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Systemic distribution of woodchuck hepatitis virus in the tissues of experimentally infected woodchucks
VIROLOGY
165, 172-181 (1988)
Systemic Distribution of Woodchuck Hepatitis Virus in the Tissues of Experimentally Infected Woodchucks BRENT E . KORBA,*' ERIC J . GOWANS,* ,2 FRANCES V . WELLS,* BUD C . TENNANT,t RICHARD CLARKE,* AND JOHN L . GERIN* *Georgetown University, Division of Molecular Virology and Immunology, Rockville, Maryland 20852, and tCornell University, College of Veterinary Medicine, Ithaca, New York 14853 Received October 76, 7987 ; accepted January 7, 1988 To better assess the extent of the tissue tropism of mammalian hepadnaviruses, 10 tissues from each of six woodchucks were examined for the presence and state of woodchuck hepatitis virus (WHV) nucleic acids 15 months after experimental WHV infection . The tissues examined were peripheral blood lymphocytes, lymph node, spleen, bone marrow, thymus, pancreas, kidney, ovary, testis, and liver . Tissue samples from three chronically infected animals and three animals with serologic patterns of recovery (serum : WHsAg , anti-WHC', anti-WHs*, WHV DNA - ) from acute WHV infection were analyzed in parallel by in situ hybridization and Southern and Northern blot techniques . WHV nucleic acids were detected in several individual tissues from each animal examined, although not all tissues in every animal contained WHV. Substantial differences were observed among the various tissues and animals with respect to the frequency, level, and intratissue distribution of WHV nucleic acids, as well as the presence of different viral genomic forms . Active WHV DNA replication was present only in the liver and spleen of the chronically infected animals . No evidence of ongoing WHV DNA replication was found in any of the tissues from the recovered animals . WHV DNA was homogeneously distributed among all hepatocytes in the livers of the chronic carriers . By contrast, WHV DNA In all the e)trahepatic tissues, and in the livers of the recovered animals, was detected only in scattered foci of cells . A 1988 Academic Press .lnc.
INTRODUCTION
Tagawa at al., 1985 ; Jilbert et al., 1987a,b ; Freiman et at, 1988) . The systemic distribution of DHBV in chronically infected Pekin ducks has been actively investigated by a number of laboratories, while studies on extrahepatic infections by the mammalian hepadnaviruses have focused primarily on PBL . The state of DHBV DNA in chronic carrier ducks appears to differ from that observed for WHV and HBV DNA in some extrahepatic tissues, such as the spleen where active replication of WHV and HBV DNA, but not DHBV DNA, is observed (Korba et al., 1987a ; Jilbert et al., 1987a ; Tagawa etal., 1985 ; Shimoda at al., 1981 ; Lieberman et at, 1987) . To better estimate the extent and pattern of systemic infection by mammalian hepadnaviruses, a variety of tissues from six woodchucks was examined for the presence and state of WHV nucleic acids 15 months following experimental WHV infection . The tissues included in this survey were liver, PBL, lymph node, bone marrow, spleen, thymus, pancreas, kidney, ovary, and testis . Three animals with chronic WHV infections were compared with a group of three identically infected animals which displayed serologic patterns (WHsAg - , anti-WHc', anti-WHs') of recovery from acute WHV infections 9 months prior to this analysis .
The woodchuck hepatitis virus (WHV) and its natural host, the Eastern woodchuck (M. marota), is the relevant experimental animal model of infection and disease, including hepatocellular carcinoma, for infection of humans by hepatitis B virus (HBV) (Popper at at, 1981, 1987) . The tissue tropism of this and other members of Hepadnaviridae has been extended by a number of studies to include several extrahepatic tissues, most notably mononuclear cells of the peripheral blood (PBL) and spleen (see Korba et al., 1986, 1987a for a review ; Jilbert at al., 1987 ; Freiman et al., 1988) . The presence of WHV in peripheral blood lymphocytes (PBL), lymph nodes, and lymphoid cells of the spleen (SLC) has been demonstrated to be an active infection as evidenced by the presence of viralspecific RNA transcripts and the replication of WHV DNA in the SLC of chronically infected woodchucks (Korba et al., 1986, 1 987a) . Active infections by duck hepatitis B virus (DHBV) have been found in nonhepatic tissues other than those of the lymphoid system, including pancreas and kidney (Halpern et al., 1983 ; ' To whom requests for reprints should be addressed . Present address : Institute of Medical and Veterinary Science, Division of Medical Virology, IMVS, Box 14, Rundle Mall PO, Adelaide, Australia, SA 5000 . 2
0042-6822/88 $3 .00 Copyright 0 1988 by Academic Press . Inc . All rights of reproduction in any form reserved .
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SYSTEMIC INFECTION BY WHV
Parallel examinations of these tissues were performed using in situ hybridization and Southern and Northern blot techniques to provide a comprehensive analysis of the state and distribution of WHV nucleic acids . This approach utilizes the unique advantages of each set of techniques to supplement and reinforce the results obtained by the alternative procedures . A number of differences were found in the state and intratissue distribution of WHV nucleic acids from those reported for DHBV and are discussed in comparative detail in this report . MATERIALS AND METHODS Source material Woodchucks were maintained in isolation and infection protocols conducted at the woodchuck breeding facility at Cornell University . Animals were experimentally infected at 3 days of age with a well-characterized virus pool, WHV7, as previously described (Popper at al ., 1987) . Lymphoid cells were prepared from PBL, mesenteric lymph node, bone marrow, spleen, and thymus as previously described (Korba et al., 1986, 1987a) . The remaining tissues were frozen immediately upon sampling in liquid nitrogen and stored at -70° . Diagnosis of the state of viral infection was based upon serologic analyses of WHV surface antigen (WHsAg), antibodies to WHV core antigen (WHcAg) and WHsAg (anti-WHc, anti-WHs) (Cote et al ., 1984 ; Ponzetto et al_ 1985) . The liver of one chronic carrier, animal 1408, was found to contain five small (0 .5 to 1 .0 cm) neoplastic nodules . No grossly identifiable neoplastic nodules or regions were found in the livers of the other five animals . Nucleic acid isolation, in situ hybridization, Southern and Northern blot analyses Whole cell DNA and RNA were prepared from isolated lymphoid cells or frozen tissue as previously described (Korba et al., 1986, 1 987a) . Analyses of viral nucleic acids by Southern and Northern blot hybridization techniques were performed as described in previous studies (Korba et al., 1986, 1 987a) . The probe used was a 3 .3-kb BamHl DNA fragment isolated from a cloned WHV genome . This WHV clone (WHV7) was isolated from the same viral infection pool used for these experiments and subsequently sequenced (Cohen et al., 1987) . In situ hybridization was performed as previously described (filbert et at, 1987b ; Gowans et al ., 1987) . Samples were fixed in ethanol :acetic acid (3 :1) and processed into paraffin wax ; 5 µM sections were postfixed as described (filbert etal ., 1 987a) and hybrid-
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ized with a WHV DNA fragment identical to that used for blot hybridization but radiolabeled by nick-translation with 125 l-dCTP (New England Nuclear) . For a hybridization background control, pBR322 DNA (labeled to the same specific activity as the WHV DNA) was used . For every sample, two adjacent tissue sections, mounted on the same slide, were hybridized in parallel : one with WHV DNA and one with pBR322 DNA . Quantitation of WHV nucleic acids by Southern and Northern blot hybridization Estimates of the overall level of WHV-specific DNA and RNA given in Table I were based upon densitometric comparison of the hybridization signals from tissue samples to that from known amounts of cloned WHV DNA coelectrophoresed in each gel . Averages of the amount of WHV nucleic acids from multiple determinations were obtained and the values were normalized to the levels of whole cell DNA or RNA in each lane . Levels of WHV nucleic acids were determined only from descrete bands which could be clearly identified as those specific for WHV . The unique patterns of heterogeneous smears representing WHV DNA replication intermediates were also included in the quantitation calculations . Homogeneous smears of degraded WHV nucleic acids, when present, were normally excluded from the calculations . These degraded nucleic acids were present in widely varying levels and, at most, represented less than 50% of the total WHV nucleic acids . Thus, the quantitation values used in this study represent the minimal amount of WHV nucleic acids present in the tissue samples . Levels of WHV DNA are presented as the number of WHV genome equivalents per cell . DNA levels are given as a threefold range and rounded to two significant figures since (i) the exact number of cells in a given tissue sample cannot be precisely determined, (ii) the exact yield of DNA on a per cell basis from these tissues cannot be precisely estimated but is assumed to be 1-3 pg DNA per cell, and (iii) the heterogeneity of the WHV DNA forms in some tissues does not permit a more accurate estimate of WHV genome equivalents per a specific amount of WHV DNA . RNA levels are presented as picograms WHV RNA per microgram whole cell RNA since the relative level of the various categories of RNA in a given cell or tissue are highly variable, depending upon such factors as the type of cell and the relative state of activity of a given tissue . Hence, it is difficult to estimate the yield of any given category of RNA on a per cell basis from tissue samples . WHV RNA levels are shown as a threefold range of values based upon the results of multiple sample analyses and rounded to two significant figures .
