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


  • Carla S. Coffin,

    1. Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3V6, Canada
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  • Tram N.Q. Pham,

    1. Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3V6, Canada
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  • Patricia M. Mulrooney,

    1. Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3V6, Canada
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  • Norma D. Churchill,

    1. Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3V6, Canada
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  • Tomasz I. Michalak

    Corresponding author
    1. Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3V6, Canada
    2. Discipline of Laboratory Medicine, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3V6, Canada
    • Molecular Virology and Hepatology Research, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John's, NL, Canada A1B 3V6
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    • fax: 709-777 8279


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. (HEPATOLOGY 2004.)

Antibodies 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 anti-HBc testing.6, 7 However, individuals with isolated anti-HBc 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 acute hepatitis B.13, 14 Moreover, transmission of HBV from blood and organ donors with isolated anti-HBc to virus-naïve individuals5, 15–21 and reactivation of the infection in anti-HBc–positive persons22, 23 have been reported. Also, it is perceived that occult HBV infection with or without anti-HBc is associated with an increased risk of developing hepatocellular carcinoma.24–27

Woodchuck hepatitis virus (WHV) is closely related to HBV in considering molecular and pathobiological properties, including similar patterns of induced liver disease.28, 29 In our previous studies, we have shown that recovery from acute WHV hepatitis is invariably followed by life-long persistence of low-level WHV replication in the liver and the lymphatic system that is accompanied by antibodies to WHV core antigen (anti-WHc).30 In contrast, offspring born to woodchuck mothers convalescent from WHV hepatitis carry comparable levels of infectious virus in the absence of anti-WHc.31 Similarly, animals inoculated with small amounts of WHV (<103 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. Hepatic and lymphoid tissues were analyzed for molecular markers of WHV replication and livers examined for histological alterations.


HBV, hepatitis B virus; anti-HBc, antibodies to HBV core antigen; HBsAg, HBV surface antigen; anti-HBs, antibodies to HBsAg; PCR, polymerase chain reaction; PBMC, peripheral blood mononuclear cells; WHV, woodchuck hepatitis virus; anti-WHc, antibodies to WHV core antigen; WHsAg, WHV surface antigen; anti-WHs, antibodies to WHsAg; vge, virus genome equivalents; ELISA, enzyme-linked immunoassay; HBSS, Hanks' balanced salt solution; rWHV DNA, recombinant WHV DNA; RT, reverse transcription; NAH, nucleic acid hybridization; SOI, secondary (residual) occult infection; POI, primary occult infection.

Materials and Methods

Animals and Sample Collection.

Thirteen adult woodchucks (7 males and 6 females) were intravenously injected with WHV at 1.1 × 1010 DNase-protected virus genome equivalents (vge) (Table 1). 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 inoculum, which had been used to infect the animal from which WHV/tm3 originated. WHV/tm2 complete sequence determined in this study was essentially identical to that of WHV/tm3 (GenBank accession numbers: AY628095 and AY628096). The woodchucks were followed 11 to 64 months postinoculation (Table 1).

Table 1. Serological and Virological Characteristics of WHV Infection and Numbers of Liver Samples Studied
Study Group and Animal No./SexObservation Period* (mo)WHV SerologyDuration of Serum WHV DNA Positivity (mo)WHV DNA Loads at the End of Follow-upPhase of WHV Infection and Number of Liver Samples Analyzed
Duration of WHsAg (mo)Duration of Anti-WHs (mo)Duration of Anti-WHc (mo)SerumPBMCLiverAnti-WHs and Anti-WHc PositivityIsolated Anti-WHc Positivity
  • Abbreviations: F, female; M, male; mo, month; ND, not detected.

  • NOTE. WHV DNA detected by dot-blot hybridization and, when negative, by PCR/NAH. The serum WHV DNA content is presented as estimated number of WHV vge per 1 mL, whereas quantities of WHV DNA in PBMC and liver samples are per 104 cells, as described by Michalak et al.30

  • *

    From time of inoculation with WHV.

