medizinische hochschule hannover zentrum innere medizin ......1.1.1 epidemiology and natural history...
TRANSCRIPT
Medizinische Hochschule Hannover
Zentrum Innere Medizin
Klinik für Gastroenterologie, Hepatologie und
Endokrinologie
(Direktor: Prof. Dr. med. Michael P. Manns)
Role of Parvovirus B19 Infection in hepatitis C
Dissertation
zur Erlangung des Doktorgrades der Medizin an der
Medizinischen Hochschule Hannover
vorgelegt von
Chun Wang
aus Shanghai China
Hannover 2008
Role of Parvovirus B19 infection in hepatitis C
Angenommen vom Senat der Medizinschen Hochschule Hannover am:
Gedruckt mit Genehmigung der Medizinschen Hochschule Hannover
Präsident: Prof. Dr. Dieter Bitter-Suermann
Betreuer: Priv. Doz. Dr. med. Heiner Wedemeyer
Referent: Prof. Dr. Thomas F. Schulz
Korreferent: Prof. Dr. Stefan Poehlmann
Tag der mündlichen Prüfung:
20. Mai 2008
Promotionsausschussmitglieder:
Prof. Dr. med. Alexander Kapp
Prof. Dr. med. Burkhard Wippermann
Prof. Dr. med. Stefan Kubicka
Role of Parvovirus B19 infection in hepatitis C
Table of contents 1. Introduction............................................................................................................. 1
1.1 Hepatitis C virus ............................................................................................1
1.2 Parvovirus B19 ...............................................................................................3
1.3 Aims.................................................................................................................6
2. Subjects and methods ............................................................................................. 8
2.1 Subjects ...........................................................................................................8
2.2 Methods...........................................................................................................9
3. Results .................................................................................................................... 12
4. Discussion............................................................................................................... 27
5. Summary................................................................................................................ 32
6. Reference ............................................................................................................... 33
7. List of abbreviations:............................................................................................ 38
8. Appendix................................................................................................................ 39
Role of Parvovirus B19 infection in hepatitis C -- Introduction
1
1. Introduction
1.1 Hepatitis C virus
1.1.1 Epidemiology and natural history
Hepatitis C virus was discovered in 1989 by M.Houghton and colleague [1].
Worldwide, approximately 170 million people (ca. 3% of the world’s population) are
infected with hepatitis C virus. HCV prevalence differ between regions from <0.4% in
northern Europe to > 10% in southern African countries [2].
Hepatitis C virus is a major causative agent of end-stage liver damage (cirrhosis)
and hepatocellular carcinoma. Alcohol consumption, virus co-infection (e.g. HBV,
HIV), genetics, and other liver disease such as NASH, are the major factors, which
can influence the natural history of HCV and will contribute to high risk of
developing liver cirrhosis in the range from 1-40% after twenty or thirty years [3].
The detailed mechanisms how these factors contribute to progression of chronic liver
disease are largely undefined. In particular, the role of coinfections with other
pathogens than HIV and HBV has rarely been studied.
1.1.2 Virology
The hepatitis C virus is an enveloped noncytopathic single-stranded RNA of ~10,
000 nucleotides, 30-60nm diameter, belonging to the Flaviviridae family, which acts
as an mRNA in the cytoplasm to translate a polyprotein. Cellular and viral proteases
are involved in the generation of structural proteins: core, envelope protein 1 (E1) and
2 (E2), p7 and nonstructural proteins (NS2, -3, -4A, -4B, -5A and -5B) [4]. Binding of
the HCV E2 protein to the second extracellular loop of CD81 and endocytosis of
HCV via the LDL (low density lipoprotein) receptor may play an important role in
proceeding infection. Recently, Claudin-1 has been reported as a co-receptor in the
late period of viral entry [5]. However, the detailed mechanism how HCV is infecting
the hepatocyte is still unknown.
Genetic heterogeneity is the most notable feature of HCV. Six major genotypes
and over 100 subtypes of HCV have been described. HCV genotypes show some
different patterns in natural history. While genotype 3 infection is associated with
steatosis [6, 7], more frequent flares of hepatitis were observed in genotype 2 infected
persons [8].
Role of Parvovirus B19 infection in hepatitis C -- Introduction
2
Recently it was suggested that the risk for the development of hepatocellular
carcinoma might be higher in genotype 1b infection [9]. Moreover, an association of
HCV genotypes with responses to antiviral therapy is well established as individuals
with HCV genotype 2 infection show the highest response rates followed by genotype
3 infected individuals. The lowest response rate are observed in genotype 1 infection
with no significant differences between genotype 1b and genotype 1a [3].
1.1.3 Immunology of HCV infection
The immune response against HCV is a crucial factor for viral replication,
elimination, as well as the outcome of virus infection. The immune response can
target all HCV structural and non-structural protein. However there is no specific
antibody pattern associated with recovery or replication.
It has been frequently demonstrated that predominant multi-epitope specific
CD4+ and CD8
+ T-cell responses are correlated with spontaneous recovery from HCV
infection. By contrast, delayed, transient or narrowly focused T-cell responses lead to
chronic hepatitis C [10-13] . Simultaneously, an early IFN-gamma response by CD8+
T cells induces resolution of HCV, whereas persistence of function impaired CD8+ T
cells leads to chronic infection. HCV-specific T cell responses have rarely been
studied in the context of viral co-infections. In individuals infected with HCV and
HIV, HCV-specific T cell responses are impaired along with the depletion of total
CD4+ T cells following the progression of HIV infection. Moreover, HIV infected
patients with spontaneous control of HCV show a high risk for HCV viremia re-
occurrence due to CD4+ T cell depletion [14] . HCV and Schistosomiasis co-
infections were studied on Egyptian individuals. This coinfection leads to high HCV
viral load, more frequently chronic HCV infection and more severe liver disease due
to impaired HCV-specific T cell responses cause by Schistosomiasis [15]. No study
yet has investigated HCV specific immunity in the context of B19-infections.
However, this may have implications as heterologous immunity has been shown to
significantly influence the outcome of various viral infections in rodents and humans
[16, 17].
Role of Parvovirus B19 infection in hepatitis C -- Introduction
3
1.1.4 Therapy
Even though the acute infection phase is usually asymptomatic, chronic HCV
could be prevented by early treatment of acute HCV infection with interferon alpha
monotherapy for just 6 months [18, 19]. Approximately 50% of genotype 1 patients
and 80-90% of genotype 2 and 3 infected patients can reach sustained virologic
response with the combination therapy of pegylated interferons and ribavirin. Patients
with high risk factor for progressive liver disease (e.g. ISHAK fibrosis score >= 2)
should be treated early [3]. Other factors being associated with a better treatment
response are younger age, female sex, low viral load before therapy, a rapid response
with HCV-RNA negativity until week 4 of therapy, a low baseline gamma GT level,
absence of hepatic steatosis and high adherence to therapy [3].
1.2 Parvovirus B19
1.2.1 Epidemiology and natural history
Parvovirus B19 (B19) was occasionally discovered by Cossart et al. during
testing for hepatitis B virus surface antigen in 1974 [20]. The infection with this virus
is worldwide especially in childhood. The prevalence of anti-B19 IgG antibody is 2-
15% in young children (1-5 years), 15-60% in school age children (6-19 years), 30-
60% in adults, and more than 85% in the senior population [21-24]. Pathways of
transmission are respiratory route, blood-derived products and vertically from mother
to fetus. B19 is a pathogen with a variety of clinical manifestations, ranging from
asymptomatic courses in immunocompetent individuals to lethal cytopenias in
immunocompromised patients [25]. The most common disease associated with B19
infection is erythema infectiosum, also termed fifth disease or “slapped-cheek” disease.
The duration of viremia has been reported from weeks to months in
immunocompetent hosts following the development of virus specific antibodies.
However, Lindblom etal [26] reported that viremia was detectable during the whole
follow-up period (128 weeks) in 4 of 5 immunocompetent individuals after acute B19
infection in spite of the resolution of clinical symptoms. Thus, the clearance of B19
viremia may be slower than previously thought. Bone marrow is the primary site of
replication [25].
Role of Parvovirus B19 infection in hepatitis C -- Introduction
4
1.2.2 Virology
B19 is a single-stranded nonenveloped DNA virus, 22-24 nm diameters,
containing 5,596 nucleotides (nt), and is member of the family Parvoviridae known to
be pathogenic in humans.
The B19 genome has two large open reading frames, encoding for the non-
structural protein (NS1) and for structural capsid proteins (VP1 and VP2). NS1
manifests site-specific DNA-binding, subserves multiple replicative functions and is
cytotoxic to host cells [27, 28]. VP2 is the major structural protein to make up 96% of
the total capsid protein [29]. VP1 accounts for the remaining 4% and is identical to
VP2 with the addition of 227 amino acids (termed the VP1 unique region, VP1u) at
the amino terminus [29, 30]. Importantly, the VP1 protein comprises a viral
phospholipase A2 activity [31] which was suggested to be critical for efficient transfer
of the viral genome to initiate replication. The Erythrocyte P antigen has been
identified as the B19 receptor for entering into cell to initiate infection [32]. In
addition, a5b1-integrin and the Ku80 autoantigen have been described as co-receptors
[33-35].
Unlike HCV, the nucleotide sequence of B19 is rather conserved, containing
only 6% divergence. So far three genotypes: B19-, LaLi-, and V9-related viruses have
been identified [36]. However, the variation of B19 sequence has no correlation with
specific disease symptoms and persistent infection [25, 37, 38].
1.2.3 Immunology of B19 infection
Humoral immune response against B19 is correlated with the eradication of the
virus and has a long lasting protection against re-infection [39]. B19-specific cellular
immune responses have been studied only in the recent decade. NS1, VP1 and VP2
can be targets of the host’s cellular immune responses. B19 specific CD8+ and CD4
+
T cell responses can be found in acute infection, persistent infection and remote
infection [40-47]. The maintenance of B19-specific T cell responses may indicate
persistence of low loads of residual virus as it has been shown in the case of recovery
after HBV infection [48]. Importantly, specific T cell epitopes have been
characterized in recent years. One group [47] has described two particularly
frequently detected epitopes in healthy B19 IgG-positive but IgM/DNA-negative
individuals. Although one epitope (LASEESAFYVLEHSSFQLLG) was DRB1*1501
restricted, positive T cell responses were detectable in DRB1*1501 negative
Role of Parvovirus B19 infection in hepatitis C -- Introduction
5
individuals too. In 6 serologically recovered B19 infected individuals with positive
responses against this epitope only 2 were DRB1*15 positive. The lack of an efficient
T cell response against B19 may lead to persistent infection [49].
B19-specific T cell responses have not been studied yet in the context of
hepatitis virus infections. There is no information on frequency, specificity and
strength of B19 specific T cell immunity in hepatitis C versus responses in healthy
controls. Thus, one aim of this thesis project was to address this question accordingly.
1.2.4 Therapy
Normally, B19 infection requires no specific antiviral treatment. In
immunocompetent individuals, some patients may need symptomatic treatment
(NSAIDs) because of B19-induced arthralgia. Erythrocyte transfusion is required in
cases of B19-induced transient aplastic crisis [50]. In immunocompromised patients,
infusion of immunoglobulin is an effective therapy to against chronic anemia or
cytopenia due to persistent B19 infection. Fetal blood transfusions is a curative
treatment in severe B19-related hydrops fetalis [25, 51, 52]. In patients with B19-
related myocarditis, interferon beta is a kind of successful treatment contributes to
virus clearance and protects left ventricular function [53]. Currently, larger trials are
exploring the efficacy of interferon beta treatment of B19-related mycoarditits.
