Infection: Molecular Virologic Features of the Pancreas, Kidney, Ovary, and Testis

Transcription

1 JOURNAL OF VIROLOGY, Sept. 199, p X/9/ $2./ Copyright C) 199, American Society for Microbiology Vol. 64, No. 9 Natural History of Experimental Woodchuck Hepatitis Virus Infection: Molecular Virologic Features of the Pancreas, Kidney, Ovary, and Testis BRENT E. KORBA,1* THOMAS L. BROWN,' FRANCES V. WELLS,' BETTY BALDWIN,2 PAUL J. COTE,' HOWARD STEINBERG,2 BUD C. TENNANT,2 AND JOHN L. GERIN' Georgetown University Medical Center, Division of Molecular Virology and Immunology, Rockville, Maryland 2852,' and College of Veterinary Medicine, Cornell University, Ithaca, New York Received 25 January 199/Accepted 7 June 199 The kinetic patterns of woodchuck hepatitis virus (WHV) infection were monitored in the pancreas, kidneys, ovaries, and testes. Groups of woodchucks experimentally infected with a standardied 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 (B. E. Korba, P. J. Cote, F. V. Wells, B. Baldwin, H. Popper, R. H. Purcell, B. C. Tennant, and J. L. Gerin, J. Virol. 63: , 1989). Tissues from an additional group of long-term (2 to 3 years) chronic WHV carriers which had been infected with the same WHV inocula were also examined. Viral DNA replication intermediates were found in all four tissues during the acute phase of WHV infection. However, WHV DNA replication intermediates were observed only in the kidneys of a small proportion of the chronically infected animals. Following the acute phase of infection, WHV DNA was present only in the pancreas, kidneys, and ovaries of the chronically infected woodchucks. A progressive evolution of different WHV genomic forms related to the replicative state of WHV was observed in these tissues. Histologic evaluation of these four tissues revealed only minimal, localied lesions which were not correlated with the state of WHV activity. The observations compiled in this study further extend the tissue tropism of WHV. The systemic nature of viral infections by several members of the Hepadnaviridae (hepatitis B virus [HBV], woodchuck hepatitis virus [WHV], and duck hepatitis B virus [DHBV]) has been documented in a number of studies (see references 18 and 19 for reviews). The hepadnavirus genomes present in several extrahepatic tissues represent active viral infections, as shown by the presence of viral DNA replication intermediates and virus-specific RNA transcripts (11, 13, 18, 2, 21, 23, 31). In some peripheral blood lymphocyte (PBL) populations, a latent form of WHV infection can be maintained, as demonstrated by culturing of these cells in the presence of mitogens which can lead to the reactivation of virus replication and the export of infectious virions (16, 17). A comprehensive analysis of WHV infection of the liver and of five primary components of the lymphoid system during the natural course of virus infection (which proceeded to either chronic infection with the development of hepatocellular carcinoma or serologic recovery) demonstrated dynamic patterns of changes in virus status (18, 19). Studies of systemic DHBV infection, monitored from the early stages of virus infection and progressing to chronic infection, have similarly revealed changing patterns of DHBV DNA in different tissues (8, 13, 31). In a continuation of a previous study (18), this report details the status of WHV in several nonhepatic tissues that have been demonstrated previously to harbor hepadnavirus genomes in both humans and ducks. MATERIALS AND METHODS Experimental design. This study was designed to examine the kinetics of WHV infection in various tissues during the * Corresponding author entire course of an experimentally induced WHV infection. The overall experimental design has been described previously (18). Briefly, woodchucks were inoculated subcutaneously with approximately 5 x 16 5% woodchuck infectious doses of a well-characteried virus pool, WHV7 (9), at 3 days of age and maintained in isolation. Infection protocols were conducted at Cornell University. At various times (see below), groups of three to five woodchucks were sacrificed and tissues were examined for evidence of WHV infection. Animals were chosen at random for the first four times (4, 8, 14, and 18 weeks postinoculation). Thereafter (at 28, 42, and 65 weeks), animals were separated on the basis of WHV serologic markers (WHV surface antigen [WHsAg], antibody to