Georgetown University Medical Center, Division ofMolecular Virology and Immunology, Rockville, ...... Chisari, F. V., K. L. Castle, C. Xavier, and D. S. Anderson.
JOURNAL OF VIROLOGY, Mar. 1989, p. 1360-1370 0022-538X/89/031360-11$02.00/0 Copyright © 1989, American Society for Microbiology
Vol. 63, No. 3
Natural History of Woodchuck Hepatitis Virus Infections during the Course of Experimental Viral Infection: Molecular Virologic Features of the Liver and Lymphoid Tissues BRENT E. KORBA,1* PAUL J. COTE,' FRANCES V. WELLS,' BETTY BALDWIN,2 HANS POPPER,3 ROBERT H.
PURCELL,4 BUD C. TENNANT,2 AND JOHN L. GERIN' Georgetown University Medical Center, Division of Molecular Virology and Immunology, Rockville, Maryland 208521; College of Veterinary Medicine, Cornell University, Ithaca, New York 148532; Stratton Laboratory for the Study of Liver Diseases, Mount Sinai School of Medicine of the City University of New York, New York, New York 100293; and National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 208924 Received 5 August 1988/Accepted 6 December 1988
In this study, the kinetic patterns of woodchuck hepatitis virus (WHV) infection were monitored in the liver and the five primary components of the lymphoid system (peripheral blood lymphocytes, lymph nodes, bone marrow, spleen, and thymus). Groups of woodchucks experimentally infected with a standardized inoculum of WHV were sacrificed at different times over a 65-week period beginning in the preacute phase of viral infection and continuing to the period of serologic recovery or the establishment of chronic infections and subsequent hepatocellular carcinoma. Infection by WHV was not limited to the liver but involved the major components of the lymphoid system during all stages of virus infection. A complex series of kinetic patterns was observed for the appearance of WHV DNA in the different lymphoid compartments and the liver during the entire course of viral infection. A progressive evolution of different WHV genomic forms related to the replicative state of WHV was also observed. Lymphoid cells of the bone marrow were the first cells in which WHV DNA was detected, followed in order by the liver, the spleen, peripheral blood lymphocytes, lymph nodes, and finally the thymus. Several differences were observed in the cellular WHV DNA patterns between woodchucks that developed chronic WHV infections and those that serologically recovered from acute WHV infections. The observations compiled in this study indicate that the host lymphoid system is intimately involved in the natural history of hepadnavirus infections from the earliest stages of virus entry.
During the past several years, the tissue tropism of the hepatitis B virus (HBV) and other members of the family Hepadnaviridae, the woodchuck hepatitis virus (WHV) and the duck hepatitis virus (DHBV), has been extended to several tissues other than the liver (5, 8, 10, 13, 16, 29, 38; for a review, see reference 19). The presence of viral-specific RNA transcripts and viral DNA replication intermediates in a number of extrahepatic tissues has demonstrated that the hepadnavirus genomes present in some extrahepatic tissues represent active viral infections (14, 17, 19, 21, 22, 24, 38, 39). Recently, reactivation of quiescent WHV infections and export of mature virions have been demonstrated in vitro after mitogen stimulation of WHV-infected woodchuck peripheral blood lymphocytes (PBL), suggesting that a latent form of WHV infection can be maintained in some lymphoid cells (18). The majority of studies on extrahepatic hepadnavirus infections have focused upon PBL from chronically infected humans and animals. Systemic infection by DHBV has been monitored over the early stages of virus infection (13, 17, 39). These studies revealed changing kinetic patterns of DHBV during the progression to chronic viral infection. However, these studies did not address the virologic events involved in animals which resolved DHBV infections. No parallel study has been reported for mammalian hepadnavi-
hepatocellular carcinoma (HCC) and lymphoid cell infection (19, 21, 22, 32). In this report, the time course of WHV infection of the liver and the five primary components of the lymphoid system (PBL, lymph nodes, bone marrow, spleen, and thymus) have been monitored over the natural course of viral infection. Woodchucks were experimentally inoculated with WHV, and tissues were examined over a 65-week period for viral nucleic acids at several stages of WHV infection, beginning with the preacute phase and continuing through to serologic recovery or to chronic infection with the development of HCC. The results compiled in this study suggest that the lymphoid system is intimately involved in the natural history of hepadnavirus infections from the earliest stages of virus entry. These observations are compared with previous, parallel studies on DHBV (12, 17, 39) and should contribute to an overall understanding of the pathobiology of hepadnaviruses. (A preliminary report on part of this study was made at the 1987 UCLA Symposium on Hepadnaviruses, Keystone, Colo. [20]). MATERIALS AND METHODS Experimental design. This study was designed to examine the kinetics of WHV infection of liver and extrahepatic tissues during the entire course of virus infection. Since the stages covering long-term WHV infections (18 to 42 months postinoculation) have been extensively studied in previous reports (19, 21, 22), this study focused primarily upon the earlier stages of virus infection, 1 to 15 months postinoculation. Woodchucks were inoculated subcutaneously with approximately 5 x 106 50% woodchuck infectious doses of a
ruses.
WHV and its natural host, the eastern woodchuck (Marmonax), constitute the relevant experimental animal model for the study of HBV-induced disease, especially
mota
*
Corresponding author. 1360
VOL. 63, 1989
KINETIC PATTERNS OF WHV INFECTION z
1361
Serum DNA
0
....WHsAg
.......anti-WHc w Q I- LU
/ |
*.
***
\
anti-WHs
uJ 4
8
12
16 20 24 28 WEEKS POST INOCULATION
40
50
60
FIG. 1. Generalized pattern of WHV serologic markers following acute experimental WHV infection of 3-day-old woodchucks. WHV serologic markers were assayed as described in Materials and Methods. This generalized scheme is accurate for over 90% of experimentally infected woodchucks and is based upon the analysis of several populations of experimentally infected animals (31; unpublished observations).
