Hepatitis B Virus Replication in Diverse Cell Types During Chronic Hepatitis B Virus Infection

ANDREW ASON, ON,'^^ MARK WICK,2 HEATHER WHITE3 AND ROBERT PERRILL01.3

'Gastroenterology Section, Veterans Affairs Medical Center, St. Louis, Missouri 631 06, and Departments of 2Pathology and 31nternal Medicine, Washington Uniuersity, St. Louis, Missouri 631 10

Hepatitis B virus-specific nucleic acid sequences and proteins have been detected in extrahepatic tissues of acutely and chronically infected patients. However, apart from peripheral blood mononuclear cells and bone marrow cells, little is known about the specific cell types that permit viral replication. In this study, we assessed the extrahepatic tissues of four patients who died with chronic hepatitis B virus infection and two uninfected controls by means of in situ hybridization and immunohistochemical study. Three of these patients had diffuse extrahepatic dis- tribution of the virus. Hepatitis B virus nucleic acid sequences and proteins were detected in the lymph nodes, spleen, bone marrow, kidney, skin, colon, stomach, testes and periadrenal ganglia. The following cell types were found to be positive for hepatitis B virus: endothelial cells, macrophages/monocytes, he- matopoietic precursors, basal keratinocytes, mucosal epithelial cells, stromal fibroblasts and sustentacular and neuronal cells. It is probable that these cells could support viral replication because hepatitis B virus DNA replicative intermediates, viral transcripts and HBsAg and HBcAg proteins were detected in most. These findings may be relevant to the initiation of extrahepatic syndromes associated with chronic hep- atitis B virus infection such as vasculitis, glomerulo- nephropathy, neuropathy and dermatitis. (HEPATOLOGY 1993;18:781-789.)

During HBV infection the viral burden and subse- quent tissue damage are mainly confined to the liver. Similar to the other hepatotrophic hepadnaviruses, however, HEV may gain access to and replicate in several different tissues. The diffuse extrahepatic life cycle of the Hepadnauiridae has been well defined in several animal models such as ducks (1-3) and wood- chucks (4, 5). In these animals, viral DNA and tran- scripts have been observed in diverse tissues, including lymphoid tissue, kidney, pancreas and gonads, sug-

Received January 28, 1993; accepted May 17, 1993. Address reprint requests to: Robert Perrillo, M.D., St. Louis Veterans Affairs

3 1/1/49 132 Medical Center (111 JC), 915 North Grand Blvd., St. Louis, MO 63106.

gesting that viral replication occurs in many extrahe- patic sites. The earliest studies documenting the extra- hepatic biology of human HBV focused on readily obtainable tissue and body fluid samples such as pe- ripheral blood mononuclear cells (PBMCs) (61, saliva, semen, urine, feces and biliary and pancreatic juices (7-10). Subsequently, HBsAg was located by means of immunohistochemical study in the pancreas (1 l), bone marrow (12) and skin (13). More recently, HBV-related nucleic acid sequences have been detected with Southern blotting in pancreas, kidney (14, 15, 161, lymphoid tissue (16, 17) thyroid and adrenal glands, gonads (15) and placenta (16).

The pathological significance of the extrahepatic distribution and biology of HBV is not well understood. Several extrahepatic syndromes associated with HBV infection have been considered to be mediated by immune complexes or other immunological mecha- nisms; these include neuropathies, glomerulonephritis, vasculitis, cryoglobulinemia, aplastic anemia and a variety of dermatological lesions (18, 19). Apart from these syndromes, however, the detection of Hepad- nauiridae in extrahepatic tissues in animals and human subjects is seldom accompanied by evidence of tissue damage (1-5,14,15,16). At present, little is known of the mechanism(s) behind this apparent lack of disease; nor have the specific cell types that support viral replication been identified. Accordingly, we assessed the mclecular forms of HBV in several nonhepatic tissues by means of in situ hybridization and immunohistochemical study in an attempt to specifically identify cells infected with replicating virus. We believe that the information so derived could further our understanding of the extra- hepatic syndromes associated with HBV infection and lead to better appreciation of the mechanisms of viral persistence and reactivation.

