Low Doses of Hepadnavirus Induce Infection of the Lymphatic System That Does Not Engage the Liver

Abstract

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...

Persistence of isolated antibodies to woodchuck hepatitis virus core antigen is indicative of occult infection

“…These levels were similar to those previously reported for woodchucks with serologically silent infection persisting after resolution of acute WHV hepatitis, 30,37 that was termed secondary (residual) occult infection (SOI). 33 The superior sensitivity of WHV genome detection (Ͻ10 vge/mL) in this and our previous studies 30 -33 over nested PCR assays used by others (sensitivity 10 2 -10 3 vge/mL) was achieved by, among other factors, routinely using PCR with primer pairs amplifying fragments of three different genes of WHV, always employing Southern blot hybridization to increase the sensitivity and specificity of virus identification, and detecting hybridization signals through phosphorimaging analysis.…”

“…All animals except 5/F and 6/F were infected with WHV/tm3 inoculum carrying wild-type WHV (GenBank accession number AY334075). 33 5/F and 6/F were infected with WHV/tm2 Abbreviations: F, female; M, male; mo, month; ND, not detected. NOTE.…”

“…The sensitivity of the nested PCR/NAH was Ͻ 10 vge/mL and that for WHV cccDNA Ϸ10 2 vge/mL, as reported. [31][32][33] Electron Microscopy. Four mL of autopsy sera from 6/F and 10/M (Table 1) were centrifuged at 200,000g for 18 hours at 5°C in an SW50.1 Beckman rotor and the resulting pellets examined by electron microscopy after negative staining with 1% phosphotungstic acid.…”

“…30,31 For PCR detecting WHV cccDNA, mung bean nuclease treatment, primers and conditions previously established were applied. 32,33 The presence of WHV mRNA was examined using total RNA extracted with Trizol (Invitrogen Life Technologies, Burlington, Ontario, Canada). Reverse transcription (RT) reaction was done as reported.…”

“…31 Similarly, animals inoculated with small amounts of WHV (Ͻ10 3 vge/mL) develop an anti-WHc-negative infection with low-level viremia. 32,33 In this study, the significance of the sole detection of anticore antibodies as an indicator of a low-rate hepadnavirus replication was explored. To address this issue, woodchucks persistently carrying isolated anti-WHc, or anti-WHc along with antibodies to WHsAg (anti-WHs), acquired due to a primary WHV surface antigen (WHsAg)-nonreactive infection or after resolved WHV hepatitis were tested for molecular and physicochemical evidence of virus occurrence.…”