WHV core antigen [anti-whc], and antibody to WHsAg [anti-whs]) to enable a comparison of persistently infected animals (chronic carriers: WHsAg+, anti-whc+, anti-whs-) and convalescent animals (animals that had serologically recovered: WHsAg-, anti-whc+, anti- WHs+). WHV serologic analyses were performed as previously described (6, 26). In addition, tissues from 11 longterm (2 to 3 years) chronic WHV carriers were examined when those animals were sacrificed because of the development of hepatocellular carcinoma. The status of WHV in PBLs and liver tissues of these 11 woodchucks has been described previously (22). The 4- and 8-week times were chosen to examine the preacute phase of WHV infection, while the 14- and 18-week times were selected to study the acute phase of WHV infection. The 28-week time was chosen to examine animals during the early stages of persistent (chronic) infection or within 7 days after the initial detection of anti-whs in the serum of woodchucks in which acute viral infections had been resolved. Weeks 4 and 65 were chosen to examine animals at two separate periods during the later stages of

2 45 KORBA ET AL. chronicity or of recovery from acute viral infections. Although WHV-infected woodchucks were selected at random for the first four times in this study, retrospective serologic analyses (18) demonstrated that these 2 animals represented the times of WHV infection predicted by the generalied serologic profile (see top panels of Fig. 2). The generalied serologic profile was developed from the analysis of over 4 WHV-infected animals (P. J. Cote, unpublished data). In this study, WHV infections in the kidneys, pancreas, ovaries, and testes were examined. Analyses of WHV infections in the liver and the lymphoid system from these same animals were reported previously (18). The selection of tissues for examination was based upon observations from several earlier studies: the pancreas and kidneys have been demonstrated to be sites of DHBV and HBV infection (7, 8, 11, 13), and vertical (maternal) and sexual transmissions of HBV have been indicated by previous reports (1, 1, 15, 25). Nucleic acid isolation and Southern and Northern (RNA) blot analyses. Tissues were froen immediately after collection in liquid nitrogen and stored at -7 C. Whole-cell DNA and RNA were prepared from froen tissue as previously described (19, 2). The analysis of viral nucleic acids by Southern and Northern blot hybridiation techniques and the criteria for quantitation have been previously described (19, 2). The 32P-labeled hybridiation 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 standardied virus pool as that used for the experimental infection; its sequence has been published (4). On each individual gel, known amounts of both positive and negative hybridiation control DNA markers (cloned WHV and pbr322) were coelectrophoresed with experimental samples. Estimates of the overall levels of WHV-specific DNA and RNA were based upon a comparison of the hybridiation signals from tissue samples to the signals from the WHV DNA markers present in the same gel by using an AMBIS beta scanner (AMBIS Systems, San Diego, Calif.). RESULTS Kinetic patterns of WHV DNA. In the four tissues examined in this study, WHV DNA first appeared during the acute phase of virus infection, approximately 12 to 14 weeks postinoculation (Fig. 1 and Tables 1 and 2). The initial levels of WHV DNA were the highest observed for each tissue. The levels of WHV DNA declined progressively in all four tissues during the course of WHV infection. The overall levels of WHV DNA observed in these tissues were approximately 1- to 1,-fold lower than that observed in the liver tissue from these same animals (18). WHV DNA levels were highest in the kidneys and pancreas (12 to 48 genomic copies per cell) (Table 1) during the acute phase of infection. WHV DNA was present in all (1 of 1) animals examined (Tables 1 and 2). In both tissues, the frequency and level of WHV DNA declined soon after the acute phase of infection (Fig. 1 and Tables 1 and 2). WHV DNA eventually became undetectable in these two tissues in all convalescent animals between 28 and 42 weeks postinoculation (Tables 1 and 2). In the pancreas of animals with persistent WHV infection, the frequency and level of WHV DNA declined less rapidly and WHV DNA was maintained in most woodchucks at a low level (less than 1. copy per cell) during