MATERIALS AND METHODS Formalin-fixed, paraffin-embedded autopsy tissues from

four patients with chronic HBV infection at the time of death and two uninfected control patients who died of alcoholic hepatitis were evaluated in this study (Table 1). All available tissue from each subject with chronic hepatitis B (Table 1) and representative tissues from the two controls were studied by

781

782 MASONETAL. HEPATOLOGY October 1993

TABLE 1. Autopsy tissues in subjects with chronic HBV infection Patient Sedage Serum HBeAg and Liver histological

no. (Yr) HBaAg status Cause of death appearance Autopsy tissues

1 F/54 HBeAgand Graft failure after liver trans-

2 M/32 HBeAgand Graft failure after liver trans- HBsAg positive plantation

HBsAg positive plantation

3 MI37 HBeAg and Kaposi's sarcoma, AIDS HBsAg positive

4 MI47 HBeAg negative Bronchopneumonia, hepatic en- HBsAg positive cephalopathy and massive

ascites

Submassive necrosis

Massive necrosis

Adrenal gland, brain, gonads, heart, kidney, lung, pancreas

Adrenal gland, brain, skin, go- nads, heart, kidney, lung, lymph nodes, pancreas, spleen

Brain, bone marrow, skin, go- nads, heart, kidney, Kaposi's sarcoma, lung, lymph nodes, spleen

Adrenal gland, skin, gut," go- nads, kidney, lung, lymph nodes, spleen, tongue

CAH

Cirrhosis

""Gut" refers to colon and stomach.

TABLE 2. Tissues and cell types associated with HBV infection

Tissues Cell types

Lymph node Spleen Periadrenal ganglion Skin

Gut

Kidney Bone marrow Testicle

Endothelium, macrophage/monocyte Endothelium, macrophagelmonocyte Neuronal cells, sustentacular cells Basal keratinocytes, dermal stromal

Endothelium, mucosal epithelium and

Endothelium Hematopoietic precursors Intertubular stromal fibroblasts

fibroblasts

stromal fibroblasts

means of both immunohistochemical methods and in situ hybridization.

In S i tu Hybridization. The methods used for in situ hybridization and riboprobe construction have been described in detail elsewhere (20). The 35S-labeled riboprobes, specific for the replicative intermediates of HBV DNA or HBV RNA, were synthesized from a 310-bp complementary DNA of the HBV (ayw) surface antigen gene (a kind donation of D. A. Shafritz, M.D., Albert Einstein College of Medicine, New York, NY). The pGem 32 plasmid construct containing the HBsAg comple- mentary DNA insert was linearized with either EcoRI or Hind111 restriction enzymes (all reagents from Promega Corp., Madison, WI). For HBV DNA riboprobe construction, 0.5 kg of EcoRI linearized template was incubated for 1 hr at 40" C in a total volume of 10 kl with 10 units of SP6 RNA polymerase, 1 unit RNAsin, 15 k1 of dried [ 35S]UTP (specific activity = 1250 Ci/mmol/L) (concentration = 8 mmol/L), NEN (Du Pont, Wilmington, DE) and 2.0 kl of cold nucleotides (10 mmol/L each) ATP, CTP and GTP and 0.1 mmol/L UTP. Likewise, the Hind 111-linearized template was incubated with 10 units of T7 polymerase at 37" C to generate an antisense riboprobe to HBV RNA. The riboprobes were incubated with DNAse at 37" C for 15 min to remove the DNA template, treated with phenoVchloroform and chloroform alone and then extracted twice in 4 m o w ammonia acetate to remove unincorporated nucleotides and low molecular weight riboprobe sequences.

Paraffin-embedded liver tissues were sectioned onto micro- scope slides, incubated in an oven at 65" C for 1 hr,

deparaffinized in xylenes, rehydrated in graded alcohols and rinsed in PBS (pH 7.4; Sigma Chemical Co., St. Louis, MO). After a 20-min digestion with 1 mg/L proteinase K at 37" C, the slides were washed with 0.25% acetic anhydride in 0.1 mol/L triethanolamine buffer, dehydrated with graded alcohols and air-dried. Hybridizations were performed overnight at 52" C with [35S]UTP-labeled riboprobes (antisense to HBsAg RNA or antisense to the single-stranded replicative intermediates of the native HBV DNA). The riboprobe was added to the hybridization solution at a concentration calculated at 10,000 cpm/kl. The probe and hybridization solution containing 50% formamide, 10% dextran sulfate, 2 x saline sodium citrate (SSC), 1 x Denhart's, 1 mmom EDTA, 20 mmol/L Tris (pH 81, 100 mmol/L dithiothreitol and yeast transfer RNA (0.5 mg/mL) (all reagents Sigma Chemical Co.), were heated to 68" C for 10 min before use to disaggregate the probe.