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RESULTS
WHV monomer genomes (3 .3 kb) with the occasional presence of molecules migrating as expected for covalently closed, superhelical monomers. The third category (replication intermediates ; RI) consists of a heterogenous distribution of low-molecular-weight (0 .5-3 .2 kb) single- and double-stranded WHV DNA molecules which represent viral DNA replication intermediates (Summers and Mason, 1982 ; Seeger et al., 1986). Integrated WHV DNA was not included as a separate category since these DNA forms were observed only in the liver of a single, recovered animal, number 1290 (see Fig . 1 D) . WHV DNA was detected in all the tissues examined, except for testes, in the chronic carrier group, whereas viral DNA showed a more limited distribution in the serologically recovered group (Table 1) . However, not every tissue from an individual animal contained WHV DNA . In addition, the level of WHV nucleic acids as well as the WHV genomic forms varied considerably
WHV nucleic acid patterns indicate widespread systemic viral infection A summary of the Southern and Northern blot hybridization analyses of WHV nucleic acids present in the various tissues is presented in Table 1 . Examples of the different WHV DNA patterns are shown in Fig . 1 (see also Korba et al., 1986, 1987a) . WHV DNA forms were classified into three general categories based upon restriction enzyme analysis . The first category (multimeric ; MM) represents nonintegrated, multimeric WHV DNA molecules (7-12 kb) (Fig . 1C) . Restriction enzyme digestion patterns suggest that these forms may represent head-to-tail genomic dimers (Korba et al., 1986, 1987a) . The two bands frequently observed may represent open-circular and linear forms . The second category of DNA molecules (monomeric ; M) are nonintegrated open-circular/linear
TABLE 1 WHV NUCLEIC ACIDS IN TISSUE OF WHV-INFECTED WOODCHUCKS A. Chronically infected (WHSAg') animals F1279
M1288
Tissue
MM
M
RI
DNA
RNA
MM
PBL LN BM Thymus Spleen Liver Pancreas Kidney Ovary Testis
+ +
+ +
-
+ + -
0 .3-1 0 .3-1 0 0 4-12 30-90 0 0 0
+ +
+ + + + + +
1-3 0 .7-2 0 0.1-0 .3 60-180 600-1800 0 .3-1 1-3 1-3
M
M1408
RI
DNA
RNA
MM
M
RI
DNA
RNA
-
0 .2-0 .6 0 .3-1 0 0 .2-0.6 50-150 700-2100 .3-1 0 0 .2-0 .6
0.1-0 .3 0.1-0 .3 0 0 3-9 40-120 0 0
+ + + -
+ + + +
-
0 .3-1 0.7-2 0 0 30-90 500-1500 0 0 .3-1
0 .2-0.6 0 .2-0 .6 0 0 3-9 36-108 0 0
0
0
0
0
DNA
RNA
0 .3-1 0 .3-1 0 .3-1 0 2-6 1-3 0 0 0
0 0 0 0 0 0 0 0 0
+ + -
+ + -
B . Recovered (anti-WHSI animals M1444
M 1290 Tissue
MM
P8L LN BM Thymus Spleen Liver Pancreas Kidney Ovary Testis
+ + + -
-
M
+ + + + -
RI
DNA
-
0.2 0 .6 0.2-0 .6 0 0 .2-0 .6 2-6 0 .3-1 0 0
-
0
RNA
MM
M
0 0 0 0 0 0 0 0
+ + +
+
0
-
+ + -
RI
F1613
DNA 0 .2-0.6 0 .3-1 0 .3-1 0 .2-0.6 1-3 0 0 0 0
RNA
MM
M
0 0 0 0 0 0 0 0
+ + -
+ +
+
+ +
-
RI
-
-
0
Note . Distribution of WHV nucleic acids in tissues of WHV-Infected woodchucks . Tissues were prepared and whole cell nucleic acids were isolated and analyzed by Southern and Northern blot hybridization techniques as described under Materials and Methods . Sex of animals is indicated by F (female) or M (male) preceding the animal number . LN, lymph node; BM, bone marrow, (+) indicates presence and (-) indicates absence of WHV DNA forms ; ND, not determined . See text for description of WHV DNA Categories (MM, M, RI) . DNA values represent copy number of WHV genomesrcell ; RNA values represent pg WHV RNA/µgwhole cell RNA . See Materials and Methods for detailed description of nucleic acid quantitation .
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SYSTEMIC INFECTION BY WHV E
D
A
B
C
I
1 2 3 4
1 2 3 4
1 2
3
1 2 3
FIG. 1 . Examples of Southern blot analysis of tissues from WHV-infected woodchucks. Whole cell DNA was extracted and analyzed by restriction enzyme digestion and Southern blot hybridization as described under Materials and Methods . Tissues used were the following : A, 25 pl WC1408 serum; B, WC1279 thymus (30 pg DNNlane) ; C, WC1408 PBL (30 pg DNNIane); D, WC1290 liver (25 pg DNA/lane): E, WC1408 spleen (5 gg DNA/lane) . For all gels, the restriction enzyme digestions were Dane 1) undigested DNA ; (lane 2)Aval (no recognition site in WHV7 (19)); (lane 3) BamHl (one site in WHV7) ; (lane 4) EcoRl/Hindlll double digest (one site for each enzyme in WHV7 producing bands of 2 .2 and 1 .1 kb) . Exposure times: (A and E) 16 hr, (C) 48 hr, (D) 72 hr, (B) 7 days .
between the various tissues and different woodchucks . However, there was an overall consistency in the WHV tissue infection patterns for the two groups of infected woodchucks (chronic carriers and recovered animals) . High levels of WHV DNA replication intermediates were observed only in the liver and spleen from the chronic carriers . Generally, levels of WHV DNA in the other tissues from both groups of animals were low or undetectable . WHV-specific RNA was detected only in the liver, spleen, PBL, and lymph nodes of the chronic carriers . Several different combinations of the three classes of WHV DNA molecules were observed in the various tissues (see Table 1 and Fig . 1) . Typical patterns of DNA replication intermediates with some monomeric molecules were observed in the livers and spleens of the chronic carriers . In some tissues (e .g ., 1408 Spleen, Fig . 1E), multimeric DNA molecules were present in addition to the DNA replication intermediates . In some cell populations (e .g ., 1288 LN, 1408 PBL, Fig . 1C) only nonintegrated multimeric, WHV DNA forms were observed . However, it is probable that monomeric DNA forms were also present but below the sensitivity levels of the assay . In general, monomeric WHV DNA forms in lymphoid cells are present as a minor species relative to the multimeric forms (Korba et al ., 1986, 1987a) .
In other cell populations (e .g ., 1279 Thymus, Fig . 1 B, 1613 BM) only monomeric WHV DNA forms were observed . These DNA molecules were present as discrete, tight bands, distinctly different from the diffuse DNA bands observed for WHV DNA extracted from serum virus particles (see Fig . 1A), indicating that this DNA population was probably double-stranded . This pattern of WHV DNA molecules was present only in those tissues which had marginal levels of WHV DNA and no WHV-specific RNA . It is possible that undetectable levels of the other categories of WHV DNA were also present . WHV-specific RNA, when present, existed primarily as 2 .3- and 3 .6-kb species in all the various tissues . The relative levels of WHV RNA, in general, correlated with the relative level of WHV DNA and DNA replication intermediates (Table 1) . Where little or no evidence of active WHV DNA replication was found, the levels of WHV RNA were very low and varied considerably . Differences in WHV nucleic acid patterns were observed in lymphoid cells between chronically infected and recovered animals WHV DNA was not present in the bone marrow of any of the chronic carriers but was present in the bone marrow from all three recovered animals . While no significant differences existed in the level or distribu-
1 76
KORBA ET AL .