well-characterized virus pool, WHV7 (8, 32), at 3 days of age and were monitored for up to 65 weeks. Woodchucks were maintained in isolation, and infection protocols were conducted at the woodchuck breeding facility at Cornell University. The generalized serologic pattern of acute WHV infection with recovery observed for experimental virus inoculation of 3-day-old woodchucks (31; unpublished observations) is shown in Fig. 1. In animals which develop chronic WHV infections, antibody to WHV surface antigen (WHsAg) (anti-WHs) does not appear in the serum and WHsAg and WHV DNA remain at high levels (31). At the points indicated by the arrows in Fig. 1, groups of three to five woodchucks were sacrificed and the different tissues were examined for evidence of WHV infection (a total of 43 animals from 19 separate litters). Groups of five woodchucks were chosen at random for the first four time points (4, 8, 14, and 18 weeks postinoculation). Thereafter, animals were separated on the basis of WHV serologic markers (WHsAg, antibody to WHV core antigen [anti-WHc], and anti-WHs) to enable a comparison of persistently infected animals (chronic carrier [WHsAg+, anti-WHc+, anti-WHs-]) and convalescent animals (serologically recovered [WHsAg-, anti-WHc+, anti-WHs+]). These comparisons were made at 28 weeks (five anti-WHs+ and four WHsAg+ woodchucks), 40 weeks (four anti-WHs+ and four WHsAg+ woodchucks), and 65 weeks postinoculation (three anti-WHs+ and three
WHsAg+ woodchucks). Selection of animals was controlled with respect to sex and litter. In the overall study design, (i) one or two animals from the same litter were chosen for examination at different time points during the course of WHV infection, (ii) both persistently infected and convalescent litter mates were examined at the same time point, and (iii) animals from different litters were included at each time point. Sexes of the individual woodchucks are indicated in Table 1. The tissues examined in this study were liver, mesenteric lymph node cells (LNC), femoral bone marrow cells, splenic lymphoid cells (SLC), and thymic lymphoid cells (TLC). For all five components of the lymphoid system, WHV DNA and RNA were extracted from isolated lymphoid cells prepared by centrifugation over Ficoll-Hypaque (see below). The selection of tissues for examination was based on observations from previous studies which demonstrated WHV infection of the PBL, lymph nodes, bone marrow, and spleen in WHV chronic carriers (21, 22). Source material and analysis of WHV serologic markers. Lymphoid cells were prepared from PBL, lymph nodes,
bone marrow, spleen, and thymus as previously described (21, 22). Samples of each lymphoid cell preparation were immediately lysed for nucleic acid isolation or cryogenically preserved in fetal bovine serum-dimethylsulfoxide freezing medium (Biofluids, Gaithersburg, Md.) and stored in liquid nitrogen. Liver tissues were immediately frozen in liquid nitrogen and stored at -70°C. Classifications of the stages of WHV infection and disease were based upon serologic analyses of WHsAg, anti-WHc, and anti-WHs (performed as previously described [21]) and by histologic examination of liver tissue samples). The liver of one chronic carrier, WC1408, taken at week 65, contained five small (0.5- to 1.0-cm) HCCs. The liver of another persistently infected animal, WC1663, taken at week 42, also contained a single, small (less than 0.5-cm) HCC. No grossly identifiable neoplastic nodules or regions were found in the livers of the other woodchucks. Nucleic acid isolation Southern and Northern (RNA) blot analyses. Whole-cell DNA and RNA were prepared from freshly isolated lymphoid cells or frozen liver tissue as previously described (21, 22). Analyses of viral nucleic acids by Southern and Northern blot hybridization techniques were performed as described in previous studies (21, 22). The 32P-labeled hybridization probe used was a 3.3-kilobase (kb) BamHI DNA fragment isolated from a cloned WHV genome. This WHV clone (WHV7) was isolated from the same standardized virus pool used for the experimental infection; its sequence has been published elsewhere (7). Estimates of the overall level of WHV-specific DNA and RNA were based upon densitometric comparison of the hybridization signals from tissue samples with those from known amounts of cloned WHV DNA coelectrophoresed in each gel as previously described (19). On each individual gel, both positive and negative hybridization control DNA markers (WHV and pBR322) were coelectrophoresed with experimental samples. RESULTS Analysis of WHV serologic markers. Time points for analysis were chosen on the basis of a generalized profile of WHV serologic markers (Fig. 1). This serologic profile was compiled from the analysis of several hundred woodchucks experimentally infected with the WHV inoculum used in this
study (31, 40; unpublished results). The 4- and 8-week time points were chosen to examine the preacute phase of WHV infection, while the 14- and 18-week
1362
J. VIROL.
KORBA ET AL.
TABLE 1. WHV serologic profiles at times of tissue analysis' Animalb F1477 M1480 F2020 F2027 M2031 F1634 M1638 F1642 M1646 F1648 F1479 F1481 M2021 F2026 F2028 F1637 F1641 M1645 M1647 F1651 M1635 F1636 M1655 M1656 M2015 M1482 F2016 M2022 F2025 M1478 M1644 M1664 M2023 F1483 M1643 M1663 M2024 F1290 M1444 M1613 F1279 M1288 M1408
Weeks
postinoculation 4 4 4 4 4 8 8 8 8 8 14 14 14 14 14 18 18 18 18 18 28 28 28 28 28 28 28 28 28 42 42 42 42 42 42 42 42 65 65 65 65 65
Presence of WHV serologic maLrkers
WI
Hsg
Anti-
WHV DNA
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ + + + +
+
_
+
_
+
_
+
_
+
+
+
+
+
+
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tions).
+ + +
+
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+
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+
20 animals represented the points of WHV infection predicted by the generalized serologic profile (Table 1). At 8 weeks postinoculation, only one animal, WC1634, displayed a low level (P/N ratio of 6.5) of WHsAg in the serum. All woodchucks were positive for serum WHV DNA at 8 weeks postinoculation, although the WHV DNA levels observed (105 to 106 WHV genome equivalents per ml) were approximately 1,000- to 10,000-fold lower than those typically found during the acute phase of WHV infection. The apparent discrepancies between the WHsAg and WHV DNA serologic assays at the 8-week time points can be accounted for by a 100-fold-greater level of sensitivity for the WHV DNA assay (unpublished observations). At 28 weeks postinoculation, all four woodchucks that had seroconverted to anti-WHs still carried similarly low levels of WHV DNA in their sera. These animals were taken for analysis within 7 to 10 days of the first detectable levels of anti-WHs antibodies. The four animals that remained WHsAg positive at 28 weeks postinoculation most likely represented woodchucks which would have remained persistently infected. In separate studies, at least 95% of WHVinfected woodchucks with the type of serologic profile observed in these four animals remained WHsAg positive and did not seroconvert to anti-WHs (unpublished observa-
+ +
_
65
'
Analysis of WHV serologic markers was performed as described in the Materials and Methods section. b Grouping of animal numbers by litter: litter 1 (L1), 1279; L2, 1288; L3, 1290; L4, 1408; L5, 1444; L6, 1613; L7, 1477, 1478, and 1479; L8, 1480, 1481, 1482, and 1483; L9, 1634, 1635, 1636, and 1637; LIO, 1638 and 1641; Lll, 1642, 1643, 1644, and 1645; L12, 1646 and 1647; L13, 1648 and 1651; L14, 1655 and 1656; L15, 1663 and 1664; L16, 2015 and 2016; L17, 2220, 2021, 2022, 2023, 2024, 2025, and 2026; L18, 2027 and 2028; L19, 2031. Sex of each animal is indicated by M or F preceding animal number.
time points were selected to study the acute phase of infection. The 28-week time point was chosen to examine animals during the early stages of persistent (chronic) infection, or 7 to 10 days after the first appearance of detectable levels of anti-WHs in the sera of woodchucks which had resolved acute viral infections. Weeks 40 and 65 were chosen to examine animals at two separate periods during the later stages of chronicity or recovery from acute infections.