After hybridization, the slides were washed in 25 mmoVL P-mercaptoethanol (Sigma Chemical Co.), 4~ SSC (15 min, twice) and 0 . 5 ~ SSC (5 min). This was followed by a high-stringency wash in 0.1% SSC for 20 min at 60" C. The samples were then digested in 20 pg/ml RNase A to remove unhybridized probe; this was followed by another high- stringency wash at 60" C in 0.1% SSC. Slides were then dehydrated in graded alcohols and air-dried. The slides were then coated with NTB emulsion (Eastman Kodak Co., Roch- ester, NY) and stored at 4" C for 6 wk for autoradiography. After developing, the slides were counterstained with hema- toxylin and eosin-Y. Some liver tissue sections were pretreated with RNAse A as a control for the viral RNA hybridizations before the proteinase K digestion to demonstrate that the viral RNA antisense probe was annealing only to viral RNA. Because riboprobes can be destroyed by any residual RNases, a 3' 35S-labeled oligonucleotide probe (see reference 21 for sequence of oligonucleotide) complementary to the HBcAg region of HBV RNA was used for the latter step instead of the riboprobe. After hybridization with the oligoprobe, three low-stringency washes in 2 x SSC for 30 min each were performed at room temperature before autoradiography.

The sensitivity and specificity of this in situ hybridization procedure has been studied extensively and described else- where for hepatic and nonhepatic tissue (22-24). This in situ hybridization procedure is capable of highlighting the occa- sional HBV-positive cell, and its results can be confirmed with the polymerase chain reaction technique (22). Also, HBV nucleic acid sequences did not nonspecifidly hybridize with

HEPATOLOGY Vol. 18, No. 4, 1993 MASONETAL. 783

FIG. 1. (a) A nodular area of the red pulp occupies most of this micrograph of the spleen (H&E; original m e c a t i o n x 100) and (b) darkiield autoradiograph of the same field demonstrates diffuse in situ hybridization signal to HBV DNA confined to the red pulp of the spleen. (c) Immunostaining for HBcAg shows intense nuclear staining of hematopoietic cells in the splenic red pulp. Some cells have the appearance of lymphocytes (small arrows), whereas others are morphologically consistent with splenic littoral cells or macrophages (large arrows) (original magnification x 400).

sense and antisense genomic riboprobes (22). Riboprobe signal to HBV RNA and DNA has only been observed in tissues of patients with serologically documented chronic hepatitis B (22-24). Using the antisense riboprobe to HBV RNA, we completely abolished the signal with RNAse A pretreatment of the tissue, confirming the specificity of the probe to HBV RNA sequences (23, 24). Because no DNA denaturing step is included in these in situ hybridization methods, the H3V DNA riboprobe specifically hybridizes to the replicative intermediates of the L (minus) strand of HBV DNA rather than denatured HBV DNA. The specificity of the HBV DNA riboprobe has been demonstrated; hepatic hy- bridization signal to replicative intermediates of HBV DNA was only detected in patients with serum HBeAg, HBV DNA or both (24).

Immunohistochemical Study. The autopsy tissues were deparafflnized in Wlenes and absolute alcohol and incubated for 30 min in 0.6% methanol and hydrogen peroxide. After rehydration in graded alcohols, distilled water and PBS, the samples were incubated in PBS with either HBsAg antibody (Biogenics, San Ramon, CA) or HBcAg antibody (Dako, Carpinteria, CA) for 16 hr at 4" C. The subsequent antibody bridge assembly was accomplished by means of the ABC method as described previously (25). The slides were developed in 0.25 mglml diaminobenzidine with 0.003% hydrogen per- oxide for up to 10 min, dipped into 0.125% osmium tetroxide and counterstained with hematoqlin. The specificity of the HBaAg and HBcAg immunohistochemical methods has been established with hepatic tissue from patients with and without serum markers for hepatitis B infection (24).

784 MASONETAL HEPATOLOGY October 1993

FIG. 2. (a) Germinal centers of a lymph node (H&E; original magnification ~ 2 0 0 ) and (b) darkfield autoradiograph of the same field demonstrating intrasinusoidal in situ hybridization signal to HBV RNA. (c) Immunostaining of lymph nodes for HBcAg (original magnification x 400). The nuclei of macrophages (arrows) are intensely stained for this marker. (d) In situ hybridization signal to HBV RNA in macrophages (Zarge arrows) and perisinusoidal lymphocytes (small arrow) (original magnification x 600).