W
Q
0
SYSTEMIC INFECTION BY WHV
Lion of WHV DNA forms in the lymphoid cells of the thymus, lymph node, or PBL between the two groups of animals, the relative level of WHV DNA was substantially higher in the latter two cell populations from the chronic carriers (see Table 1) . The significantly lower levels of WHV DNA and the inability to detect viral-specific RNA in the SLC of the recovered animals correlated with the loss of active WHV DNA replication . In the chronic carriers, the overall level of WHV DNA and RNA in the SLC was 10- to 30-fold less than that observed in hepatocytes (see also Korba et at, 1987a) . However, there was a consistently higher level of WHV nucleic acids in the SLC, relative to the liver, in the recovered animals . WHV DNA was detected in scattered foci in the extrahepatic tissues Samples of the various tissues, adjacent to those examined by blot hybridization techniques, were analyzed by in situ hybridization for WHV DNA (see Figs . 2 and 3) . WHV DNA in the livers of the chronic carriers was homogeneously distributed among all hepatocytes (Fig . 2A) . Bile duct, vascular elements, and infiltrates of lymphoid cells rarely contained WHV DNA . In the livers of the recovered animals, low levels of WHV DNA were observed in occasional, scattered foci or in isolated, single cells . WHV DNA in the spleens of the chronic carriers was present in a homogenous distribution in the red pulp with intense focal concentrations of WHV DNA in many of the germinal centers (Fig . 3A) . WHV DNA in the spleens of the recovered animals was distributed in rare, scattered single cells or small foci primarily in the germinal centers . In the remaining tissues, WHV DNA was found in only a few scattered, isolated cells (Figs . 2 and 3) . No obvious differences were noted in the general in situ hybridization patterns or types of WHV-carrying cells between the chronic carriers and the recovered animals . The only consistently detectable variations between these two groups were in the intracellular levels of WHV DNA and the relative number of WHV-positive cells in these different tissues, However, based upon comparative grain counts, WHV DNA levels in these scattered cells were relatively high (approximately 100 genome copies/cell) . In Table 1, quantitation of WHV
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DNA is displayed as an overall average level in whole tissues . Thus, in general, only 0 .1 to 1 .0% of the cells in these extrahepatic tissues carried WHV DNA (Figs . 2 and 3) . In some cells, such as in the germinal centers of the spleen (Fig . 3A), WHV DNA levels in individual cells were comparable to those observed in the hepatocytes of the chronic carriers (>1000 copies/cell) . In all tissues, WHV DNA was primarily located in the cell cytoplasm with a small proportion of grains distributed in the nuclei . WHV DNA was concentrated in the germinal centers of the lymph nodes (Fig . 3D) and in scattered cells of lymphoid appearance in the thymus (Fig . 2C) . WHV DNA in the kidney was present at moderate levels in the cells associated with the glomeruli or extraglomerular apparatus (Fig . 28) . In the pancreas, WHV DNA was present in scattered foci of heavily infected cells which appeared to be acinar cells or in cells which appeared to be tubular, ductal epithelium (Fig . 2D) . No WHV DNA was observed in cells associated with the islets . In the ovary, WHV DNA was present at moderate levels in a few, scattered, generalized epithelial cells in the outer shell of the developing follicles (Fig . 3B), Some scattered cells inside the follicles also carried WHV DNA . No consistent, specific hybridization was observed for WHV DNA in the testes . In the PBL, approximately 1% of the cells of lymphoid appearance harbored WHV DNA . Most of the infected cells carried low to moderate levels of WHV DNA, with approximately 10% being heavily infected (0-1% of the total PBL population) (Fig . 3C) . In the PBL, hybridization backgrounds with control DNA (pBR322) were unusually high . Due to a lack of antibodies to cell type-specific markers in the woodchuck system, it was not possible to identify which subpopulations of lymphoid cells carried WHV . Isolated bone marrow cells were not examined . DISCUSSION Evidence for the presence of hepadnavirus in tissues other than the liver has appeared in a number of reports over the past several years . Initially, such observations were limited to reports of HBV surface antigen (HBsAg) in extrahepatic tissues (Yoshimura et at, 1981 ; Shimoda et al., 1981) . With the advent of im-
Fic . 2 . Examples of in situ hybridization for WHV DNA in woodchuck tissues . Ethanobacetic acid-fixed tissues were prepared, hybridized, and stained with hematoxylin and eosin as described under Materials and Methods . The ' 251-labeled DNA probe used was the same as that used for 38 P-labeled, blot hybridization . Tissues used were (A. left) uninfected control liver (WC160) ; (A, right) WHV chronic carrier, WC12B8, liver ; (B) WC1279 kidney ; (C) WC1279 thymus ; (D) WC1279 pancreas . All panels photographed at 400X . Exposure times : (A, right) 16 hr, (A, left) and (9-D) 72 hr .
1 78
KORBA ET AL .
U
SYSTEMIC INFECTION BY WHV
proved technology, hepadnaviral DNA was detected in nonhepatic sites (Lie-Injo et al., 1983 ; Halpern et at, 1983) thereby confirming the presence of virus . In situ hybridization techniques lend further opportunities toward the study of hepadnavirus infection of extrahepatic tissues by demonstrating the specific cellular location of viral nucleic acids . In this report, the tissue tropism of WHV in chronically infected woodchucks has been substantially extended . The coupling of Southern, Northern, and in situ hybridization techniques provides a unique, comprehensive analysis of the state of viral nucleic acids and their intratissue distribution . Systemic infection by WHV is extensive, with viral DNA residing in the liver, pancreas, kidney, ovary, and all primary components of the lymphoid system . However, in individual animals the extent of extrahepatic infection differs considerably . As observed in previous reports (Korba et al ., 1986, 1987a), evidence of active WHV DNA replication in chronic carriers was present only in the liver and spleen . Viral DNA was homogeneously distributed among all hepatocytes in the liver, while in the spleen concentrated foci of WHV DNA were observed in the germinal centers against a lower level, widespread, homogenous background of viral DNA in the remainder of the tissue . In contrast to the liver and spleen only scattered foci of WHV DNA-containing cells were observed in the other extrahepatic tissues . Despite the lack of current WHV DNA replication, many of the scattered cells in these foci contained relatively high levels of viral DNA (greater than 100 copies/cell) . It is possible that some of these cells may have harbored actively replicating virus which, due to sensitivity levels, would not be detected by Southern blot analysis . In the DHBV model, in situ hybridization analysis has demonstrated widespread virus replication in tissues during the acute phase of infection which progresses to a localized, focal distribution of DHBV DNA in chronically infected ducks (Freiman et al., 1988) . Complete clearance of WHV DNA from the various tissues examined requires an extensive period of time following loss of serologic markers of active viral replication (usually occurring approximately 6 months postinfection in the woodchuck (Cote at al., 1984)) . The serologically recovered (WHsAg - , anti-WHc', antiWHs', WHV DNA- ) animals in this study continued to carry some viral DNA in scattered foci within several of
179
the tissues examined, including liver. However, this DNA was probably not actively replicating and, in most cases, no evidence of WHV-specific RNA was found . Previously, nonreplicating WHV DNA (but no RNA) was found in the PBL and liver tissue of some anti-WHs' woodchucks 12 to 25 months following seroconversion (Korba at al ., 1986, 1987a,b) . HBV DNA has been detected previously in the seminal fluid of chronically infected humans lending to the speculation of a direct mode of virus transfer via the germ line (Hadchouel et al ., 1985 ; Karayiannis at al,, 1985) . While seminal fluid from the chronic carrier woodchucks was not examined, no evidence of WHV DNA could be found in the testes, either by in situ or by Southern blot hybridization . However, the ovary of the single, female chronic carrier in this study clearly harbored nonreplicating WHV DNA in the cells in or near the developing follicles . A number of differences exist between the distribution and physiologic state of WHV and DHBV DNA in extrahepatic tissues . WHV and HBV DNA are actively replicating in the spleens of chronically infected woodchucks and chimpanzees (Table 1 ; Korba at al., 1987a ; Lieberman et at, 1987) . DHBV replication is usually not observed in the spleens of chronically infected Pekin ducks (Tagawa at al., 1985 ; Jilbert et al., 1987a), but does occur during the acute phase of infection (Halpern et at, 1983 ; Tawaga at al ., 1985 ; Freiman et al., 1988) . While both duck and woodchuck chronic virus carriers harbor high concentrations of viral DNA in the splenic germinal centers, DHBV DNA is believed to be present primarily in dendritic cells (Halpern et al., 1987) . Currently, it is not possible to determine specific cell identities in the woodchuck spleen due to a lack of the appropriate cellular phenotypic markers . In the pancreas of acutely infected ducks, replicating DHBV DNA is located in acinar cells (Jilbert et at, 1987b ; Freiman et at, 1988), while in chronically infected ducks DHBV DNA is associated with the islets (Halpern at al., 1983, 1985 ; Jilbert et al., 1987b ; Freiman at al., 1988) . In chronically infected woodchucks, nonreplicating WHV DNA is present only in acinar cells or in cells suspected to be tubular, ductal epithelium . Similarly, HBsAg has been reported to be concentrated in acinar cells of chronically infected human patients (Shimoda at al., 1981 ; Yoshimura et at, 1981) . However, during the acute phase of DHBV infection,
FiG . 3 . Examples of in situ hybridization for WHV DNA in infected woodchuck tissues . (A) WC1279 spleen ; (B) WC1279 ovary ; (C) WC1288 PBL ; (D) WC1279 lymph node . (A, left) and (D, left) photographed at 200X . All other panels photographed at 400X . Exposure times : (A) 40 hr, (B-D) 72 hr .