  • Anti-WHc antibody lost 5.5 months postinoculation with WHV; 3 liver biopsies were collected during the period of anti-WHc negativity.

Group A: Primary WHsAg-Negative Infection
5/F39ND35.538.51020.05–0.50.2–200 (3)
        Subtotal510 (3)
Group B: Resolved Acute Hepatitis
        Subtotal815 (3)
        Total1325 (3)

Sera were collected before injection with WHV, biweekly for up to 6 months postinoculation and then monthly. PBMC were isolated monthly.32 Liver biopsies were obtained by laparotomy as described.30 The first biopsy was taken 4 to 6 weeks before WHV inoculation, the second 2 to 6 weeks postinoculation, and subsequent at about yearly intervals. In total, 62 liver specimens were examined (Table 1). Ten biopsies were collected prior to injection, 11 in early (acute or presumably acute) phase, 13 during follow-up when both anti-WHc and anti-WHs were detected, and 25 when only isolated anti-WHc occurred. In addition, 3 biopsies were obtained from 5/F after spontaneous clearance of anti-WHc at 5.5 months postinoculation (Table 1). At autopsy, fragments of liver, lymphatic organs and other tissues, and serum and PBMC were preserved. The study protocol was approved by the Institutional Presidents Committee on Animal Bioethics and Care.

Serological Assays and Categories of WHV Infection.

The serological status of WHV infection was determined by routine testing of sera for WHsAg, anti-WHc, and anti-WHs. WHsAg was assayed by an in-house specific enzyme-linked immunoassay (ELISA) or by a cross-reactive radioimmunoassay (Ausria II; Abbott Laboratories, North Chicago, IL) (before June 1998), both with relative sensitivity of 3.25 ng WHsAg/mL. Anti-WHs was measured by specific ELISA34 or a cross-reactive kit (Ausab EIA; Abbott). Anti-WHc was detected by a specific competition ELISA.30 Based on the serological data, the animals were divided into 2 groups: (A) with a primary serum WHsAg-negative infection (n = 6), and (B) with resolved serologically evident acute hepatitis (n = 7) (Table 1). 5/F was included in group A since it had serum WHsAg-negative infection and was initially reactive for anti-WHc.

Serum Ultracentrifugation and DNase Digestion.

To determine the presence of envelope-protected WHV particles, 4 mL of sera from 6/F and 10/M obtained at 41 or 36 months postinoculation (test samples), respectively, and at 1.5 months postinoculation (positive controls) were centrifuged over a 0.5-mL 60% (w/v) sucrose cushion at 200,000g for 22 hours at 5°C in an SW50.1 rotor (Beckman Instruments Inc., Palo Alto, CA). Two 350-μL bottom fractions were collected from each tube and pooled. Then, two 300-μL samples of each pool were supplemented with 40 μL of DNase digestion buffer (100 mmol/L MgCl2 in 500 mmol/L Tris-HCl buffer, pH 8.0) and 5 μL of DNase (2 U/μL; deoxyribonuclease I; Sigma, St. Louis, MO) was added to one of the samples and 5 μL of Hanks' balanced salt solution (HBSS, Gibco, Grand Island, NY) to the other (untreated control) and incubated for 1 hour at 37°C. In addition, full-length recombinant WHV DNA (rWHV DNA) (100 pg/sample),35 treated with DNase or HBSS, was used as positive and negative enzyme reactivity controls, respectively.

In an attempt to enhance detection of WHsAg, 200 μL of selected sera were spun through 4.5 mL of 15% (w/v) sucrose in TN buffer (140 mmol/L NaCl in 10 mmol/L Tris-HCl buffer, pH 8.0) over a 0.5-mL 60% sucrose cushion, as described.12 Samples from 4/F, 6/F, 7/M and 10/M with isolated anti-WHc or anti-WHc/anti-WHs (test samples) and those obtained prior to WHV inoculation (negative controls) and during early phase of WHV infection (positive controls) from the same animals were examined. Following previous findings,12 200 μL of the two bottom 350-μL fractions were tested for WHsAg and 100 μL used to extract DNA.

