Hepatitis B virus nucleic acids associated with human peripheral blood mononuclear cells do not originate from replicating virus

Köck, Josef; Theilmann, Lorenz; Galle, Peter R.; Schlicht, H J

doi:10.1002/hep.510230303

Cited by 68 publications

(59 citation statements)

References 30 publications

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“…31 These methods, however, hardly distinguish input viruses from newly synthesized particles. Because nuclear entry of viral DNA is a prerequisite for successful HBV infection of a cell and results in the formation of HBV ccc DNA, 15,32 Köck et al 16 developed a PCR assay that amplifies cccDNA more efficiently than relaxed circular DNA present in HBV particles, and tested whether PBMC can be infected by HBV. They found no evidence that PMBC can be infected with HBV and concluded that adsorbed virus can explain detection of HBV DNA or HBV RNA by PCR.…”

Section: Discussionmentioning

confidence: 99%

“…To detect HBV ccc DNA, wedesignedprimersHBVccc2760fw(5Ј-GACTCTCTC-GTCCCCTTCTC-3Ј) and HBVccc156rev (5Ј-ATG-GTGAGGTGAACAATGCT-3Ј) (580-bp fragment), which span the gap and the nick in the rc form of the HBV genome as principally described earlier. 16 Using optimized PCR conditions on a Light Cycler instrument (95°C for 5Ј, 45 cycles at 95°C for 15Љ, 60°C for 4Љ, 72°C for 25Љ, and detection at 88°C for 2Љ after each cycle), we determined the specificity to amplify ccc DNA over rc DNA to be 10 4 to1.…”

Section: Methodsmentioning

confidence: 99%

“…9,10,12,13 However, this conclusion can be questioned, because the detection of HBV-DNA and even HBV-RNA in peripheral blood mononuclear cells (PBMC) can be explained by adsorbed virus and does not necessarily correlate with detection of cccDNA and thus with infection. 16 Therefore, whether HBV can infect DC, induce expression of viral antigens, and initiate replication of the viral genome remains unknown.…”

Section: H Epatitis B Virus (Hbv) Infection Represents Amentioning

confidence: 99%

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Dendritic cells take up viral antigens but do not support the early steps of hepatitis B virus infection

et al. 2006

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Dendritic cells (DC) of hepatitis B virus (HBV) carriers have been reported to exhibit functional impairment. Possible explanations for this phenomenon are infection of HBV by DC or alteration of DC function by HBV. We therefore analyzed whether DC support the different steps of HBV infection and replication: uptake, deposition of the HBV genome in the nucleus, antigen expression, and progeny virus release. When HBV genomes were artificially introduced into monocyte-derived DC by adenoviral vectors, low-level expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) but no HBV replication was detected. When monocyte-derived DC were subjected to wild-type HBV or a recombinant HBV expressing Renilla luciferase under a non-liver-specific promoter, intracellular HBV DNA was detected in a low percentage of cells. However, neither nuclear cccDNA was formed nor luciferase activity was detected, indicating that either uncoating or nucleocytoplasmic transport were blocked. To verify our observation in the in vivo situation, myeloid and plasmacytoid DC were isolated from blood of high viremic HBV carriers, and analyzed by quantitative polymerase chain reaction (PCR) and electron microscopy. Although circulating DC had in vivo been exposed to more than 10 4 HBV virions per cell, HBV genomic DNA was hardly detected, and no nuclear cccDNA was detected at all. By using electron microscopy, subviral particles were found in endocytic vesicles, but virions were undetectable as were viral capsids in the cytoplasm. H epatitis B virus (HBV) infection represents a major health problem worldwide. Over 350 million individuals are chronically infected with HBV and are at high risk to develop liver cirrhosis or hepatocellular carcinoma. To eliminate the virus after infection, a strong humoral and cellular immune response is required. 1 Thus, control of HBV infection is associated with a multispecific and polyclonal cytotoxic T-cell response and a strong type 1 T helper cell response. 2,3 In contrast, chronically infected patients display oligoclonal T helper cell responses with weak or undetectable cytotoxic T-cell activity. 4 Dendritic cells (DC) are the most important professional antigen-presenting cells. They act as key players in initiating virus-specific T-cell responses. 5 This is reflected by the fact that viruses can evade immune responses by impairing DC function. [6][7][8] The role of DC during HBV infection was intensively studied during the last years, but whether total numbers of DC are reduced during chronic infection is discussed controversary. [9][10][11] In some studies, functional deficits of these cells were reported on contact of moDC with HBV, 14 whereas they remained minor in others. 12 Whether the weak or absent T-cell response described in chronic hepatitis B patients results from a defect in the DC compartment, which is caused by the virus itself, is unclear. Theoretically, numbers or functionality of DC subsets could be affected by interaction of surface receptors on DC with either vi...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: H Epatitis B Virus (Hbv) Infection Represents Amentioning