Although WHV-infected woodchucks were selected at random for the first four time points in this study, the results of retrospective serologic analyses demonstrated that these
Kinetic patterns of WHV DNA. Southern blot hybridization analysis of WHV DNA revealed that the kinetic patterns of WHV DNA varied considerably for the different tissues and cell populations during the course of WHV infection (Tables 2 and 3; Fig. 2). These patterns demonstrated that a complex series of infections in these different cell populations occurred as the overall WHV infection proceeded through the acute phase and either resolved or continued into a chronic infection. Lymphoid cells of the bone marrow from four of five woodchucks contained the first detectable levels of WHV DNA (0.2 to 6 copies per cell) approximately 4 weeks after virus inoculation (Fig. 2). All five animals were negative at 4 weeks for all WHV serologic markers, including WHV DNA. No other tissues, including the liver, contained positive hybridization signals for WHV DNA at this point. The sensitivity limit for detection of WHV DNA was approximately 0.01 to 0.001 WHV genome equivalents per cell. WHV DNA levels in the bone marrow progressively declined during the next several weeks and became undetectable by the peak of the acute phase of infection, 14 to 18 weeks postinoculation (Fig. 2). WHV DNA remained at undetectable levels for the duration of the study period in woodchucks that remained persistently infected (Fig. 2, dashed line). However, in those animals that resolved acute WHV infections, as judged by the disappearance of WHsAg from the serum and the appearance of anti-WHs, WHV DNA reappeared in bone marrow at low levels (two to six copies per cell) at 26 to 28 weeks postinoculation in four of five anti-WHs+ animals (Fig. 2, solid line). These DNA signals then progressively declined to fewer than 0.5 copies per cell over the remainder of the study period. The second tissue to acquire detectable levels of WHV DNA was the liver, in which WHV DNA appeared at moderate levels (15 to 40 copies per cell) 6 to 8 weeks postinoculation in five of five animals (Fig. 2). These signals coincided with the appearance of low levels of WHV DNAcontaining particles in the serum (approximately 105 to 106 per ml) in all five animals. However, low levels of WHsAg were detectable in the serum of only one animal, WC1634, at this time point (Table 1). All five animals were still negative
1363
KINETIC PATTERNS OF WHV INFECTION
VOL. 63, 1989
TABLE 2. Levels of WHV nucleic acids in tissues at different times during the
course
of viral infection
Levels of WHV DNA and RNA in the following tissues as detected by Southern and Northern hybridization analysesa
Weeks
postinoculation and
Lymph node
PBL
Thymus (TLC)b
Bone marrow
Liver
Spleen (SLC)
serologic state
RNA
DNA
RNA
DNA
RNA
DNA
RNA
DNA
RNA
DNA
RNA
0
ND ND
1-3 1-3
3-9 3-9
NDC ND 3-9 3-9
0.2-0.6
0.2-0.6
0 0
0.2-0.6
0
0 0 0 0
0
0 0.3-1 0.2-0.6
0
0 0 0 0
0 0 60-180 55-160
0 0 1-3 1-3
0 15-45 300-900 600-1,800
0 3-9 40-1 70-2
anti-WHs+ WHsAg+
0.3-1 0.5-2
0.1-0.3 0.1-0.3
0.5-2 1-3
0.1-0.3 0.1-0.3
2-6 0
0 0
4-12 0.1-0.3
0 0
14-42 45-140
0.3-1 2-6
55-160 500-1,500
0.5-2 55-1
anti-WHs+ WHsAg+
0.1-0.3 0.5-2
0 0.1-0.3
0.1-0.3 0.5-2
0 0.1-0.3
1-3 0
0 0
0.5-2 0.1-0.3
0 0
2-6 35-110
0.1-0.3 2-6
2-6 600-1,800
0.2-0 50-1
anti-WHs+ WHsAg+
0.1-0.3 1-3
0 0.1-0.3
0.2-0.6 0.5-2
0 0.1-0.3
0.1-0.3 0
0 0
0.2-0.6 0.1-0.3
0 0
1-3 30-90
0 3-9
0.5-2 600-1,800
0 40-1
DNA
4 8 14 18 28
0 0
42 65 a WHV DNA and RNA were analyzed by Southern and Northern blot hybridization techniques as described in Materials and Methods. Values presented are the average nucleic acid levels in the individual tissues for all the animals in each group (three to five woodchucks per group). In Fig. 2, the DNA levels for each animal are plotted to illustrate the range of WHV DNA levels observed. Levels of WHV DNA are presented as WHV genomic equivalents per cell. WHV RNA levels are presented as picograms of WHV RNA per microgram of whole-cell DNA. A zero value denotes undetectable levels of WHV DNA (less than 0.001 copies per cell) or WHV RNA (less than 0.03 pg/p.g of whole-cell RNA). b In F1634 and F2026, distinct thymus tissue could not be distinguished from surrounding tissue. ' ND, Not determined. Distinct mesenteric lymph nodes could not be accurately identified in the 4- or 8-week-old woodchucks.
