of the baseline) (6). A standard graph was
constructed from the threshold cycle values obtained
from serially diluted positive control plasmid (8, 18,
19). The threshold cycle values from clinical samples
were plotted on the standard curve, and the copy number
was automatically calculated by the use of a Sequence
Detector v1.6 (PE Applied Biosystems), a software
package for data analysis. Samples were defined as
negative when the threshold cycle values exceeded 50
cycles. The copy number of each virus DNA was
The Characteristics of the Cases in Which Viral DNA
Was Detected
Table 1 shows the clinical and virological character-
Table 1. Characteristics of 4 cases positive for EBV-DNA
Antibody
Viral load
Anti-VCA Anti-EBNA EBV-DNA
a)
No. Age Sex
Symptoms
IgG IgM
(Copies/ml)
Diagnosis
Date of sampling
2
1
2
3
4
2 yr
2 yr
2 yr
2 yr
M
M
M
F
fever, cough
fever, cough
fever, cough
CLN swelling
＋
＋
＋
＋
－
－
＋
＋
＋
＋
＋
－
3.0×10
Mycoplasma pneumoniae
Viral bronchitis
Unknown hepatitis
6
8
13
1
2
2.6×10
2
5.5×10
3
3.3×10
Primary EBV infection
M: male, F: female, CLN: cervical lymph node.
a)
The first day of symptoms was assigned as day 0.

HERPESVIRUS DNA IN THE SERUM
179
Table 2. Characteristics of 6 cases positive for HHV6/7-DNA
Viral load
Antibody
HHV6-DNA HHV7-DNA Anti-HHV6 Anti-HHV7
Date of
sampling
a)
No. Age Sex
Symptoms
fever, rash
fever
fever
(Copies/ml) (Copies/ml) IgG IgM IgG IgM
Diagnosis
3
5
6
7
8
9
9 mo
2 mo
5 mo
2 yr
M
F
M
M
1.7×10
1.0×10
1.2×10
1.2×10
undetected ＜8 ＜8 ＜8 n.d. Exanthem subitum
undetected ＜8 ＜8 16 n.d. Unknown fever
undetected ＜8 ＜8 ＜8 n.d. Unknown fever
undetected 64 ＜8 ＜8 n.d. Salmonella infection
＜8 n.d. ＜8 ＜8 Exanthem subitum
64 n.d. 64 Mycoplasma pneumoniae
2
15
3
2
5
4
4
4
vomiting, diarrhea
3
11 mo M fever, rash
3 yr fever, cough
M: male, F: female, n.d.: not done.
undetected
undetected
1.1×10
2.7×10
2
1
0
M
8
4
a)
The first day of symptoms was assigned as day 0.
istics of the 4 cases in which EBV-DNA was detected.
The clinical symptoms in cases 1 and 2 did not seem to
be due to EBV. They were diagnosed as mycoplasma
pneumoniae and viral bronchitis, respectively, both of
which are unrelated to EBV. Case 3 had mild liver dys-
function of unknown etiology. The detection of both
anti-VCA IgG antibody and anti-EBNA antibody indi-
cated that this patient had been infected with EBV pre-
viously, but he also had anti-VCA IgM antibody, sug-
gesting that he might have been experiencing a reacti-
vation of EBV. Any relationship of his infection to
other hepatitis viruses, such as hepatitis A, B, and C, or
to CMV was discounted, because of negative results
with virus-specific antigens or antibodies. Based on
clinical and serological characteristics, case 4 was diag-
nosed as a primary EBV infection, there were 10 times
more EBV-DNA copies than found in the other 3 cases.
Table 2 shows the clinical and virological character-
istics of the 4 cases with HHV6-DNA and the 2 cases
with HHV7-DNA. Cases 5 and 9 were clinical cases of
exanthem subitum. In cases 6 and 7, fever was the only
clinical finding, and no skin rash was found after the
fever subsided. In each case, neither antibody titers to
HHV6 nor the patient’s age could be used to rule out the
possibility of primary HHV6 infection. Case 8 was a sal-
monella infection, and case 10 was a mycoplasma infec-
tion.
firmed that the presence of EBV-DNA in their serum was
not due to contamination or technical problems. The
final case (case 3) had mild hepatitis of unknown origin.
Serological examinations indicated that this patient had
been previously infected with EBV, but a positive anti-
VCA IgM antibody suggested that he might be experi-
encing a reactivation of EBV. It is unclear whether the
liver dysfunction was associated with EBV in this patient.