the later stages of infection (Tables 1 and 2 and Fig. 1). In the kidneys of the persistently infected woodchucks, the WHV DNA level did not begin to decline until J. VIROL. after 28 weeks and WHV DNA was thereafter maintained at the same low level (Table 1 and Fig. 1). Peak levels of WHV DNA in the ovaries and testes were very low, approximately one to three copies per cell. WHV DNA was present in these tissues in all 1 animals examined during the acute phase of infection (Tables 1 and 2). The kinetic patterns of WHV DNA in the ovaries were similar to those in the pancreas and kidneys: a progressive decline following the acute phase of infection to a level of less than 1. copy per cell during the later stages of infection in the persistently infected animals (Tables 1 and 2 and Fig. 1). WHV DNA became undetectable in the ovaries of the animals that had serologically recovered between 18 and 28 weeks postinoculation. By contrast, WHV DNA present in the testes became undetectable in all persistently infected woodchucks between 28 and 42 weeks postinoculation (Tables 1 and 2). Samples of kidneys, pancreas, ovaries, and testes from the 11 long-term chronic WHV carriers sacrificed because of the development of hepatocellular carcinomas were also examined: 3 at 24 months postinoculation, 5 at 26 months, 2 at 28 months, and 1 each at 34 and 38 months. WHV DNA was present at low levels (1 to 3 copies per cell) in the kidneys and pancreas from 9 of the 11 animals and in the ovaries (.2 to.6 copy per cell) from 2 of 5 of the female woodchucks in this group (Tables 1 and 2). All 11 animals carried WHV in at least one of these three tissues. No WHV DNA was present in the testes of the six male animals in this group. No obvious correlations between age and the level or frequency of WHV DNA in the various tissues were observed. Progression of WHV DNA forms. As observed in liver and lymphoid tissues (18), a complex progression of WHV genomic forms was observed in these four tissues (Table 3 and Fig. 2). These different viral DNA forms were resolved by Southern blot hybridiation analysis following restriction enyme digestion and agarose gel electrophoresis of wholecell DNA. In all tissues in all animals, WHV DNA was episomal, as defined by previously established criteria (18). On the basis of analyses utiliing these criteria, WHV DNA forms were divided into three classes of molecules: (i) multimeric, episomal DNA molecules 7 to 12 kb in sie (18, 19); (ii) monomeric, episomal WHV genomes 3.3 kb in sie; and (iii) a heterogeneous population of single- and doublestranded WHV DNA fragments 3.3 to.5 kb in sie, representing WHV DNA replication intermediates (27, 3). In all four tissues, WHV DNA replication intermediates were present in essentially all the animals examined during the acute phase of infection (Table 3). WHV DNA replication had ceased in all four tissues between 18 and 28 weeks postinoculation in the convalescent animals and in the ovaries and testes of the persistently infected woodchucks (Table 3). WHV DNA replication intermediates were present for a longer period in the pancreas and kidneys of the persistently infected animals. At 42 weeks postinoculation, 5% (two of four) of the animals still harbored WHV DNA replication intermediates in the kidneys. WHV DNA replication in the kidneys persisted into the later stages of virus infection in 3 of the 11 long-term chronic WHV carriers (Table 3). By contrast, only one of four persistently infected woodchucks carried WHV DNA replication intermediates in the pancreas at 28 weeks postinoculation, and no WHV DNA replication intermediates were detected in the pancreas at the later stages of virus infection (Table 3 and Fig. 2). As observed in the liver and lymphoid tissues of these woodchucks (18), episomal multimeric genomic forms were

3 VOL. 64, 199 KINETIC PATTERNS OF WHV INFECTION J C.) -I ~1 a OVARY * I * I i- A a1 I I- I p1 p. I WEEKS POST-INOCULATION FIG. 1. Kinetics of the appearance of WHV DNA in tissues. Values plotted are the WHV DNA levels observed in the different tissues from each individual woodchuck. Lines indicate the average values for WHV DNA levels in each group of woodchucks with the same serologic profile at each time. At 4, 8, 14, and 18 weeks postinoculation, animals were chosen at random (O). At the later times, animals were separated into WHsAG+ (O and - - -) and anti-whs+ (-and -) groups.