for anti-WHc antibodies, which are normally not present at detectable levels in serum until 12 to 14 weeks postinoculation (31, 40) (Table 1 and Fig. 1). WHV DNA levels in the liver exceeded 1,000 copies per cell by the peak of acute infection (14 to 18 weeks postinoculation) and remained at these levels in the animals which developed chronic infections (Fig. 2). In the anti-WHs+ animals, WHV DNA rapidly declined in both the serum and liver (Fig. 2). WHV DNA was still present in the liver at one to three copies per cell in the anti-WHs+ woodchucks at 65 weeks postinoculation, although the sera of these three animals did not contain detectable levels of WHV DNA (Table 1). WHV DNA appeared simultaneously in the PBL, SLC, and LNC at the peak of the acute infection in all animals examined (Fig. 2). Distinct lymph nodes could not be reliably identified in the 4- and 8-week old animals. The levels of WHV DNA detected during the acute phase of viral infection were in the maximum ranges observed for these cell populations, 2 to 8 copies per cell for PBL and LNC and 40
to 200 copies per cell for the SLC (Table 2). As observed for the liver, WHV DNA in the SLC remained at high levels in
those animals with persistent WHV infections and rapidly declined to two to six copies per cell at 65 weeks postinoculation in those animals which developed anti-WHs (Fig. 2). In the PBL and LNC, WHV DNA levels remained at moderate levels throughout the acute stage of infection and then rapidly declined to approximately 0.3 to 1 copies per cell in both the persistently infected and convalescent groups by 65 weeks postinoculation (Fig. 2). Although the persistently infected animals generally appeared to harbor more WHV DNA in the PBL and LNC (Table 2), there were no significant differences in the WHV DNA kinetic patterns for these cell populations between the two classes of WHVinfected woodchucks. WHV DNA was not observed in the TLC until 26 to 28 weeks postinoculation (Fig. 2). This event coincided with the first appearance in convalescent animals of anti-WHs antibodies in the serum and the reappearance of WHV DNA in
TABLE 3. Frequency of WHV nucleic acids in the tissues of WHV-infected woodchucks Percentage of infected woodchucks with WHV nucleic acids in:
Weeks
postinoculation and serologic state
Lymph node
PBL
DNA
RNA
DNA
RNA
RNA
DNA
RNA
DNA
RNA
0 0 20
0
0 0
0 100
0 0
0 100
0 100
100 100
0 0 0 0
0
100 100
80 60 60 0
100
100
100
0
0
100
100
100
100
40 25
100 75
40 25
80 0
0 0
100 75
0 0
100 100
100 100
100 100
100 100
50 75
0 50
50 75
0 25
75 0
0 0
75 50
0 0
100 100
50 100
100 100
50 100
100 100
0 100
100 100
0 100
67
0 0
67
0
100
0
100
0
67
0
100
100
100
100
DNA
RNA
0 0 100 100
0 0 100 100
anti-WHs+ WHsAg+
100 100
anti-WHs+ WHsAg+ anti-WHs+ WHsAg+
4 8 14 18 28
Liver
Spleen
Thymus
Bone marrow
DNA
NDa ND
42 65 a ND, Not determined. Distinct mesenteric
0
lymph nodes could not be accurately identified in the 4- or 8-week-old woodchucks.
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the bone marrow. WHV DNA levels in the TLC from four of five anti-WHs+ woodchucks were substantially higher (up to 10 copies per cell) than the levels observed in the TLC from three of four WHsAg+ animals (a maximum of 0.6 copies per cell) (Table 2; Fig. 2). WHV DNA levels in the TLC declined rapidly in the convalescent animals during the remainder of the study. Both persistently infected and convalescent woodchucks harbored approximately 0.2 WHV DNA copies per cell in the TLC at 65 weeks postinoculation. WHV DNA hybridization signals, representing approximately 0.1 copies per cell, were detected in the TLC from one of five woodchucks (WC2028) examined at week 14. It is not readily apparent at this time whether these hybridization signals represented an early, low-level entry into the TLC (as in the bone marrow) or an anomaly of the particular WHV infection in this woodchuck. Since these signals were especially weak and appeared in only 1 of 20 animals examined at weeks 4 to 18, the generalized model developed for the WHV kinetic infection pattern of the thymus (Fig. 3) does not currently include this single case observation. WHV-specific RNA species. In general, the levels of WHVspecific RNA transcripts were correlated well with the overall levels of WHV DNA and WHV DNA replication intermediates (Tables 2 and 3). The highest levels of WHV RNA were observed in the liver. Compared with WHV RNA levels in the liver, WHV RNA levels in the SLC were approximately 100-fold lower and WHV RNA levels in the PBL and LNC were approximately 1,000-fold lower (Table 2). During the acute stage of viral infection, WHV-specific RNA transcripts were observed at relatively high levels in the liver, SLC, PBL, and LNC. As the course of viral infection proceeded, WHV RNA was observed less frequently in the PBL and LNC in the persistently infected group, while high levels of WHV RNA were maintained in the SLC and liver (Tables 2 and 3). In the convalescent group of woodchucks, WHV RNA appeared progressively less often with increasing time postinfection in all tissues. No WHV-specific RNA was observed at any stage of the viral infection in either the bone marrow or the thymus. Analysis of WHV RNA by Northern blot hybridization techniques demonstrated that, in all tissues, the majority of the viral-specific RNA transcripts were the 3.6- and 2.3-kb classes of RNA species commonly observed in hepadnavirus-infected hepatocytes (21, 26, 41), with approximately two-thirds of the WHV RNA hybridization signal present as the 2.3-kb species. WHV RNA transcripts other than the 2.3- and 3.6-kb species were occasionally observed at different stages of the viral infection. These viral RNA transcripts represented only a minor component of the WHV RNA transcripts observed (less than 5% of the total autoradiographic signal from any given tissue sample) and were found in only 18 of the 246 individual tissues examined from the 43 WHV-infected woodchucks examined in this study. The appearance of these additional viral RNA transcripts did not appear to correlate with any specific tissue, particular stage of viral infection, serologic pattern, or gross histologic appearance of the tissues. Progression of WHV DNA forms. As described above, the overall levels of WHV DNA changed dramatically in the various tissues examined during the natural course of WHV infection. A complex progression of WHV genomic forms was also observed in these tissues (Table 4). These different viral DNA forms were resolved by Southern blot hybridization analysis after restriction enzyme digestion and agarose gel electrophoresis of whole-cell DNA as described below. In all tissues and in almost all animals, WHV DNA was
KINETIC PATTERNS OF WHV INFECTION
1365
episomal. The single exception was liver tissue from WC1290, an anti-WHs+ woodchuck taken at 65 weeks postinoculation, in which WHV DNA was found to be integrated as discrete fragments into the cellular genome. WHV DNA was designated episomal by the following criteria: (i) no change in the electrophoretic migration pattern between undigested DNA and DNA digested with a restriction enzyme which does not cut WHV7 (AvaI in this study [7]), (ii) an electrophoretic DNA pattern containing only a single, 3.3-kb WHV DNA fragment after digestion with a restriction enzyme (BamHI) which makes a single cut in WHV7 (hepadnaviruses are circular genomes [41]), and (iii) after digestion with restriction enzymes which make multiple cuts in WHV7, an electrophoretic DNA pattern containing only the expected WHV subgenomic fragments (2.2 and 1.1 kb, resulting from a double digestion of WHV7 with HindIIl and EcoRI). On the basis of analyses utilizing all of these criteria, WHV DNA forms were divided into three classes of molecules: (i) MM (multimeric, episomal DNA molecules 7 to 12 kb in size [2, 3]), (ii) M (monomeric, episomal WHV genomes 3.3 kb in size), and (iii) RI (a heterogeneous population of single- and double-stranded WHV DNA fragments, 3.3 to 0.5 kb in size, which represent WHV DNA replication intermediates [34, 37]). In the lymphoid cells of the bone marrow and thymus, the predominant WHV DNA molecules were monomeric genomes (Table 4). In the bone marrow only monomeric WHV genomes were detected, while some multimeric WHV DNA molecules were occasionally found in the TLC. However, the multimeric WHV DNA molecules were present in the TLC of only 3 of 18 woodchucks, 2 at week 42 (WC1664 in the anti-WHs+ group and WC1663 in the WHsAg+ group) and 1 at week 65 (WC1444 in the anti-WHs+ group). No WHV DNA replication intermediates were found in the thymus or bone marrow. The monomeric WHV genomes in these cells migrated as discrete, sharp bands. These DNA molecules were distinctly different from the characteristic, diffuse DNA bands observed in the analysis of serum virion genomes, which are a result of the circular, partially singlestranded DNA molecules present in hepadnavirus virions (19, 41). The discrete, monomer-sized WHV DNA bands observed in the lymphoid cells indicated that these DNA molecules were most likely completely double stranded and suggest that completion of the positive DNA strand in the WHV genomes had occurred upon cellular entry. In contrast to the WHV DNA patterns observed in the bone marrow and thymus, the other tissues examined, including liver, displayed a complex progression of the different classes of WHV DNA forms (Table 4). This progression of WHV DNA forms was observed in all tissues of the animals with serologic patterns indicative of self-limited WHV infections and in the PBL and lymph nodes of the woodchucks which developed chronic WHV infections. Whereas a single, generalized pattern of progression was observed for all these tissues, the stage of WHV infection in which changes in the WHV DNA patterns occurred and the duration of any given class of WHV DNA molecules were different for each of the various tissues. In Fig. 3, a comprehensive model is presented which illustrates the kinetics of WHV infection and the progression of the different WHV DNA forms throughout the natural course of viral infection. The top panels of Fig. 3 display the patterns of WHV serologic markers as a guide to the different, serologically defined stages of viral infection. Examples of these different WHV DNA patterns are displayed in Fig. 4 to illustrate the
1366
KORBA ET AL.