1.2.5 B19 infection and liver disease
In the past decade, a growing number of studies have been published to reveal
an association between B19 and liver disease especially in the etiology of fulminant
liver failure and the persistence of B19 in liver [54-63]. The summary of these studies
is shown in Table 1. Recently, an investigation aimed to evaluate the effect of B19-
infection on the course of HBV-associated liver disease by Toan and colleague
demonstrated that B19-infection was not only frequent in HBV-infected Vietnamese
patients but also obviously associated with severe hepatitis B-associated liver disease.
In this study, total of 463 Vietnamese individuals was recruited. Of these, 399 were
HBV-infected patients (311 symptomatic HBV-infected patients with well-
characterized clinical profiles, 88 asymptomatic chronic HBV carriers with no liver
disease) and 64 were healthy individuals. Sera of these individuals were tested for the
presence of B19-DNA by nPCR and quantity real-time PCR and DNA-sequencing.
Paralleled liver biochemical and serological testing were also assayed. The prevalence
Role of Parvovirus B19 infection in hepatitis C -- Introduction
6
of B19 DNA was significantly higher in HBV-infected patients compared to the
healthy control group (24.1% vs 4.7%, p<0.001). Moreover, it showed a significantly
higher prevalence of B19 DNA in the HCC subgroup compared with no-HCC
subgroup (38.6% vs 18.5%, p<0.001). The prevalence of B19 DNA in “severe (LC
and HCC)” patients group and “mild (AHB, CHB, and ASYM)” patients group were
29.9% and 18.5%, respectively (p=0.008). By using multivariate analysis they also
demonstrated the serum B19 viral load is correlated with the HBV viral load and
serum ALT levels. Based on these findings the authors concluded that B19 may be
persistent in HBV patients and B19 may play an important role in the pathogenesis of
HBV in Vietnamese.
1.3 Aims
Based on the recent findings of B19 being possibly involved in disease
progression in Asian hepatitis B patients, we here aimed to investigate whether B19
infection may be a co-factor for disease progression in European patients with chronic
hepatitis C. Therefore, the following questions were addressed:
(i) What is the prevalence of B19 infection in German patients with chronic HCV
and HBV infection versus healthy individuals?
(ii) If B19 can be detected in hepatitis C patients – how does antiviral therapy with
interferon alpha and ribavirin influence B19 viremia?
(iii) Does B19 persist in blood and/or liver of immunocompetent hosts after
apparent recovery from B19 infection?
(iv) Is any virological or serological marker of B19 infection associated with the
outcome of chronic hepatitis C infection?
(v) What is frequency and strength of B19-specific T cell responses in
serologically recovered healthy controls vs. patients with chronic hepatitis virus
infection?
Role of Parvovirus B19 infection in hepatitis C -- Introduction
7
Table 1: Summary of studies on B19 and fulminant hepatitis or non-A-E
hepatitis
Study group Subjects Conclusion
Eis-Huebinger
(Germany)[54]
Explanted liver
tissue, liver and
bone marrow from
autopsied adults and
serum samples
B19 DNA was present frequently in livers
of adults with severe liver damage
indicating the persistence of B19 in the
liver
Abe (Japan) [55] Explanted liver
tissue and serum
samples
B19 may act as an causative agent of
fulminant hepatitis
He (China) [56] Serum samples B19 is not associated with non-A-E
hepatitis. The prevalence of B19 infection
in patients with non-A-E hepatitis is
similar to that in patients with A-E
hepatitis. B19 and HBV coinfection does
not lead to more severe liver damage
Wong
(USA)[57]
Explanted liver
tissue
The prevalence of B19 DNA in livers from
patients with fulminant hepatitis (FH) or
hepatitis-associated aplastic anemia (HAA)
is similar to livers from patients with HBV
or HCV infection. Thus, B19 is not
associated with FH or HAA.
Karetnyi
(USA)[60]
Explanted liver
tissues
B19 may play a role in liver damage in
patients with non-A-E fulminant liver
failure.
Sokal (Belgium)
[61]
Serum samples The evidence of acute B19 infection in
children younger than 5 years with FH of
unknown etiology showed an eusemia
Yoto (Japan)[62] Serum samples B19 may be the causative agent of acute
hepatitis
So. K (Australia)
[63]
Drago (Italy)
[58]
Hillingso
(Denmark)[59]
Case reports Acute B19 infection leaded to abnormal
liver parameters. B19 may be a causative
agent of fulminant liver failure or
fulminant hepatitis.
Role of Parvovirus B19 infection in hepatitis C -- Subjects and Methods
8
2. Subjects and methods
2.1 Subjects
2.1.1 Hepatitis C patients. Stored serum samples were studied from 75 patients with
chronic hepatitis C infection who have been treated with interferon alpha and ribavirin
combination therapy. All sera were taken before treatment was started. All patients
were treated in the outpatient clinic of the Department of Gastroenterology,
Hepatology and Endocrinology of Medical School Hannover, Germany. Another 16
sera and paired peripheral blood mononuclear cells (PBMCs) were collected from
patients with chronic hepatitis C infection without antiviral therapy for evaluating
cellular immune responses. These 91 patients had a mean age of 46 years ranging
from 19-73 years (male: 49; female: 42). Characteristics are summarized in Table 4.
2.1.2 Hepatitis B patients. Serum samples were taken from randomly selected 50
patients with chronic hepatitis B infection (mean age: 47 years, range: 15-72, male 33;
female: 17). The presence of hepatitis B surface antigen (HBsAg) for more than 6
months was required for the diagnosis of chronic HBV infection.
2.1.3 Liver tissue samples
Explanted liver tissues were obtained at the time of liver transplantation from 50
patients with end-stage liver damage undergoing orthotopic liver transplantation for
various reasons from 1993 to 2000 at Medical School Hanover (mean age: 47 years,
range: 8-69). Of these, 19 had HCV related end-stage liver disease, while 31 had non-
HCV liver cirrhosis (e.g. primary biliary cirrhosis, primary sclerosing cholangitis,
Morbus Wilson and Polycystic liver disease).
Routine liver biopsy samples were obtained from 32 patients (mean age: 46
years, range: 33-75). Of these, 13 had chronic hepatitis C. 19 biopsies were taken
from patients with no evidence of HCV infection.
All of these samples were stored at -20°C until used.
All patients were recruited at Medical School Hannover, Germany.
Role of Parvovirus B19 infection in hepatitis C -- Subjects and Methods
9
2.1.4 Healthy individuals
Sera and paired PBMCs were collected from 30 laboratory and clinic healthy
volunteers (mean age: 37.5 years, range: 25-65, male: 16, female: 14). Among them,
19 were B19-IgG antibody positive while 11 were negative.
2.2 Methods
2.2.1 Serological tests.
Anti-B19 IgM and anti-B19 IgG antibodies were tested using the Parvovirus B19
IgM/IgG Enzyme Immunoassay (Biotrin, Ireland) according to the manufacture’s
instruction. Anti-HCV antibodies were tested using ARCHITECT Anti-HCV assay
(Abbott, USA).
2.2.2 Extraction of nucleic acids from liver tissue
DNAs were extracted from 20-30mg frozen liver tissue using the QIAamp DNA
mini kit (Qiagen, Germany) according to the manufacture’s instruction.
Contamination was excluded by re-extraction of these same samples in another
laboratory by a different person.
DNAs from liver biopsy samples were extracted by a person who had never
contacted with B19 virus in another laboratory.
2.2.3 Polymerase chain reaction (PCR).
Nested PCR (nPCR) and quantitative real time PCR (qPCR) were performed to
detect B19 DNA. Primers specific for the VP1/VP2 structural sequence were
employed in nPCR as described elsewhere [64]. Briefly, the primer pairs for the first
round of PCR were as follows: sense 5’-AGCATGTGGAGTGAGGGGGC-3’ and
anti-sense 5’-AAAGCATCAGGAGCTATACTTCC-3’. The primers for second
round were sense 5’-GCTAACTCTGTAACTTGTAC-3’ and anti-sense 5’-
AAATATCTCCATG GGGTTGAG (NCBI GenBank accession No. U38509). The
whole reaction consisted of two 35-cycle programs (1 cycle= 94°C for 30 seconds,
50°C for 30 seconds and 72°C for 45 seconds)
Quantitative real-time PCR (qPCR) was performed using the Parvo B19 artus
LC-PCR kit (Qiagen, Germany) according to the manufacture’s instruction. A qPCR
of genomic C-reactive protein (CRP) DNA was performed with same samples in
Role of Parvovirus B19 infection in hepatitis C -- Subjects and Methods
10
order to determine the amount of human CRP DNA representing the actual amount of
amplifiable cellular DNA in each sample [65, 66]. B19 copy numbers per cell were
calculated from the amplification of B19 divided by the amount of human CRP DNA.
Samples were defined as a “serologically recovered” cohort by the presence of
anti-B19 IgG with the absence of IgM and DNA.
2.2.4 Isolation of PBMCs and carboxy fluorescein succinimidyl ester (CFSE)-
based T cell proliferation assay.
PBMCs were separated from heparinized blood samples by gradient
centrifugation on Ficoll-Paque and stored in liquid nitrogen until used.
CFSE-based T cell proliferation assay was performed as described previously
[11]. Briefly, frozen PBMCs were thawed and suspended at 107/ml in PBS plus 0.2%
BSA and incubated at 37°C for 7 min with 2.5ųM CFSE (Sigma, USA). An equal
volume of FCS was added thereafter and cells were incubated on ice for 5 min to stop
reaction, followed by 3 times washing. Then, labelled cells were resuspended in
medium (RPMI 1640 supplemented with 10% inactive AB serum and penicillin),
plated at 0.3*106 cells per 200ul per well in round-bottom 96-well microtiter plates
and cultured with DMSO alone (background), synthetic peptides (VP1/2 7.2 and
VP1/2 4.7 [47], ProImmune, UK, Table 2) at a final concentration of 1ųg/ml and
5ųg/ml, tetanus toxoid (TT, 3ųg/ml) and PHA (6ųg/ml) as positive controls at 37°C
with 5% CO2 for 7 days. Each condition was duplicated.
PBMCs from patients with chronic HCV infection were stimulated also with
recombinant genotype 1a-derived HCV core, NS3 and NS4 protein (Microgen,
Germany) at a final concentration of 1ųg/ml.
Flow cytometric analysis: at the time of harvest, CFSE-labelled PBMCs were
washed in PBS containing 2% FCS and 0.05% sodium azide, and stained with the
following antibodies: anti-human CD4-APC, CD3-PE (Becton Dickson, Germany) at
4°C. Flow cytometric data (100,000 nongated events) were acquired on a BD
FACSCalibur 4-color flow cytometer using BD Cellquest software (both from BD
Biosciences). For analysis, BD FlowJo 6.1.1 (Treestar, Ashland, OR, USA) was used
to gate on CD4+CD3
+ T cell populations. The number of cells that had proliferated
was determined by gating on the lineage-positive CFSElow
and CFSEhigh
subset. The
background of CD4+ T cells proliferative frequency (%) was calculated as the number
of CFSElow
CD4+ T cells /(numbers of CFSE
low CD4
+ T cells + numbers of CFSE
high
Role of Parvovirus B19 infection in hepatitis C -- Subjects and Methods
11
CD4+ T cells) *100 in the absence of antigen. The PF was calculated by subtracting
the mean background proliferation from the proliferating fraction in response to
specific antigen. The SI was calculated by dividing the antigen-induced PF by the
background PF. Both a PF of 1.0% or more and an SI of 2.0 or more are considered
as a positive response, as previously defined [67, 68] .