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KORBA ET AL .
DHBV DNA is located primarily in acinar cells (A . Jilbert and J . Freiman, personal communication) . In the kidney of chronically infected woodchucks nonreplicating viral DNA is concentrated in cells that are in or near the glomeruli . While nonreplicating HBV DNA has been found in the kidney during chronic infection, the location of the viral DNA-carrying cells has yet to be clarified (Dejean et al., 1984 ; Naumova et at., 1985) . Both DHBsAg and DHBV DNA have been detected in the glomeruli (Halpern at al., 1983 ; Jilbert et al., 1987a) . In some cases, replicating DHBV DNA has been found in the kidney of chronically infected ducks (Halpern et al., 1983 ; Tagawa et al., 1985) . The nature of the differences in extrahepatic infection patterns between WHV, HBV, and DHBV is unclear at the present time . Most likely, these relate to fundamental differences in host physiology and not to inherent variations in the primary characteristics of these highly related viruses . The gross observational differences reported to date should not be interpreted as inconsistencies in contrasting models of viral hepatitis, but as fortuitous clues into the interactions of host cell and virus factors . Further definition of the normal physiologic functions of the various types of virus-carrying cells in the woodchuck and duck will enhance the understanding of the cellular functions required for hepadnavirus propagation . The role of hepadnavirus infection of cells other than hepatocytes in virus-induced disease remains to be elucidated . The infection of lymphoid cells may modulate responses of the immune system to these viruses . Alternatively, these infected cells could serve as additional sources of virus pools . The lack of active viral replication at many of the extrahepatic sites does not mean that these viral genomes are replication incompetent . The hepadnavirus genomes in PBL from chronically infected chimpanzees and woodchucks have been shown to be transcriptionally active (Korba et al., 1986, 1987a) . PBL from HBV-infected humans have not been examined for virus-specific RNA . The multimeric viral DNA forms observed in extrahepatic tissues are not found in serum virions and are thus a product of active intracellular processing (e .g ., replication, recombination) . In addition, active replication of HBV and WHV has been observed in the spleen (Lieberman et al., 1987 ; Korba at al., 1 987a) and replication of DHBV has been observed in several tissues at different times during the course of virus infection (Halpern et al., 1983 ; Tagawa at al., 1985 ; Freiman et at, 1988) . This study serves as a basis for more extensive analysis of the kinetics of WHV infection of various tissues during the natural course of viral infection . The systemic movement of virus within the tissues examined
here during the course of infection is critical to understanding of the overall pathologic mechanisms of hepadnavirus-induced disease. Correlations of the state of virus in extrahepatic tissues with serologic markers of virus infection and the state of liver disease may provide the necessary information for improved prognosis and the development of more efficient antiviral therapy . ACKNOWLEDGMENTS The authors gratefully acknowledge the contributions of B . Baldwin, W . Hornbuckle, A . Glasser, W . Sherman (Cornell University), and their colleagues for animal maintenance and assistance in the collection of tissues . 1 . King (Cornell University) kindly provided the histologic analysis of the different tissues and P . Cote (Georgetown University) provided all serologic analyses . The authors also thank 1 . Freiman for corn rnunication of results prior to publication . This manuscript was prepared with the assistance of C . Culp . This work was supported by Contracts NO 1-Al-22665 and NO 1-Al-52585 between the National Institute of Allergy and Infectious Diseases and Georgetown University and Cornell Universities, respectively .
REFERENCES COHEN, 1 ., MILLER, R . H ., ROSENBLUM, B ., DENNISTON . K ., GERIN, 1 . L ., and PURCELL, R. H . (1987) . Fidelity of hepadnavirus polymerase : Sequence variation of woodchuck hepatitis virus genomes . Virology 162, 12-20 . COTE, P . J ., JR ., DAPOLITO, G . M ., SHIH, 1 . W .-K ., and GERIN, 1 . L . (1984) . Antigenic analysis of woodchuck hepatitis virus surface antigen with site-specific radioimmunoassays . J. Virol. 49, 701-708 . y~ DEJEAN, A ., LUGASSY, C ., ZAFRANI, S ., TIOLLAIS, P., and BRECHOT, C . (1984) . Detection of hepatitis B virus DNA in pancreas, kidney and skirt of two human carriers of the virus . ). Gen. Virol. 65, 651-655 . FREIMAN, 1. S ., JILBERT, A . R, DIXON, R. 1 ., HOLMES, M ., GowANs, E . 1 ., BURRELL, C . J ., WILLS, E . 1 ., and COSSART, Y . E . (1988) . Experimental duck hepatitis B virus infection : Pathology and evolution of hepatic and extrahepatic infection . Hepatology, in press . GOWANS, E . 1 ., NEGRO, F ., BAROUDY, B . M ., KOREA, B . E ., BONING, F . . PoNZETTO, A . . PURCELL, R . H ., and GERIN, 1 . L . (1987) . Molecular markers of WHV and HDV in single cells . In "Hepadnaviruses" (W . Robinson . K . Koike, and H . Will, Eds .), pp . 591-600 . A . R . Liss, New York. HAOCHOUEL, M ., SCOTTO, 1 ., HURET, 1 . L ., MOLINE, C ., VILLA, E ., DEGOS, F ., and BRECHOT, C . (1985) . Presence of HBV DNA in spermatazoa : A possible vertical transmission of HBV via the germ line . ). Med. Virol, 16, 61-66 . HALPERN, M . S., EGAN, J ., McMAHON, and ERwERT, D . L . (1985). Duck hepatitis B virus is tropic for exocrine cells of the pancreas . Virology 146, 157-161 . HALPERN, M . S ., ENGLAND, 1 . M ., DEERY, D . T ., PETCU, 0 . J ., MASON, W . S ., and MOLNAR-KiMBER, K . L . (1983) . Viral nucleic acid synthesis and antigen accumulation in pancreas and kidney of Pekin ducks infected with duck hepatitis B virus . Proc . NaU. Aced. Sci. USA 80,4865-4869 . HALPERN, M . S ., MASON, W . S., ERWERT, D ., O'CONNELL, A. P ., and ENGLAND, 1 . M . (1987). DHBV infection of ducks and associated immunity . Proc . UCLA Symp. Cell . Mol. Biol., in press . JILBERT, A . R ., FREIMAN, 1, S ., BURRELL, C . J ., HOLMES, M ., GOWANS, E . ) ., ROwLAND, R ., ITALL, P., and CossART, Y . E. (1987a) . Early virus cell interactions in duck hepatitis 8 virus infection : A time
SYSTEMIC INFECTION BY WHV
course study of tissue tropism and sites of virus production . In "Viral Hepatitis and Liver Disease ." A. R. Liss, New York, in press . JILBERT . A . R ., FREIMAN, J . S ., GOWANS, E . J ., HOLMES, M ., COSSART, Y . E ., and BURELL, C . J . (1987b) . Duck hepatitis B virus DNA in liver, spleen and pancreas : Analysis by in situ and Southern blot hybridization . Virology 158, 330-338 . KARAYIANNIS, P ., NOVICK, D . M ., L0K, A . S . F ., FOWLER, M . J . F ., MONJARDINO, J ., and THOMAS, H . C . (1985) . Hepatitis B virus DNA in saliva, urine, and seminal fluid of carriers of hepatitis B e antigen . Brit. Med. J. 290, 1853-1855 . KOREA, B . E ., WELLS, F . V ., TENNANT, B . C ., COTE, P . J ., and GERIN, 1 . L . (1987a) . Lymphoid cells in the spleen of woodchuck hepatitis virus-infected woodchucks are a site of active viral replication . J. Virol. 61, 1318-1324 . KOREA, B . E ., WELLS, F . V ., TENNANT, B . C ., COTE, P . J ., and GERIN, J . L . (1 987b) . Presence of WHV DNA in hepatocellular carcinoma tissue from woodchucks which have recovered from active viral infections . In "Viral Hepatitis and Liver Disease ." A . R . Liss, New York, in press . KOREA, B . E ., WELLS, F . V ., TENNANT, B . C ., YOAKUM, G . H ., PURCELL, R . H ., and GERIN, J . L . (1986). Hepadnavirus infection of peripheral blood lymphocytes in vivo: Woodchuck and chimpanzee models of viral hepatitis . J. Virol. 58, 1-8 . LIEBERMAN . H . M ., TUNG, W . W ., and SHAERITZ, D . A . (1987) . Splenic replication of hepatitis B virus in the chimpanzee chronic carrier . J. Med. Virol . 21, 347-359 . LIE-INJO, L. F ., BALASEGARAM, C . G ., LOPEZ, C . G ., and HERREA, A . R . (1983) . Hepatitis B virus DNA in liver white blood cells of patients with hepatoma . DNA 2, 301-308 . NAUMOVA, A . K ., FAvonvo, M . 0 ., KETELADZE, E . S ., NOSIKOV, V . V .,
