Hindawi Publishing Corporation
Advances in Virology
Volume 2012, Article ID 205085, 7 pages
Associationof Active HumanHerpesvirus-6, -7 and
Parvovirus B19 InfectionwithClinicalOutcomesinPatients with
Maksims Chistjakovs,1AlinaSultanova,1LudmilaViksna,2and ModraMurovska1
1August Kirchenstein Institute of Microbiology and Virology, Riga Stradins University, Ratsupites Street 5, LV-1067 Riga, Latvia
2Department of Infectology and Dermatology, Riga Stradins University, Linezera Street 3, LV-1006 Riga, Latvia
3Department of Neurology and Neurosurgery, Riga Stradins University, Pilsonu Street 13, LV-1002 Riga, Latvia
Correspondence should be addressed to Angelika Krumina, email@example.com
Received 2 March 2012; Revised 21 June 2012; Accepted 28 June 2012
Academic Editor: Julia G. Prado
Copyright © 2012 Svetlana Chapenko et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Frequency of active human herpesvirus-6, -7 (HHV-6,HHV-7) and parvovirus B19 (B19) infection/coinfection and its association
with clinical course of ME/CFS was evaluated. 108 ME/CFS patients and 90 practically healthy persons were enrolled in
the study. Viral genomic sequences were detected by PCR, virus-specific antibodies and cytokine levels—by ELISA, HHV-6
variants—by restriction analysis. Active viral infection including concurrent infection was found in 64.8% (70/108) of patients
and in 13.3% (12/90) of practically healthy persons. Increase in peripheral blood leukocyte DNA HHV-6 load as well as in
active betaherpesvirus infection and subfebrility, lymphadenopathy and malaise after exertion, and between active B19 infection
and multijoint pain. Neuropsychological disturbances were detected in all patients. The manifestation of symptoms was of more
frequent occurrence in patients with concurrent infection. The high rate of active HHV-6, HHV-7 and B19 infection/coinfection
with the simultaneous increase in plasma proinflammatory cytokines’ level as well as the association between active viral infection
and distinctive types of clinical symptoms shows necessity of simultaneous study of these viral infections for identification of
possible subsets of ME/CFS.
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/
CFS) is a disease characterized by profound disabling fatigue
lasting at least 6 months and accompanied by a combination
of nonspecific symptoms. According to the 1994 US Center’s
for Disease Control and Prevention (CDC) case definition,
which at present, is widespread in research and clinical
practice, at least four out of eight symptoms (impaired
memory or concentration, sore throat, tender cervical or
axillary lymph nodes, muscle pain, multi-joint pain, new
headaches, sleep disturbances and post-exertion malaise)
should be present in cases of ME/CFS . During the
clinical course of disease multiple body systems are affected
by immune, neuroendocrine, musculoskeletal as well as
psychiatric factors that reflect on the heterogeneity of the
disease. Because fatigue is a common symptom of many
diseases, a wide differential diagnosis needs to be done. The
observation that many cases of the disease begin with a flu-
like illness has prompted the hypothesis that viral infections
are implicated in this disorder.
Infections of human β-herpesviruses-human herpesvi-
rus-6 and -7 (HHV-6, HHV-7), cytomegalovirus (CMV) [2–
4], Epstein-Barr virus [5, 6], parvovirus B19 (B19) [7–9],
and enterovirus  are suggested as etiological agents for
ME/CFS. The major hypothesis of the pathogenesis of the
disease is that persistent viral infections may trigger and lead
to chronic activation of the immune system with abnormal
2Advances in Virology
regulation of cytokine production . However, until now
the role of viral infections as etiological agents for ME/CFS
has been evaluated inconsistently [12–15]. Identification of
specific biomarkers for differential diagnosis of ME/CFS has
intensively been studied [16–19].