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VOL. 63, 1989
KINETIC PATTERNS OF WHV INFECTION
1367
TABLE 4. Frequency of WHV DNA forms in the tissues of WHV-infected woodchucks
Percentage of different WHV DNA forms in the following tissues of WHV DNA-carrying woodchucksa
Weeks postinoculation and serologic state
PBL
Lymph node
Bone marrow
Thymus (TLC)
Spleen (SLC)
Liver
MMb
M
RI
MM
M
RI
MM
M
RI
MM
M
RI
MM
M
RI
MM
0 0 0 100
0 0 100 100
0 0 100 100
0 0 0 80
0 0 100 100
0 0 100 100
0 0 0 0
100 100 100 0
0 0 0 0
0 0 0 0
0 0 100 0
0 0 0 0
0 0 0 0
0 0 100 100
0 0 100 100
anti-WHs+ WHsAg+
80 100
80 100
0 0
60 67
80 100
0 0
0 0
100 0
0 0
0 0
100 100
0 0
80 0
100 100
anti-WHs+ WHsAg+
100 100
100 100
0 0
50 100
100 67
0 0
0 0
100 0
0 0
33 50
100 100
0 0
75 0
anti-WHs+ WHsAg+
100 100
67 33
0 0
100 100
33 67
0 0
0 0
100 0
0 0
50
100 100
0 0
33 33
4 8 14
18 28
M
RI
0 0 0 0
0 100 100 100
0 100 100 100
60 100
100 0
100 100
40 100
100 100
0 100
25 0
100 100
0 100
100 100
0 100
33 0
100 100
0 100
42 65 0
a Resolution of the different WHV DNA forms was determined by Southern blot hybridization analysis of whole-cell DNA after restriction enzyme digestions and agarose gel electrophoresis. Numbers reflect the percentage of the different classes of WHV DNA forms in those woodchucks carrying WHV DNA in the indicated tissues. Episomal WHV DNA forms were distinguished from integrated WHV DNA (see text) by a comparison of the WHV DNA patterns in undigested cell DNA with the WHV DNA patterns after separate digestions with AvaI and BamHI and a double digestion with Hindlll and EcoRI. b The classes of WHV DNA observed were: MM (episomal, multimeric WHV DNA forms 7 to 12 kb in size), M, (monomeric, episomal WHV genomes 3.3 kb in size), and RI (heterogenous smears of single and double-stranded WHV DNA fragments, 3.3 to 0.5 kb in size, which represent WHV DNA replication intermediates [34, 37]). See Table 2 footnotes for other details.
progression from a replicative state (A) to a nonreplicative state (C). With the exceptions of the bone marrow and thymus, WHV DNA first appeared in all of the different cell populations (PBL, lymph node, spleen, and liver) during the acute phase of viral infection in a pattern representing active virus replication (Fig. 3 and 4A). High levels of WHV RNA were also observed, but no multimeric WHV DNA molecules
A
C
B
1 2 3
1 12.1
12.1
11.1
11.1
10 1
10.1
_4.
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-'-w3.1.0
4.1-a.-
2. 0
3.0-E .62.0-_ 1.6-1. O -
2 3
0.5
1.0-
8.1 7.1----.6.1-
5.1 4.1 3.0--
2.0-X4 1.6--
1.0o
0.5-
FIG. 4. Examples of the progression of WHV DNA patterns. Analysis of WHV DNA by Southern blot hybridization anlaysis is described in Materials and Methods. Lanes 1, undigested DNA; lanes 2, DNA digested with AvaI (no recognition sequence in WHV7 [7]); lanes 3, DNA digested with BamHI (one recognition site in WHV7, producing a single band of 3.3 kb). (A) WC1481 PBL DNA (14 weeks postinoculation; 10 ,ug per lane). (B) WC1651 PBL DNA (18 weeks postinoculation; 10 ,ug per lane). (C) WC1482 PBL DNA (28 weeks postinoculation, WHsAg+; 30 ,ug per lane). Exposure times: panel A, 3 days; panel B, 7 days; panel C, 10 days.
were detected at this point. Several weeks later, the multimeric WHV DNA molecules appeared. These viral DNA forms were superimposed upon patterns of WHV DNA replication intermediates (Fig. 3 and 4B). WHV RNA was still present at elevated levels at this point. The next stage in this progression to recovery was characterized by a loss of the WHV DNA replication intermediates and a dramatic decrease in, and eventual loss of, WHV RNA. At this point, only the multimeric and monomeric WHV DNA molecules were observed (Fig. 3 and 4C). This stage appeared to span a relatively prolonged period, during which the stoichiometric ratios of the multimeric and monomeric DNA molecules varied considerably. In some cases, only one or the other of these two classes of WHV DNA molecules was observed (Table 4). In the lymphoid cells, the predominant WHV DNA forms were the multimeric molecules. In the liver and the spleen, the multimeric WHV DNA forms were less frequently observed, and the predominant remaining WHV DNA molecules were the monomeric genomes (Table 4). In the livers and spleens of the chronically infected animals, WHV replication intermediates and high levels of WHV RNA persisted throughout the entire course of viral infection (Fig. 3). As in the convalescent animals, WHV DNA replication was not observed in the PBL or LNC of the persistently infected woodchucks after the end of the acute phase of infection, 28 weeks postinoculation (Fig. 3). The multimeric WHV DNA molecules were not generally observed in the liver or splenic tissues of the persistently infected animals. However, the spleen of one chronically infected woodchuck, WC1408, taken at 65 weeks, contained episomal, multimeric WHV DNA forms superimposed over a pattern of WHV DNA replication intermediates (Table 4). DISCUSSION In this study, a comprehensive analysis was made of the progression of WHV infections in the liver and the primary components of the lymphoid system during the natural course of WHV infection. The uniformity of response to experimental WHV infections with standardized virus inocula (Table 1) permitted the detailed analysis of several
1368
J. VIROL.
KORBA ET AL.