Table 2: Synthetic peptide specification
B19 region CD4+ T-cell-restricted peptide sequence peptide no
VP1/2 FLIPYDPEHHYKVFSPAASS 4.7
VP1/2 LASEESAFYVLEHSSFQLLG 7.2
Role of Parvovirus B19 infection in hepatitis C -- Results
12
3. Results
3.1 Prevalence of B19 serologically recovered infection and the presence of B19
DNA in serum samples from patients with chronic hepatitis C and B infection.
Anti-B19 IgG antibodies were found in 67/91 (74%) of the chronic HCV
infected patients and in 19/30 (63%) healthy individuals, only one HCV sample tested
positive for B19 IgM antibody. The percentage of patients with serological evidence
for a previous B19 infection was similar in these two groups (Fig. 1).
Figure 1
Figure 1, Prevalence of anti-B19 IgG antibody positive in European individuals
Legend: Anti-B19 IgG antibodies were tested in 30 healthy individuals and 91 chronic
HCV infected patients. The frequency of positive samples is similar in these two
groups.
63
74
37
26
0
10
20
30
40
50
60
70
80
healthy control (n=30) chronic HCV patients (n=91)
freq
uen
cy
(%)
IgG
pos
IgG
neg
Role of Parvovirus B19 infection in hepatitis C -- Results
13
In contrast to previous studies on Asian HCV infected patients [69, 70], all but
one HCV serum were negative for B19 DNA by qPCR. This unique B19 DNA
positive serum sample was also positive for both anti-B19 IgM and IgG. Consecutive
serum samples of the same patient were collected for up to 48 months. The B19
viremia lasted at least one year even under combination antiviral therapy of daily
injections with recombinant interferon alpha-2b plus ribavirin and was not associated
with ALT level or HCV viremia (Fig. 2). However, the patient became B19-negative
during a second course of pegylated interferon alpha 2b plus ribavirin therapy.
Importantly, B19 DNA was also undetectable in all 50 sera collected from HBsAg
positive patients (Table 3). All sera were collected in Germany. Those data are in
contrast to recent data from Vietnamese patients [69]. Thus, B19 viremia is an
extremely rare finding in German patients with viral hepatitis B and C.
Role of Parvovirus B19 infection in hepatitis C -- Results
14
Figure 2
Figure 2: Fluctuation of ALT, HCV-RNA and B19 viremia in the single HCV-
infected patient with B19 viremia.
The patient was followed for 48 months and treated twice with interferon alpha and
ribavirin. B19 viremia lasted at least one year even during the first combination
therapy.
The first combination therapy (from follow-up month 0 to 5) consisted of interferon
alpha-2b plus ribavirin for 22 weeks. The dose of interferon alpha-2b was 10 MU per
day in the first two weeks followed by 3 MU per day for 8 weeks followed by 3 MU
every 2nd
day. The second combination therapy (from follow-up month 13 to 19)
consisted of PEG-interferon alpha-2b (100 µg qw) plus ribavirin for 20 weeks.
Table 3: Frequency of B19 viremia in patients with chronic hepatitis C or B
infection
Number of patients B19 viremia
chronic hepatitis C 91 1
chronic hepatitis B 50 0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 1 2 3 4 5 6 13 15 16 17 18 19 20 27 48
follow-up month
AL
T x
UL
N
0.1
1
10
100
1000
10000
HC
V R
NA
(lo
g IE
/ml)
HCV RNA
1sttherapy 2ndtherapy
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 1 2 3 4 5 6 13 15 16 17 18 19 20 27 48
0.1
1
10
100
1000
10000
ALTALT HCV RNA
pos pos pos pos neg neg neg
1sttherapy 2ndtherapy
B19
DNA
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 1 2 3 4 5 6 13 15 16 17 18 19 20 27 48
follow-up month
AL
T x
UL
N
0.1
1
10
100
1000
10000
HC
V R
NA
(lo
g IE
/ml)
HCV RNA
1sttherapy 2ndtherapy
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 1 2 3 4 5 6 13 15 16 17 18 19 20 27 48
0.1
1
10
100
1000
10000
ALTALTALTALT HCV RNA
pos pos pos pos neg neg neg
1sttherapy 2ndtherapy
B19
DNA
Role of Parvovirus B19 infection in hepatitis C -- Results
15
3.2 Retrospective analysis of Clinical characteristics in relation to B19 serology
Clinical characteristics, histological staging and grading of hepatitis C patients
are shown in Table 4. These 91 chronic HCV infected patients were divided into two
groups based on the serological testing for B19. In addition, the histological staging
and inflammation score of these two groups are shown in Figure 3. Importantly, there
was no significant difference in any of the clinic investigated parameters. Also
histological staging did not differ between the two groups. Thus, past B19 infection
had no apparent effect on the course of HCV infection in this cross-sectional study.
Table 4: Characteristics of HCV-infected patients with or without anti-B19 IgG
antibodies
anti-B19 IgG pos
(n=67)
anti-B19 IgG neg
(n=24)
p
value
Age(years, mean±SD,
range:19-73)
48±10 44±8.7 0.26
Male: 38 Male: 12 Gender
Female: 29 Female: 12
0.5
F score(mean±SD,
range:0-6)
2.6±2.1 2.2±1.5 0.34
Percentage of live
cirrhosisa(%)
23 8
Activity score(mean±SD,
range:0-18)
5.2±2.0 5.2±2.2 1
ALT ULN(times) 3.1±2.3 3.2±2.6 0.91
AST ULN(times) 2.4±1.8 2.3±1.5 0.71
gGT ULN(times) 2.1±2.2 2.2±2.8 0.85
Total bilirubin(umol/l) 12±7.9 13±5.6 0.74
Albumin(g/l) 44±3.4 45±3.1 0.2
Prothrombin time
(Quick value %)
98±23 96±8.6 0.43
Platelets(Tsd/ul) 187±56 187±93 0.97
Presence of B19 viremia 1 0 0.55
Presence of anti-B19 IgM 1 0 0.55
Legend: a, F score >=5. There were no significant differences of clinic characteristics
in these two groups.
Role of Parvovirus B19 infection in hepatitis C -- Results
16
Figure 3: Histological grading and staging chronic hepatitis C patients with and
without anti-B19 IgG antibodies.
Histological staging was available for 85/91 chronic HCV infected patients.
Inflammation scores was available for 82/91 patients. There were no significant
differences between the two groups.
0
1
2
3
4
5
6
Anti-B19 IgG pos Anti-B19 IgG neg
F s
co
re (
ISH
AK
)
0
1
2
3
4
5
6
Anti-B19 IgG pos Anti-B19 IgG neg
F s
co
re (
ISH
AK
)
0
2
4
6
8
10
12
14
16
18
Anti-B19 IgG pos Anti-B19 IgG neg
Infl
am
mati
on
sco
re
0
2
4
6
8
10
12
14
16
18
Anti-B19 IgG pos Anti-B19 IgG neg
Infl
am
mati
on
sco
re
Role of Parvovirus B19 infection in hepatitis C -- Results
17
3.3 Presence of B19 DNA in explanted livers and routine liver biopsy samples
The presence of B19 DNA in liver by qPCR is shown in Figure 4. Surprisingly,
B19 DNA was amplified from more than half of the liver tissues studied and more
frequently detectable in explanted end-stage liver tissues (74%) than in biopsy
samples (44%)(p<0.05). This held also true for non-HCV subgroups (non-HCV
explanted liver vs. non-HCV liver biopsy samples, p<0.05). Importantly, there was no
difference in frequency of B19-DNA detection between HCV-positive and HCV-
negative samples for both explanted livers and routine liver biopsy samples.
In biopsy samples, the presence of B19 DNA was similar in severe liver disease
(F score >=5) and moderate liver disease (F score <5). Moreover, in the same group,
there was no difference of grading (inflammatory activity) in B19 DNA positive
samples versus negative samples.
There was no significant difference of virus copy number per cell both between
explanted liver and routine biopsy samples and HCV-positive compared with HCV-
negative liver samples (Fig. 5). The level of viral load was relatively low.
In the same samples, none was B19 DNA positive by nPCR.
The result of qPCR was reliable as similar results were obtained using re-
extracted DNA from the same samples in different lab by a different person (Part of
data shown in Table 5). Values depicted are the average of two independent qPCR
tests.
Role of Parvovirus B19 infection in hepatitis C -- Results
18
Figure 4
Figure 4: Presence of B19 DNA in end-stage liver disease (explanted livers) and
routine liver biopsy samples.
B19 DNA was investigated in 50 explanted livers and 32 routine liver biopsy samples
by qPCR. B19 DNA was amplified more frequently in explanted end-stage liver
tissues (74%) than in biopsy samples (44%)(p<0.05).
74
44
68
46
77
42
6459
0
10
20
30
40
50
60
70
80
90
exp
lan
ted
liv
ers(
n=
50
)
rou
tin
e li
ver
bio
psy
sam
ple
s(n
=3
2)
exp
lan
ted
liv
ers-
HC
V
sub
gro
up
(n=
19
)
bio
psy
sa
mp
les-
HC
V
sub
gro
up
(n=
13
)
exp
lan
ted
liv
ers-
no
n-H
CV
sub
gro
up
(n=
31
)
bio
psy
sa
mp
les-
no
n-H
CV
sub
gro
up
(n=
19
)
HC
V s
ub
gro
up
(n=
32
)
non
-HC
V
sub
gro
up
(n=
50
)
Pre
se
nce o
f B
19 D
NA
%p<0.05 p<0.05
74
44
68
46
77
42
6459
0
10
20
30
40
50
60
70
80
90
exp
lan
ted
liv
ers(
n=
50
)
rou
tin
e li
ver
bio
psy
sam
ple
s(n
=3
2)
exp
lan
ted
liv
ers-
HC
V
sub
gro
up
(n=
19
)
bio
psy
sa
mp
les-
HC
V
sub
gro
up
(n=
13
)
exp
lan
ted
liv
ers-
no
n-H
CV
sub
gro
up
(n=
31
)
bio
psy
sa
mp
les-
no
n-H
CV
sub
gro
up
(n=
19
)
HC
V s
ub
gro
up
(n=
32
)
non
-HC
V
sub
gro
up
(n=
50
)
Pre
se
nce o
f B
19 D
NA
%p<0.05 p<0.05
Role of Parvovirus B19 infection in hepatitis C -- Results
19
Figure 5
Figure 5: B19 DNA copy numbers per cell in routine liver biopsy samples and
end-stage liver disease (explanted livers)
B19 DNA copy numbers/cell of 14 B19 positive routine liver biopsy samples and 37
explanted livers was calculated from the amplification of B19 divided by the amount
of human CRP DNA representing the actual amount of amplifiable cellular DNA in
each sample. The median of copy numbers/cell in routine liver biopsy samples was
8.29E-05 (in chronic hepatitis C subgroup was 1.38E-04, while in non-HCV liver
disease subgroup was 7.18E-05). The median of copy numbers/cell in explanted livers
was 9.63E-05 (in end-stage hepatitis C subgroup was 6.95E-05, while in non-HCV
end-stage liver disease subgroup was 2.42E-04). There was no significant difference
of virus load in these groups.