confidence: 99%

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Dendritic cells take up viral antigens but do not support the early steps of hepatitis B virus infection

et al. 2006

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“…[11][12][13][14][15][16] Quantitation of cccDNA in human peripheral blood mononuclear cells and liver biopsies has been performed. [17][18][19][20] In these studies, primers spanning across the gap in the minus strand and corresponding to the variable region on the plus strand were used to amplify across noninterrupted cccDNA. It should be noted that, even with selective polymerase chain reaction (PCR) methods, amplification of residual rcDNA can still occur after selfannealing of the partial elongation products.…”

Section: Hronic Hepatitis B Virus (Hbv) Infection Affectsmentioning

confidence: 99%

Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients

et al. 2004

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This study examined a signal amplification assay, the Invader assay, for the quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in liver biopsies and sera. DNA was extracted from liver biopsy and serum samples were collected from 16 hepatitis B e antigen (HBeAg)-positive and 36 antibody-to-HBeAg-positive (anti-HBe-positive) chronic hepatitis B patients. The amount of total HBV DNA and cccDNA was measured using the Invader assay. Anti-HBe-positive patients had lower median total intrahepatic HBV DNA (P < .001) and intrahepatic cccDNA levels (P ‫؍‬ .001) than HBeAg-positive patients. Intrahepatic cccDNA correlated positively with the total intrahepatic HBV DNA (r ‫؍‬ 0.950, P < .001). However, the proportion of intrahepatic HBV DNA in the form of cccDNA was inversely related to the amount of total intrahepatic HBV DNA (r ‫؍‬ ؊0.822, P < .001). A small amount of cccDNA was detected in 39 of 52 (75%) serum samples. Anti-HBe-positive patients had lower median serum cccDNA levels than HBeAg-positive patients (P ‫؍‬ .002). Serum HBV DNA correlated positively with intrahepatic total HBV DNA (r ‫؍‬ 0.778, P < .001) and intrahepatic cccDNA (r ‫؍‬ 0.481, P ‫؍‬ .002). In conclusion, the Invader assay is a reliable assay for the quantitation of cccDNA. vitro studies have shown that lamivudine has a profound effect on relaxed circular DNA (rcDNA) while having little or no effect on cccDNA. 2,3 This is one possible reason for the rebound of HBV DNA to pretreatment levels often seen after lamivudine withdrawal. 4 Another nonreplicative form of HBV DNA is the double-stranded linear (DL) form produced by in situ priming during HBV replication. 5 DL DNA is a possible precursor to HBV DNA integration. 6 -8 It can also form cccDNA through nonhomologous recombination at its ends via a process called illegitimate replication. 9,10 cccDNA monitoring and the development of an accurate quantitative assay for cccDNA are becoming important in the understanding of the natural history and management of chronic hepatitis B (CHB). Most attempts for the quantitation of cccDNA have been made with liver biopsies from ducks or woodchucks. [11][12][13][14][15][16] Quantitation of cccDNA in human peripheral blood mononuclear cells and liver biopsies has been performed. [17][18][19][20] In these studies, primers spanning across the gap in the minus strand and corresponding to the variable region on the plus strand were used to amplify across noninterrupted cccDNA. It should be noted that, even with selective polymerase chain reaction (PCR) methods,

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“…5) provides direct evidence for viral replication in the engrafted human hepatocytes. 29 The capacity of anti-HBsAg to precipitate HBV particles from HBV-Trimera mice sera (Fig. 6, lane 3) clearly shows the presence of fully assembled virions in the blood circulation.…”

Section: Discussionmentioning

confidence: 94%

The Hepatitis B Virus-Trimera Mouse: A Model for Human Hbv Infection and Evaluation of Anti–Hbv Therapeutic Agents

et al. 1999

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Previous studies have demonstrated the feasibility of implantation of human blood cells or tissues in lethally irradiated mice or rats, radioprotected with SCID mouse bone marrow cells: The Trimera system. In the present study, we describe the development of a mouse Trimera model for human hepatitis B virus (HBV) infection. In this model, viremia is induced by transplantation of ex vivo HBV-infected human liver fragments. Engraftment of the human liver fragments, evaluated by hematoxylin-eosin staining and human serum albumin mRNA expression, was observed in 85% of the transplanted animals 1 month postimplantation. Viremia levels were determined in these mice by measuring serum HBV DNA using polymerase chain reaction (PCR), followed by dot-blot hybridization. HBV DNA is first detected 8 days after liver transplantation. Viremia attains a peak between days 18 and 25 when HBV infection is observed in 85% of the transplanted animals. Hepatitis B virus (HBV) infection is a major public health problem affecting millions of people worldwide. 1 Following acute HBV infection, 5% to 10% of the adult patients will develop persistent infection that may lead to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. 2-4 Whereas considerable progress has been achieved regarding the identification and characterization of the virus, the development of new, effective therapies has been impeded because of the lack of a practical small HBV animal model. Attempts to establish animal models to study HBV infection in rats, 5 nude mice, 6 and transgenic mice 7-9 have been described. Other animal models, based on HBV-related hepadnaviruses that infect nonprimates, were developed and successfully used for assessment of antiviral drugs. These models, however, involve relatively large animals that are difficult to handle in most laboratories. In addition, testing of antiviral agents such as nucleoside analogs could produce aberrant results as a consequence of virus-specific differential susceptibility of the viral polymerase. 10 Chimpanzees provide a good HBV animal model in which effects of vaccines and therapeutic agents can be evaluated. 11 Nonetheless, the limited availability and the high cost of these primates severely restrict their use for such purposes.Recently, we have developed a human-mouse radiation chimera in which normal mice, preconditioned by lethal total body irradiation and radioprotected with SCID mouse bone marrow cells, are permissive for engraftment of human hematopoietic cells and tissues. [12][13][14][15][16] This resulting humanmouse model that comprises three genetically disparate sources of tissue is therefore termed ''Trimera.'' The Trimera mouse, engrafted with human peripheral blood lymphocytes, has been adapted successfully to generate human monoclonal antibodies. 17 Likewise, transplantation of Trimera mice with hepatitis C virus-infected human liver tissue was used for the development of an hepatitis C virus infection model. 14 In the present study, we describe in detail the development of an HBV ani...

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Hepatitis B virus nucleic acids associated with human peripheral blood mononuclear cells do not originate from replicating virus

Cited by 68 publications

References 30 publications

Dendritic cells take up viral antigens but do not support the early steps of hepatitis B virus infection

Dendritic cells take up viral antigens but do not support the early steps of hepatitis B virus infection

Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients

The Hepatitis B Virus-Trimera Mouse: A Model for Human Hbv Infection and Evaluation of Anti–Hbv Therapeutic Agents

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