critical stages of hepadnavirus infections, especially the preacute phase and the early recovery phase, in which accurate timing of the appearance of the first detectable levels of anti-WHs antibodies was required. The uniformity of response to WHV infections was further demonstrated by (i) the levels of WHV nucleic acids (Table 2), (ii) the kinetics of the appearance and loss of WHV DNA (Table 3), and (iii) the different patterns of WHV DNA molecules (Table 4) observed in the individual tissues. The movement of WHV through different tissues and cell populations of the immune system during the overall course of viral infection demonstrates the complexity of the pathobiology of hepadnaviruses. Overall, there appeared to be four distinct systemic movements of WHV. The first appearance of cellular WHV DNA occurred in lymphoid cells of the bone marrow, although no evidence of viral replication was observed. Active replication of WHV in the liver occurred next, followed by active viral infection of the spleen, PBL, and lymph nodes and loss of WHV from the bone marrow. The last stage was characterized by the appearance of nonreplicating WHV DNA in lymphoid cells of the thymus and, in anti-WHs+ animals only, a reappearance of WHV in the bone marrow. It is not clear at this time whether these cycles of WHV infection represent parallel infections of the liver and immune system or a pattern of sequential WHV infections of the various tissues. These different virus infection cycles may reflect the interdependence of developmental interactions between the immune system and the liver. The entry of virus into these different cell populations is most likely an important criterion in determining the progress and eventual outcome of the viral infection. Entry of WHV into the spleen, PBL, and lymph nodes appears to represent secondary viral infections. These infections may be a result of either direct infection by WHV or seeding of these compartments of the immune system with WHV-bearing lymphoid cells originating in the bone marrow. Fetal liver is a source of hematopoietic stem cells that seed into the bone marrow (28). Since woodchucks are relatively immature at birth (14), it is possible that both hepatocytes and hematopoietic cells are infected in situ in the liver following inoculation at 3 days of age. The rapid loss of replicating WHV from the circulating lymphoid cell populations indicates that these cells probably do not have the capacity to support WHV replication for prolonged periods of time. However, recent in vitro experiments have demonstrated that the nonreplicating WHV genomes in the PBL of chronically infected woodchucks are replication competent under certain environmental conditions and thus may represent latent WHV infections of these cells (18). The cellular mechanisms involved in the specific shutdown of WHV replication in circulating lymphoid cells of the chronically infected woodchucks remain to be elucidated. One possibility involves the specific cytosine methylation of viral DNA, which has been directly implicated in the regulation of HBV core antigen gene expression (23, 25). Alternatively, cellular factors involved in the control of expression of hepadnavirus genes may be lacking or currently unavailable in these cells. Transactivating cellular factors have been shown to be required for efficient activity of the HBV enhancer element, while tissue-specific expression of HBV surface and presurface genes has been observed in transgenic mouse models (2, 3, 16). WHV DNA in the bone marrow cells may represent some stage in active viral infection or nonspecific uptake of WHV virions from the original inoculum. The lack of multimeric
tively rapid loss of viral DNA from the bone marrow cells indicate that these WHV genomes were probably not extensively active in this compartment of the lymphoid system. However, cultures of human bone marrow cells have been shown to harbor HBV genomes with transcriptional activity, as evidenced by the intracellular expression of HBV surface and core antigens and the extracellular export of 22-nm surface antigen particles (33). Early exposure of bone marrow cells to WHsAg may ultimately influence the ability of the host to raise anti-WHs antibodies, possibly through mechanisms such as those related to immune tolerance in neonatal animals (36). However, this study demonstrated that reappearance of WHV in the bone marrow is correlated with the production of high levels of anti-WHs antibodies and the ability to resolve acute WHV infections successfully. The resolution of acute WHV infections, with the appearance of anti-WHs antibodies, was also correlated with the appearance of relatively high levels of nonreplicating WHV DNA genomes in lymphoid cells of the thymus. It is possible that the presence of WHV DNA in both the thymus and bone marrow of the serologically recovered woodchucks is related to the complex response of the immune system to acute WHV infection superimposed upon normal developmental processes (4, 35, 43). Although WHV DNA was detected in the thymus at the later stages of viral infection whether or not anti-WHs antibodies were produced, the successful resolution of acute viral infection was correlated with higher relative levels of WHV DNA. WHV DNA was found to persist in a nonreplicating state in the tissues of convalescent woodchucks for several months after seroconversion to anti-WHs. In previous studies, a significant fraction (approximately 25%) of woodchucks which had serologically recovered (anti-surface and anti-core seropositive) from HBV or WHV infections were found to carry low levels of hepadnaviral DNA in both PBL and hepatocytes (19, 21, 22; B. E. Korba, F. V. Wells, B. Baldwin, P. J. Cote, B. C. Tennant, H. Popper, and J. L. Gerin, Hepatology, in press). These observations indicate that some individuals may never totally clear all hepadnavirus genomes after resolution of acute viral infections. In addition, anti-WHs-positive woodchucks have been shown to be at increased risk for the development of HCC (Korba et al., Hepatology, in press). HBV DNA has been detected in the PBL of both acutely and chronically infected individuals (8, 9, 15, 29). However, HBV DNA in the PBL during the acute phase of infection was found to be in a nonreplicating state. These different observations may reflect the intensity of splenic viral infection and the relative rates of maturation, turnover, and trafficking of cells among the various lymphoid compartments (35). Many of the individuals examined in the HBV studies were infected as adults, whereas the woodchucks examined in this study were infected as newborns. The age at which hepadnaviral infections are acquired is a critical factor in the development of chronicity (11, 27, 40). Multimeric hepadnavirus DNA forms are commonly observed in lymphoid cells (5, 9, 10, 15, 29; for a review, see reference 19). The evolution of such DNA molecules in this study indicates that this class of viral DNA molecules is probably a normal consequence of the life cycle of WHV. These DNA molecules appeared near the termination of WHV DNA replication in both lymphoid cells and liver tissue and may represent an attempt at viral lysogeny either as precursors to integrated forms or as stable episomal
WHV DNA forms and WHV-specific RNA and the rela-
genomes.
