Medizinische Hochschule Hannover Zentrum Innere Medizin ......1.1.1 Epidemiology and natural history Hepatitis C virus was discovered in 1989 by M.Houghton and colleague [1]. Worldwide,

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].


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].


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].


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


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


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?


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.


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


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


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


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.


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


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


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


B19

DNA


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.


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


F s

co

re (

ISH

AK

)

0

2

4

6

8

10

12

14

16

18


Infl

am

mati

on

sco

re

0

2

4

6

8

10

12

14

16

18


Infl

am

mati

on

sco

re


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.


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


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


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


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.


22

Figure 6

Figure 6 A1 & A2

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34.83.27

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35.21.88

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33.84.55

DMSO

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14.623.1

Tetanus (3μμμμ g/ml)

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7.1240.5

PHA (6μμμμ g /ml)

HCV NS3 (1μμμμ g/ml)

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30.94.09

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34.93.53

VP1/2 4.7 (5μμμμ g /ml)

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33.92.3

VP1/2 4.7 (1μμμμ g /ml)

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31.76.95

VP1/2 7.2 (1μμμμ g /ml)

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7.32 55.8

31.85.03

VP1/2 7.2 (5μμμμ g /ml)

CFSE

CD4+T

cell

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VP1/2 4.7 (5μμμμ g /ml)

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5.98 57.8

33.92.3

VP1/2 4.7 (1μμμμ g /ml)

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8.15 53.2

31.76.95

VP1/2 7.2 (1μμμμ g /ml)

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7.32 55.8

31.85.03

VP1/2 7.2 (5μμμμ g /ml)

CFSE

CD4+T

cell

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VP1/2 4.7 (5μμμμ g /ml)

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VP1/2 7.2 (5μμμμ g /ml)

CFSE

CD4+T

cell

-1

0

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DMSO VP1/2 4.7

5 g/m l

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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


23

Figure 6 B1 & B2

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Figure 6 C1 & C2

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CD4+T cellCD4+T cell

CFSECFSE

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l p

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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


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


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


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,


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


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

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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

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