HHV-6 and HHV-7 are lymphotropic, neurotropic,
and immunomodulating viruses which primary infection
is followed by lifelong persistency. Reactivation of viruses
can provoke the development of abnormalities involving
the immune system and nervous system and probably may
trigger ME/CFS . Two distinct variants of HHV-6 (A and
B) have been described  and the human glycoprotein
CD46 has been recorded as a receptor molecule for both
variants . Clinical features of HHV-6A infection remain
Human parvovirus B19 was first discovered by Cossart
et al.  in the sera of healthy blood donors. The virus
is ubiquitous and course of infection depends on the
host’s hematological status and immune response. Cellular
receptor for B19 is globoside (blood group P antigen) .
Tissue distribution of P antigen may explain the clinical
manifestation of the viral infection .
The appearance of antibodies to B19 is associated with
clearance of the virus from the bloodstream; however, the
persons under the presence of anti-B19 antibodies indicates
occurred . It is unclear how often ME/CFS appears after
The aim of this study was to determine frequency of
active HHV-6, HHV-7, and B19 infection/coinfection and to
evaluate association of active single and concurrent infection
with clinical outcomes in cases of ME/CFS.
2.1. Patients. One hundred eight randomly selected patients
(66 females, 42 males; mean age 37 years) with clinically
diagnosed ME/CFS after rigorous examination of criteria
(fatigue for at least six months and at least four of eight
symptoms: postexertional malaise, impaired memory or
concentration, unrefreshing sleep, muscle pain, multijoint
pain, tender cervical or axillary lymph nodes, sore throat,
headache) corresponding to the 1994 CDC definition and 90
practically healthy persons (55 females, 35 males; mean age
39 years) were investigated for evidence of HHV-6, HHV-7
and B19 infection/coinfection. The presence and frequency
of clinical features in ME/CFS patients were examined in
relation to active viral infection. Severity of fatigue was
evaluated by Fatigue Severity Scale (FSS) with maximal
score of 72 points . The cohort was established with
the approval of the Ethics Committee of the Riga Stradins
University and all participants gave their informed consent
prior to the examination. Ninety practically healthy persons
(55 females, 35 males; mean age 39 years) were included in
this examination as a control group.
2.2. HHV-6 and B19 Serology. Plasma samples were tested
by ELISA kits (Panbio, Sinnamon Park, OLD, Australia) for
specific anti-HHV-6 IgM and IgG class antibodies according
manufacturer’s protocols. Tests for antibodies against B19
were carried out using B19 IgG and IgM the anti-VP2
enzymatic immunoassay kits (Biotrin, Dublin, Ireland) in
accordance with the manufacturer’s recommendations.
2.3. Nested Polymerase Chain Reaction (nPCR). The tech-
nique of nPCR was used to detect viral genomic sequences
in DNA isolated from peripheral blood leukocytes (PBLs)
and cell free plasma (markers of persistent and active
infection, resp.). Total DNA was isolated from 0.5ml of fresh
whole blood by phenol-chloroform extraction. The QIAamp
Blood Kit (QIAGEN, Hilden, Germany) was used to purify
DNA from 200μL of cell-free blood plasma. The plasma
samples were treated with DNase I (Fermentas, Vilnius,
Lithuania) before DNA purification. To assure the quality
of the PBL DNA and to exclude contamination of plasma
recognized the globin gene. PCR amplification of viral DNA
was carried out in the presence of 1μg of PBL DNA or 10μL
of plasma DNA (which corresponded to 100μL of plasma).
HHV-6, HHV-7, and B19 DNA were detected in accordance
with Secchiero et al. , Berneman et al. , and
Cavallo et al. , respectively. Positive controls (HHV-
6 and HHV-7 genomic DNA; Advanced Biotechnologies
Inc, Columbia, MD, USA and B19 genomic DNA isolated
from viremic serum kindly provided by Prof. K.Hedman,
Department of Virology, Heartman Institute, University of
Helsinki) and negative controls (DNA obtained from practi-
cally healthy HHV-6, HHV-7 and B19 negative blood donor
and no template DNA) were included in each experiment.