The result of qPCR was reliable as similar results were obtained using re-extracted
DNA from the same samples in different lab by a different person. Values depicted
are the average of two independent qPCR tests.
1.00E-06
1.00E-05
1.00E-04
1.00E-03
1.00E-02
1.00E-01
1.00E+00
Co
py
nu
mb
ers/
cell
HCV (n=6) Non-HCV (n=8) HCV (n=13) Non-HCV (n=24)
routine liver biopsy samples explanted livers
Role of Parvovirus B19 infection in hepatitis C -- Results
20
Table 5: Virus copy numbers of B19 DNA in explanted liver tissues using qPCR
with DNA templates extracted in 2 different labs
patient copy numbers/cell(1st qPCR) copy numbers/cell(2
nd qPCR)
PED 0 0
DJN 2.14E-05 2.88E-05
BHN 0 0
MHA 0 0
REH 1.39E-05 2.74E-04
BAD 1.48E-04 1.06E-04
IFA 1.10E-04 1.04E-03
SDR 3.14E-05 8.87E-05
CGO 1.97E-01 2.89E+00
SD 5.33E-01 3.94E-05
BAA 1.81E-05 8.43E-05
EKH 0 0
MVR 7.49E-06 2.09E-04
KTT 0 0
LAE 4.43E-03 2.19E-03
MKT 0 0
KVR 3.33E-05 1.09E-05
BRH 6.38E-05 8.09E-05
MGD 1.24E-03 1.67E-04
DHS 5.48E-05 6.39E-05
GMS 0 2.55E-05
GMA 0 3.15E-05
POA 1.71E-05 0
MKZ 4.61E-05 3.16E-05
WKS 3.35E-03 1.88E-03
FDH 4.39E-04 2.13E-03
SRA 5.29E-05 1.13E-04
SBD 2.64E-04 5.23E-04
BOR 0 0
SAE 8.26E-05 0
FWR 0 0
FAA 3.19E-04 1.16E-04
BMN 0 0
RMS 2.14E-03 5.64E-04
RHD 2.90E-05 1.03E-04
SCA 0 0
LHA 0 0
Role of Parvovirus B19 infection in hepatitis C -- Results
21
3.4 Proliferation of B19 specific CD4+ T cells in chronic HCV infected patients
and healthy individuals with B19 serologically recovered infection
A flow cytometry-CFSE assay was performed to evaluate the proliferation of
B19 specific CD4+ T cell after stimulation of PBMCs with synthetic peptides. In B19
serologically recovered healthy individuals, 3/19 (16%) previous showed a positive
CD4+ T cell responses to the peptide VP1/2 7.2. In chronic HCV infected patients
with B19 serologically recovered 3/13 (23%) patients had positive responses to
peptide VP1/2 7.2 and/or peptide 4.7. Thus the frequency of positive responses was
similar in these two groups. Representative examples of three individuals are shown
in figure 6.
In addition, the positive responses of HCV-specific CD4+ T cells against NS3,
NS4 and core were detectable in 2/13, 5/13 and 0/13 chronic HCV infected patients,
respectively. The summary of CD4+ T cells responses after stimulating with specific
antigen is shown in Table 6.
Role of Parvovirus B19 infection in hepatitis C -- Results
22
Figure 6
Figure 6 A1 & A2
1 10 100 1000 10000
1
10
100
1000
10000
2.98 58.9
34.83.27
1 10 100 1000 10000
1
10
100
1000
10000
2.62 60.3
35.21.88
1 10 100 1000 10000
1
10
100
1000
10000
2.43 59.2
33.84.55
DMSO
1 10 100 1000 10000
1
10
100
1000
10000
39.1 23.2
14.623.1
Tetanus (3μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
41.6 10.7
7.1240.5
PHA (6μμμμ g /ml)
HCV NS3 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
10.3 54.7
30.94.09
HCV NS4 (1μμμμ g/ml) HCV core (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
2.65 58.9
34.93.53
VP1/2 4.7 (5μμμμ g /ml)
1 10 100 1000 10000
1
10
100
1000
10000
5.98 57.8
33.92.3
VP1/2 4.7 (1μμμμ g /ml)
1 10 100 1000 10000
1
10
100
1000
10000
8.15 53.2
31.76.95
VP1/2 7.2 (1μμμμ g /ml)
1 10 100 1000 10000
1
10
100
1000
10000
7.32 55.8
31.85.03
VP1/2 7.2 (5μμμμ g /ml)
CFSE
CD4+T
cell
1 10 100 1000 10000
1
10
100
1000
10000
2.98 58.9
34.83.27
1 10 100 1000 10000
1
10
100
1000
10000
2.98 58.9
34.83.27
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
2.98 58.9
34.83.27
2.982.98 58.958.9
34.834.83.273.27
1 10 100 1000 10000
1
10
100
1000
10000
2.62 60.3
35.21.88
1 10 100 1000 10000
1
10
100
1000
10000
2.62 60.3
35.21.88
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
2.62 60.3
35.21.88
2.622.62 60.360.3
35.235.21.881.88
1 10 100 1000 10000
1
10
100
1000
10000
2.43 59.2
33.84.55
DMSO
1 10 100 1000 10000
1
10
100
1000
10000
39.1 23.2
14.623.1
Tetanus (3μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
41.6 10.7
7.1240.5
PHA (6μμμμ g /ml)
HCV NS3 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
10.3 54.7
30.94.09
HCV NS4 (1μμμμ g/ml) HCV core (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
2.65 58.9
34.93.53
VP1/2 4.7 (5μμμμ g /ml)
1 10 100 1000 10000
1
10
100
1000
10000
5.98 57.8
33.92.3
VP1/2 4.7 (1μμμμ g /ml)
1 10 100 1000 10000
1
10
100
1000
10000
8.15 53.2
31.76.95
VP1/2 7.2 (1μμμμ g /ml)
1 10 100 1000 10000
1
10
100
1000
10000
7.32 55.8
31.85.03
VP1/2 7.2 (5μμμμ g /ml)
CFSE
CD4+T
cell
1 10 100 1000 10000
1
10
100
1000
10000
2.43 59.2
33.84.55
1 10 100 1000 10000
1
10
100
1000
10000
2.43 59.2
33.84.55
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
2.43 59.2
33.84.55
2.432.43 59.259.2
33.833.84.554.55
DMSO
1 10 100 1000 10000
1
10
100
1000
10000
39.1 23.2
14.623.1
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
39.1 23.2
14.623.1
39.139.1 23.223.2
14.614.623.123.1
Tetanus (3μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
41.6 10.7
7.1240.5
1 10 100 1000 10000
1
10
100
1000
10000
41.6 10.7
7.1240.5
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
41.6 10.7
7.1240.5
41.641.6 10.710.7
7.127.1240.540.5
PHA (6μμμμ g /ml)
HCV NS3 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
10.3 54.7
30.94.09
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
10.3 54.7
30.94.09
10.310.3 54.754.7
30.930.94.094.09
HCV NS4 (1μμμμ g/ml) HCV core (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
2.65 58.9
34.93.53
1 10 100 1000 10000
1
10
100
1000
10000
2.65 58.9
34.93.53
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
2.65 58.9
34.93.53
2.652.65 58.958.9
34.934.93.533.53
VP1/2 4.7 (5μμμμ g /ml)
1 10 100 1000 10000
1
10
100
1000
10000
5.98 57.8
33.92.3
1 10 100 1000 10000
1
10
100
1000
10000
5.98 57.8
33.92.3
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
5.98 57.8
33.92.3
5.985.98 57.857.8
33.933.92.32.3
VP1/2 4.7 (1μμμμ g /ml)
1 10 100 1000 10000
1
10
100
1000
10000
8.15 53.2
31.76.95
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
8.15 53.2
31.76.95
8.158.15 53.253.2
31.731.76.956.95
VP1/2 7.2 (1μμμμ g /ml)
1 10 100 1000 10000
1
10
100
1000
10000
7.32 55.8
31.85.03
1 10 100 1000 10000
1
10
100
1000
10000
7.32 55.8
31.85.03
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
7.32 55.8
31.85.03
7.327.32 55.855.8
31.831.85.035.03
VP1/2 7.2 (5μμμμ g /ml)
CFSE
CD4+T
cell
-1
0
1
2
3
4
5
6
7
8
9
10
DMSO VP1/2 4.7
5 g/m l
VP1/2 4.7
1 g/m l
VP1/2 7.2
5 g/m l
VP1/2 7.2
1 g/m l
NS3
1 g/m l
NS4
1 g/m l
Core
1 g/m l
TT
3 g/m l
CD
3+C
D4+
T c
ell
pro
life
rati
on
s
SI
PF
Role of Parvovirus B19 infection in hepatitis C -- Results
23
Figure 6 B1 & B2
1 10 100 1000 10000
1
10
100
1000
10000
0.18 52.2
47.40.22
DMSO
1 10 100 1000 10000
1
10
100
1000
10000
4.01 46.4
42.57.07
Tetanus (3μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
25.6 29.2
20.824.5
PHA (6μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
1.31 51.3
46.41.02
HCV NS4 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.32 53.5
45.90.23
HCV core (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.9 51.2
46.91
HCV NS3 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.27 52.8
46.60.3
VP1/2 4.7 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.52 52.5
46.60.45
VP1/2 4.7 (5μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.28 52.8
46.70.21
VP1/2 7.2 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.29 52.3
46.90.46
VP1/2 7.2 (5μμμμ g/ml)
CD4+T
cell
CFSE
1 10 100 1000 10000
1
10
100
1000
10000
0.18 52.2
47.40.22
DMSO
1 10 100 1000 10000
1
10
100
1000
10000
0.18 52.2
47.40.22
1 10 100 1000 10000
1
10
100
1000
10000
0.18 52.2
47.40.22
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
0.18 52.2
47.40.22
0.180.18 52.252.2
47.447.40.220.22
DMSO
1 10 100 1000 10000
1
10
100
1000
10000
4.01 46.4
42.57.07
Tetanus (3μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
4.01 46.4
42.57.07
1 10 100 1000 10000
1
10
100
1000
10000
4.01 46.4
42.57.07
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
4.01 46.4
42.57.07
4.014.01 46.446.4
42.542.57.077.07
Tetanus (3μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
25.6 29.2
20.824.5
PHA (6μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
25.6 29.2
20.824.5
1 10 100 1000 10000
1
10
100
1000
10000
25.6 29.2
20.824.5
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
25.6 29.2
20.824.5
25.625.6 29.229.2
20.820.824.524.5
PHA (6μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
1.31 51.3
46.41.02
HCV NS4 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
1.31 51.3
46.41.02
1 10 100 1000 10000
1
10
100
1000
10000
1.31 51.3
46.41.02
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
1.31 51.3
46.41.02
1.311.31 51.351.3
46.446.41.021.02
HCV NS4 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.32 53.5
45.90.23
HCV core (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.32 53.5
45.90.23
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
0.32 53.5
45.90.23
0.320.32 53.553.5
45.945.90.230.23
HCV core (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.9 51.2
46.91
HCV NS3 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.9 51.2
46.91
1 10 100 1000 10000
1
10
100
1000
10000
0.9 51.2
46.91
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
0.9 51.2
46.91
0.90.9 51.251.2
46.946.911
HCV NS3 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.27 52.8
46.60.3
VP1/2 4.7 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.27 52.8
46.60.3
1 10 100 1000 10000
1
10
100
1000
10000
0.27 52.8
46.60.3
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
0.27 52.8
46.60.3
0.270.27 52.852.8
46.646.60.30.3
VP1/2 4.7 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.52 52.5
46.60.45
VP1/2 4.7 (5μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.52 52.5
46.60.45
1 10 100 1000 10000
1
10
100
1000
10000
0.52 52.5
46.60.45
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