Detection of WHV DNA, cccDNA, and mRNA.

DNA from 100 μL of serum or sucrose fractions, from DNase-treated or untreated serum pellets, or from ≈1 × 106 naïve (i.e., mitogen unstimulated) PBMC or 1-4 μg DNA from liver or lymphoid tissues was isolated by the proteinase K-phenol-chloroform method.30 For PCR, WHV DNA was amplified using three sets of direct or nested oligonucleotide primers specific for WHV core (C), envelope (surface, S) and X gene sequences, as reported.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.30, 31 In all instances, DNA and RNA isolations were carried out in parallel with mock samples containing TE buffer (1 mmol EDTA in 10 mmol/L Tris-HCl buffer, pH 8.0) instead of nucleic acid. In addition, DNA or RNA from serum, PBMC, or liver tissue of a healthy animal and from a woodchuck with WHsAg-positive chronic WHV hepatitis were included as negative and positive controls, respectively. In the case of RT-PCR, test RNA samples not treated with RT were included to rule out potential WHV DNA carryover.30, 31 The final PCR contamination controls consisted of water added to the direct or nested PCR cocktails instead of template DNA, cDNA or direct PCR product. Nucleic acid hybridization (NAH), i.e., Southern blot analysis, of the PCR products to a rWHV DNA probe was routinely used to verify specificity of detection and validity of controls. The sensitivity of the nested PCR/NAH was < 10 vge/mL and that for WHV cccDNA ≈102 vge/mL, as reported.31–33

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.

Liver Histology.

Histological examination of liver samples was done after routine processing to paraffin and staining.36 Morphological alterations encountered in hepatocellular, extrahepatocellular intralobular, and portal compartments of hepatic parenchyma were graded on a numerical scale from 0-3, as described,36–38 and referred herein as the histological degree of residual hepatitis.


WHV Serological Markers in Primary WHsAg-Negative and Resolved Infections.

The serological data for 13 woodchucks infected with WHV, which developed either a primary serum WHsAg-negative infection (group A) or serum WHsAg-reactive self-limited acute hepatitis (group B), are summarized in Table 1. WHsAg remained negative in group A animals by standard assays, although the antigen was detectable in some sera after ultracentrifugation (Table 2). In animals from group B, WHsAg was transiently reactive during a 1 to 2.5 months (mean 1.9 ± SEM 0.3) period. Considering both groups, anti-WHc were detected at as early as 0.5 to 1.5 months postinoculation (mean 0.9 ± SEM 0.1) and persisted to the end of follow-up in all except 5/F, which lost the antibody after 5.5 months postinoculation (Table 1). Overall, excluding 5/F the mean duration of anti-WHc positivity was 26 months (± SEM 5). Anti-WHs could be identified in all cases for a variable length of time ranging from 1 to 19 months (mean 8.4 ± SEM 1.5). They declined to undetectable levels during follow-up, creating a window lasting between 2.5 and 60 months (mean 16 ± SEM 4.7) where anti-WHc existed as the sole serological indicator of past WHV exposure.

Table 2. Detection of WHV DNA and WHsAg in Sucrose Fractions of Ultracentrifuged Sera From Woodchucks With Anti-WHc and Anti-WHs or Isolated Anti-WHc Antibodies
Animal No./SexTime of Acquisition of Serum Before or After Inoculation with WHV (mo)WHV Serology*Fraction 1Fraction 2
  • NOTE. WHV DNA detected by direct or nested PCR/NAH with WHV C gene-specific primers using DNA extracted from 100 μL of each fraction, whereas WHsAg was determined by ELISA using 200 μL of each fraction.

  • *

    Evaluated at the time of serum collection.

