Integration Of Woodchuck Hepatitis Virus DNA Research Articles

Primary seronegative but molecularly evident hepadnaviral infection engages liver and induces hepatocarcinoma in the woodchuck model of hepatitis B.

Hepadnavirus at very low doses establishes in woodchucks asymptomatic, serologically undetectable but molecularly evident persistent infection. This primary occult infection (POI) preferentially engages the immune system and initiates virus-specific T cell response in the absence of antiviral antibody induction. The current study aimed to determine whether POI with time may culminate in serologically identifiable infection and hepatitis, and what are, if any, its pathological consequences. Juvenile woodchucks were intravenously injected with inocula containing 10 or 100 virions of woodchuck hepatitis virus (WHV) to induce POI and followed for life or up to 5.5 years thereafter. All 10 animals established molecularly detectable infection with virus DNA in serum (<100–200 copies/mL) and in circulating lymphoid cells, but serum WHV surface antigen and antibodies to WHV core antigen remained undetectable for life. By approximately 2.5–3.5 years post-infection, circulating virus transiently increased to 103 copies/mL and virus replication became detectable in the livers, but serological markers of infection and biochemical or histological evidence of hepatitis remained undetectable. Nonetheless, typical hepatocellular carcinoma (HCC) developed in 2/10 animals. WHV DNA integration into hepatic and lymphatic system genomes was identified in 9/10 animals. Virus recovered from the liver virus-negative or virus-positive phases of POI displayed the wild-type sequence and transmitted infection to healthy woodchucks causing hepatitis and HCC. In summary, for the first time, our data demonstrate that an asymptomatic hepadnaviral persistence initiated by very small amounts of otherwise pathogenic virus, advancing in the absence of traditional serological markers of infection and hepatitis, coincides with virus DNA integration into the host's hepatic and immune system genomes, retains liver pro-oncogenic potency and is capable of transmitting liver pathogenic infection. This emphasizes the role for primary occult hepatitis B virus infection in the development of seemingly cyptogenic HCC in seronegative but virus DNA reactive patients.

Liver repopulation with xenogenic hepatocytes in B and T cell-deficient mice leads to chronic hepadnavirus infection and clonal growth of hepatocellular carcinoma.

To investigate host and viral mechanisms determining hepadnaviral persistence and hepatocarcinogenesis, we developed a mouse model by transplanting woodchuck hepatocytes into the liver of mice that contain the urokinase-type plasminogen activator transgene (uPA) and lack mature B and T lymphocytes due to a recombination activation gene 2 (RAG-2) gene knockout. The woodchuck hepatocytes were transplanted via intrasplenic injection and were found to integrate into the recipient mouse liver cord structure. Normal adult woodchuck hepatocytes proliferated and reconstituted up to 90% of the uPA/RAG-2 mouse liver. uPA/RAG-2 mice containing woodchuck hepatocytes were infectable with woodchuck hepatitis virus (WHV) and showed WHV replication for at least 10 months with titers up to 1 x 10(11) virions per ml in the peripheral blood. WHV-infected hepatocytes from chronic carrier woodchucks also established a persistent infection in uPA/RAG-2 mice after an 8- to 12-week lag period of viremia. Although WHV envelope, core, and X proteins were produced in the uPA/RAG-2 mice, no inflammatory host immune response was observed in the liver of WHV-replicating mice. A first antiviral test demonstrated a greater than four orders of magnitude drop in WHV titer in response to interferon alpha treatment. WHV replication was up-regulated by dexamethasone treatment. Comparison of precancerous lesions in donor woodchucks versus recipient uPA/RAG-2 mice revealed an enrichment of dysplastic precancerous hepatocytes in transplanted mice. Clonal amplification of hepatocytes from a woodchuck with hepatocellular carcinomas was demonstrated by the detection of unique WHV DNA integration patterns in hepatocellular carcinomas that arose in uPA/RAG-2 mice. In the absence of B or T cell-mediated immune responses, WHV establishes a persistent noncytotoxic infection of woodchuck hepatocytes in uPA/RAG-2 chimeric mouse livers. Further studies of the kinetics of hepadnavirus infection and replication in quiescent and proliferating hepatocytes should increase our understanding of hepadnavirus spread and aid in the design of therapies to block or cure persistent infection.

Woodchuck hepatitis virus DNA integration in a common chromosomal region of the woodchuck genome in two independent hepatocellular carcinomas

In woodchuck hepatocellular carcinoma (HCC) the myc-oncogene family (particularly N-myc2) and the win locus of cellular genome have been reported as frequent targets for integration of woodchuck hepatitis virus (WHV) DNA. In this paper a further cellular locus, b3n, is reported as recurrent target for WHV integration in woodchuck HCC. Cloning and sequencing of a WHV-DNA integration and its cellular flanking regions showed that viral DNA was inserted in a chromosomal region already described for WHV integration in another single HCC. The two integration sites are only 0.5 kb apart. A link between WHV integration in b3n and HCC development may be postulated. Careful analysis of the sequence of the unoccupied locus revealed that, in addition to Alu-like repeats and a gag-like coding region, already described, several features of Matrix Attachment Region (MAR) sequences are present. Thus (part of) b3n might be a previously unrecognized MAR. Organization of the chromatin in functional domains and regulation of gene expression are some functions attributed to MAR sequences. The occurrence of WHV-DNA integration close to the same putative MAR in two different HCCs suggests that a mechanism of deregulation of MAR functions by WHV insertion might act in some liver tumors.