0.52 52.5
46.60.45
0.520.52 52.552.5
46.646.60.450.45
VP1/2 4.7 (5μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.28 52.8
46.70.21
VP1/2 7.2 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.28 52.8
46.70.21
1 10 100 1000 10000
1
10
100
1000
10000
0.28 52.8
46.70.21
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
0.28 52.8
46.70.21
0.280.28 52.852.8
46.746.70.210.21
VP1/2 7.2 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.29 52.3
46.90.46
VP1/2 7.2 (5μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
0.29 52.3
46.90.46
1 10 100 1000 10000
1
10
100
1000
10000
0.29 52.3
46.90.46
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
0.29 52.3
46.90.46
0.290.29 52.352.3
46.946.90.460.46
VP1/2 7.2 (5μμμμ g/ml)
CD4+T
cell
CD4+T
cell
CFSECFSE
-1
0
1
2
3
4
5
6
7
8
9
10
DMSO VP1/2 4.7
5 g/ml
VP1/2 4.7
1 g/ml
VP1/2 7.2
5 g/ml
VP1/2 7.2
1 g/ml
NS3
1 g/ml
NS4
1 g/ml
Core
1 g/ml
TT
3 g/ml
CD
3+C
D4
+ T
cell
pro
life
ra
tio
n
SI
PF
Role of Parvovirus B19 infection in hepatitis C -- Results
24
Figure 6 C1 & C2
1 10 100 1000 10000
1
10
100
1000
10000
3.84 55.7
37.13.37
DMSO
1 10 100 1000 10000
1
10
100
1000
10000
29 26.5
14.729.8
PHA (6μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
5.41 52.3
35.86.55
VP1/2 4.7 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
6.71 53.5
35.64.19
VP1/2 4.7 (5μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
10.1 44.7
30.814.4
VP1/2 7.2 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
5.01 52.9
36.35.76
VP1/2 7.2 (5μμμμ g/ml)
CD4+T cell
CFSE
1 10 100 1000 10000
1
10
100
1000
10000
3.84 55.7
37.13.37
DMSO
1 10 100 1000 10000
1
10
100
1000
10000
3.84 55.7
37.13.37
1 10 100 1000 10000
1
10
100
1000
10000
3.84 55.7
37.13.37
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
3.84 55.7
37.13.37
3.843.84 55.755.7
37.137.13.373.37
DMSO
1 10 100 1000 10000
1
10
100
1000
10000
29 26.5
14.729.8
PHA (6μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
29 26.5
14.729.8
1 10 100 1000 10000
1
10
100
1000
10000
29 26.5
14.729.8
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
29 26.5
14.729.8
2929 26.526.5
14.714.729.829.8
PHA (6μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
5.41 52.3
35.86.55
VP1/2 4.7 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
5.41 52.3
35.86.55
1 10 100 1000 10000
1
10
100
1000
10000
5.41 52.3
35.86.55
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
5.41 52.3
35.86.55
5.415.41 52.352.3
35.835.86.556.55
VP1/2 4.7 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
6.71 53.5
35.64.19
VP1/2 4.7 (5μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
6.71 53.5
35.64.19
1 10 100 1000 10000
1
10
100
1000
10000
6.71 53.5
35.64.19
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
6.71 53.5
35.64.19
6.716.71 53.553.5
35.635.64.194.19
VP1/2 4.7 (5μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
10.1 44.7
30.814.4
VP1/2 7.2 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
10.1 44.7
30.814.4
1 10 100 1000 10000
1
10
100
1000
10000
10.1 44.7
30.814.4
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
10.1 44.7
30.814.4
10.110.1 44.744.7
30.830.814.414.4
VP1/2 7.2 (1μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
5.01 52.9
36.35.76
VP1/2 7.2 (5μμμμ g/ml)
1 10 100 1000 10000
1
10
100
1000
10000
5.01 52.9
36.35.76
1 10 100 1000 10000
1
10
100
1000
10000
5.01 52.9
36.35.76
1 10 100 1000 10000
1
10
100
1000
10000
1 10 100 1000 100001 10 100 1000 100001 10 100 1000 10000
1
10
100
1000
10000
1
10
100
1000
10000
1
10
100
1000
10000
5.01 52.9
36.35.76
5.015.01 52.952.9
36.336.35.765.76
VP1/2 7.2 (5μμμμ g/ml)
CD4+T cellCD4+T cell
CFSECFSE
-1
0
1
2
3
4
5
6
7
8
9
10
DMSO VP1/2 4.7
5 g/ml
VP1/2 4.7
1 g/ml
VP1/2 7.2
5 g/ml
VP1/2 7.2
1 g/ml
PHA 6 g/ml
CD
3+C
D4
+ T
cel
l p
roli
fera
tio
n
SI
PF
Role of Parvovirus B19 infection in hepatitis C -- Results
25
Figure 6: Proliferation of CD4+ T cells in HCV-infected patients and healthy
individuals with B19 serologically recovered infection.
Dot plots (A1, B1, C1) show the flow cytometric analysis of the proportion of CD4+ T
cells proliferating in response to stimulation of PBMCs with no stimulus (DMSO, as
background), phytohemagglutinin (PHA, as positive control), tetanus toxoid (TT, as
positive control), two synthetic B19 peptides (VP1/2 4.7, VP1/2 7.2) in two different
concentrations (1ųg/ml and 5ųg/ml) or recombinant HCV NS3, NS4 and core protein.
Values in the upper left quadrants of dot plots represent the proportion of CFSElow
CD4+ T cells (daughter cells), which have proliferated during the 7-day culture.
Values in the upper right quadrants of dot plots are CFSEhigh
CD4+ T cells (parent
cells). Column figures (A2, B2, C2) represent the stimulation index (SI) and
proliferating fraction ( PF) of the respective individual. Both a PF of 1.0% or more
and an SI of 2.0 or more were required for a positive response. The PF was
calculated by subtracting the mean background proliferation from the proliferating
fraction in response to specific antigen. The SI was calculated by dividing the antigen-
induced PF by the background PF.
A1 and A2 show the positive responses of CD4+ T cells in an anti-B19 IgG antibody
positive chronic hepatitis C patient after stimulation of PBMCs with VP1/2 4.7,
VP1/2 7.2 and HCV NS4. B1 and B2 show the negative responses of CD4+ T cells in
an anti-B19 IgG antibody positive chronic hepatitis C patient after stimulation of
PBMCs with B19 peptides, while positive with HCV antigen (NS3 and NS4). C1 and
C2 show the positive responses of CD4+ T cells in an anti-B19 IgG antibody positive
healthy control after stimulation of PBMCs with VP1/2 7.2
Role of Parvovirus B19 infection in hepatitis C -- Results
26
Table 6: Overview of CD4+ T cells responses determined by CFSE assay of
PBMCs in the presence or absence of antigen in anti-B19 IgG antibody positive
individuals.
VP1/2 4.7 VP1/2 7.2 HCV NS3 HCV NS4 Core
Anti-B19 IgG antibody positive healthy individuals
HI 1 - - NT NT NT
HI 2 - - NT NT NT
HI 3 - - NT NT NT
HI 4 - - NT NT NT
HI 5 - - NT NT NT
HI 6 - - NT NT NT
HI 7 - + (SI=2.3; PF=9) NT NT NT
HI 8 - - NT NT NT
HI 9 - - NT NT NT
HI 10 - - NT NT NT
HI 11 - - NT NT NT
HI 12 - - NT NT NT
HI 13 - - NT NT NT
HI 14 - + (SI=2.4; PF=6.3) NT NT NT
HI 15 - - NT NT NT
HI 16 - - NT NT NT
HI 17 - + (SI=2; PF=2.3) NT NT NT
HI 18 - - NT NT NT
HI 19 - - NT NT NT
Anti-B19 IgG antibody positive chronic hepatitis C patients
HCV 1 - - - - -
HCV 2 - - - - -
HCV 3 - - + (SI=3.6; PF=1) + (SI=5; PF=1.5)
HCV 4 - - - - -
HCV 5 - - - - -
HCV 6 + (SI=2.2; PF=3.6) + (SI=3.7; PF=7.8) - + (SI=6.8; PF=16)
HCV 7 + (SI=2.6; PF=1) - - + (SI=6; PF=3)
HCV 8 - - - - -
HCV 9 - + (SI=2.3; PF=1.6) + (SI=2.7; PF=2) + (SI=2.3; PF=1.6)
HCV 10 - - - - -
HCV 11 - - - + (SI=2.6; PF=1.5) -
HCV 12 - - - - -
HCV 13 - - - - -
A positive response is designated both a PF of 1.0% or more and an SI of 2.0 or more.
NT, not tested.
Role of Parvovirus B19 infection in hepatitis C -- Discussion
27
4. Discussion
Parvovirus B19 is the causative agent of erythema infectiosum (fifth disease) [71,
72]. Clinical manifestations of B19 infection can range from mild disease with or
without immune-mediated symptoms (e.g. rash or arthralgia) to severe symptoms,
such as acute or persistent arthralgia [73], hydrops fetalis, intrauterine fetal death [74,
75], myocarditis [76, 77]. Moreover a number of hematologic disorders (e.g. aplastic
anemia and congenital anemia) has been linked to B19 [78]. However, in an
immunocompromised host, B19 infection may result in persistent infection with
chronic pure red cell aplasia and virus-associated hemophagocytic syndrome (VAHS)
[79] .
An overview over B19 antibody prevalence shows[23] an age-related difference.
However, there is no epidemiological difference within continentals. It is observed
that from May 2004 to Jan. 2006 was a high incidence period in Germany, by testing
2.8 million donations from Germany and Austria for B19 DNA since 2000. The
positive frequency of B19 DNA was 274 per 100,000 donations [80].
B19 is thought to be a “hit and run” virus which can be completely eliminated.
However, it was reported that B19 may persist in bone marrow [81-84], muscle [85],
synovia [86] and skin [87] in immunocompetent individuals with the absence of
viremia or symptomatic infection. Likewise, three groups demonstrated the
persistence of B19 DNA in the liver [54, 55, 57]. Whether B19 is a pathogenic agent
of fulminant liver failure and non-A-E hepatitis or a major risk factor to worsen liver
function and accelerate disease progression is still controversial. One study from
China suggested that the co-infection of B19 and HBV did not lead to more severe
liver damage [56]. Nevertheless, two other studies on B19 infection in patients with
chronic viral hepatitis revealed different conclusions. Hsu and colleagues [70] from
Taiwan detected B19 DNA in serum samples from HBV patients (37%) and HCV
patients (24%) by nPCR. Furthermore, IgM antibodies were detected in HBV- (35%)
and HCV- (16%) infected individuals respectively. However, the co-infection of B19
with HBV or HCV did not increase the frequency of liver dysfunction in patients with
chronic hepatitis. In contrast, Toan and colleagues [69] demonstrated that B19
infection was not only frequent in HBV-infected Vietnamese patients but also
obviously associated with severe hepatitis B-associated liver disease. They also
showed that B19 DNA may be persistent in hepatitis B patients. By using multivariate
analysis they also demonstrated a positive correlation between the serum B19 viral
Role of Parvovirus B19 infection in hepatitis C -- Discussion
28
load with the HBV viral load and serum ALT. From this they concluded that B19
might play an important role in the pathogenesis of HBV in Vietnamese patients.