Criteria to define persistent viral infection were the
presence of virus-specific IgG class antibodies in blood
plasma and viral genomic sequences in DNA isolated from
the whole blood. The presence of HHV-6-specific IgM class
antibodies in blood plasma and viral genomic sequence in
plasma DNA samples, as well as elevated titer of virus-
specific IgG class antibodies, without IgM antibodies and
viral genomic sequence in plasma DNA samples was defined
as active HHV-6 infection. The cases with HHV-7-specific
sequence in DNA isolated from cell free blood plasma were
defined as active HHV-7 infection cases. The presence of
HHV-7-specific antibodies was not examined due to the lack
of commercial test systems for IgM class antibody detection.
The cases with persistent infection and without markers of
active viral infection were defined as latent stage of persistent
infection (latent infection) cases. B19 genomic sequence in
plasma DNA samples with or without the presence of IgM
class specific antibodies and the presence of viral sequence in
in blood plasma we defined as active B19 infection.
2.4. Quantitative Real-Time PCR. The viral load of HHV-6
in PBL DNA samples from patients with latent and active
viral infections was determined using the HHV-6 Real-
Time Alert Q-PCR kit (Nanogen Advanced Diagnostics,
Advances in Virology3
Buttigliera Alta, Italy) and an Applied Biosystems 7500 Real-
time PCR System (Applied Biosystems, Carlsbad, CA, USA),
in accordance with the manufacturer’s recommendations.
2.5. Restriction Endonuclease Analysis. Restriction endonu-
clease analysis was carried out using the restriction enzyme
HindIII (Fermentas, Vilnius, Lithuania), which cleaves the
163bp HHV-6B amplimer into two fragments of 66 and
97bp but does not cleave HHV-6A amplimer.
2.6. Assay for Cytokine Determination. Endogen Human
ELISA kits (Pierce Biotechnology, Rockford, IL, USA) were
used to detect the level of tumor necrosis factor (TNF)-α,
interleukin (IL)-6 and IL-4 in plasma samples from ME/CFS
patients according to the manufacturer’s recommendations.
The sensitivity of the ELISAs was 2pg/mL for TNF-α and
<1pg/mL for IL-6, and <4pg/mL for IL-4. All samples
were tested in duplicate. Plasma samples were processed
immediately after collection and then stored at −70◦C.
of active HHV-6, HHV-7, and B19 infection/coinfection
between tested groups was assessed by Odds ratio, 95% CI
values and Fisher’s exact test and in the levels of cytokines-
by Student’s t-test, using MedCalc software for Windows,
version 12.2.1; a value of P < 0.05 was considered to be
3.1. HHV-6 and B-19 Serology. Specific anti-HHV-6 anti-
bodies were detected in 87/108 (80.6%) plasma samples
(IgG-71, IgM-3, IgM + IgG-13) from the ME/CFS patients
versus 69/90 (76.7%) practically healthy persons’ plasma
samples (IgG-67, IgM + IgG-2) and specific anti-B19
antibodies in 92/108 (85.2%) plasma samples (IgG-62, IgM-
6, IgM + IgG-24) from the ME/CFS patients versus 55/90
the practically healthy persons.
3.2. Prevalence of Active HHV-6, HHV-7, and B19 Infections.
Active viral infection/coinfection was detected in 70 (64.8%)
and latent—in 32 (29.6%) out of 108 patients with ME/CFS.
Six (5.6%) patients were negative for viral infection (Table
1). The rate of active viral infection was significantly higher
in patients comparing to the rate in the practically healthy
persons (70/108 and 12/90, resp.; Odds ratio 0.14, 95% CI
0.07–0.26, P = 0.0001). In the patients significant difference
was detected between the frequency of single active and
concurrent active viral infection (41/70, 58.6% and 29/70,
41.4%, resp.; Odds ratio 2.0, 95% CI 1.02–3.92, P = 0.044).
Among the patients who were infected with a single virus,
the rate of HHV-7 active infection (40%) was significantly
higher in comparison with B19 (15.7%, Odds ratio 3.58,
95% CI 1.60–7.97, P = 0.002) and HHV-6 active infection
(2.9%, Odds ratio 22.7, 95% CI 5.13–100.1, P < 0.0001). No
significant difference was detected between the frequency of
active concurrent dual HHV-6 + HHV-7 and dual HHV-7 +
Table 1: Frequency of Active HHV-6, HHV-7 and B19 Infection in
ME/CFS Patients and Practically Health Persons.