Multiple rearrangements and activated expression of c- myc induced by woodchuck hepatitis virus integration in a primary liver tumour

Woodchuck hepatitis virus (WHV) is a small, partially double-stranded DNA virus. Like the related human hepatitis B virus (HBV), WHV induces acute and chronic hepatitis and hepatocellular carcinoma (HCC) in its natural host. WHV DNA integration into c-myc and N-myc, resulting in deregulated expression of these genes, has been described previously in woodchuck HCC. We have analysed a woodchuck liver tumour in which WHV DNA was integrated in the c-myc gene. The virus insertion provoked multiple alterations in one c-myc allele, probably involving secondary deletions and mutations. Integrated viral DNA, including promotor and enhancer sequences, acted as an insertional mutagen, leading to enhanced expression of heterogenous c-myc transcripts ranging from 7.2 to 14 kb in size, strikingly longer than normal 2.3-kb c-myc RNA. These results provide an additional example in which the oncogenic activation of a myc gene by cis-acting effect of WHV insertion may play a critical role in virus-induced woodchuck HCC.

Integration of hepatitis virus DNA near c-myc in woodchuck hepatocellular carcinoma

A total of 33 hepatocellular carcinomas, induced in woodchucks by chronic infection with woodchuck hepatitis virus (WHV), a virus closely related to the human hepatitis B virus, were analyzed for the state of viral DNA, the expression of viral genes and of different cellular proto-oncogenes. Low levels of viral replication and presence of integrated viral forms including sequences of the enhancer element, appeared as a general rule in these tumors. Enhanced expression of one or more of the nuclear protooncogenes: c-myc, N-myc, c-fos, c-jun and jun-B was frequently observed. In two hepatomas, elevated expression and allelic alterations of c-myc were subsequent to integration of WHV DNA near the c-myc coding domain. The viral strategy for insertional activation of c-myc in these tumors appeared basically identical to that of mammalian retroviruses in T-cell lymphomas of mice and rats. Whether insertional mutagenesis of different oncogenes may be more generally linked to liver oncogenesis induced by WHV and hepatitis B viruses remains to be determined.

Clonal origin of mammalian hepatitis B virus‐related hepatocellular carcinoma

The clonal origins of 20 multifocal hepatocellular carcinomas (HCCs) in four woodchucks were analyzed by the Southern blot hybridization technique. The woodchucks were divided into two groups according to the morphological classification of multifocal tumors: 1) three woodchucks had multifocal tumors that were widely separated and similar in size, which suggests a multiclonal origin of the tumors; and 2) one woodchuck had ten small multifocal tumors surrounding two large main tumors, which indicated intrahepatic metastasis from an original tumor. Results from the first group demonstrated that the number of integrated woodchuck hepatitis virus (WHV) DNAs differed from tumor to tumor, and none of the bands were the same size. In the second group, eight of the ten small tumors surrounding the two large tumors showed the same pattern of WHV DNA integration. One demonstrated an additional band and also shared the same bands with the other tumors, and one small tumor had a different pattern of integration from the others. It was concluded that the clone dissimilarity demonstrated by hybridization patterns does not necessarily mean that HCCs originate independently from different clones, because genetic changes may occur after or at the time of metastasis.

Characterization of woodchuck hepatitis virus DNA and RNA in the hepatocellular carcinomas of woodchucks

Integration and transcription of woodchuck hepatitis virus DNA were studied by Southern and Northern blot analysis in 26 hepatocellular carcinomas and in adjacent nontumor tissue of woodchucks (Marmota monax). All liver tissue chronically infected with woodchuck hepatitis virus contained various amounts of episomal and replicative forms of woodchuck hepatitis virus DNA: episomal and replicative forms of woodchuck hepatitis virus DNA without integration were found in six tumors, episomal and integrated woodchuck hepatitis virus DNA was observed in 18 tumors and exclusively integrated woodchuck hepatitis virus DNA was found in two tumors. In most tumors and in all of the liver tissues chronically infected with woodchuck hepatitis virus, two major woodchuck hepatitis virus RNA species (3.7 and 2.1 kilobases) were detected. In tumors of two other animals (HW76 and HW89) with integrated woodchuck hepatitis virus DNA, only single major transcripts of 3.5 and 2.5 kilobases, respectively, were detected. Hybridization with subcloned woodchuck hepatitis virus DNA probes showed that both aberrant transcripts lacked the C gene and a part of the pre-S1 gene; characterization of corresponding integrated woodchuck hepatitis virus DNA sequences revealed that the C gene was deleted in one tumor, although not in the other. In agreement with the nucleic acid data, we found expression of core protein by Western blotting only in chronically infected liver tissue of these animals, but not in the corresponding tumors. Deletion of the C gene in mRNA may be due to deletion of this gene in the integrated sequences or due to transcriptional regulation.