In order to find out whether B19 acts indeed as a bystander or a risk factor in the
progression of liver disease, we assessed the prevalence of B19 infection in chronic
hepatitis C and B patients, as well as healthy individuals in Germany. The frequency
of serologically recovered B19 infection was similar in HCV patients and healthy
individuals, and corresponded to the average level reported previously [51].
Compared with anti-B19 seronegative chronic HCV infected patients, biochemical
parameters indicating liver disease (e.g. ALT, AST, gGT, total bilirubin, albumin,
prothrombin time and platelets) as well as histological staging and grading of chronic
HCV infected patients with serologically recovered B19 infection were not
aggravated. This finding was consistent with two Asian studies [56, 70], but in
contrast to the previous study on Vietnamese HBV-patients [69]. Our study is the first
study investigating the role of B19 infection in European patients. Our findings
clearly suggest that B19 does neither worsen liver disease nor accelerate disease
progression of chronic hepatitis C in German patients.
The present study gave no evidence for frequent B19 viremia in chronic C and B
infected patients (only one patient was positive for viremia) by the most sensitive
method (qPCR). By contrast, the study on Taiwanese hepatitis B and C patients using
nPCR and southern blot analysis [69] and the study on Vietnamese hepatitis B
patients using nPCR and qPCR [69] reported a high prevalence of B19 viremia. We
do not think that a technical problem to amplify B19 DNA was responsible for the
low frequency of B19 DNA in our study. First, the positive control worked always
well and secondly, the technique applied was able to detect B19 DNA at high
frequencies in liver tissue samples. Therefore, the most likely explanation for the
different results of different studies is that B19 viremia is more frequent in East-Asia
than in Germany. Further studies on additional cohorts in Europe and other regions of
the world need to clarify this issue.
The only patient, who had viremia for more than one year even during antiviral
therapy, was chronically infected with HCV and had no B19-related symptoms.
Prolonged B19 infection seems possible because the clearance of B19 viremia from
healthy hosts has been reported to be more slower than previously considered, despite
early resolution of symptoms [26]. Thus our case would be in line with this report.
Whether the second course of high dose antiviral therapy with interferon alpha-2b
Role of Parvovirus B19 infection in hepatitis C -- Discussion
29
(Pegintron) and ribavirin has contributed to clearance of B19 viremia remains
speculative. In this respect it is worth noting that treatment trials for infectious
myocarditis with interferon beta are ongoing [88]. It could well be that interferon
alpha-2b could also have a significant antiviral effect against B19. However, despite
clearance of B19 during the PEG-interferon alpha-2b treatment, our patient was a
B19-interferon nonresponder to the first course of high-dose interferon alpha-2b
therapy.
Furthermore, we could amplify B19 DNA from more than half of the liver
tissues studied. B19 DNA was found more frequently in explanted liver tissues
undergoing orthotopic liver transplantation than in routine biopsy samples, although
there was no significant difference in virus copy number per cell between these two
groups. The presence of B19 DNA in explanted liver for different kinds of liver
disease has been reported previously ranging from 24%-66%[54, 55, 57, 89]. Eis-
Huebinger and colleagues [54] demonstrated that B19 DNA was present frequently in
livers of adults with severe liver damage by comparing randomly selected liver tissues
of 43 patients undergoing orthotopic transplantation for various reasons with 23
autopsied adults without liver disease (only one alcoholic liver cirrhosis). B19 DNA
was also detected in one liver biopsy sample from an anti-B19 IgM antibody positive
Brazilian patient with non-A-E hepatitis [90]. In our study, the frequency of B19
DNA in explanted liver was higher than in previous studies. Moreover, this is the first
study to report the persistent B19 DNA in routine biopsy liver samples. Overall, our
findings are in line with the Eis-Huebinger study [54] as we also found B19 DNA
more often in end-stage liver disease. However, whether detection of B19 DNA is
cause or consequence of progressive liver disease is unknown. Only a prospective
study comparing the outcome of liver disease in individuals with detectable and
undetectable B19 DNA in liver biopsies would be able to answer this question.
As reported previously, B19 DNA were frequently detected in liver while
negative for both viremia and anti-B19 IgM antibody [54, 55]. In our study, B19 DNA
was detectable in more than half of studied livers, while only one of 91 chronic HCV
infected patients was anti-B19 IgM positive accompanied by B19 viremia. Thus, and
importantly the absence of viremia cannot be designated as the proof of the clearance
of B19.
B19 specific CD4+ T cell responses have been first described in 1996 using
recombinant protein in IgG positive but IgM negative individuals [40]. Thereafter,
Role of Parvovirus B19 infection in hepatitis C -- Discussion
30
several studies [40, 91, 92] reported that in serologically recovered individuals,
compared with NS1, recombinantly expressed VP1, VP2, VP1/2 and VP1u were
major targets of CD4+ T cells that showed vigorous proliferation. Franssila and his
group[44] reported that CD4+ Th cells were the main responding cells and could
establish memory. In 2006, Kasprowicz and colleagues [47] characterized the first
B19 CD4+ T cell epitopes using overlapping peptides.
We employed two key immunodominant peptides identified by Kasprowicz etal
to analyze B19 specific CD4+ T cell proliferation in B19 serologically recovered
individuals using a sensitive flow cytometry-based CFSE-assay which can investigate
T cell proliferation on a single-cell basis. Few positive responses were detected in
both chronic HCV infected patients and healthy individuals with B19 serologically
recovered at the same frequency. Thus our study did not reveal an impairment of
memory T cell responses to a pathogen the patient had been exposed to in the past.
This finding is of importance as HCV may alter the function of several immune cells
including dendritic cells [4, 93] (Ciesek S. et al, Impaired TRAIL-dependent
cytotoxicity of CD1c-positive dendritic cells in chronic hepatitis C virus infection
J.Virol. Hepatitis, in press). In our study, HCV-specific CD4+ T cell responses against
recombinant HCV protein NS3, NS4 and core were detectable in 2/13, 5/13 and 0/13
chronic HCV infected patients with serologically recovered B19 infection,
respectively. This frequency of HCV-specific CD4+ T cell response was similar to
most previous studies on chronic HCV infected patients before therapy [94] but lower
than in patients after spontaneous HCV clearance [95]. Aberle and coworker [94]
analyzed CD4+ T cells responses in patients with chronic HCV infection and reported
that before treatment, HCV-specific Th1 responses against NS3-4 were detected in
only 4 (13%) of the 31 patients. Meyer and colleagues [95] reported that weak
transient HCV-specific CD4+ T cell responses against at least one HCV protein (core,
NS3 and NS4) were detected in five of the seven patients with spontaneous HCV
clearance, which had the same level of magnitude and extent in age- and sex-matched
chronic hepatitis C and long-term recovered patients.
Previous infections and ongoing infections with third pathogens may have
significant consequences for the outcome of a viral infection. This phenomenon is
called “heterologous immunity” [16]. The consequence of heterologous immunity can
be both more frequent recoveries but also more severe pathology. For hepatitis C,
Wedemeyer and colleagues [96] demonstrated that cross-reactive CD8+ T cells
Role of Parvovirus B19 infection in hepatitis C -- Discussion
31
recognizing the HCV epitope NS3-1073 can be induced by influenza virus infection.
It is not known whether there is cross-reactivity between B19 and HCV. In our study,
B19 specific CD4+ T cells positive responses were only detected in anti-B19 IgG
positive individuals. All B19 seronegative individuals had no positive CD4+ T cells
responses against the B19 peptides (data not shown). Moreover, B19-specific T cell
responses were not different between HCV-patients and healthy controls as discussed
above. Thus, at least for the two B19 peptides studied here there is no obvious cross-
reactivity with HCV. However, more studies investigating potential T cell cross
reactivities more systematically and also with different read-outs (cytokine secretion;
cytotoxicity) are necessary. It would also be interesting to investigate T cell responses
in chronic HCV carriers who experience a B19 superinfection.
In summary, B19 DNA may persist in the liver at a low level for a long period
after acute infection. However, even though B19-DNA can be detected
intrahepatically, there is no evidence that B19 is a “hepatitis virus” worsening liver
disease and accelerating disease progression of chronic hepatitis C in European
patients. T cell responses to B19 are also not affected by HCV infection.
Role of Parvovirus B19 infection in hepatitis C -- Summary
32
5. Summary
Parvovirus B19 is the causative agent for fifth disease. Recently, it was reported
that B19 may play an important role in the pathogenesis of HBV in Vietnamese
patients. We here aimed to investigate whether B19 infection may be a co-factor for
disease progression in European patients with chronic hepatitis C.
In this study, 91 serum samples from well characterized and histologically
staged chronic hepatitis C patients and 50 serum samples from chronic hepatitis B
patients were investigated for the presence of B19 antibodies and B19 DNA (real-time
PCR). Moreover, 50 explanted livers and 32 liver biopsy samples (obtained for
routine staging of chronic liver disease) were tested for B19 DNA. Finally, B19-
specific CD4+ T cell responses were studied in 13 anti-B19-positive hepatitis C
patients and 19 serologically recovered B19 infected healthy individuals.
In contrast to the previous study on Vietnamese HBV-patients, we detected B19
DNA in only 1/ 91 serum samples from chronic hepatitis C patients and in none of the
serum samples from chronic hepatitis B patients. B19 IgG antibodies were found in
67/91 (74%) HCV patients, while only the B19 DNA-positive hepatitis C patient was
anti-B19-IgM positive. Clinical and histological characteristics did not differ between
anti-B19-IgG-positive and IgG-negative patients. Surprisingly, B19 DNA was
amplified from more than half of the liver tissues studied and more frequently
detectable in explanted end-stage liver tissues (74%) than in biopsy samples
(44%)(p<0.05). However, there was no significant difference in virus copy number
per cell between these two groups. Finally, B19-specific CD4+ T cell responses were
detected in a similar frequency in healthy anti-B19-positive individuals (3/19; 16%)
and chronic hepatitis C patients (3/13; 23%).
In conclusion, this study gives evidence that B19 may persist in the liver.
However, even though B19-DNA can be detected intrahepatically, there is no
evidence that B19 is a “hepatitis virus” worsening liver disease and accelerating
disease progression of chronic hepatitis C in European patients.
Role of Parvovirus B19 infection in hepatitis C -- Reference
33
6. Reference
1. Choo, Q.L., et al., Isolation of a cDNA clone derived from a blood-borne non-
A, non-B viral hepatitis genome. Science, 1989. 244(4902): p. 359-62.
2. Touzet, S., et al., Epidemiology of hepatitis C virus infection in seven
European Union countries: a critical analysis of the literature. HENCORE
Group. (Hepatitis C European Network for Co-operative Research. Eur J
Gastroenterol Hepatol, 2000. 12(6): p. 667-78.