  • Two bottom fractions collected after serum ultracentrifugation through 15% sucrose layered over a 60% sucrose cushion, as described in Materials and Methods.

Group A: Primary WHsAg-Negative Infection
 2anti-WHs+; anti-WHc+++++
 3anti-WHs+; anti-WHc+++
Group B: Resolved Acute Hepatitis
 3anti-WHs+; anti-WHc++++
 1.5WHsAg+; anti-WHc+++++

Detection of Anti-WHc is Invariably Accompanied by WHV DNA Presence.

Serum WHV DNA became detectable from 0.5 to 1.5 months postinoculation (mean 0.6 ± SEM 0.1) and persisted until autopsy in all animals (Table 1). Overall, the mean time period of WHV DNA positivity for group A and B animals was 27.2 months (± SEM 4.7). Figure 1 illustrates a typical pattern of WHV DNA detection in a woodchuck with a silent infection followed for more than 3 years postinoculation. In this case, sequential sera were positive for WHV DNA by direct or nested PCR/NAH with primers specific for at least two of three WHV sequences routinely examined. In all animals, the levels of circulating WHV DNA, at the time when anti-WHc alone or anti-WHc/anti-WHs occurred, did not exceed 102 vge/mL and these loads were comparable to those detected at autopsy (Table 1). Of note is that WHV DNA loads in sera with WHsAg detectable by standard ELISAs were normally greater than 103 to 104 vge/mL, but they could be as high as 1010 to 1011 vge/mL.

Figure 1.

Analysis of WHV DNA presence in sequential sera from woodchuck 5/F with a primary WHsAg-negative infection and with loss of anti-WHc antibody during follow-up. Total DNA extracted from 100-μL serum samples collected at approximately 6-month intervals between 2 weeks and 39 months postinoculation was tested for WHV DNA by PCR using WHV C, S, and X gene-specific primers. The direct or nested PCR amplicons were detected by NAH. Contamination controls included mock (M) treated exactly as test DNA and water added instead of DNA and amplified by a direct (DW) or a nested (NW) PCR. The negative and positive controls consisted of DNA extracted from 100 μL serum of healthy (H) and WHsAg-positive chronically infected (CH) woodchucks, respectively. Positive samples showed the expected sizes (bp) of direct or nested PCR amplicons.

The presence of WHV genomes was analyzed during follow-up at three sites, i.e., serum, PBMC, and liver. WHV DNA was detected in all three locations at the majority of the time points tested. Occasionally, the intensity of WHV DNA signals varied significantly between the sites at a given time point of follow-up, i.e., the signal could be readily detected in the liver and serum, but not in PBMC, as shown in Fig. 2. However, the liver always remained WHV DNA reactive.

Figure 2.

Detection of WHV DNA in parallel sequential sera, PBMC and liver biopsies from 10/M with 36-month follow-up after recovery from self limiting acute WHV hepatitis. DNA was tested by PCR/NAH with WHV C gene-specific primers. Samples of serum, PBMC and liver obtained before inoculation with WHV (−1 month) and from healthy (H) and WHsAg-positive chronically infected (CH) animals were used as negative and positive controls, respectively. Contamination controls were described in legend to Fig. 1. Shown are direct (623-bp) and nested (428-bp) WHV C gene amplicons.

Examination of autopsy samples, which were obtained at the time when only anti-WHc occurred, demonstrated WHV DNA in the liver and lymphatic organs of all animals (13/13), as well as in sera (12/13) and PBMC (11/13) of the majority of the woodchucks, as shown in Table 1. At autopsy, the estimated levels of WHV DNA, as described elsewhere,30 ranged between 0.005 to 0.5 vge/104 PBMC and 0.02 to 2 vge/104 hepatocytes (Table 1).

Analysis of WHV cccDNA and mRNA in autopsy liver and lymphoid tissues by PCR-based methods showed that virus replication continued, although at a low level, during the time period when only anti-WHc were detected. Figure 3 shows expression of WHV cccDNA in liver, spleen, and bone marrow and WHV mRNA in liver and spleen of 10/M, in which anti-WHc persisted for 2 years before autopsy as the only serological marker of past WHV exposure.