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and KISSELEv, L . L . (1985) . Nucleotide sequences in human chromosomal DNA from nonhepatic tissues homologous to the hepatitis B virus genome . Gene 35, 19-25 . PONZETTO, A ., COTE, P . J ., JR ., FORD, E . C ., ENGLE, R ., CICMANEC, D ., SHAPRO, M ., PURCELL, R . H ., and GERIN, J . L . (1985) . Radioimmunoassay and characterization of woodchuck hepatitis virus core antigen and antibody . Virus Res . 2, 301-315 . POPPER, H ., ROTH, L., PURCELL, R . H ., TENNANT, B . C ., and GERIN, J . L . (1987) . Hepatocarcinogenicity of the woodchuck hepatitis virus . Proc . Natl. Aced. Sci. USA 84, 866-870 . POPPER, H ., SHIH, J . W . K ., GERIN, J . L ., WONG, D . C ., HOVER, B . H ., LONDON, W . T ., SLY, D . L., and PURCELL, R . H . (1981). Woodchuck hepatitis and hepatocellular carcinoma : Correlation of histologic with virologic observations . Hepatology 1, 91-98 . SEEGER, C ., GANEM, D ., and VARMUS, H . E . (1986) . Biochemical and genetic evidence for the hepatitis B virus replication strategy . Science 232, 477-484 . SHIMODA . T ., SHIKATA, T ., KARASAWA, T ., TZUKAGOSHI, S ., YOSIMURA, M ., and SAKURAI, I . (1981) . Light microscopic localization of hepatitis B virus antigens in the human pancreas . Gastroenterology 81, 998-1005 . SUMMERS, J ., and MASON, W . S . (1982) . Replication of the genome of a hepatitis-B-like virus by reverse transcription of an RNA intermediate . Cell 29, 403-415 . TAGAWA, M ., OMATA, M ., YOKOSUKA, K ., UCHIUMI, F ., IMAZEKI, F ., and OKUDA, K. (1985) . Early events in duck hepatitis B virus infection . Gastroenterology 84, 1224-1229 . YOSHIMURA, M ., SAKURAI, I ., SHIMODA, T., ABE, K ., OKANO, T ., and SHIKATA, T . (1981) . Detection of HBsAg in the pancreas . Acre . Pathol. Japan 31, 711-717 .
165, 172-181 (1988)
Systemic Distribution of Woodchuck Hepatitis Virus in the Tissues of Experimentally Infected Woodchucks BRENT E . KORBA,*' ERIC J . GOWANS,* ,2 FRANCES V . WELLS,* BUD C . TENNANT,t RICHARD CLARKE,* AND JOHN L . GERIN* *Georgetown University, Division of Molecular Virology and Immunology, Rockville, Maryland 20852, and tCornell University, College of Veterinary Medicine, Ithaca, New York 14853 Received October 76, 7987 ; accepted January 7, 1988 To better assess the extent of the tissue tropism of mammalian hepadnaviruses, 10 tissues from each of six woodchucks were examined for the presence and state of woodchuck hepatitis virus (WHV) nucleic acids 15 months after experimental WHV infection . The tissues examined were peripheral blood lymphocytes, lymph node, spleen, bone marrow, thymus, pancreas, kidney, ovary, testis, and liver . Tissue samples from three chronically infected animals and three animals with serologic patterns of recovery (serum : WHsAg , anti-WHC', anti-WHs*, WHV DNA - ) from acute WHV infection were analyzed in parallel by in situ hybridization and Southern and Northern blot techniques . WHV nucleic acids were detected in several individual tissues from each animal examined, although not all tissues in every animal contained WHV. Substantial differences were observed among the various tissues and animals with respect to the frequency, level, and intratissue distribution of WHV nucleic acids, as well as the presence of different viral genomic forms . Active WHV DNA replication was present only in the liver and spleen of the chronically infected animals . No evidence of ongoing WHV DNA replication was found in any of the tissues from the recovered animals . WHV DNA was homogeneously distributed among all hepatocytes in the livers of the chronic carriers . By contrast, WHV DNA In all the e)trahepatic tissues, and in the livers of the recovered animals, was detected only in scattered foci of cells . A 1988 Academic Press .lnc.
INTRODUCTION
Tagawa at al., 1985 ; Jilbert et al., 1987a,b ; Freiman et at, 1988) . The systemic distribution of DHBV in chronically infected Pekin ducks has been actively investigated by a number of laboratories, while studies on extrahepatic infections by the mammalian hepadnaviruses have focused primarily on PBL . The state of DHBV DNA in chronic carrier ducks appears to differ from that observed for WHV and HBV DNA in some extrahepatic tissues, such as the spleen where active replication of WHV and HBV DNA, but not DHBV DNA, is observed (Korba et al., 1987a ; Jilbert et al., 1987a ; Tagawa etal., 1985 ; Shimoda at al., 1981 ; Lieberman et at, 1987) . To better estimate the extent and pattern of systemic infection by mammalian hepadnaviruses, a variety of tissues from six woodchucks was examined for the presence and state of WHV nucleic acids 15 months following experimental WHV infection . The tissues included in this survey were liver, PBL, lymph node, bone marrow, spleen, thymus, pancreas, kidney, ovary, and testis . Three animals with chronic WHV infections were compared with a group of three identically infected animals which displayed serologic patterns (WHsAg - , anti-WHc', anti-WHs') of recovery from acute WHV infections 9 months prior to this analysis .
The woodchuck hepatitis virus (WHV) and its natural host, the Eastern woodchuck (M. marota), is the relevant experimental animal model of infection and disease, including hepatocellular carcinoma, for infection of humans by hepatitis B virus (HBV) (Popper at at, 1981, 1987) . The tissue tropism of this and other members of Hepadnaviridae has been extended by a number of studies to include several extrahepatic tissues, most notably mononuclear cells of the peripheral blood (PBL) and spleen (see Korba et al., 1986, 1987a for a review ; Jilbert at al., 1987 ; Freiman et al., 1988) . The presence of WHV in peripheral blood lymphocytes (PBL), lymph nodes, and lymphoid cells of the spleen (SLC) has been demonstrated to be an active infection as evidenced by the presence of viralspecific RNA transcripts and the replication of WHV DNA in the SLC of chronically infected woodchucks (Korba et al., 1986, 1 987a) . Active infections by duck hepatitis B virus (DHBV) have been found in nonhepatic tissues other than those of the lymphoid system, including pancreas and kidney (Halpern et al., 1983 ; ' To whom requests for reprints should be addressed . Present address : Institute of Medical and Veterinary Science, Division of Medical Virology, IMVS, Box 14, Rundle Mall PO, Adelaide, Australia, SA 5000 . 2
0042-6822/88 $3 .00 Copyright 0 1988 by Academic Press . Inc . All rights of reproduction in any form reserved .