3. Manns, M.P., H. Wedemeyer, and M. Cornberg, Treating viral hepatitis C:
efficacy, side effects, and complications. Gut, 2006. 55(9): p. 1350-9.
4. Rehermann, B. and M. Nascimbeni, Immunology of hepatitis B virus and
hepatitis C virus infection. Nat Rev Immunol, 2005. 5(3): p. 215-29.
5. Evans, M.J., et al., Claudin-1 is a hepatitis C virus co-receptor required for a
late step in entry. Nature, 2007. 446(7137): p. 801-5.
6. Matos, C.A., et al., Steatosis in chronic hepatitis C: relationship to the virus
and host risk factors. J Gastroenterol Hepatol, 2006. 21(8): p. 1236-9.
7. Lonardo, A., et al., Hepatitis C and steatosis: a reappraisal. J Viral Hepat,
2006. 13(2): p. 73-80.
8. Rumi, M.G., et al., Hepatitis C reactivation in patients with chronic infection
with genotypes 1b and 2c: a retrospective cohort study of 206 untreated
patients. Gut, 2005. 54(3): p. 402-6.
9. Bruno, S., et al., Hepatitis C virus genotype 1b as a major risk factor
associated with hepatocellular carcinoma in patients with cirrhosis: a
seventeen-year prospective cohort study. Hepatology, 2007. 46(5): p. 1350-6.
10. Thimme, R., et al., Determinants of viral clearance and persistence during
acute hepatitis C virus infection. J Exp Med, 2001. 194(10): p. 1395-406.
11. Wedemeyer, H., et al., Impaired effector function of hepatitis C virus-specific
CD8+ T cells in chronic hepatitis C virus infection. J Immunol, 2002. 169(6):
p. 3447-58.
12. Diepolder, H.M., et al., Possible mechanism involving T-lymphocyte response
to non-structural protein 3 in viral clearance in acute hepatitis C virus
infection. Lancet, 1995. 346(8981): p. 1006-7.
13. Lechner, F., et al., Analysis of successful immune responses in persons
infected with hepatitis C virus. J Exp Med, 2000. 191(9): p. 1499-512.
14. Kim, A.Y., et al., Impaired hepatitis C virus-specific T cell responses and
recurrent hepatitis C virus in HIV coinfection. PLoS Med, 2006. 3(12): p.
e492.
15. Strickland, G.T., Liver disease in Egypt: hepatitis C superseded
schistosomiasis as a result of iatrogenic and biological factors. Hepatology,
2006. 43(5): p. 915-22.
16. Welsh, R.M., et al., The privacy of T cell memory to viruses. Curr Top
Microbiol Immunol, 2006. 311: p. 117-53.
17. Selin, L.K., et al., CD8 memory T cells: cross-reactivity and heterologous
immunity. Semin Immunol, 2004. 16(5): p. 335-47.
18. Jaeckel, E., et al., Treatment of acute hepatitis C with interferon alfa-2b. N
Engl J Med, 2001. 345(20): p. 1452-7.
19. Wiegand, J., et al., Early monotherapy with pegylated interferon alpha-2b for
acute hepatitis C infection: the HEP-NET acute-HCV-II study. Hepatology,
2006. 43(2): p. 250-6.
20. Cossart, Y.E., et al., Parvovirus-like particles in human sera. Lancet, 1975.
1(7898): p. 72-3.
Role of Parvovirus B19 infection in hepatitis C -- Reference
34
21. Anderson, L.J., et al., Detection of antibodies and antigens of human
parvovirus B19 by enzyme-linked immunosorbent assay. J Clin Microbiol,
1986. 24(4): p. 522-6.
22. Cohen, B.J. and M.M. Buckley, The prevalence of antibody to human
parvovirus B19 in England and Wales. J Med Microbiol, 1988. 25(2): p. 151-3.
23. Kelly, H.A., et al., The age-specific prevalence of human parvovirus immunity
in Victoria, Australia compared with other parts of the world. Epidemiol
Infect, 2000. 124(3): p. 449-57.
24. Tsujimura, M., et al., Human parvovirus B19 infection in blood donors. Vox
Sang, 1995. 69(3): p. 206-12.
25. Broliden, K., T. Tolfvenstam, and O. Norbeck, Clinical aspects of parvovirus
B19 infection. J Intern Med, 2006. 260(4): p. 285-304.
26. Lindblom, A., et al., Slow clearance of human parvovirus B19 viremia
following acute infection. Clin Infect Dis, 2005. 41(8): p. 1201-3.
27. Ozawa, K., et al., The gene encoding the nonstructural protein of B19 (human)
parvovirus may be lethal in transfected cells. J Virol, 1988. 62(8): p. 2884-9.
28. Moffatt, S., et al., Human parvovirus B19 nonstructural (NS1) protein induces
apoptosis in erythroid lineage cells. J Virol, 1998. 72(4): p. 3018-28.
29. Ozawa, K. and N. Young, Characterization of capsid and noncapsid proteins
of B19 parvovirus propagated in human erythroid bone marrow cell cultures.
J Virol, 1987. 61(8): p. 2627-30.
30. Shade, R.O., et al., Nucleotide sequence and genome organization of human
parvovirus B19 isolated from the serum of a child during aplastic crisis. J
Virol, 1986. 58(3): p. 921-36.
31. Zadori, Z., et al., A viral phospholipase A2 is required for parvovirus
infectivity. Dev Cell, 2001. 1(2): p. 291-302.
32. Brown, K.E., S.M. Anderson, and N.S. Young, Erythrocyte P antigen: cellular
receptor for B19 parvovirus. Science, 1993. 262(5130): p. 114-7.
33. Weigel-Kelley, K.A., M.C. Yoder, and A. Srivastava, Recombinant human
parvovirus B19 vectors: erythrocyte P antigen is necessary but not sufficient
for successful transduction of human hematopoietic cells. J Virol, 2001. 75(9):
p. 4110-6.
34. Weigel-Kelley, K.A., M.C. Yoder, and A. Srivastava, Alpha5beta1 integrin as
a cellular coreceptor for human parvovirus B19: requirement of functional
activation of beta1 integrin for viral entry. Blood, 2003. 102(12): p. 3927-33.
35. Munakata, Y., et al., Ku80 autoantigen as a cellular coreceptor for human
parvovirus B19 infection. Blood, 2005. 106(10): p. 3449-56.
36. Servant, A., et al., Genetic diversity within human erythroviruses:
identification of three genotypes. J Virol, 2002. 76(18): p. 9124-34.
37. Umene, K. and T. Nunoue, The genome type of human parvovirus B19 strains
isolated in Japan during 1981 differs from types detected in 1986 to 1987: a
correlation between genome type and prevalence. J Gen Virol, 1990. 71 (Pt 4):
p. 983-6.
38. Umene, K. and T. Nunoue, Genetic diversity of human parvovirus B19
determined using a set of restriction endonucleases recognizing four or five
base pairs and partial nucleotide sequencing: use of sequence variability in
virus classification. J Gen Virol, 1991. 72 (Pt 8): p. 1997-2001.
39. Kurtzman, G.J., et al., Immune response to B19 parvovirus and an antibody
defect in persistent viral infection. J Clin Invest, 1989. 84(4): p. 1114-23.
Role of Parvovirus B19 infection in hepatitis C -- Reference
35
40. von Poblotzki, A., et al., Lymphoproliferative responses after infection with
human parvovirus B19. J Virol, 1996. 70(10): p. 7327-30.
41. Tolfvenstam, T., et al., Mapping of B-cell epitopes on human parvovirus B19
non-structural and structural proteins. Vaccine, 2000. 19(7-8): p. 758-63.
42. Isa, A., et al., Prolonged activation of virus-specific CD8+T cells after acute
B19 infection. PLoS Med, 2005. 2(12): p. e343.
43. Norbeck, O., et al., Sustained CD8+ T-cell responses induced after acute
parvovirus B19 infection in humans. J Virol, 2005. 79(18): p. 12117-21.
44. Franssila, R., et al., T helper cell-mediated interferon-gamma expression after
human parvovirus B19 infection: persisting VP2-specific and transient VP1u-
specific activity. Clin Exp Immunol, 2005. 142(1): p. 53-61.
45. Franssila, R. and K. Hedman, T-helper cell-mediated interferon-gamma,
interleukin-10 and proliferation responses to a candidate recombinant vaccine
for human parvovirus B19. Vaccine, 2004. 22(27-28): p. 3809-15.
46. Lindner, J., et al., CD4(+) T-cell responses against the VP1-unique region in
individuals with recent and persistent parvovirus B19 infection. J Vet Med B
Infect Dis Vet Public Health, 2005. 52(7-8): p. 356-61.
47. Kasprowicz, V., et al., Tracking of peptide-specific CD4+ T-cell responses
after an acute resolving viral infection: a study of parvovirus B19. J Virol,
2006. 80(22): p. 11209-17.
48. Rehermann, B., et al., The hepatitis B virus persists for decades after patients'
recovery from acute viral hepatitis despite active maintenance of a cytotoxic
T-lymphocyte response. Nat Med, 1996. 2(10): p. 1104-8.
49. Isa, A., et al., Aberrant cellular immune responses in humans infected
persistently with parvovirus B19. J Med Virol, 2006. 78(1): p. 129-33.
50. Harris, J.W., Parvovirus B19 for the hematologist. Am J Hematol, 1992. 39(2):
p. 119-30.
51. Young, N.S. and K.E. Brown, Parvovirus B19. N Engl J Med, 2004. 350(6): p.
586-97.
52. Heegaard, E.D. and K.E. Brown, Human parvovirus B19. Clin Microbiol Rev,
2002. 15(3): p. 485-505.
53. Kuhl, U., et al., Interferon-beta treatment eliminates cardiotropic viruses and
improves left ventricular function in patients with myocardial persistence of
viral genomes and left ventricular dysfunction. Circulation, 2003. 107(22): p.
2793-8.
54. Eis-Hubinger, A.M., et al., Evidence for persistence of parvovirus B19 DNA in
livers of adults. J Med Virol, 2001. 65(2): p. 395-401.
55. Abe, K., et al., Characterization of erythrovirus B19 genomes isolated in liver
tissues from patients with fulminant hepatitis and biliary atresia who
underwent liver transplantation. Int J Med Sci, 2007. 4(2): p. 105-9.
56. He, Z., et al., Retrospective analysis of non-A-E hepatitis: possible role of
hepatitis B and C virus infection. J Med Virol, 2003. 69(1): p. 59-65.
57. Wong, S., N.S. Young, and K.E. Brown, Prevalence of parvovirus B19 in liver
tissue: no association with fulminant hepatitis or hepatitis-associated aplastic
anemia. J Infect Dis, 2003. 187(10): p. 1581-6.
58. Drago, F., et al., Parvovirus B19 infection associated with acute hepatitis and
a purpuric exanthem. Br J Dermatol, 1999. 141(1): p. 160-1.
59. Hillingso, J.G., I.P. Jensen, and L. Tom-Petersen, Parvovirus B19 and acute
hepatitis in adults. Lancet, 1998. 351(9107): p. 955-6.
Role of Parvovirus B19 infection in hepatitis C -- Reference
36
60. Karetnyi, Y.V., et al., Human parvovirus B19 infection in acute fulminant liver
failure. Arch Virol, 1999. 144(9): p. 1713-24.