KINETIC PATTERNS OF WHV INFECTION
VOL. 63, 1989
The overall patterns of WHV RNA species did not appear to provide any immediate new information on the biologic mechanisms of WHV. While some different viral RNA species were occasionally observed, these did not appear to correlate with the composition of WHV DNA forms or the stage of the overall virus infection and appeared in only a small minority of the tissue samples. However, further analysis of the WHV RNA transcripts in these tissues may provide important insights into host-virus interactions. Some differences exist between the kinetics of viral infection among the different extrahepatic tissues for DHBV and WHV. In Pekin ducks experimentally infected at 1 day of age, replicating DHBV DNA is first detected in the liver and then appears simultaneously in the spleen, pancreas, and kidney (12, 13, 17, 39). DHBV replication proceeds in all tissues during the acute phase of infection. DHBV replication ceases in the spleens of chronically infected ducks but continues in the liver, pancreas, and possibly the kidney (12, 13, 17, 39). By contrast, active replication of WHV and HBV is observed only in the liver and spleen of chronic virus carriers (19, 21, 23). An analysis of WHV nucleic acids in the pancreas and kidney during the early stages of WHV infection has not yet been conducted. The nature of the differences between DHBV and WHV probably relate to fundamental differences in host physiology rather than to differences in the general characteristics of these closely related viruses. The precise role of hepadnavirus infection of lymphoid cells remains to be elucidated. It is reasonable to suspect that hepadnavirus infection of lymphoid cells may alter host immune responses. A number of reports have documented alterations in several immunologic responses in chronic HBV carriers (1, 6, 43). The persistence of virus in circulating lymphoid cells could be involved in the reactivation of infection and exacerbation of liver disease in long-term asymptomatic HBV carriers (10, 30, 42). A correlation has recently been observed between the presence of HBV DNA in cord blood leukocytes (in the absence of serum virions) and the failure of vaccine administration in newborn infants to prevent chronic HBV infections (5). The types of observations compiled for WHV in this study and DHBV in previous studies (12, 17, 39) add a high level of complexity to the pathobiologic mechanisms of hepadnaviruses. Infection by these viruses involves the systemic movement of virus through many different cell populations during the natural course of virus infection. Exact identification of the cell populations involved will be necessary to better comprehend the roles of these cells in the overall course of virus-induced disease. When further correlated with serologic markers of virus infection and the state of liver disease, the present observations should help to elucidate the mechanisms of hepadnavirus pathogenesis and provide information for improved prognosis and the development of more efficient antiviral therapy. ACKNOWLEDGMENTS
The support and assistance of W. Hornbuckle, J. Wright, W. Sherman, A. Glasser, T. Manwarren, S. Gomez, L. Fullam, E. DeLeo (Cornell University), R. Engle, M. Rochee (Georgetown University), and their colleagues is greatly appreciated. The manuscript was prepared for publication by C. Culp and Y. Bellinger. This work was supported by Public Health Service contracts N01-AI-22665, N01-AI-72623, and N01-A1-52585 between the National Institute of Allergy and Infectious Diseases and Georgetown and Cornell Universities.
1369
LITERATURE CITED 1. Alexander, G. J. M., M. Mondelli, N. V. Naumov, K. T. Nouri-Aria, D. Vergani, D. Lowe, A. L. W. F. Eddleston, and R. Williams. 1986. Functional characterization of peripheral blood lymphocytes in chronic HBsAg carriers. Clin. Exper. Immunol.
63:498-507. 2. Burk, R. D., J. A. Deloia, M. K. Elawady, and J. Geachart. 1988. Tissue preferential expression of the hepatitis B virus (HBV) surface antigen gene in two lines of HBV transgenic mice. J. Virol. 62:649-654. 3. Cabinet, C., H. Farza, D. Morello, M. Hadchovel, and C. Pourcel. 1985. Specific expression of hepatitis B surface antigen (HBsAg) in transgenic mice. Science 230:1160-1163. 4. Celis, E., K. G. Abraham, and R. W. Miller. 1987. Modulation of the immunological response to hepatitis B virus by antibodies. Hepatology 7:563-568. 5. Chen, H.-D., K.-B. Choo, T.-C. Wu, H.-T. Ng, and S.-H. Han. 1987. Hepatitis B infection of cord blood leukocytes. J. Med. Virol. 22:211-216. 6. Chisari, F. V., K. L. Castle, C. Xavier, and D. S. Anderson. 1981. Functional properties of lymphocyte subpopulations in HBV infection. I. T suppressor control of T-lymphocyte responsiveness. J. Immunol. 126:38-45. 7. Cohen, J., R. H. Miller, B. Rosenblum, K. Denniston, J. L. Gerin, and R. H. Purcell. 1988. Sequence comparison of woodchuck hepatitis virus replicative forms shows conservation of the genome. Virology 162:12-20. 8. Davidson, F., G. J. M. Alexander, C. Anatassakos, E. A. Fagan, and R. Williams. 1987. Leucocyte hepatitis B virus DNA in acute and chronic hepatitis B virus infection. J. Med. Virol. 22:379-389. 9. Davidson, F., G. J. M. Alexander, R. Trowbridge, E. Fagan, and R. Williams. 1987. Detection of HBV DNA in spermatozoa, urine, saliva and leucocytes of chronic HBsAg carriers. A lack of relationship with serum markers of replication. J. Hepatol. 4:37-44. 10. Davis, G. L., J. H. Hoofnagle, and J. G. Waggoner. 1987. Spontaneous reactivation of chronic hepatitis B virus infection. Gastroenterology 86:230-235. 11. Fukuda, R., S. Fukumoto, and Y. Shimada. 1987. A sequential study of viral DNA in serum in experimental transmission of duck hepatitis B virus. J. Med. Virol. 21:311-320. 12. Freiman, J. S., A. R. Jilbert, R. J. Dixon, M. Holmes, E. J. Gowans, C. J. Burell, E. J. Wills, and Y. E. Cossart. 1988. Experimental duck hepatitis B virus infection: pathology and evolution of hepatic and extrahepatic infection. Hepatology 8:507-513. 13. Halpern, M. S., J. M. England, D. T. Deery, D. J. Petcu, W. S. Mason, and K. L. Molnar-Kimber. 1983. Viral nucleic acid synthesis and antigen accumulation in pancreas and kidney of Peking ducks infected with duck hepatitis B virus. Proc. Natl. Acad. Sci. USA 80:4865-4869. 14. Hamilton, W. J., Jr. The life history of the rufescent woodchuck Marmota monax rufescens howell 1934. Ann. Carnegie Mus. 23:85-178. 15. Hoar, D. I., T. Bowen, D. Matheson, and M. C. Poon. 1985. Hepatitis B virus DNA is enriched in polymorphonuclear cells. Blood 66:1251-1253. 16. Jameel, S., and A. Siddiqui. 1986. The human hepatitis B virus enhancer requires trans-activating cellular factor(s) for activity. Mol. Cell. Biol. 6:710-715. 17. Jilbert, A. R., J. S. Freiman, E. J. Gowans, M. Holmes, Y. E. Cossart, and C. J. Burell. 1987. Duck hepatitis B virus DNA in liver, spleen and pancreas: analysis by in situ and Southern blot hybridization. Virology 158:330-338. 18. Korba, B. E., P. J. Cote, and J. L. Gerin. 1988. Mitogen-induced replication of woodchuck hepatitis virus in cultured peripheral blood lymphocytes. Science 241:1213-1216. 19. Korba, B. E., E. J. Gowans, F. V. Wells, B. C. Tennant, R. Clark, and J. L. Gerin. 1988. Systemic distribution of woodchuck hepatitis virus in the tissues of experimentally infected woodchucks. Virology 165:172-181. 20. Korba, B. E., B. C. Tennant, E. Gowans, P. J. Cote, F. Wells, B.
1370
21.
22.
23.
24.
25.
26. 27.
28. 29.
30.
KORBA ET AL.
Baldwin, and J. L. Gerin. 1987. Infection and tissue tropism of WHV during the natural course of infection of woodchucks, p. 419-428. In W. Robinson, K. Koike, and H. Will (ed.), Hepadna viruses. Alan R. Liss, Inc., New York. Korba, B. E., F. V. Wells, B. C. Tennant, P. J. Cote, and J. L. Gerin. 1987. Lymphoid cells in the spleen of woodchuck hepatitis virus-infected woodchucks are a site of active viral replication. J. Virol. 61:1318-1324. Korba, B. E., F. V. Wells, B. C. Tennant, G. H. Yoakum, R. H. Purcell, and J. L. Gerin. 1986. Hepadnavirus infection of peripheral blood lymphocytes in vivo: woodchuck and chimpanzee models of viral hepatitis. J. Virol. 58:1-8. Korba, B. E., V. L. Wilson, and G. H. Yoakum. 1985. Induction of HBV core gene in human cells by cytosine demethylation in the promoter. Science 238:1103-1105. Lieberman, H. M., W. W. Tung, and D. A. Shafritz. 1987. Splenic replication of hepatitis B virus in the chimpanzee chronic carrier. J. Med. Virol. 21:347-359. Miller, R. A., and W. S. Robinson. 1983. Integrated hepatitis B virus DNA sequences specifying the major viral core polypeptide are methylated in PLC/PRF/5 cells. Proc. Natl. Acad. Sci. USA 80:2534-2538. Moroy, T., J. Etiemble, C. Trepo, P. Tiollais, and M.-A. Buendia. 1985. Transcription of woodchuck hepatitis virus in the chronically infected liver. EMBO J. 4:1507-1514. Okada, K., I. Kamiyama, M. Inomata, M. Imai, Y. M. Yakawa, and M. Mayumi. 1976. E antigen and anti-e in the serum of asymptomatic carrier mothers indicates of positive and negative transmission hepatitis B virus to infants. New Engi. J. Med. 294:746-749. Owen, J. J. T. 1977. Ontogenesis of lymphocytes, p. 21-33. In F. Loor and G. E. Roelants (ed.), B and T cells in immune recognition. John Wiley & Sons, Inc., New York. Pasquinelli, C., F. Laure, L. Chatenoud, G. Beaurin, C. Gazengel, H. Bismuth, F. Degos, P. Tiollais, J. F. Bach, and C. Brechot. 1986. Hepatitis B virus DNA in mononuclear blood cells. J. Hepatol. 3:95-103. Perillo, R. P., C. R. Campbell, and G. E. Sanders. 1984. Spontaneous clearance and reactivation of hepatitis B virus infection among male homosexuals with chronic type B hepatitis. Ann. Intern. Med. 100:43-46.
J. VIROL. 31. Ponzetto, A., P. J. Cote, E. C. Ford, R. H. Purcell, and J. L. Gerin. 1984. Core antigen and antibody in woodchucks after infection with woodchuck hepatitis virus. J. Virol. 52:70-76. 32. Popper, H., L. Roth, R. H. Purcell, B. C. Tennant, and J. L. Gerin. 1987. Hepatocarcinogenicity of the woodchuck hepatitis virus. Proc. Natl. Acad. Sci. USA 84:866-870. 33. Romet-Lemmone, J. L., M. F. McLane, E. Elfassi, W. Haseltine, J. Azocar, and M. Essex. 1983. Hepatitis B virus infection in cultured human lymphoblastoid cells. Science 221:667-669. 34. Seeger, C., D. Ganem, and H. E. Varmus. 1986. Biochemical and genetic evidence for the hepatitis B virus replication strategy. Science 232:477-484. 35. Sprent, J. 1977. Migration and life span of lymphocytes, p. 59-78. In F. Loor and G. E. Roelants (ed.), B and T cells in immune recognition. John Wiley & Sons, Inc., New York. 36. Strober, S. 1984. Natural suppressor (NS) cells, neonatal tolerance and total lymphoid irradiation. Annu. Rev. Immunol. 2:219-237. 37. Summers, J., and W. S. Mason. 1982. Replication of the genome of a hepatitis-B-like virus by reverse transcription of an RNA intermediate. Cell 29:403-415. 38. Tagawa, M., W. S. Robinson, and P. L. Marion. 1987. Duck hepatitis B virus replicates in the yolk sac of developing embryos. J. Virol. 61:2273-2279. 39. Tagawa, M., M. Omata, K. Yokosuka, F. Uchiumi, F. Imazeki, and K. Okuda. 1985. Early events in duck hepatitis B virus infection. Gastroenterology 84:1224-1229. 40. Tennant, B., W. E. Hornbuckle, B. H. Baldwin, J. M. King, P. Cote, H. Popper, R. H. Purcell, and J. L. Gerin. 1988. Influence of age on the response to experimental woodchuck hepatitis virus infection, p. 462-465. In A. J. Zuckerman (ed.), Viral hepatitis and liver disease. Alan R. Liss, Inc., New York. 41. Tiollais, P., C. Pourcel, and A. Dejean. 1985. The hepatitis B virus. Nature (London) 317:489-495. 42. Tong, M. J., R. E. Sampliner, S. Govindarajan, and R. L. Co. 1987. Spontaneous reactivation of hepatitis B in Chinese patients with HBsAg-positive chronic active hepatitis. Hepatology 7:713-718. 43. Vento, S., S. Ranier, R. Williams, E. G. Rondanelli, C. J. O'Brian, and A. L. W. F. Eddleston. 1987. Prospective study of cellular immunity to hepatitis B virus antigens from the early incubation phase of acute hepatitis B. Lancet ii:119-122.