RESULTS In Situ Hybridization and Immunohistochemical

Study. Positive hybridization signals to HBV tran- scripts, DNA replicative intermediates or both were observed in the liver of each patient in the study group (Table 1) but in neither of the two control livers. No specific hybridization signal was observed in any of the samples pretreated with RNAse A, in the extrahepatic tissues from case 1 or in the two control patients without serum markers of HBV infection. However, hybrid- ization signals to both HBV DNA and RNA were observed in a variety of extrahepatic tissues and cell types of patients 2, 3 and 4 (Table 2). Several tissues (spleen, lymph nodes, periadrenal ganglia and skin) had diffuse HBV DNA and RNA hybridization signals, and

all tissues but skin displayed immunostaining for HBsAg and HBcAg (Figs. 1-4). In contrast, no specific viral protein staining was observed in the tissues in which only an occasional cell had a positive HBV RNA signal such as bone marrow (data not shown), testis (data not shown) and kidney (Fig. 5 ) . In the gut, an occasional cell exhibited an in situ hybridization signal, whereas diffuse staining for HBsAg was evident in the vascular endothelium (Fig. 6).

Patient 2, who experienced HBV reinfection of his hepatic allograft and then fibrosing cholestatic hepatitis, had diffuse HBsAg and HBcAg staining and strong HBV DNA and RNA signals in the spleen (Fig. 11, lymph nodes (Fig. 2) and periadrenal ganglia (Fig. 3). In this patient only an occasional cell in the kidney had detectable HBV

.

HEPATOLOGY Vol. 18, No. 4, 1993 MASON ET AL. 785

FIG. 3. (a) Periadrenal ganglion seen adjacent to the fibrous adrenal capsule (bottom) (original magnification x 200). (b) Corresponding immunostain for HBcAg showing intense labeling of ganglion constituents. The adjacent adrenal capsule is unstained. (c) Darkfield autoradiograph of in situ hybridization demonstrating diffuse signal to HBV RNA in the ganglion elements; adrenal capsule is negative. (d) Higher-power photomicrograph of HBcAg immunostain in the periadrenal ganglion. Neurons (large arrows) and smaller sustentacular cells (small arrows) both demonstrate strong nuclear immunoreactivity (original magnification x 400). (e) Corresponding autoradiograph of HBV RNA in situ hybridization showing similar distribution of signal in neurons (large arrows) and sustentacular cells (small arrow) (original magnification x 400).

RNA (Fig. 5). HBV DNA and RNA were observed in the bone marrow and spleen of patient 3, who was positive for antibody to human immunodeficiency virus-1. Pa- tient 4, who was seronegative for HBV DNA and HBeAg at the time of death, demonstrated a weak RNA signal in liver (data not shown) but diffuse distribution in the dermis, epidermis (Fig. 4) and periadrenal ganglia. In this patient, HBV RNA was detected in a focal distri- bution in the colon (Fig. 6b) and testicular tissue, but diffuse HBsAg staining was observed in the endothelium of the stomach (Fig. 6a).

Histological Appearance and Cell Types. HBV was detected in the tissues and specific cell types listed in

Table 2. These primarily included the sustentacular and neuronal elements of periadrenal ganglia; capillary endothelia of lymph nodes, gastric submucosa, peritu- bular renovascular rete and renal glomeruli; littoral (sinusoidal lining) cells of the spleen; interfollicular macrophages in lymph nodes; fibroblasts in the dermis, gastrointestinal tract, and intertubular testicular stroma; and basal epithelium of the epidermis.

Infected endothelial cells in all sites were identified by their clear relationship to vascular lumina of varying calibers (Figs. 5 and 6a); they were cuboidal, and lined continuous tubular channels filled at least partially with erythrocytes. Neurons were clustered in juxtaadrenal

786 MASONETAL HEPATOLOGY October 1993

FIG. 4. Autoradiograph of skin demonstrating in situ hybridization signal to HBsAg RNA in the dermis (large arrow) and epidermis (small arrows) (H&E; original magnification x 400).

ganglia, with large vesicular nuclei, prominent nucleoli and abundant cytoplasm containing lipofuscin (Fig. 3d and e). Fusiform sustentacular elements occupied the interneuronal tissue spaces; they showed compact, bluntly spindled nuclei with scant cytoplasm (Fig. 3d and e).