4 452 KORBA ET AL. J. VIROL. TABLE 1. Levels of WHV nucleic acids in tissues at different times during the course of viral infectiona Levels of WHV nucleic acids as detected by Southern and Northern blot analyses in: Wk postinoculation Pancreas Kidneys Ovaries Testes and group DNA RNA DNA RNA DNA RNA DNA RNA Anti-WHs WHsAg Anti-WHs+ ND ND WHsAg Anti-WHs+ WHsAg WHsAg awhv nucleic acid levels were determined from Southern and Northern blot hybridiation analyses as described in Materials and Methods. WHV DNA levels are presented as the number of genomic copies per cell, and WHV RNA levels are presented as picograms of WHV RNA per microgram of whole-cell RNA, as defined by previously established criteria (18). The sensitivities of detection were approximately.1 copy per cell for WHV DNA and.1 pg/,ug of whole-cell RNA for WHV RNA. ND, Not determined. also observed in these four tissues (Table 3 and Fig. 2). In some tissues, these WHV DNA molecules were present at the initial appearance of WHV DNA (Table 3). As observed in the lymphoid tissues (18), these multimeric WHV DNA molecules, along with episomal monomeric genomes, were TABLE 2. Frequency of WHV nucleic acids in the tissues of WHV-infected woodchucksa % of infected woodchucks with WHV nucleic acids in: Wk postinoculation and Pancreas Kidneys Ovaries Testes group DNA RNA DNA RNA DNA RNA DNA RNA Anti-WHs+ 6 8 WHsAg Anti-WHs+ ND ND WHsAg Anti-WHs+ WHsAg , WHsAg+ a ND, Not determined. the primary WHV DNA forms which persisted during the later stages of virus infection (Table 3 and Fig. 2). In the liver and lymphoid tissues of these animals, WHV DNA replication intermediates, when present, represented 8 to 9% of the total WHV DNA signal (on a per-cell basis) and were observed in essentially all animals with the same serologic profile at a specific time (18). In the four, tissues examined in this study, however, WHV DNA replication intermediates represented a much smaller proportion (1 to 3%, on a per-cell basis) of the WHV DNA signal (data not shown). These lower levels of WHV DNA replication were observed at all stages of virus infection, including the acute phase of virus infection, in which all animals carried replicating virus in each of these four tissues (Table 3). During the later stages of WHV infection (28 weeks and later), the disappearance of WHV DNA replication intermediates was not synchronous, since a small proportion of the woodchucks examined retained replicating virus in some tissue at each of several times (Table 3). WHV-specffic RNA species. The frequency and level of WHV-specific RNA transcripts correlated well with the overall levels of WHV DNA and WHV DNA replication intermediates (Tables 1 to 3 and Fig. 2). As compared with the WHV RNA level in the livers of these same animals (18), the WHV RNA levels in the four tissues examined here were approximately 1- to 1,-fold lower. Analysis of WHV RNA by Northern blot hybridiation demonstrated that, in all tissues, the majority of the virusspecific RNA transcripts were the 3.6- and 2.3-kb classes of RNA species commonly observed in WHV-infected hepatocytes (2, 24), with approximately two-thirds of the WHV RNA hybridiation signal being present as the 2.3-kb species (data not shown). WHV RNA transcripts other than the 2.3- and 3.6-kb species were occasionally observed. These transcripts represented less than 5% of the total autoradio-

5 VOL. 64, 199 KINETIC PATTERNS OF WHV INFECTION 453 TABLE 3. Frequency of WHV DNA forms in tissues of WHV-infected woodchucksa % of different DNA forms in the following tissue in WHV DNA-carrying woodchucks: Wk postinoculation and Pancreas Kidneys Ovaries Testes group MM M RI MM M RI MM M RI MM M RI Anti-WHs WHsAg Anti-WHs+ ND ND ND WHsAg Anti-WHs+ WHsAg , WHsAg+ a The different WHV genomic forms were identified following restriction enyme digestion and Southem blot hybridiation analysis as described in Materials and Methods. MM, Episomal, multimeric molecules 7 to 12 kb in sie (19); M, episomal, open circular or linear monomeric genomes 3.3 kb in sie (19); RI, heterogeneous single-stranded and partially double-stranded WHV DNA molecules representing WHV DNA replication