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SYSTEMIC INFECTION BY WHV
Parallel examinations of these tissues were performed using in situ hybridization and Southern and Northern blot techniques to provide a comprehensive analysis of the state and distribution of WHV nucleic acids . This approach utilizes the unique advantages of each set of techniques to supplement and reinforce the results obtained by the alternative procedures . A number of differences were found in the state and intratissue distribution of WHV nucleic acids from those reported for DHBV and are discussed in comparative detail in this report . MATERIALS AND METHODS Source material Woodchucks were maintained in isolation and infection protocols conducted at the woodchuck breeding facility at Cornell University . Animals were experimentally infected at 3 days of age with a well-characterized virus pool, WHV7, as previously described (Popper at al ., 1987) . Lymphoid cells were prepared from PBL, mesenteric lymph node, bone marrow, spleen, and thymus as previously described (Korba et al., 1986, 1987a) . The remaining tissues were frozen immediately upon sampling in liquid nitrogen and stored at -70° . Diagnosis of the state of viral infection was based upon serologic analyses of WHV surface antigen (WHsAg), antibodies to WHV core antigen (WHcAg) and WHsAg (anti-WHc, anti-WHs) (Cote et al ., 1984 ; Ponzetto et al_ 1985) . The liver of one chronic carrier, animal 1408, was found to contain five small (0 .5 to 1 .0 cm) neoplastic nodules . No grossly identifiable neoplastic nodules or regions were found in the livers of the other five animals . Nucleic acid isolation, in situ hybridization, Southern and Northern blot analyses Whole cell DNA and RNA were prepared from isolated lymphoid cells or frozen tissue as previously described (Korba et al., 1986, 1 987a) . Analyses of viral nucleic acids by Southern and Northern blot hybridization techniques were performed as described in previous studies (Korba et al., 1986, 1 987a) . The probe used was a 3 .3-kb BamHl DNA fragment isolated from a cloned WHV genome . This WHV clone (WHV7) was isolated from the same viral infection pool used for these experiments and subsequently sequenced (Cohen et al., 1987) . In situ hybridization was performed as previously described (filbert et at, 1987b ; Gowans et al ., 1987) . Samples were fixed in ethanol :acetic acid (3 :1) and processed into paraffin wax ; 5 µM sections were postfixed as described (filbert etal ., 1 987a) and hybrid-
173
ized with a WHV DNA fragment identical to that used for blot hybridization but radiolabeled by nick-translation with 125 l-dCTP (New England Nuclear) . For a hybridization background control, pBR322 DNA (labeled to the same specific activity as the WHV DNA) was used . For every sample, two adjacent tissue sections, mounted on the same slide, were hybridized in parallel : one with WHV DNA and one with pBR322 DNA . Quantitation of WHV nucleic acids by Southern and Northern blot hybridization Estimates of the overall level of WHV-specific DNA and RNA given in Table I were based upon densitometric comparison of the hybridization signals from tissue samples to that from known amounts of cloned WHV DNA coelectrophoresed in each gel . Averages of the amount of WHV nucleic acids from multiple determinations were obtained and the values were normalized to the levels of whole cell DNA or RNA in each lane . Levels of WHV nucleic acids were determined only from descrete bands which could be clearly identified as those specific for WHV . The unique patterns of heterogeneous smears representing WHV DNA replication intermediates were also included in the quantitation calculations . Homogeneous smears of degraded WHV nucleic acids, when present, were normally excluded from the calculations . These degraded nucleic acids were present in widely varying levels and, at most, represented less than 50% of the total WHV nucleic acids . Thus, the quantitation values used in this study represent the minimal amount of WHV nucleic acids present in the tissue samples . Levels of WHV DNA are presented as the number of WHV genome equivalents per cell . DNA levels are given as a threefold range and rounded to two significant figures since (i) the exact number of cells in a given tissue sample cannot be precisely determined, (ii) the exact yield of DNA on a per cell basis from these tissues cannot be precisely estimated but is assumed to be 1-3 pg DNA per cell, and (iii) the heterogeneity of the WHV DNA forms in some tissues does not permit a more accurate estimate of WHV genome equivalents per a specific amount of WHV DNA . RNA levels are presented as picograms WHV RNA per microgram whole cell RNA since the relative level of the various categories of RNA in a given cell or tissue are highly variable, depending upon such factors as the type of cell and the relative state of activity of a given tissue . Hence, it is difficult to estimate the yield of any given category of RNA on a per cell basis from tissue samples . WHV RNA levels are shown as a threefold range of values based upon the results of multiple sample analyses and rounded to two significant figures .
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KORBA ET AL.
RESULTS
WHV monomer genomes (3 .3 kb) with the occasional presence of molecules migrating as expected for covalently closed, superhelical monomers. The third category (replication intermediates ; RI) consists of a heterogenous distribution of low-molecular-weight (0 .5-3 .2 kb) single- and double-stranded WHV DNA molecules which represent viral DNA replication intermediates (Summers and Mason, 1982 ; Seeger et al., 1986). Integrated WHV DNA was not included as a separate category since these DNA forms were observed only in the liver of a single, recovered animal, number 1290 (see Fig . 1 D) . WHV DNA was detected in all the tissues examined, except for testes, in the chronic carrier group, whereas viral DNA showed a more limited distribution in the serologically recovered group (Table 1) . However, not every tissue from an individual animal contained WHV DNA . In addition, the level of WHV nucleic acids as well as the WHV genomic forms varied considerably
WHV nucleic acid patterns indicate widespread systemic viral infection A summary of the Southern and Northern blot hybridization analyses of WHV nucleic acids present in the various tissues is presented in Table 1 . Examples of the different WHV DNA patterns are shown in Fig . 1 (see also Korba et al., 1986, 1987a) . WHV DNA forms were classified into three general categories based upon restriction enzyme analysis . The first category (multimeric ; MM) represents nonintegrated, multimeric WHV DNA molecules (7-12 kb) (Fig . 1C) . Restriction enzyme digestion patterns suggest that these forms may represent head-to-tail genomic dimers (Korba et al., 1986, 1987a) . The two bands frequently observed may represent open-circular and linear forms . The second category of DNA molecules (monomeric ; M) are nonintegrated open-circular/linear
TABLE 1 WHV NUCLEIC ACIDS IN TISSUE OF WHV-INFECTED WOODCHUCKS A. Chronically infected (WHSAg') animals F1279
M1288
Tissue
MM
M
RI
DNA
RNA
MM
PBL LN BM Thymus Spleen Liver Pancreas Kidney Ovary Testis
+ +
+ +
-
+ + -
0 .3-1 0 .3-1 0 0 4-12 30-90 0 0 0
+ +
+ + + + + +
1-3 0 .7-2 0 0.1-0 .3 60-180 600-1800 0 .3-1 1-3 1-3
M
M1408
RI
DNA
RNA
MM
M
RI
DNA
RNA
-
0 .2-0 .6 0 .3-1 0 0 .2-0.6 50-150 700-2100 .3-1 0 0 .2-0 .6
0.1-0 .3 0.1-0 .3 0 0 3-9 40-120 0 0
+ + + -
+ + + +
-
0 .3-1 0.7-2 0 0 30-90 500-1500 0 0 .3-1
0 .2-0.6 0 .2-0 .6 0 0 3-9 36-108 0 0
0
0
0
0
DNA
RNA
0 .3-1 0 .3-1 0 .3-1 0 2-6 1-3 0 0 0
0 0 0 0 0 0 0 0 0
+ + -
+ + -
B . Recovered (anti-WHSI animals M1444
M 1290 Tissue
MM
P8L LN BM Thymus Spleen Liver Pancreas Kidney Ovary Testis
+ + + -
-
M
+ + + + -
RI
DNA
-
0.2 0 .6 0.2-0 .6 0 0 .2-0 .6 2-6 0 .3-1 0 0
-
0
RNA
MM
M
0 0 0 0 0 0 0 0
+ + +
+
0
-
+ + -
RI
F1613
DNA 0 .2-0.6 0 .3-1 0 .3-1 0 .2-0.6 1-3 0 0 0 0
RNA
MM
M
0 0 0 0 0 0 0 0
+ + -
+ +
+
+ +
-
RI
-
-
0
Note . Distribution of WHV nucleic acids in tissues of WHV-Infected woodchucks . Tissues were prepared and whole cell nucleic acids were isolated and analyzed by Southern and Northern blot hybridization techniques as described under Materials and Methods . Sex of animals is indicated by F (female) or M (male) preceding the animal number . LN, lymph node; BM, bone marrow, (+) indicates presence and (-) indicates absence of WHV DNA forms ; ND, not determined . See text for description of WHV DNA Categories (MM, M, RI) . DNA values represent copy number of WHV genomesrcell ; RNA values represent pg WHV RNA/µgwhole cell RNA . See Materials and Methods for detailed description of nucleic acid quantitation .
