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
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
(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
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 extentin 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
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