Infected cells were dispersed throughout the red pulp of the spleen, where they clustered near sinusoidal lumina but were clearly situated in the splenic cords (Fig. 1). Some had a compact structure similar to that of lymphocytes, with round, hyperchromatic nuclei and scant cytoplasm (Fig. lc). Others were larger, with reniform vesicular nuclei and abundant cytoplasm (Fig. lc); these were difficult to identify definitively as macrophages or littoral cells, particularly because the latter cell types are probably interrelated. On the other hand, positive cells in lymph nodes were intrasinusoidal and had abundant cytoplasm, typical of macrophages (Fig. 2c and d).

Finally, elongated spindle cells in the skin (Fig. 4) and testis (data not shown) were surrounded by abundant, mature collagenous matrix. These cells showed fusiform nuclear contours, dispersed chromatin and indistinct cell boundaries, typical of fibroblastic elements. Of note was that none of the infected tissues in this study demonstrated evidence of inflammation.

DISCUSSION Although the extrahepatic biology of HBV and other

hepadnaviruses has been amply demonstrated (1-17), this study differs from previous reports in that it describes the many cell types that harbor HBV in patients with chronic hepatitis B. The detection of HBV RNA and replicative intermediates of HBV DNA, HBsAg and HBcAg in most of the various cell types harboring HBV suggests that these cells can support viral repli- cation as previously demonstrated in hepadnavirus

FIG. 5. Intense in situ hybridization signal to HBV RNA in a swollen endothelial cell fZarge arrow) of a renal afferent glomerular arteriole. A portion of the corresponding glomerulus is shown at the bottom of the figure (small arrows) (H&E; original magnification x 400).

animal models (1-5). These findings do not prove that infectious virus is being synthesized or secreted by these cells, however; defective viral processing and assembly cannot be detected with the methods we used in this study. Of note was that the infected cells were not associated with an inflammatory response. These findings have potential implications in the extrahepatic syndromes associated with HBV infection, reactivation of infection after loss of serum HBsAg and recurrence of infection after liver transplantation for hepatitis B.

In this study, HBV was detected in a variety of cell types, including vascular endothelium in various tis- sues: macrophage, monocyte, lymphoid and hemopoetic stem cells in lymphoid tissue; neuronal and susten- tacular cells in the periadrenal ganglion; mucosal epi- thelium in the gut; and dermal, epidermal and testicular stromal cells. It is interesting to note that most of the cell types that we have found to harbor HBV are also associated with HBV-related extrahepatic syndromes such as the glomerulonephritis, vasculitides, neuropa- thies, aplastic anemia and a variety of dermatologic lesions. For example, hepatitis B can cause aplastic anemia (19,26) and is trophic for lymphoid, myeloid and hemopoetic stem cells. Likewise, HBV nucleic acids and proteins were detected in nerve ganglions in amounts comparable to those noted in the liver during viral replication, and HBV infection has been associated with the Guillain-Barr6 syndrome, isolated neuropathies and mononeuritis multiplex of polyarteritis nodosa (19). Also, the presence of HBV in testicular tissue may be related to the semen abnormalities observed in patients with chronic HBV infection (19). Many HBV-related der- matological syndromes have previously been assumed to be mediated by immune mechanisms such as immune complex disease (13, 18, 19). But the demonstration of HBV nucleic acids and proteins in the dermis, epidermis and skin capillary endothelium suggests that the various

HEPATOLOGY Vol. 18, No. 4, 1993 MASONETAL. 787

FIG. 6. (a) HBsAg immunostaining demonstrating HBV in the capillary endothelium of the stomach farrows). (b) Autoradiograph of in situ hybridization demonstrating focal signal in stromal cells of the colonic mucosa to HBV RNA (H&E; original magnification x 400).

vasculitic and other HBV-related skin lesions are asso- ciated with local viral replication as well (13).