175
SYSTEMIC INFECTION BY WHV E
D
A
B
C
I
1 2 3 4
1 2 3 4
1 2
3
1 2 3
FIG. 1 . Examples of Southern blot analysis of tissues from WHV-infected woodchucks. Whole cell DNA was extracted and analyzed by restriction enzyme digestion and Southern blot hybridization as described under Materials and Methods . Tissues used were the following : A, 25 pl WC1408 serum; B, WC1279 thymus (30 pg DNNlane) ; C, WC1408 PBL (30 pg DNNIane); D, WC1290 liver (25 pg DNA/lane): E, WC1408 spleen (5 gg DNA/lane) . For all gels, the restriction enzyme digestions were Dane 1) undigested DNA ; (lane 2)Aval (no recognition site in WHV7 (19)); (lane 3) BamHl (one site in WHV7) ; (lane 4) EcoRl/Hindlll double digest (one site for each enzyme in WHV7 producing bands of 2 .2 and 1 .1 kb) . Exposure times: (A and E) 16 hr, (C) 48 hr, (D) 72 hr, (B) 7 days .
between the various tissues and different woodchucks . However, there was an overall consistency in the WHV tissue infection patterns for the two groups of infected woodchucks (chronic carriers and recovered animals) . High levels of WHV DNA replication intermediates were observed only in the liver and spleen from the chronic carriers . Generally, levels of WHV DNA in the other tissues from both groups of animals were low or undetectable . WHV-specific RNA was detected only in the liver, spleen, PBL, and lymph nodes of the chronic carriers . Several different combinations of the three classes of WHV DNA molecules were observed in the various tissues (see Table 1 and Fig . 1) . Typical patterns of DNA replication intermediates with some monomeric molecules were observed in the livers and spleens of the chronic carriers . In some tissues (e .g ., 1408 Spleen, Fig . 1E), multimeric DNA molecules were present in addition to the DNA replication intermediates . In some cell populations (e .g ., 1288 LN, 1408 PBL, Fig . 1C) only nonintegrated multimeric, WHV DNA forms were observed . However, it is probable that monomeric DNA forms were also present but below the sensitivity levels of the assay . In general, monomeric WHV DNA forms in lymphoid cells are present as a minor species relative to the multimeric forms (Korba et al ., 1986, 1987a) .
In other cell populations (e .g ., 1279 Thymus, Fig . 1 B, 1613 BM) only monomeric WHV DNA forms were observed . These DNA molecules were present as discrete, tight bands, distinctly different from the diffuse DNA bands observed for WHV DNA extracted from serum virus particles (see Fig . 1A), indicating that this DNA population was probably double-stranded . This pattern of WHV DNA molecules was present only in those tissues which had marginal levels of WHV DNA and no WHV-specific RNA . It is possible that undetectable levels of the other categories of WHV DNA were also present . WHV-specific RNA, when present, existed primarily as 2 .3- and 3 .6-kb species in all the various tissues . The relative levels of WHV RNA, in general, correlated with the relative level of WHV DNA and DNA replication intermediates (Table 1) . Where little or no evidence of active WHV DNA replication was found, the levels of WHV RNA were very low and varied considerably . Differences in WHV nucleic acid patterns were observed in lymphoid cells between chronically infected and recovered animals WHV DNA was not present in the bone marrow of any of the chronic carriers but was present in the bone marrow from all three recovered animals . While no significant differences existed in the level or distribu-
1 76
KORBA ET AL .
W
Q
0
SYSTEMIC INFECTION BY WHV
Lion of WHV DNA forms in the lymphoid cells of the thymus, lymph node, or PBL between the two groups of animals, the relative level of WHV DNA was substantially higher in the latter two cell populations from the chronic carriers (see Table 1) . The significantly lower levels of WHV DNA and the inability to detect viral-specific RNA in the SLC of the recovered animals correlated with the loss of active WHV DNA replication . In the chronic carriers, the overall level of WHV DNA and RNA in the SLC was 10- to 30-fold less than that observed in hepatocytes (see also Korba et at, 1987a) . However, there was a consistently higher level of WHV nucleic acids in the SLC, relative to the liver, in the recovered animals . WHV DNA was detected in scattered foci in the extrahepatic tissues Samples of the various tissues, adjacent to those examined by blot hybridization techniques, were analyzed by in situ hybridization for WHV DNA (see Figs . 2 and 3) . WHV DNA in the livers of the chronic carriers was homogeneously distributed among all hepatocytes (Fig . 2A) . Bile duct, vascular elements, and infiltrates of lymphoid cells rarely contained WHV DNA . In the livers of the recovered animals, low levels of WHV DNA were observed in occasional, scattered foci or in isolated, single cells . WHV DNA in the spleens of the chronic carriers was present in a homogenous distribution in the red pulp with intense focal concentrations of WHV DNA in many of the germinal centers (Fig . 3A) . WHV DNA in the spleens of the recovered animals was distributed in rare, scattered single cells or small foci primarily in the germinal centers . In the remaining tissues, WHV DNA was found in only a few scattered, isolated cells (Figs . 2 and 3) . No obvious differences were noted in the general in situ hybridization patterns or types of WHV-carrying cells between the chronic carriers and the recovered animals . The only consistently detectable variations between these two groups were in the intracellular levels of WHV DNA and the relative number of WHV-positive cells in these different tissues, However, based upon comparative grain counts, WHV DNA levels in these scattered cells were relatively high (approximately 100 genome copies/cell) . In Table 1, quantitation of WHV
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DNA is displayed as an overall average level in whole tissues . Thus, in general, only 0 .1 to 1 .0% of the cells in these extrahepatic tissues carried WHV DNA (Figs . 2 and 3) . In some cells, such as in the germinal centers of the spleen (Fig . 3A), WHV DNA levels in individual cells were comparable to those observed in the hepatocytes of the chronic carriers (>1000 copies/cell) . In all tissues, WHV DNA was primarily located in the cell cytoplasm with a small proportion of grains distributed in the nuclei . WHV DNA was concentrated in the germinal centers of the lymph nodes (Fig . 3D) and in scattered cells of lymphoid appearance in the thymus (Fig . 2C) . WHV DNA in the kidney was present at moderate levels in the cells associated with the glomeruli or extraglomerular apparatus (Fig . 28) . In the pancreas, WHV DNA was present in scattered foci of heavily infected cells which appeared to be acinar cells or in cells which appeared to be tubular, ductal epithelium (Fig . 2D) . No WHV DNA was observed in cells associated with the islets . In the ovary, WHV DNA was present at moderate levels in a few, scattered, generalized epithelial cells in the outer shell of the developing follicles (Fig . 3B), Some scattered cells inside the follicles also carried WHV DNA . No consistent, specific hybridization was observed for WHV DNA in the testes . In the PBL, approximately 1% of the cells of lymphoid appearance harbored WHV DNA . Most of the infected cells carried low to moderate levels of WHV DNA, with approximately 10% being heavily infected (0-1% of the total PBL population) (Fig . 3C) . In the PBL, hybridization backgrounds with control DNA (pBR322) were unusually high . Due to a lack of antibodies to cell type-specific markers in the woodchuck system, it was not possible to identify which subpopulations of lymphoid cells carried WHV . Isolated bone marrow cells were not examined . DISCUSSION Evidence for the presence of hepadnavirus in tissues other than the liver has appeared in a number of reports over the past several years . Initially, such observations were limited to reports of HBV surface antigen (HBsAg) in extrahepatic tissues (Yoshimura et at, 1981 ; Shimoda et al., 1981) . With the advent of im-
Fic . 2 . Examples of in situ hybridization for WHV DNA in woodchuck tissues . Ethanobacetic acid-fixed tissues were prepared, hybridized, and stained with hematoxylin and eosin as described under Materials and Methods . The ' 251-labeled DNA probe used was the same as that used for 38 P-labeled, blot hybridization . Tissues used were (A. left) uninfected control liver (WC160) ; (A, right) WHV chronic carrier, WC12B8, liver ; (B) WC1279 kidney ; (C) WC1279 thymus ; (D) WC1279 pancreas . All panels photographed at 400X . Exposure times : (A, right) 16 hr, (A, left) and (9-D) 72 hr .