intermediates (27, 29). ND, Not determined. graphic signal from any given tissue sample and were found in only 6 of the 148 individual tissue samples examined from the 54 WHV-infected woodchucks tested in this study. These tissue samples consisted of three samples of splenic tissue, one sample of kidney tissue, and two samples of pancreatic tissue taken from six different animals at'different times during the course of infection. Histologic analysis. Formalin-fixed samples of the tissues from these animals were examined for pathologic consequences of WHV infection. Overall, the histologic evaluation of these tissues (conducted under code) revealed only minimal, localied lesions which were not correlated with the state of WHV activity. No significant lesions were observed in any of the pancreatic tissues obtained from the young animals in this study. Minimal fatty infiltrates were found in 8 of 11 pancreatic tissues obtained from the long-term chronic WHV carriers, but no evidence of inflammation, fibrosis, or atrophy was present. No significant histologic changes were observed in any of the ovarian or testicular tissues from any of the woodchucks examined in this study. There was no evidence of glomerulonephritis in any of the woodchucks in this study. The kidney tissues from 9 of the 11 long-term chronic WHV carriers had mild, focal histologic changes, which included (in decreasing order of frequency) extramedullary hematopoiesis, nonsuppurative interstitial nephritis, and subacute tubular nephrosis. Similar renal changes which represented all the different stages of WHV infection examined, including the preacute phase, were noted in 5 of 43 young WHV-infected woodchucks. DISCUSSION In this study, a comprehensive analysis of the progression of WHV infections in the pancreas, kidneys, ovaries, and testes during the natural course of WHV infection was made. The changing status of WHV in these four tissues further demonstrates the complexity of systemic WHV infections. Cells in the pancreas and especially in the kidneys appeared to be able to support WHV replication for substantial but variable periods during the course of virus infection. During the later stages of virus infection, however, a majority of the animals did not harbor replicating WHV in these two tissues. The percentage of the total WHV DNA signal present as WHV DNA replication intermediates within these two tissues was especially low and variable throughout the entire course of virus infection. By contrast, the patterns of WHV genomic forms, especially WHV DNA replication intermediates, observed in the liver and lymphoid tissues were essentially identical for an individual tissue or cell population in all animals with the same WHV serologic profile at a specific time (16-19, 22). The variations in WHV replicative status in the pancreas and kidneys may be a result of a continuous cycle of infection of a small population of susceptible cells or the reactivation of quiescent WHV infections. In vitro experiments have demonstrated that the nonreplicating WHV genomes in the PBL of chronically infected woodchucks are replication competent and can produce infectious virus under certain environmental conditions (16, 17). Analysis of pancreas and kidney tissues from the persistently infected animals by in situ hybridiation 65 weeks postinoculation revealed a scattered and focal distribution of WHV-infected cells (19). The rapid loss of replicating WHV from the ovaries and testes may indicate that cells in these tissues have the capacity to support WHV replication for only short periods of time. The replicating viral genomes observed in these tissues may be due to a low percentage of contaminating PBL, since WHV replication in PBL was also limited to only

6 454 KORBA ET AL. J. VIROL. CIO Co ct la I- IL. cc x F C) LLI U- UJ - -J 'I) to in.x a 39 c nuj ( x L u L (I,~~~~~~r 4~~ >, 2 > -Jll ol~i c Z Co I- Ln Z o-1 In r 2 3 J o ( I.- L M N Z L 2 o F o UJ C.!1 ULu W W( Co. L 2 j a E (An 4c a C.) rc w F o -J 44 IL 4c C rc 2 -J 44 M Z M CO cn > u 3: _~ o uo <N -o cis 4U, *Ef oo; ' 3-vo -C cts 44 L rx, < ' - C * (- ~1- (AW Q.> 'y U, - = c 4 C,) UI > C r m > 4c.7i IL Y Ot I-.