Figure 3.

Detection of WHV cccDNA and mRNA in the liver and lymphatic organs obtained from 10/M with isolated anti-WHc carried for ≈2 years prior to autopsy. (A) Total DNA from liver or lymphoid tissues was digested with single strand-specific mung bean nuclease and amplified with PCR primers spanning the nick region of the WHV genome, as described in Materials and Methods. Nested PCR products were analyzed by NAH. Contamination controls included were described in legend to Fig. 1. (B) Total RNA was transcribed in RT reaction (RT+) and cDNA amplified by nested PCR with WHV C gene-specific primers. Each RNA sample was also subjected to the same treatment in the absence of RT (RT−). Nucleic acid-free sample (M) and liver RNA from a WHsAg-positive chronic carrier were included as negative and positive controls, respectively. Contamination controls were described in legend to Fig. 1. Positive signals showed the expected 428-bp amplicons.

The longevity of anti-WHc and the duration of WHV DNA detected by PCR/NAH lasted for the same time period in 7 of the 13 animals (Table 1). In 5 woodchucks, WHV DNA detection preceded the appearance of anti-WHc by 0.5 to 1 months, but both markers remained positive up to the end of follow-up. In 5/F, WHV DNA persisted for more than 2.5 years after loss of anti-WHc. In general, the existence of anti-WHc was intimately linked with the detection of WHV DNA by nested PCR/NAH. In fact, there was no time point during follow-up where isolated anti-WHc or anti-WHc/anti-WHs did not coexist with WHV DNA in the liver and serum and/or PBMC. However, detection of WHV DNA was not always accompanied by anti-WHc, as it was seen before the appearance of anti-WHc in 5 of 13 animals and after 5.5 months postinoculation in 5/F (Table 1).

Properties of WHV DNA-Reactive Particles in Sera With Isolated Anti-WHc.

To determine if WHV DNA reactivity in sera with isolated anti-WHc was contained within the envelope-protected virions, selected sera were spun down and subjected to DNase digestion. As shown in Fig. 4, the fractions from sera with isolated anti-WHc, collected at 41 months (6/F) and at 36 months (10/M) postinoculation remained reactive for WHV DNA after DNase treatment, similar to control sera obtained from an early phase of WHV infection from the same animals. Hybridization signals were evidently less intense after DNase treatment, suggesting that both virions and unenveloped viral genomes or their fragments occurred. DNase treatment of protein-free rWHV DNA completely eliminated the signal (Fig. 4).

Figure 4.

Effect of DNase on WHV DNA-reactive particles circulating in 6/F with persistent isolated anti-WHc after a primary WHsAg-negative infection and in 10/M that resolved acute hepatitis and remained anti-WHc reactive. Serum samples collected at the early phase of infection (1.5 months) or at the end of follow-up were ultracentrifuged and the recovered bottom fraction subjected to DNase digestion (D) or not (ND). WHV DNA signals were detected by direct PCR/NAH with WHV C gene-specific primers. rWHV DNA treated with DNase or untreated was used as enzyme reactivity controls, whereas water and mock as contamination controls.

To assess whether the animals with primary WHsAg-negative infection were truly serum WHsAg nonreactive and to test if WHsAg could be detected in sera with isolated anti-WHc, relevant serum samples from 4/F, 6/F, 7/M and 10/M were centrifuged through a sucrose gradient and the bottom fractions were tested for WHV DNA and WHsAg. As shown in Table 2, WHV DNA was detected in all sera obtained after inoculation with WHV, but not in those collected prior to injection. WHsAg was occasionally identified, predominantly in fraction 1, in sera from animals with a primary WHsAg-nonreactive infection (4/F and 6/F) and in some sera from animals with isolated anti-WHc persisting after recovery from acute hepatitis (7/M and 10/M).