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U
SYSTEMIC INFECTION BY WHV
proved technology, hepadnaviral DNA was detected in nonhepatic sites (Lie-Injo et al., 1983 ; Halpern et at, 1983) thereby confirming the presence of virus . In situ hybridization techniques lend further opportunities toward the study of hepadnavirus infection of extrahepatic tissues by demonstrating the specific cellular location of viral nucleic acids . In this report, the tissue tropism of WHV in chronically infected woodchucks has been substantially extended . The coupling of Southern, Northern, and in situ hybridization techniques provides a unique, comprehensive analysis of the state of viral nucleic acids and their intratissue distribution . Systemic infection by WHV is extensive, with viral DNA residing in the liver, pancreas, kidney, ovary, and all primary components of the lymphoid system . However, in individual animals the extent of extrahepatic infection differs considerably . As observed in previous reports (Korba et al ., 1986, 1987a), evidence of active WHV DNA replication in chronic carriers was present only in the liver and spleen . Viral DNA was homogeneously distributed among all hepatocytes in the liver, while in the spleen concentrated foci of WHV DNA were observed in the germinal centers against a lower level, widespread, homogenous background of viral DNA in the remainder of the tissue . In contrast to the liver and spleen only scattered foci of WHV DNA-containing cells were observed in the other extrahepatic tissues . Despite the lack of current WHV DNA replication, many of the scattered cells in these foci contained relatively high levels of viral DNA (greater than 100 copies/cell) . It is possible that some of these cells may have harbored actively replicating virus which, due to sensitivity levels, would not be detected by Southern blot analysis . In the DHBV model, in situ hybridization analysis has demonstrated widespread virus replication in tissues during the acute phase of infection which progresses to a localized, focal distribution of DHBV DNA in chronically infected ducks (Freiman et al., 1988) . Complete clearance of WHV DNA from the various tissues examined requires an extensive period of time following loss of serologic markers of active viral replication (usually occurring approximately 6 months postinfection in the woodchuck (Cote at al., 1984)) . The serologically recovered (WHsAg - , anti-WHc', antiWHs', WHV DNA- ) animals in this study continued to carry some viral DNA in scattered foci within several of
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the tissues examined, including liver. However, this DNA was probably not actively replicating and, in most cases, no evidence of WHV-specific RNA was found . Previously, nonreplicating WHV DNA (but no RNA) was found in the PBL and liver tissue of some anti-WHs' woodchucks 12 to 25 months following seroconversion (Korba at al ., 1986, 1987a,b) . HBV DNA has been detected previously in the seminal fluid of chronically infected humans lending to the speculation of a direct mode of virus transfer via the germ line (Hadchouel et al ., 1985 ; Karayiannis at al,, 1985) . While seminal fluid from the chronic carrier woodchucks was not examined, no evidence of WHV DNA could be found in the testes, either by in situ or by Southern blot hybridization . However, the ovary of the single, female chronic carrier in this study clearly harbored nonreplicating WHV DNA in the cells in or near the developing follicles . A number of differences exist between the distribution and physiologic state of WHV and DHBV DNA in extrahepatic tissues . WHV and HBV DNA are actively replicating in the spleens of chronically infected woodchucks and chimpanzees (Table 1 ; Korba at al., 1987a ; Lieberman et at, 1987) . DHBV replication is usually not observed in the spleens of chronically infected Pekin ducks (Tagawa at al., 1985 ; Jilbert et al., 1987a), but does occur during the acute phase of infection (Halpern et at, 1983 ; Tawaga at al ., 1985 ; Freiman et al., 1988) . While both duck and woodchuck chronic virus carriers harbor high concentrations of viral DNA in the splenic germinal centers, DHBV DNA is believed to be present primarily in dendritic cells (Halpern et al., 1987) . Currently, it is not possible to determine specific cell identities in the woodchuck spleen due to a lack of the appropriate cellular phenotypic markers . In the pancreas of acutely infected ducks, replicating DHBV DNA is located in acinar cells (Jilbert et at, 1987b ; Freiman et at, 1988), while in chronically infected ducks DHBV DNA is associated with the islets (Halpern at al., 1983, 1985 ; Jilbert et al., 1987b ; Freiman at al., 1988) . In chronically infected woodchucks, nonreplicating WHV DNA is present only in acinar cells or in cells suspected to be tubular, ductal epithelium . Similarly, HBsAg has been reported to be concentrated in acinar cells of chronically infected human patients (Shimoda at al., 1981 ; Yoshimura et at, 1981) . However, during the acute phase of DHBV infection,
FiG . 3 . Examples of in situ hybridization for WHV DNA in infected woodchuck tissues . (A) WC1279 spleen ; (B) WC1279 ovary ; (C) WC1288 PBL ; (D) WC1279 lymph node . (A, left) and (D, left) photographed at 200X . All other panels photographed at 400X . Exposure times : (A) 40 hr, (B-D) 72 hr .
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DHBV DNA is located primarily in acinar cells (A . Jilbert and J . Freiman, personal communication) . In the kidney of chronically infected woodchucks nonreplicating viral DNA is concentrated in cells that are in or near the glomeruli . While nonreplicating HBV DNA has been found in the kidney during chronic infection, the location of the viral DNA-carrying cells has yet to be clarified (Dejean et al., 1984 ; Naumova et at., 1985) . Both DHBsAg and DHBV DNA have been detected in the glomeruli (Halpern at al., 1983 ; Jilbert et al., 1987a) . In some cases, replicating DHBV DNA has been found in the kidney of chronically infected ducks (Halpern et al., 1983 ; Tagawa et al., 1985) . The nature of the differences in extrahepatic infection patterns between WHV, HBV, and DHBV is unclear at the present time . Most likely, these relate to fundamental differences in host physiology and not to inherent variations in the primary characteristics of these highly related viruses . The gross observational differences reported to date should not be interpreted as inconsistencies in contrasting models of viral hepatitis, but as fortuitous clues into the interactions of host cell and virus factors . Further definition of the normal physiologic functions of the various types of virus-carrying cells in the woodchuck and duck will enhance the understanding of the cellular functions required for hepadnavirus propagation . The role of hepadnavirus infection of cells other than hepatocytes in virus-induced disease remains to be elucidated . The infection of lymphoid cells may modulate responses of the immune system to these viruses . Alternatively, these infected cells could serve as additional sources of virus pools . The lack of active viral replication at many of the extrahepatic sites does not mean that these viral genomes are replication incompetent . The hepadnavirus genomes in PBL from chronically infected chimpanzees and woodchucks have been shown to be transcriptionally active (Korba et al., 1986, 1987a) . PBL from HBV-infected humans have not been examined for virus-specific RNA . The multimeric viral DNA forms observed in extrahepatic tissues are not found in serum virions and are thus a product of active intracellular processing (e .g ., replication, recombination) . In addition, active replication of HBV and WHV has been observed in the spleen (Lieberman et al., 1987 ; Korba at al., 1 987a) and replication of DHBV has been observed in several tissues at different times during the course of virus infection (Halpern et al., 1983 ; Tagawa at al., 1985 ; Freiman et at, 1988) . This study serves as a basis for more extensive analysis of the kinetics of WHV infection of various tissues during the natural course of viral infection . The systemic movement of virus within the tissues examined
here during the course of infection is critical to understanding of the overall pathologic mechanisms of hepadnavirus-induced disease. Correlations of the state of virus in extrahepatic tissues with serologic markers of virus infection and the state of liver disease may provide the necessary information for improved prognosis and the development of more efficient antiviral therapy . ACKNOWLEDGMENTS The authors gratefully acknowledge the contributions of B . Baldwin, W . Hornbuckle, A . Glasser, W . Sherman (Cornell University), and their colleagues for animal maintenance and assistance in the collection of tissues . 1 . King (Cornell University) kindly provided the histologic analysis of the different tissues and P . Cote (Georgetown University) provided all serologic analyses . The authors also thank 1 . Freiman for corn rnunication of results prior to publication . This manuscript was prepared with the assistance of C . Culp . This work was supported by Contracts NO 1-Al-22665 and NO 1-Al-52585 between the National Institute of Allergy and Infectious Diseases and Georgetown University and Cornell Universities, respectively .
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