7 VOL. 64, 199 the acute phase in these same animals (18). However, PBL contamination of the ovarian tissues was not observed during in situ hybridiation analysis 65 weeks postinoculation, demonstrating that the WHV genomes were located in cells in or near developing follicles (19). Sexual transfer of HBV has been demonstrated, and HBV DNA has been detected in the seminal fluid of chronically infected individuals (1, 1, 15). Whether such a mode of transfer of WHV could occur is problematic. In the present study, WHV infection of woodchuck testicular tissue was relatively short, being confined to the acute phase of virus infection; therefore, any WHV genomes present in the seminal fluid of chronically infected woodchucks would most likely have come from alternative sources. The persistence of WHV DNA in ovarian cells for at least 38 months postinoculation suggests a potential for the involvement of these infections in the maternal transmission of hepadnaviruses (25, 28). The observations presented in this report and previous reports (7, 8, 11, 18, 21, 31) emphasie the importance of host cell factors in the control of hepadnavirus infections at the individual cell level. The cellular mechanisms involved in the termination of WHV replication in certain cell types in chronically infected woodchucks remain to be elucidated. Cellular factors involved in the expression of hepadnavirus genes may be lacking or only transiently available in some types of 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, 12). The transactivating activity of the HBV X gene, which also appears to enhance the level of virus replication, has been shown to exhibit cell type specificity (29). The presence of WHV, either in a quiescent or replicating state, was not associated with any obvious pathologic consequence in the extrahepatic tissues examined in this study or previous studies (18, 19). It is unclear at the present time why chronic carriage and replication of hepadnaviruses produce dramatic patterns of histologic changes in the liver but not in other infected tissues. Factors such as the ability of different cell types to tolerate viral infections, the patterns or levels of expression of viral gene products in different cells, or the pattern of presentation of viral antigens to the host immune system by different cells may be involved. There was no evidence of glomerulonephritis in the WHVinfected animals in this study, although this type of kidney disease has been associated with long-term carriage of HBV in some studies (5, 14). It is not clear if the minor renal changes observed in woodchucks carrying WHV in this study are a consequence of long-term viral infection, since no age-matched, uninfected animals were studied in parallel. Many similarities, as well as some differences, exist between the kinetics of WHV and DHBV infections of the pancreas and kidneys (DHBV infection of the ovaries and testes has not been reported). In Pekin ducks experimentally infected at 1 day of age, replicating DHBV DNA was detected in the pancreas and kidneys after infection of the liver had occurred (8, 13). DHBV replication proceeds in all tissues during the acute phase of infection and continues at high levels in the liver and pancreas and at variable levels and frequencies in the kidneys (8, 11, 13, 31). In chronically infected ducks, DHBV DNA is present primarily in the islet cells of the pancreas, while in chronically infected woodchucks and humans (no replication of HBV in pancreatic tissue has been reported), viral DNA is located primarily in KINETIC PATTERNS OF WHV INFECTION 455 acinar cells (8, 11, 13, 31). In both ducks carrying DHBV and woodchucks carrying WHV, viral DNA is located in a small 11, 19). The nature of