In addition, an electron microscopic examination of the pellet recovered after ultracentrifugation of serum collected at 41 months postinoculation from 6/F showed singular spherical and short tubular WHsAg particles (Fig. 5), although virions were not detected. However, no apparent viral particles could be seen in autopsy serum from 10/M.

Figure 5.

Ultrastructural identification of singular spherical and short tubular WHsAg particles in autopsy serum of 6/F woodchuck with anti-WHc-positive occult WHV infection lasting about 3.5 years after a primary-WHsAg negarive infection. Serum pellet recovered after ultracentrifugation was negatively stained with 1% phosphotungstic acid (original magnification, × 40,000).

Liver Alterations Associated With Isolated Anti-WHc Response.

Histological examination of liver samples, at the time when either anti-WHc/anti-WHs or anti-WHc alone occurred, revealed minimal-to-mild inflammatory alterations (rarely exceeding grade 1). These lesions were frequently interrupted by periods of normal or nearly normal liver morphology and their features were identical to those previously described in woodchucks with remote resolved acute hepatitis.30, 37 Statistical analysis of the mean grades of morphological lesions found in the individual compartments of liver parenchyma in samples collected in the anti-WHc/anti-WHs- and anti-WHc-positive phases from group A or B animals showed no significant differences (P < .13), except in hepatocellular changes in the anti-WHc-positive phase seen between groups A and B. Overall, the existence of isolated anti-WHc was accompanied by features of intermittent minimal to mild liver inflammation independent of whether the primary infection was serum WHsAg-negative or -positive.


Analysis of serial sera, circulating lymphoid cells and liver biopsies using a highly sensitive PCR/NAH showed WHV genome in all animals in which anti-WHc persisted as the sole marker of exposure to WHV. Similarly, anti-WHc occurring along with anti-WHs were concomitant with WHV DNA detection. The liver samples obtained during the anti-WHc-positive phase were consistently WHV DNA reactive regardless of the time period that elapsed after inoculation with WHV. Also, the vast majority of sera (≈70%) and PBMC (≈80%) obtained during the anti-WHc- and anti-WHc/anti-WHs-positive periods were WHV DNA reactive (data not shown). The estimated WHV copy numbers occurring in these periods were very low and essentially stable, usually not exceeding 102 vge/mL of serum and 0.5 to 2 vge/104 hepatocytes or PBMC. 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 (<10 vge/mL) in this and our previous studies30–33 over nested PCR assays used by others (sensitivity 102-103 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.

WHV cccDNA and pregenomic RNA were detected in both the liver and peripheral and organ lymphoid cells in the late observation period or at autopsy when only isolated anti-WHc occurred. They were also identified at the time when both anti-WHc and anti-WHs were present. Detection of these two replicative intermediates by sensitive PCR-based techniques indicates that low-level WHV replication progressed at these sites known to be naturally targeted by WHV.32, 39, 40 These findings are consistent with our earlier reports of SOI continuing for life after termination of acute WHV hepatitis,30, 32, 37 along with documented infectivity and liver pathogenic competence of the silently carried virus.30, 32 The current results also agree with the data from the limited studies of apparently healthy individuals reactive for anti-HBc alone or anti-HBc and anti-HBs, which showed presence of HBV cccDNA and HBV RNA in their liver8, 41 and transmission of HBV by liver grafts15–21 or blood42–44 to virus-naïve recipients.