It is well known that both HHV6 and HHV7 cause
exanthem subitum. Of 6 cases in which HHV6 or HHV7
was detected, 2 (cases 5 and 9) were clinically diag-
nosed exanthem subitum. Their blood samples may
have been negative for IgG and IgM antibodies to HHV6
or HHV7 because they were taken during a febrile peri-
od. It has been reported that specific antibodies for
HHV6 and HHV7 are not detected during febrile periods
and that IgM antibodies appear 3 to 5 days after the
onset of exanthem subitum (10, 16). Cases 6 and 7,
which presented with fever but no rash, might also have
had a primary HHV6 infection because primary infection
of HHV6 does not necessarily elicit a rash (13). Case 6
had neither IgG nor IgM antibodies to HHV6, fifteen
days after the onset of symptoms. Since this case was a
very young infant (2 months), the appearance of virus-
specific antibodies might have been delayed because of
the immaturity of the immune system. The other two
cases (cases 8 and 10) had bacterial infections. The
existence of IgG antibodies in the acute phase suggests
that they had been infected in the past. It is possible that
a subclinical reactivation of HHV6 or HHV7 had
occurred and that cell-free viral DNA was detected in the
serum of these patients. It has been reported that neither
HHV6 nor HHV7 is detected in the serum of healthy
adults (4, 14).
Discussion
Using a real-time PCR assay, we detected EBV-DNA
in serum from 4 immunocompetent children. The detec-
tion rate of EBV-DNA was about 3.4% in samples from
EBV-seropositive children. Two previous studies, though
small, detected EBV-DNA in the serum of 7
―
10% of
As we have shown in this study, herpesvirus DNA can
be detected in the serum of immunocompetent children,
at low frequency. Although some cases were thought to
have a primary infection, there were others in whisch we
could find no relationship between clinical manifestations
and the detection of viral DNA. Therefore a careful
healthy adults (5, 12). Our results for immunocompetent
children are in general accord with these previous find-
ings. Of 4 cases with EBV-DNA, one was diagnosed as
a primary EBV infection, but 2 had no serological or clin-
ical findings suggestive of active EBV infection. We con-

1
80
S. HARA ET AL
diagnosis is required subsequent to the detection of her-
pesvirus DNA. It is unclear why virus DNA was detect-
ed in the cases without virus-specific symptoms. A
reactivation might occur in tandem with other infec-
tions. In this study, 4 of 10 cases co-occurred with
other infections, such as mycoplasma and salmonella.
There is a report that Salmonella typhimurium activates
human immunodeficiency virus type 1 in chronically
infected promonocytic cells by inducing tumor necrosis
factor-alpha production (1). Through such a mecha-
nism, bacterial infection might cause the reactivation
of herpesviruses.
yama, Y., and Morishima, T. 2000. Monitoring of cell-free
viral DNA in primary Epstein-Barr virus infection. Med.
Microbiol. Immunol. (Berl.) 188: 197―202.
1
0) Kondo, K., Hayakawa, Y., Mori, H. Sato, S., Kondo, T.,
Takahashi, K., Minamishima, Y., Takahashi M., and
Yamanishi, K. 1990. Detection by polymerase chain reaction
amplification of human herpesvirus 6 DNA in peripheral
blood of patients with exanthem subitum. J. Clin. Microbiol.
28: 970
11) Kwok, S., and Higuchi, R. 1989. Avoiding false positives
with PCR. Nature 339: 237 238.
―974.
―
1
1
1
2) Lo, Y.M., Chan, L.Y., Lo, K.W., Leung, S.F., Zhang, J.,
Chan, A.T., Lee, J.C., Hjelm, N.M., Johnson, P.J., and
Huang, D.P. 1999. Quantitative analysis of cell-free Epstein-
Barr virus DNA in plasma of patients with nasopharyngeal
We thank Dr. K. Watanabe, professor of the Department of
Pediatrics, Nagoya University Graduate School of Medicine, for
his critical reading of the manuscript and valuable suggestions.
This work was supported by a grant from the Japan Society for the
Promotion of Science (JSPS-RFTF97L00703).
carcinoma. Cancer Res. 59: 1188―1191.
3) Pruksananonda, P., Hall, C.B., Insel, R.A., McIntyre, K.,
Pellett, P.E., Long, C.E., Schnabel, K.C., Pincus, P.H.,
Stamey, F.R., Dambaugh, T.R., et al. 1992. Primary human
herpesvirus 6 infection in young children. N. Engl. J. Med.
3
26: 1445―1450.
References
4) Secchiero, P., Carrigan, D.R., Asano, Y., Benedetti, L.,
Crowley, R.W., Komaroff, A.L., Gallo, R.C., and Lusso, P.
1995. Detection of human herpesvirus 6 in plasma of children
with primary infection and immunosuppressed patients by
1
) Andreana, A., Gollapudi, S., Kim, C.H., and Gupta, S. 1994.