61. Sokal, E.M., et al., Acute parvovirus B19 infection associated with fulminant
hepatitis of favourable prognosis in young children. Lancet, 1998. 352(9142):
p. 1739-41.
62. Yoto, Y., et al., Human parvovirus B19 infection associated with acute
hepatitis. Lancet, 1996. 347(9005): p. 868-9.
63. So, K., et al., Urgent liver transplantation for acute liver failure due to
parvovirus B19 infection complicated by primary Epstein-Barr virus and
cytomegalovirus infections and aplastic anaemia. Intern Med J, 2007. 37(3): p.
192-5.
64. Bultmann, B.D., et al., Fatal parvovirus B19-associated myocarditis clinically
mimicking ischemic heart disease: an endothelial cell-mediated disease. Hum
Pathol, 2003. 34(1): p. 92-5.
65. Wandinger, K., et al., Association between clinical disease activity and
Epstein-Barr virus reactivation in MS. Neurology, 2000. 55(2): p. 178-84.
66. Gan, Y.J., et al., A defective, rearranged Epstein-Barr virus genome in EBER-
negative and EBER-positive Hodgkin's disease. Am J Pathol, 2002. 160(3): p.
781-6.
67. Crawford, M.P., et al., High prevalence of autoreactive, neuroantigen-specific
CD8+ T cells in multiple sclerosis revealed by novel flow cytometric assay.
Blood, 2004. 103(11): p. 4222-31.
68. Semmo, N., et al., Analysis of the relationship between cytokine secretion and
proliferative capacity in hepatitis C virus infection. J Viral Hepat, 2007. 14(7):
p. 492-502.
69. Toan, N.L., et al., Co-infection of human parvovirus B19 in Vietnamese
patients with hepatitis B virus infection. J Hepatol, 2006. 45(3): p. 361-9.
70. Hsu, T.C., et al., Human parvovirus B19 infection in patients with chronic
hepatitis B or hepatitis C infection. J Gastroenterol Hepatol, 2005. 20(5): p.
733-8.
71. Anderson, M.J., et al., Experimental parvoviral infection in humans. J Infect
Dis, 1985. 152(2): p. 257-65.
72. Anderson, M.J., et al., Human parvovirus, the cause of erythema infectiosum
(fifth disease)? Lancet, 1983. 1(8338): p. 1378.
73. Naides, S.J., Infection with Parvovirus B19. Curr Infect Dis Rep, 1999. 1(3): p.
273-278.
74. Katz, V.L., N.C. Chescheir, and M. Bethea, Hydrops fetalis from B19
parvovirus infection. J Perinatol, 1990. 10(4): p. 366-8.
75. Gilbert, G.L., Parvovirus B19 infection and its significance in pregnancy.
Commun Dis Intell, 2000. 24 Suppl: p. 69-71.
76. Orth, T., et al., Human parvovirus B19 infection associated with severe acute
perimyocarditis in a 34-year-old man. Eur Heart J, 1997. 18(3): p. 524-5.
77. Papadogiannakis, N., et al., Active, fulminant, lethal myocarditis associated
with parvovirus B19 infection in an infant. Clin Infect Dis, 2002. 35(9): p.
1027-31.
78. Brown, K.E., Haematological consequences of parvovirus B19 infection.
Baillieres Best Pract Res Clin Haematol, 2000. 13(2): p. 245-59.
79. Koch, W.C., et al., Manifestations and treatment of human parvovirus B19
infection in immunocompromised patients. J Pediatr, 1990. 116(3): p. 355-9.
Role of Parvovirus B19 infection in hepatitis C -- Reference
37
80. Schmidt, M., et al., Blood donor screening for parvovirus B19 in Germany
and Austria. Transfusion, 2007. 47(10): p. 1775-82.
81. Cassinotti, P., et al., Evidence for persistence of human parvovirus B19 DNA
in bone marrow. J Med Virol, 1997. 53(3): p. 229-32.
82. Heegaard, E.D., et al., Prevalence of parvovirus B19 and parvovirus V9 DNA
and antibodies in paired bone marrow and serum samples from healthy
individuals. J Clin Microbiol, 2002. 40(3): p. 933-6.
83. Lundqvist, A., et al., Clinical and laboratory findings in immunocompetent
patients with persistent parvovirus B19 DNA in bone marrow. Scand J Infect
Dis, 1999. 31(1): p. 11-6.
84. Lundqvist, A., et al., Prevalence of parvovirus B19 DNA in bone marrow of
patients with haematological disorders. Scand J Infect Dis, 1999. 31(2): p.
119-22.
85. Chevrel, G., et al., Dermatomyositis associated with the presence of
parvovirus B19 DNA in muscle. Rheumatology (Oxford), 2000. 39(9): p.
1037-9.
86. Nikkari, S., et al., Persistence of parvovirus B19 in synovial fluid and bone
marrow. Ann Rheum Dis, 1995. 54(7): p. 597-600.
87. Vuorinen, T., et al., Presence of parvovirus B19 DNA in chronic urticaric and
healthy human skin. J Clin Virol, 2002. 25(2): p. 217-21.
88. Kuhl, U., Antiviral treatment of myocarditis and acute dilated cardiomyopathy.
Heart Fail Clin, 2005. 1(3): p. 467-74.
89. Langnas, A.N., et al., Parvovirus B19 as a possible causative agent of
fulminant liver failure and associated aplastic anemia. Hepatology, 1995.
22(6): p. 1661-5.
90. Pinho, J.R., et al., Detection of human parvovirus B19 in a patient with
hepatitis. Braz J Med Biol Res, 2001. 34(9): p. 1131-8.
91. Franssila, R., K. Hokynar, and K. Hedman, T helper cell-mediated in vitro
responses of recently and remotely infected subjects to a candidate
recombinant vaccine for human parvovirus b19. J Infect Dis, 2001. 183(5): p.
805-9.
92. Mitchell, L.A., R. Leong, and K.A. Rosenke, Lymphocyte recognition of
human parvovirus B19 non-structural (NS1) protein: associations with
occurrence of acute and chronic arthropathy? J Med Microbiol, 2001. 50(7):
p. 627-35.
93. Bain, C., et al., Impaired allostimulatory function of dendritic cells in chronic
hepatitis C infection. Gastroenterology, 2001. 120(2): p. 512-24.
94. Aberle, J.H., et al., CD4+ T cell responses in patients with chronic hepatitis C
undergoing peginterferon/ribavirin therapy correlate with faster, but not
sustained, viral clearance. J Infect Dis, 2007. 195(9): p. 1315-9.
95. Meyer, M.F., et al., Clearance of low levels of HCV viremia in the absence of
a strong adaptive immune response. Virol J, 2007. 4: p. 58.
96. Wedemeyer, H., et al., Cross-reactivity between hepatitis C virus and
Influenza A virus determinant-specific cytotoxic T cells. J Virol, 2001. 75(23):
p. 11392-400.
Role of Parvovirus B19 infection in hepatitis C -- Abbreviations
38
7. List of abbreviations:
B19 Parvovirus B19
HCV Hepatitis C virus
qPCR Quantity Polymerase chain reaction
nPCR Nested Polymerase chain reaction
FH Fulminant hepatitis
PBMC Peripheral blood mononuclear cell
ALT alanine aminotransferase
AST aspartate aminotransferase
gGT gamma-glutamyltransferase
ULM Upper limited of normal
AHB Acute hepatitis B
CHB Chronic hepatitis B
LC Liver cirrhosis
HCC Hepatocellular carcinoma
VAHS Virus-associated-hemophagocytic
syndrome
NASH Nonalcoholic steatohepatitis
DMSO dimethyl sulfoxide
PHA phytohemagglutinin
TT Tetanus toxoid
Role of Parvovirus B19 infection in hepatitis C -- Acknowledgements
39
8. Appendix
Acknowledgements
I would like to acknowledge numerous people for their kindness and support in the
past two years. Without their priceless help, this thesis could not be finished.
At the very beginning, I’m honored to express my appreciation to Prof. Dr. med.
Michael P. Manns, who gave me great support during my two-year advanced study in
his Department.
Here I must express the deepest gratitude to my supervisor, Priv. Doz. Dr. med.
Heiner Wedemeyer. He gave me the opportunity to join his lab. He offered me
valuable ideas, suggestions and guidance with his professional attainment in
Hepatology and Immunology. His patience and kindness are greatly appreciated.
Moreover, he always puts high priority on my project and has provided me with good
communication whenever he is available. I’m very much obliged to his efforts of
helping me complete the dissertation.
I’m also grateful to PD. Dr. med. Albert Heim and PD. Dr. rer. nat. C.-Thomas Bock
who gave me patient guidance and many useful advices and supports.
I owe special thanks to all members of our group: Markus, Katja, Sandra, Evi, Verana,
Kerstin, Suneetha. All of them gave me great supports and showed warmest concern
not only for my study but also for my daily life in Germany.
I wish to extend my gratitude to Hong Jiang, Regina, Peter, Birgit, Ursel, Nicole and
many many faculty members and staffs of the department assisted and encouraged me
in various ways during my study.
At last, I would like to say thanks to my family and my husband for their support and
love.
Role of Parvovirus B19 infection in hepatitis C -- CV
40
Curriculum vitae
Personal information:
Name: Wang
First name: Chun
Gender: Female
Birthday: 06.06.1970
Birthplace: Shanghai, P.R. China
Nation: Chinese
Education:
Sept. 1977- July. 1982 Elementary education
Sept. 1982- July.1988 High school
Study:
Sept.1988- July.1993 Shanghai Tiedao Medical College, Shanghai, China
Completion: medical bachelor’s degree
Sept.2001- July. 2004 Tongji University, Shanghai, China
Completion: medical master’s degree
Occupation:
Aug. 1993- Aug. 2001 Shanghai Railway Bureau Central Hospital
Dept. Internal medicine and Dept. Endocrinology
Shanghai, China
Aug. 2004-Sept. 2005 Tongji University affiliated Shanghai Tenth People´s
Hospital.
Dept. Endocrinology
Shanghai, China
Oct. 2005-Sept. 2007 Medical school Hannover,
Dept. Gastroenterology, Hepatology and
Endocrinology
Hannover, Germany
Role of Parvovirus B19 infection in hepatitis C -- Erklärung
41
Erklärung
Laut Paragraf 2, Absatz 2, Nummern 5 und 6 der Promotionsordnung der
Medizinischen Hochschule Hannover.
Ich erkläre, dass ich die der Medizinischen Hochschule Hannover zur Promotion
eingereichte Dissertation mit dem Titel „Parvo-Virus B19 Infection: Evidence for
intrahepatic long-term persistence but no association with disease progression in
chronic hepatitis C" in der Klinik für Gastroenterologie, Hepatologie und
Endokrinologie
der Medizinischen Hochschule Hannover unter Leitung von Priv. Doz. Dr. med.
Heiner Wedemeyer ohne sonstige Hilfe selbst durchgeführt und bei der Abfassung der
Dissertation keine anderen als die dort aufgeführten Hilfsmittel benutzt habe.
Ich habe diese Dissertation bisher an keiner in- oder ausländischen Hochschule zur
Promotion eingereicht. Weiterhin versichere ich, dass ich den beantragten Titel bisher
noch nicht erworben habe.
Hannover, den 3.2.2008
Chun Wang