these proportion of the glomeruli (8, differences between DHBV and WHV probably relates to fundamental differences in host response rather than to differences in the general characteristics of these closely related viruses. The combined observations of WHV and DHBV tissue infection patterns in this study and previous studies demonstrate the complexity of the pathobiologic mechanisms of hepadnaviruses. Infection by these viruses involves the systemic spread of virus through many different cell populations during the natural course of virus infection. Identification of the cell populations involved and their physiologic functions in the different host species will be necessary for a more complete understanding of the roles of these cellular infections in the overall course of virus-induced disease. When correlated with serologic markers of virus infection and the state of liver disease, the present observations may provide additional information for improved prognosis and the development of more efficient antiviral therapies. ACKNOWLEDGMENTS The support and assistance of W. Hornbuckle, J. Wright, W. Sherman, A. Glasser, T. Manwarren, S. Gome, L. Fullan, and E. Deleo (Cornell University), R. Engle, M. Rochee, and K. Cass (Georgetown University), and their colleagues are greatly appreciated. This work was supported by Public Health Service contracts between the National Institutes of Allergy and Infectious Diseases and Georgetown University (NO1-AI and N1-AI and Cornell University N1-AI-52585). LITERATURE CITED 1. Alter, H. J., R. H. Purcell, J. L. Gerin, W. T. London, P. M. Kaplan, V. J. McAuliffe, J. Wagner, and P. V. Holland Transmission of hepatitis B to chimpanees by hepatitis B surface antigen-positive saliva and semen. Infect. Immun. 16: Burk, R. D., J. A. DeLoia, M. K. ElAwady, and J. D. Gearhart Tissue preferential expression of the hepatitis B virus (HBV) surface antigen gene in two lines of HBV transgenic mice. J. Virol. 62: Cabinet, C., H. Fara, D. Morello, M. Hadchovel, and C. Pourcel Specific expression of hepatitis B surface antigen (HBsAg) in transgenic mice. Science 23: Cohen, J. I., R. H. Miller, B. Rosenblum, K. Denniston, J. L. Gerin, and R. H. Purcell Sequence comparison of woodchuck hepatitis virus replicative forms demonstrates conservation of the genome. Virology 162: Combes, B., P. Stastny, E. H. Eigenbrodt, A. Barrera, A. R. Hull, and N. W. Carter Glomerulonephritis with deposition of Australian antigen-antibody complexes in glomerular basement membrane. Lancet ii: Cote, P. J., M. Shapiro, R. E. Engle, H. Popper, R. H. Purcell, and J. L. Gerin Protection of chimpanees from type B hepatitis by immuniation with woodchuck hepatitis virus surface antigen. J. Virol. 59: Dejean, A., C. Lugassy, S. Zafrani, P. Tiollais, and C. Brechot Detection of hepatitis B virus DNA in pancreas, kidney and skin of human carriers of the virus. J. Gen. Virol. 65: Freiman, J. S., A. R. Jilbert, R. J. Dixon, M. Holmes, E. J. Gowans, C. J. Burrell, E. J. Wills, and Y. E. Cossart Experimental duck hepatitis B virus infection: pathology and evolution of hepatic and extrahepatic infection. Hepatology 8: Gerin, J. L., P. J. Cote, B. E. Korba, and B. C. Tennant Hepadnavirus-induced liver cancer in woodchucks. Cancer Prevent. Detect. 14:

8 456 KORBA ET AL. 1. Hadchouel, M., J. Scotto, J. L. Huret, C. Moline, E. Villa, F. Degos, and C. Brechot Presence of HBV DNA in spermatooa: a possible vertical transmission of HBV via the germ line. J. Med. Virol. 16: Halpern, M. S., J. M. England, D. T. Deery, D. J. Petcu, W. S. Mason, and K. L. Molnar-Kimber Viral nucleic acid synthesis and antigen accumulation in pancreas and kidney of Pekin ducks infected with duck hepatitis B virus. Proc. Natl. Acad. Sci. USA 8: Jameel, S., and A. Siddiqui The human hepatitis B virus enhancer requires trans-acting cellular factor(s) for activity. Mol. Cell. Biol. 6: Jilbert, R., J. S. Freiman, E. J. Gowans, M. Holmes, Y. E. Cossart, and C. J. Burrell Duck hepatitis B virus DNA in liver, spleen, and pancreas: analysis in situ and Southern blot hybridiation. Virology 158: Kar, N. L., F. M. Lai, K. W. Chan, C. B. Chow, K. L. Tong, and J. Vallence-Owen The clinico-pathologic features of hepatitis B virus-associated glomerulonephritis. Q. J. Med. 63: Karayiannis, P., D. M. Novick, A. S. Lok, M. J. Fowler, J. Monjardino, and H. C. Thomas Hepatitis B virus DNA in saliva, urine, and seminal fluid of hepatitis B e antigen carriers. Br. Med. J. Clin. Res. 29: Korba, B. E., P. J. Cote, and J. L. Gerin Mitogen-induced replication of woodchuck hepatitis virus in cultured peripheral blood lymphocytes. Science 241: Korba, B. E., P. J. Cote, M. Shapiro, and J. L. Gerin In vitro production of infectious woodchuck hepatitis B virus by lipopolysaccharide-stimulated peripheral blood lymphocytes. J. Infect. Dis. 16: Korba, B. E., P. J. Cote, F. V. Wells, B. Baldwin, H. Popper, R. H. Purcell, B. C. Tennant, and J. L. Gerin Natural history of woodchuck hepatitis virus infections during the course of experimental viral infection: molecular virologic features of the liver and lymphoid tissues. J. Virol. 63: Korba, B. E., E. J. Gowans, F. V. Wells, B. C. Tennant, R. Clarke, and J. L. Gerin Systemic distribution of woodchuck hepatitis virus in the tissues of experimentally infected woodchucks. Virology 165: Korba, B. E., F. Wells, B. C. Tennant, P. J. Cote, and J. L. Gerin Lymphoid cells in the spleens of woodchuck hepatitis virus-infected woodchucks are a site of active viral J. VIROL. replication. J. Virol. 61: Korba, B. E., F. Wells, B. C. Tennant, G. H. Yoakum, R. H. Purcell, and J. L. Gerin Hepadnavirus infection of peripheral blood lymphocytes in vivo: woodchuck and chimpanee models of viral hepatitis. J. Virol. 58: Korba, B. E., F. V. Wells, B. Baldwin, P. J. Cote, B. C. Tennant, H. Popper, and J. L. Gerin Hepatocellular carcinoma in woodchuck hepatitis virus-infected woodchucks: presence of viral DNA in tumor tissue from chronic carriers and animals serologically recovered from acute infections. Hepatology 9: Lieberman, H. M., W. W. Tung, and D. A. Shafrit Splenic replication of hepatitis B virus in the chimpanee chronic carrier. J. Med. Virol. 21: Moroy, T., J. Etiemble, C. Trepo, P. Tiollais, and M. A. Buendia Transcription of woodchuck hepatitis virus in the chronically infected liver. EMBO J. 4: Okada, K., I. Kamiyama, M. Inomata, Y. M. Yakawa, and M. Mayumi e antigen and anti-e in the serum of asymptomatic carrier mothers indicates positive and negative transmission of hepatitis B virus to infants. N. Engl. J. Med. 294: Ponetto, A., P. J. Cote, E. C. Ford, R. H. Purcell, and J. L. Gerin Core antigen and antibody in woodchucks after infection with woodchuck hepatitis virus. J. Virol. 52: Seeger, C., D. Ganem, and H. E. Varmus Biochemical and genetic evidence for the hepatitis B virus replication strategy. Science 232: Shen, H. D., K. B. Choo, T. C. Wu, H. T. Ng, and S. H. Han Hepatitis B virus infection of cord blood leukocytes. J. Med. Virol. 22: Spandau, D. F., and C.-H. Lee trans-activation of viral enhancers by the hepatitis B virus X protein. J. Virol. 62: Summers, J., and W. S. Mason Replication of the genome of a hepatitis-b-like virus by reverse transcription of an RNA intermediate. Cell 29: Tagawa, M., M. Omata,. Yokosuka, K. Uchiumi, F. Imaeki, and K. Okuda Early events in duck hepatitis B virus infection. Sequential appearance of viral deoxyribonucleic acid in the liver, pancreas, kidney, and spleen. Gastroenterology 89: