It is presumed that resolution of hepatitis C, as evidenced by normalization of liver function tests and disappearance of hepatitis C virus (HCV) RNA from serum, as determined by conventional laboratory assays, reflects virus eradication. In this study, we examined the expression of the HCV genome in the sera, peripheral blood mononuclear cells (PBMC), and, on some occasions, monocyte-derived dendritic cells (DC) long after resolution of hepatitis C by using a highly sensitive reverse transcription ( Hepatitis C virus (HCV) is a small positive-strand RNA virus of approximately 9,400 nucleotides that chronically infects an estimated 170 to 350 million people worldwide. Of those acutely afflicted, only 15% recover, while the remaining 85% succumb to chronic hepatitis (4). Furthermore, up to onefifth of the individuals with chronic hepatitis C progress to cirrhosis, and these patients are at a greater risk of developing hepatocellular carcinoma (9).It is generally accepted that HCV replicates by making a cRNA strand known as the negative or replicative strand. Although the liver is the main site of virus replication, there is an increasing body of evidence for virus propagation in extrahepatic locations, including cells of the lymphatic and the central nervous systems (17,28). In regard to infection of lymphoid cells, HCV positive and negative strands were detected in the peripheral blood mononuclear cells (PBMC) and the bone marrow from chronically infected patients (26,29,37). It was also shown that HCV can propagate in lymphoid cell cultures and that the virus derived is infectious (33, 34). The notion of natural HCV tropism for lymphoid cells is supported by a significant overrepresentation of certain lymphoproliferative disorders in the HCV-infected population. For instance, type II mixed cryoglobulinemia occurs 11 times more frequently in patients with HCV than in those without (6). Also, nonHodgkin lymphoma appears to be, albeit less strongly, associated with HCV infection (24).The current RT-PCR-based assay approved for clinical diagnostics, i.e., the Amplicor HCV v2.0 assay (Roche Molecular Diagnostics, Pleasanton, Calif.), detects HCV RNA with a sensitivity of 1,000 virus genomic equivalents (vge) per ml (or 500 IU/ml). Other assays can identify HCV RNA at 52 vge/ml (or 10 IU/ml) (i.e., the Versant HCV RNA qualitative assay; Bayer Corp., Tarrytown, N.J.). This implies that small quantities of HCV occurring either in serum or within cells may escape detection. Therefore, considering the natural history of HCV infection, there exists a possibility that the virus may not be completely eradicated at the time of clinical and serological resolution of hepatitis. This situation may occur following spontaneous recovery or antiviral therapy.Lymphotropism is a characteristic of many DNA and RNA viruses capable of inducing persistent infection (8,27). A number of studies, including those with highly hepatotropic hepatitis B virus (19, 30) and woodchuck hepatitis virus (3,21,22), have demonstrated that pathogenic vir...

Woodchuck hepatitis virus (WHV), which is closely related to human hepatitis B virus and is considered to be principally hepatotropic, invades the host's lymphatic system and persists in lymphoid cells independently of whether the infection is symptomatic and serologically evident or concealed. In this study, we show, with the woodchuck model of hepatitis B, that hepadnavirus can establish an infection that engages the lymphatic system, but not the liver, and persists in the absence of virus serological markers, including antiviral antibodies. This primary occult infection is caused by wild-type virus invading the host at a quantity usually not greater than 10 3 virions. It is characterized by trace virus replication progressing in lymphatic organs and peripheral lymphoid cells that, with time, may also spread to the liver. The infection is transmissible to virus-naive hosts as an asymptomatic, indefinitely long, occult carriage of small amounts of biologically competent virus. In contrast to residual silent WHV persistence, which normally endures after the resolution of viral hepatitis and involves the liver, primary occult infection restricted to the lymphatic system does not protect against reinfection with a large, liver-pathogenic WHV dose; however, the occult infection is associated with a swift recovery from hepatitis caused by the superinfection. Our study documents that the lymphatic system is the primary target of WHV infection when small quantities of virions invade a susceptible host.Replication and retention of virus in cells of the immune system characterize many persistent viral infections and are a major hindrance to sterilizing antiviral therapy. Human hepatitis B virus (HBV) and its close relative woodchuck hepatitis virus (WHV), infecting the eastern American woodchuck (Marmota monax), are noncytopathic hepadnaviruses which cause similar courses and outcomes of liver disease (7,27,29,41,42). It is estimated that 350 to 400 million people worldwide are chronically infected with HBV (43). This lifelong infection, with which the patient is serologically HBV surface antigen (HBsAg) reactive, is accompanied by chronic hepatitis that frequently advances to cirrhosis and hepatocellular carcinoma (HCC). It is now evident that HBV also elicits occult long-term persistence, as has been determined from the detection of the virus by HBV DNA PCR but not by the current and otherwise sensitive immunoassays for HBsAg (1-3, 7, 10, 22, 25, 34, 36, 38, 45, 46). The epidemiological and pathogenic importance of this silent HBV carriage is increasingly evident, particularly in regard to (i) the transmission of virus traces through seemingly HBV-negative blood transfusion, hemodialysis, or organ transplantation (5, 12, 37); (ii) cytotoxic or immunosuppressive therapy (13, 24); and (iii) the pathogenesis of liver diseases considered to be cryptogenic, e.g., HCC (4,6,21,44). In the woodchuck model of hepatitis B, which is the closest natural animal model for the study of HBV pathobiology (27,29,41,42), the lifelong s...

Antibodies against virus nucleocapsid (anticore) normally accompany hepadnaviral hepatitis but they may also occur in the absence of symptoms and other serological indicators of the infection. This situation can be encountered following a clinically and serologically unapparent exposure to hepatitis B virus (HBV) or after recovery from hepatitis B. In this study, woodchucks inoculated with woodchuck hepatitis virus (WHV) were investigated to determine the relationship between anticore detection and the molecular status of virus replication in a primary WHV surface antigen (WHsAg)-negative infection or long-after resolution of WHV hepatitis. Serial, parallel samples of sera, peripheral blood mononuclear cells (PBMC) and liver tissue, collected for more than 5 years after inoculation with virus, were examined for WHV DNA by highly sensitive polymerase chain reaction (PCR)/nucleic acid hybridization assays. Sera were also tested for WHV DNA after DNase treatment and for WHV DNA and WHsAg after concentration in sucrose. Liver and PBMC were examined for WHV covalently closed circular DNA and viral RNA transcripts by PCR-based techniques to assess virus replication status. The study showed that anticore antibodies existing in the absence of other serological markers are a reliable indicator of occult WHV infection. This state can be accompanied by traces of circulating particles behaving as intact virions and by intermittent minimal-to-mild liver inflammation. In conclusion, the long-term presence of anticore antibodies alone is a consequence of sustained restimulation of the immune system by virus nucleocapsid produced during low-level hepadnaviral assembly. ( A ntibodies to hepatits B virus (HBV) core antigen (anti-HBc) coexist with HBV surface antigen (HBsAg) in symptomatic infection and can persist along with antibodies to HBsAg (anti-HBs) or without them for life after recovery from hepatitis B. 1,2 They may also occur in the pre-acute phase of hepatitis prior to the appearance of HBsAg. 3 Absence of anti-HBc is rare in HBV infection and has been attributed to aberrant immunological response to the virus or to infection with viral variants. The detection of anti-HBc as the only serological marker of HBV exposure ("isolated anti-core" or "anti-core alone") has been found in up to 10%-20% of blood donors in endemic areas. 4,5 Since anti-HBc-like reactivity was occasionally seen after HBV vaccination, this result has raised doubts as to the reliability of antiHBc testing. 6,7 However, individuals with isolated antiHBc showed the highest rates of HBV DNA detection by specific polymerase chain reaction (PCR) assays, 5,8 -11 suggesting ongoing low-level HBV infection despite the absence of classical serological markers. This outcome was consistent with the earlier findings of anti-HBc along with HBV DNA in serum and peripheral blood mononuclear cells (PBMC) 12 and a vigorous cytotoxic T-lymphocyte response to HBV antigens in apparently completely healthy individuals years after recovery from