(n = 108)
(n = 90)
Active viral infection
Dual HHV-6 + HHV-7
Dual HHV-7 + B19
Triple HHV-6 + HHV-7 + B19
Latent viral infection
Without viral infection
B19 infection (Odds ratio 0.61, 95% CI 0.25–1.47, P =
HHV-6B was identified in 15 and HHV-6A in one out of
16PBL and plasma DNA samples.
3.3. HHV-6 DNA Load in PBL DNA Samples . The number
of HHV-6 DNA copies in PBL DNA of the ME/CFS patients
with and without HHV-6 viremia was compared. A clear
increase of HHV-6 load in PBL DNA was detected in 16
patients with plasma viremia in comparison with seven
patients without it (132.61 ± 41.38 × 103and 8.73 ± 3.96 ×
103copies/μg DNA, resp.).
3.4. Relationship between Plasma Level of TNF-α, IL-6, and
IL-4 and Active Viral Infection/Coinfection. To investigate the
relationship between the active viral infections and plasma
cytokine levels, the levels of proinflammatory (TNF-α, IL-
6) and anti-inflammatory (IL-4) cytokines were measured
in 106ME/CFS patients (Table 2). The mean levels of TNF-
α and IL-6 cytokines were significantly higher in patients
with active viral infection/coinfection (52.51 ± 15.13pg/mL,
18.59 ±3.56pg/mL, resp.) than in those with latent (18.81 ±
2.52pg/mL, 2.56±1.02pg/mL, resp.; P < 0.0001) or without
viral infections (7.71 ±3.07pg/mL, 1.32 ±3.07pg/mL, resp.;
P < 0.0001). No significant difference was detected between
expression levels of TNF-α in the patients with active single
HHV-7 and active single B19 infection, as well as with dual
active HHV-6 + HHV-7 and dual active HHV-7 + B19
coinfection (Table 2).
The highest level of TNF-α was detected in patients with
activetriple HHV-6+HHV-7 +B19coinfection.Significantly
higher level of IL-6 expression was detected in plasma
samples of the patients with single active B19 infection
in comparison with the patients with single active HHV-
7 infection (P < 0.001) (Table 2). The mean levels of this
cytokine were also significantly higher in patients with active
concurrent HHV-7 + B19, as well as in patients with triple
HHV-6 + HHV-7 + B19 infection (29.19 ± 6.26pg/mL, P <
0.001) in comparison with the level in patients with dual
HHV-6 + HHV-7 infection (11.22 ± 3.14pg/mL). None of
the ME/CFS patients had increased plasma level of IL-4.
4 Advances in Virology
Table 2: Plasma cytokine levels in patients with ME/CFS.
7.71 ± 3.07
18.81 ± 2.52 2.56 ± 1.02
44.81 ± 10.56 7.76 ± 2.45
44.12 ± 11.67 15.62 ± 4.63
50.38 ± 8.74 11.22 ± 3.14
1.32 ± 0.43
Without (n = 6)
Latent (n = 32)
Active (n = 68)
Single HHV-7 (n = 28)
Single B19 (n = 11)
Dual HHV-6 + HHV-7
(n = 10)
Dual HHV-7 + B19
(n = 15)
Triple HHV-6 + HHV-7 +
B19 (n = 4)
49.89 ± 16.1 28.70 ± 3.05
73.33 ± 18.76 29.67 ± 4.52
3.5. Assessment of Active Betaherpesviruses and B19 Infec-
tion/Coinfection in Association with Clinical Outcomes in
ME/CFS Patients. Severe chronic fatigue for at least six
months or longer was observed in all patients irrespective of
the causation of the active infection (total FSS scores 58.89–
60.99, P < 0.05). Subfebrility, tender cervical or axillary
lymph nodes, and postexertional malaise were not revealed
in the patients with single B19 active infection but were
detected in patients with single HHV-7 active (50%, 75%,
100%, resp.), dual HHV-6 + HHV-7 (70%, 80%, 90%, resp.)
as well as triple HHV-6 + HHV-7 + B19 coinfection (74.1%,
68.4%, 74.1%) (Table 3).
Although muscle pain was observed in all patients, the
frequency of multijoint pain was more clearly displayed in all
patients with active B19 infection, as in cases of single as well
as in cases of coinfection with β-herpesviruses.
Severe postexertional malaise corresponding to “Exercise
brings on my fatigue” by FSS was detected in all patients
(mean score 6.94 ± 0.243 from 7 maximum) with single
HHV-6, HHV-7, and in 9/10 with dual HHV-6 + HHV-7
coinfection as well as in 14/19 with triple HHV-6 + HHV-
7 + B19 coinfection (90% and 74%, resp.).
Neuropsychological disturbances were observed in all 70
patients. Impaired memory was detected in 22 out of
57 (38.6%) patients with active β-herpesviruses infection/
coinfection but not observed in patients with single HHV-
6 and single B19 infection. Impaired concentration was
detected in 34 out of 70 patients (48.6%), more frequently in
patients with B19 infection. Sleep disturbances were revealed
in 49 out of 70 (70%) patients, the sleepiness was more
characteristic for patients with single HHV-6, B19, and dual
HHV-7+B19 coinfection (2/2, 11/11 and 15/15, resp.).
Headaches of new type were observed in 16 out of 30
(53.3%) patients with B19 infection/coinfection and in 14
out of 38 (36.8%) patients with HHV-7 and dual HHV-6 +
Chronic fatigue (for at least 6 months or longer period)
was observed also in all 38 patients with latent infection
and without infection (32 with latent infection and 6 with-
out infection). Postexertional malaise (23/38, 60.5%, mean
score 5.23 ± 0.135 from 7 maximum), impaired memory
(34/38, 89.5%), decreased concentration (32/38, 84.2%),
and sleep disturbances (24/38, 63.2%) were predominant
symptoms in these patients. Subfebrility (10/38, 26.3%)
and lymphadenopathy (11/38, 29%) were observed only in
with β-herpesviruses infection/coinfection (11/14) as well as
with B19 infection/coinfection (3/14). Multijoint pain was
observed in 15 out of 38 (39.5%) patients and in 10 of them
B19 infection/coinfection was found. Headaches of new type
were observed in 13 out of 38 (34.2%) patients. Among
these 13 patients β-herpesviruses infection/coinfection was
detected in 9 patients and B19 infection/coinfection in 4
patients. Clinical manifestations of the above-mentioned
symptoms were not severe (total FSS scores were 42.83–
48.90, P < 0.05).
Subfebrility, lymphadenopathy, malaise after exertion,
muscle pain, multijoint pain, sleep disturbances, and
headaches of new type were more frequent in patients
with active viral infection/coinfection than in patients with
latent infection and without infection. Whereas presence of
impaired memory and impaired concentration was more
frequent in patients with latent infection and without
infection in comparison to the patients with active infection.
ME/CFS is heterogeneous disorder with common set of
symptoms that follows a viral infection. Despite efforts in
the development of standardized research criteria to define
ME/CFS [1, 11, 29, 30] progress in diagnosis and elucidation
of the role of viral infections is slow, due to a lack of
common standard clinical definition and specific biomarkers
This study is the continuation of research of HHV-6
and HHV-7 as triggering factors for ME/CFS . In the
HHV-6, HHV-7 and parvovirus B19 infection/coinfection
and potential relationship of active viral infection with
different clinical symptoms in 108 patients with clinically
The results of our study showed a high rate of HHV-6
and B19 seroprevalence among our patients and practically
healthy persons. The positive rates of anti-HHV-6 and anti-
B19 IgG class antibodies are similar in ME/CFS patients and
practically healthy persons. At the same time, the elevated
frequency of anti-HHV-6 IgM class antibodies is found in
ME/CFS patients which is in concordance with previously
observation by Patnaik et al.  and Ablashi et al. .
In contrast, Koelle et al.  and Cameron et al. 
do not support these data. Also the elevated frequency of
anti-B19 IgM class antibodies is found in ME/CFS patients
in comparison with the practically healthy persons (Odds
ratio 0.36, 95% CI 0.17–0.77, P = 0.009). High rates of
active viral infection/coinfection in ME/CFS patients might
be associated with insufficiency of the humoral immune
response to the viruses.
We identified active viral infection in 70 (65%) ME/CFS
patients, 41 of them had active single HHV-6, HHV-7, and
Advances in Virology5
Table 3: Symptoms of ME/CFS in patients with active viral infection/coinfection.
Active viral infection
(n = 2)
(n = 28)
(n = 11)
HHV-6 + HHV-7
(n = 10)
HHV-7 + B19 and
HHV-6 + HHV-7 +
(n = 19)
Malaise after exertion
B19 infection and 29-concurrent (dual HHV-6 + HHV-7,
HHV-7 + B19 and triple HHV-6 + HHV-7 + B19) infection.
The active single HHV-7 and B19 infection, but not active
single HHV-6 or active concurrent infection, has been
previously detected in Latvian blood donors . The
active HHV-6 infection was confirmed by the detection of
viral sequence in plasma DNA samples and a concomitant
increase of HHV-6 load in PBLs. This corresponds with the
data demonstrated by Ihira et al. .
Although HHV-6A is predominant in ME/CFS patients,
it was detected in only one patient that might be limited to
persistency sites other than the peripheral blood .
Despite the fact that the precise mechanism by which
betaherpesviruses and B19 impair immunological function
are not completely clear, previous investigations have shown
that these viruses are effective modulators of the immune
response, mainly by modulating the production of proin-
flammatory cytokines, including TNF-α and IL-6 [35, 36].
We detected the presence of significantly higher levels of
TNF-α and IL-6 in plasma samples from patients with active
viral infection. This finding confirms the immunomodu-
lating properties of these viruses and is in concordance
with the data of Fletcher et al. . However, our data do
not corroborate with the findings of Vollmer-Conna et al.
 that have found no significant difference of cytokine
production in patients with postinfection fatigue syndrome.
The relationship between active β-herpesviruses and B19
infection/coinfection and ME/CFS clinical symptoms is not
particularly studied in detail specially in subgroups of 70
diagnosis of ME/CFS was confirmed in the presence of four
from eight analyzed symptoms  in all patients including
those with latent viral infection and without infection. The
analysis of our results showed definite relationship between
active single HHV-6 and HHV-7 infection and presence of
subfebrility, lymphadenopathy, and malaise after exertion,
and single B19 infection with multijoint pain. By concurrent
infection no clear differences in manifestations of the
symptoms were detected. Neuropsychological disturbances
were detected in all ME/CFS patients. Neuropsychological
disturbances were detected in all ME/CFS patients.
Results of our study correspond to the newest Inter-
national Consensus Criteria for clinical case definition 
which proposed several subsets of myalgic encephalomyelitis
such as neurological, immune, metabolism/cardiorespira-
The association between occurrence of ME/CFS clinical
symptoms, HHV-6, HHV-7 and B19 infection/coinfection
are involved in ME/CFS etiopathogenesis. Their role as
trigger factors could not be excluded. The correlation of
distinctive active viral infection with various types of clinical
symptoms shows necessity of simultaneous study of these
viral infections for identification of possible subsets of
This work was supported partly by the National Research
Program in Medicine, “Multi-Disciplinary Research Consor-
tium on Major Pathologies Threatening the Life Expectancy
and Quality of life of Latvian Population” Project no. 11,
the Grant from the Latvian Council of Sciences no. 09.1394
“Investigation of etiopathogenesis of chronic fatigue syn-
drome, post-infection and unspecified origin encephalopa-
thy and elaboration of diagnostic criterions”.
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