Analysis of WHV DNA in sera after treatment with DNase obtained after ≈3 year follow-up of a primary WHsAg-negative infection or SOI showed that at least a fraction of the DNA molecules was resistant to the digestion. This property was comparable to that observed in WHV DNA-positive sera collected in the early phase of infection from the same animals, providing strong evidence for circulating intact virus. This finding is analogous to results from a similar type of analysis performed on sera from offspring born to woodchuck dams recovered from WHV hepatitis31 and individuals with a past history of resolved acute hepatitis B.12 Detection of traces of WHsAg in concentrated sera simultaneously with the presence of isolated anti-WHc or anti-WHc/anti-WHs suggested that both WHV DNA and WHV envelope proteins were persistently produced, and it further supports the possibility that complete virions were assembled. This was not unexpected, since a similar finding was made previously in individuals with a past history of resolved acute hepatitis B.12

Two groups of animals infected with identical doses of the same inocula developed a primary WHV infection that was either serum WHsAg positive or negative by standard assay protocols. It is of note that the seemingly WHsAg-negative infection occurs in about 10% of woodchucks infected as adults with massive WHV doses (1.1–6.8 × 1010 DNase-protected WHV vge).39 As this study showed, WHsAg was in fact detectable in these animals but only after concentration of their sera by ultracentrifugation. The pattern of anti-WHc and anti-WHs responses in this low-level WHsAg-positive infection was comparable to those observed in animals with a classical WHsAg-positive infection associated with self-limiting hepatitis. However, serum WHV DNA load was usually 10- to 100-fold lower in early infection and liver inflammation was much milder than that at the time of acute hepatitis in high-level WHsAg-positive woodchucks (data not shown). Since the animals with low-level WHs antigenemia were infected with the same wild-type virus as the animals with a subsequent WHsAg-positive infection and only wild-type WHV sequences have been detected in their sera so far (data not shown), the development of these two patterns of primary WHV infection appears to be related to host milieu but not to virus. Possibly, this difference could be related to a lower susceptibility of some animals to WHV infection or to a swift and highly effective innate immune response, which both could be due to the individual host intrinsic makeup, such as genetic factors. At this stage, we cannot offer any experimental data supporting or arguing against these eventualities. Nevertheless, the long-term consequences of this primary serologically concealed infection, for example, WHV DNA levels, persistence of replication in the liver and the lymphatic system, and features of liver histology, were identical to those found in animals with remote self limiting hepatitis. In general, our study showed that high doses of infectious hepadnavirus may occasionally trigger serologically undetectable primary infection that invariably leads to occult virus persistence associated with circulation of isolated anti-WHc.

All animals, except 5/F, remained anti-WHc positive until the end of follow-up. Spontaneous clearance of anti-WHc is very rare in anti-WHc positive infection and occurs in no more than 1% of woodchucks in our colony inoculated with a massive virus dose (<1010 vge). The sustained loss of anti-WHc in 5/F was not linked to elimination of WHV DNA, since it persisted for more than 2.5 years thereafter. This finding indicates that SOI may occasionally progress in the absence of anti-WHc. In contrast to SOI, a primary occult infection (POI), described in our previous works,30–33 is characterized by the complete lack of anti-WHc. Other characteristics of POI include: (1) virus dose causing infection usually does not exceed 103 vge; (2) infection is limited to the lymphatic system but with time may engage the liver, and (3) it does not induce immunoprotection against challenge with a large pathogenic virus dose (<104 vge) causing hepatitis.33 However, in both SOI and POI, WHV DNA persists at comparable levels in serum (usually >102 vge) and in lymphoid cells (>103 vge/μg of total DNA).

In conclusion, the present study indicates that the long-term occurrence of isolated anti-WHc is a consequence of a low-level continuing WHV assembly. Their protracted existence appears to be driven by a sustained antigenic stimulus, for example, synthesis of core antigen, as it is likewise required to maintain strong HBV-specific cytotoxic T-cell and T helper lymphocyte responses years after resolution of acute hepatitis B.13, 14 The high degree of immunovirological compatibility between WHV and HBV infections and the data from studies analyzing healthy anti-HBc-positive individuals suggest that the occurrence of isolated anti-HBc could be important in identifying occult HBV persistence.


The authors thank Colleen L. Trelegan for expert assistance, Luke Grenning for technical support during laparotomies, and Judy Foote and Mike Goldsworthy for preparation and staining of paraffin sections for histological examinations.