Immune complexes have a predilection for vessels with fenestrated endothelial cell lining such as spleen, lymph nodes, arterioles and glomeruli, and they may cause damage by activating inflammatory mediators (27). The observation of HBsAg, HBcAg and HBeAg proteins in the basement membranes of patients with HBV-related glomerulonephritis has led physicians to conclude that the damage is mediated by immune complexes (28). However, it is possible that viral replication in the kidney itself also contributes to the renal disease associated with chronic hepatitis B. This hypothesis was recently suggested by Lisker-Melman and associates, who noted a relationship between de- creased levels of serum HBV DNA polymerase during interferon-a therapy and improvement in kidney function (29). Our observation of HBV DNA replicative intermediates and viral transcripts in the periglo- merular and peritubular endothelium provides further support for this hypothesis. Similarly, the documen- tation of HBV in skin, gut, renal, lymph node and splenic capillary endothelium as well as testicular, neuronal and sustentacular cells may be relevant to the pathogenesis of polyarteritis nodosa in HBsAg-seropositive patients (18). These patients classically suffer from vasculitic lesions in the gut, skin, testes and kidney and may also have isolated neuropathy (18). It is interesting to speculate that once the HBV-related immune complexes are captured by the vascular endothelium the vasculitic damage might also be mediated by viral replication and immune mechanisms.

The normal histological appearance and lack of in- flammatory response seen in the extrahepatic tissues of our patients is of particular interest. This phenomenon has also been reported in patients with acute hepatitis B infection (14-16) and in the duck and woodchuck models (1-5). Although we do not understand the mechanism(s) behind this lack of an inflammatory response, it is possible that the viral burden is not sufficient to be detected in these extrahepatic tissues or that the virus prevents immune recognition by the host. The first possibility does not appear tenable in all circumstances; the viral signal detected in some of the extrahepatic tissues in this study (e.g., Fig. 3) is comparable to amounts observed in the liver in chronic infection (data not shown). Similar observations were reported by Yoffe et al. in patients with acute hepatitis B (15). Viruses have evolved several mechanisms to avoid immune detection in specific cell types, including microbial mutation, disruption of the host’s immune response and molecular mimicry of the host’s proteins. For example, lytic lesions are found in the brains of mice infected with lymphocytic choriomeningitis virus, which undergoes a cell-specific mutation to adopt a persistent, noncyto- pathic phenotype in lymphocytes (30). There are many examples of reduced cell expression of human leukocyte antigen (HLA), adhesion molecules and cytokines during viral infection (31). Forster et al. suggested that HBV DNA polymerase decreases interferon-a-induced HLA class 1 expression in the infected cell by binding to and inhibiting the response element subject to stimu- lation by interferon-a (32). Viruses may also evade the host immune response in specific cell types by sharing

788 MASONETAL HEPATOLOGY October 1993

amino acid sequences that mimic their host’s immu- nodominant epitopes (33). In support of the mechanism of molecular mimicry, the proteins of many neu- rotrophic viruses such as poliomyelitis, Epstein-Barr virus, influenza and polyoma viruses share amino acid sequences with the immunodominani myelin basic protein (MBP) epitopes (33). When the homologous viral amino acid sequences are presented by the HLA class 1 infected neurons, the surveying cytotoxic lymphocytes may recognize these analogous MBP amino acid se- quences as self; consequently, an immune response is avoided (33). I t is interesting to speculate that HBV uses a similar mechanism to avoid immune recognition in neuronal tissue; HBV DNA polymerase shares marked homology of amino acid sequences with the encepha- litogenic site of MBP (33).

PBMCs and other extrahepatic tissue reservoirs are thought to provide the source of HBV for allograft reinfection in patients subjected to liver transplan- tation for chronic hepatitis B. It is interesting that patient 4 in this study was seronegative for HBV DNA and HBeAg at the time of study and, although he had an appreciable viral burden in his extrahepatic tissues (Figs. 4 and 6) , we saw minimal evidence of viral replication in the liver, with only an occasional cell positive for HBV RNA signal but not HBV DNA. This phenomenon has also been observed in patients with fulminant liver failure (16) and in chronic hepatitis B after the loss of HBeAg and HBV DNA (10, 34). Thus it appears that HBV may persist for longer periods in tissues that are not the site of an immmological response by the host. Recently it was found that HBV DNA can be detected in peripheral lymphocytes and liver with polymerase chain reaction years after the loss of HBsAg from serum (22, 23). On the basis of these reports and the findings of this study, it is reasonable to be concerned that organs from patients with his- tories of chronic hepatitis B have an increased risk of viral transmission and, therefore, may not be suitable for elective transplantation.

1.

2.

3.

4.

5.

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