The detection of small amounts of viral pathogens in infected cells by classical PCR is hampered by a partial loss of virus nucleic acid due to extraction and by difficulties in discrimination between truly intracellular virus genome material and that possibly adhered to the cell surface. These impediments limit reliable identification of virus traces within infected cells, which are typically encountered in latent and persistent occult infections. In this study, hepadnavirus-specific in situ PCR combined with the enzymatic elimination of extracellular virus and flow cytometry permitted detection of viral genomes in lymphoid cells without nucleic acid isolation and allowed quantification of infected cells during the course of persistent infection with woodchuck hepatitis virus (WHV). The validity of the procedure was confirmed by hybridization analysis of the in situ-amplified viral sequences. The results showed that hepadnavirus can be directly detected within lymphoid cells not only in serologically accountable infection, but also years after recovery from viral hepatitis and in the course of primary occult virus carriage. Percentages of infected peripheral lymphoid cells in symptomatic WHV hepatitis fluctuate between 3.4 and 20.4% (mean ؎ standard error of the mean, 9.6% ؎ 1.7%), whereas those in persistent, serologically mute WHV infection range from 1.1 to 14.6% (mean ؎ standard error of the mean, 4.8% ؎ 0.8%) (P ‫؍‬ 0.005). The data obtained provide further evidence that WHV infection continues indefinitely in the lymphatic system independently of whether it is symptomatic or concealed. They document that hepadnavirus can be detected in a significant proportion of circulating lymphoid cells in both immunovirologically apparent as well as occult persistent infection.

Woodchuck hepatitis virus (WHV), which is closely related to human hepatitis B virus and is considered to be principally hepatotropic, invades the host's lymphatic system and persists in lymphoid cells independently of whether the infection is symptomatic and serologically evident or concealed. In this study, we show, with the woodchuck model of hepatitis B, that hepadnavirus can establish an infection that engages the lymphatic system, but not the liver, and persists in the absence of virus serological markers, including antiviral antibodies. This primary occult infection is caused by wild-type virus invading the host at a quantity usually not greater than 10 3 virions. It is characterized by trace virus replication progressing in lymphatic organs and peripheral lymphoid cells that, with time, may also spread to the liver. The infection is transmissible to virus-naive hosts as an asymptomatic, indefinitely long, occult carriage of small amounts of biologically competent virus. In contrast to residual silent WHV persistence, which normally endures after the resolution of viral hepatitis and involves the liver, primary occult infection restricted to the lymphatic system does not protect against reinfection with a large, liver-pathogenic WHV dose; however, the occult infection is associated with a swift recovery from hepatitis caused by the superinfection. Our study documents that the lymphatic system is the primary target of WHV infection when small quantities of virions invade a susceptible host.Replication and retention of virus in cells of the immune system characterize many persistent viral infections and are a major hindrance to sterilizing antiviral therapy. Human hepatitis B virus (HBV) and its close relative woodchuck hepatitis virus (WHV), infecting the eastern American woodchuck (Marmota monax), are noncytopathic hepadnaviruses which cause similar courses and outcomes of liver disease (7,27,29,41,42). It is estimated that 350 to 400 million people worldwide are chronically infected with HBV (43). This lifelong infection, with which the patient is serologically HBV surface antigen (HBsAg) reactive, is accompanied by chronic hepatitis that frequently advances to cirrhosis and hepatocellular carcinoma (HCC). It is now evident that HBV also elicits occult long-term persistence, as has been determined from the detection of the virus by HBV DNA PCR but not by the current and otherwise sensitive immunoassays for HBsAg (1-3, 7, 10, 22, 25, 34, 36, 38, 45, 46). The epidemiological and pathogenic importance of this silent HBV carriage is increasingly evident, particularly in regard to (i) the transmission of virus traces through seemingly HBV-negative blood transfusion, hemodialysis, or organ transplantation (5, 12, 37); (ii) cytotoxic or immunosuppressive therapy (13, 24); and (iii) the pathogenesis of liver diseases considered to be cryptogenic, e.g., HCC (4,6,21,44). In the woodchuck model of hepatitis B, which is the closest natural animal model for the study of HBV pathobiology (27,29,41,42), the lifelong s...

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