Salmonella typhimurium activates human immunodeficien-
cy virus type 1 in chronically infected promonocytic cells by
inducing tumor necrosis factor-alpha production. Biochem.
polymerase chain reaction. J. Infect. Dis. 171: 273―280.
15) Spector, S.A., Merrill, R., Wolf, D., and Dankner, W.M.
1992. Detection of human cytomegalovirus in plasma of
AIDS patients during acute visceral disease by DNA ampli-
Biophys. Res. Commun. 201: 16―23.
2
) Brytting, M., Xu, W., Wahren, B., and Sundqvist, V.A. 1992.
Cytomegalovirus DNA detection in sera from patients with
active cytomegalovirus infections. J. Clin. Microbiol. 30:
fication. J. Clin. Microbiol. 30: 2359―2365.
16) Suga, S., Yoshikawa, T., Asano, Y., Nakashima, T., Yazaki,
T., Fukuda, M., Kojima, S., Matsuyama, T., Ono, Y., and
Oshima, S. 1992. IgM neutralizing antibody responses to
human herpesvirus-6 in patients with exanthem subitum or
organ transplantation. Microbiol. Immunol. 36: 495―506.
17) Suga, S., Yoshikawa, T., Asano, Y., Yazaki, T., and Hirata, S.
1989. Human herpesvirus-6 infection (exanthem subitum)
1
937―1941.
3
) Clark, D.A., Kidd, I.M., Collingham, K.E., Tarlow, M.,
Ayeni, T., Riordan, A., Griffiths, P.D., Emery, V.C., and
Pillay, D. 1997. Diagnosis of primary human herpesvirus 6
and 7 infections in febrile infants by polymerase chain reac-
tion. Arch. Dis. Child. 77: 42―45.
4
) Drago, F., Ranieri, E., Malaguti, F., Battifoglio, M.L., Losi,
E., and Rebora, A. 1997. Human herpesvirus 7 in patients
with pityriasis rosea. Electron microscopy investigations
and polymerase chain reaction in mononuclear cells, plasma
without rash. Pediatrics 83: 1003―1006.
18) Tanaka, N., Kimura, H., Hoshino, Y., Kato, K., Yoshikawa,
T., Asano, Y., Horibe, K., Kojima, S., and Morishima, T.
2000. Monitoring four herpesviruses in unrelated cord blood
and skin. Dermatology 195: 374
―378.
transplantation. Bone Marrow Transplant. 26: 1193―1197.
5
) Gan, Y.J., Sullivan, J.L., and Sixbey, J.W. 1994. Detection of
cell-free Epstein-Barr virus DNA in serum during acute
19) Tanaka, N., Kimura, H., Iida, K., Saito, Y., Tsuge, I., Yoshimi,
A., Matsuyama, T., and Morishima, T. 2000. Quantitative
analysis of cytomegalovirus load using a real-time PCR
infectious mononucleosis. J. Infect. Dis. 170: 436
) Heid, C.A., Stevens, J., Livak, K.J., and Williams, P.M.
996. Real time quantitative PCR. Genome Res. 6: 986 994.
―439.
6
7
assay. J. Med. Virol. 60: 455―462.
1
―
20) Yamamoto, M., Kimura, H., Hironaka, T., Hirai, K.,
Hasegawa, S., Kuzushima, K., Shibata, M., and Morishima,
T. 1995. Detection and quantification of virus DNA in plas-
ma of patients with Epstein-Barr virus-associated diseases. J.
) Huang, L.M., Kuo, P.F., Lee, C.Y., Chen, J.Y., Liu, M.Y., and
Yang, C.S. 1992. Detection of human herpesvirus-6 DNA by
polymerase chain reaction in serum or plasma. J. Med.
Virol. 38: 7―10.
Clin. Microbiol. 33: 1765―1768.
8
) Kimura, H., Morita, M., Yabuta, Y., Kuzushima, K., Kato, K.,
Kojima, S., Matsuyama, T., and Morishima, T. 1999. Quan-
titative analysis of Epstein-Barr virus load by using a real-
21) Yoshikawa, T., Asano, Y., Kobayashi, I., Nakashima, T.,
Yazaki, T., Suga, S., Ozaki, T., Wyatt, L.S., and Frenkel, N.
1993. Seroepidemiology of human herpesvirus 7 in healthy
time PCR assay. J. Clin. Microbiol. 37: 132
―136.
children and adults in Japan. J. Med. Virol. 41: 319―323.
9
) Kimura, H., Nishikawa, K., Hoshino, Y., Sofue, A., Nishi-