Chromosomal assignment of woodchuck hepatitis virus (WHV) dna integration sites in a woodchuck hepatocellular carcinoma‐derived cell line (WH257GE10)

The chromosomal sites of woodchuck hepatitis virus (WHV) DNA integration were identified in a woodchuck hepatocellular carcinoma-derived cell line (WH257GE10) by the in situ hybridization technique using 3H-labelled WHV whole genome (WHV 2) as a probe. The G-banded chromosome spreads from WH257GE10 were identified and diagrammed schematically according to their band patterns. WHV DNA was integrated into 2 sites: 33 region of the long arm of chromosome 6 (6q33) and 31 region of the long arm of chromosome 8 (8q31). Chromosomal sites of WHV DNA integration were stable during successive passage periods analyzed in vitro.

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

During long-term studies of the natural history of woodchuck hepatitis virus infection, five cases of histologically confirmed, primary hepatocellular carcinoma were observed in a total of 92 woodchucks which had recovered, by analysis of viral serologic markers (WHsAg-, anti-WHc+, anti-WHs+), from experimental acute woodchuck hepatitis virus infections 20 to 30 months prior to the detection of hepatocellular carcinoma. No hepatocellular carcinoma was observed in 167 uninfected controls at least 3 years of age and held in the same laboratory environment. Southern blot hybridization analysis of liver tissue taken from four of these recovered woodchucks revealed the presence of low levels (0.1 to 0.3 copies per cell) of integrated woodchuck hepatitis virus DNA in hepatocellular carcinoma (four of four animals) and nonneoplastic tissue (three of four animals). Similarly, hepatocellular carcinoma tissue obtained from two wild-caught, naturally infected and serologically recovered woodchucks also contained low levels of integrated woodchuck hepatitis virus DNA. Liver tissues from another 27 of these 92 recovered woodchucks (without hepatocellular carcinoma) were examined for woodchuck hepatitis virus nucleic acids 13 to 31 months following experimental woodchuck hepatitis virus infection. Nonreplicating woodchuck hepatitis virus DNA was present in the liver of eight (30%) and in the peripheral blood lymphocytes from eight (30%) of these 27 animals. These results were in marked contrast to the analysis of woodchuck hepatitis virus DNA in the liver tissue of chronic woodchuck hepatitis virus carriers (20 experimentally infected and nine naturally infected). In these animals, high levels of replicating woodchuck hepatitis virus DNA (up to 2,000 copies per cell) were observed in all hepatocellular carcinoma and nonneoplastic liver tissue. Integrated woodchuck hepatitis virus DNA was found in eight of 60 individual hepatocellular carcinomas detected in 29 chronic carriers, 15 to 40 months postinfection. Integrated woodchuck hepatitis virus DNA was present in the nonneoplastic tissue from four of these 29 chronic woodchuck hepatitis virus carriers.

Insulinlike growth factor II expression and oval cell proliferation associated with hepatocarcinogenesis in woodchuck hepatitis virus carriers

Insulinlike growth factor II (IGF-II) is a highly mitogenic fetal growth factor suspected of regulating the growth of a wide spectrum of tissues via an autocrine or paracrine mode of action or both. High steady-state levels of IGF-II RNA were detected in 45% of hepatocellular carcinomas (HCCs) arising from woodchuck livers with persistent woodchuck hepatitis virus (WHV) infection. Analysis of WHV RNA in the same HCCs revealed that HCCs with high levels of IGF-II RNA contained low or undetectable levels of WHV RNA and HCCs with low levels of IGF-II RNA contained high levels of WHV RNA. Integrated WHV DNA was present in HCCs from both groups, but viral DNA replicating forms were present, predominantly in HCCs with low levels of IGF-II. Several IGF-II RNAs, the most prominent of which were poly(A) species of approximately 3.75 and 1.1 to 1.3 kilobases, were detected only in precancerous nodules and HCCs. Levels of IGF-II were elevated two- to three-fold in the serum of woodchucks with chronic active hepatitis preceding the occurrence of HCC. Proliferation of a population of oval cells, which arise from portal tract regions in the liver, preceded the development of HCC and was a prominent feature of livers from which tumors with high levels of IGF-II occurred. The HCCs tended to have distinct histological features according to their growth factor status. Tumors with low levels of IGF-II were generally highly differentiated acinar-trabecular HCCs, whereas tumors with high levels of IGF-II were more anaplastic, with regions of fibrosis and fatty accumulation. A model to relate the pathology of WHV infection to oval cell proliferation and IGF-II expression in the development of these heterogeneous HCCs is presented.

Establishment and Characterization of a Woodchuck Hepatocellular Carcinoma Cell Line (WH44KA)

A continuous cell line was established from a hepatocellular carcinoma obtained from a woodchuck that was sero‐positive for woodchuck hepatitis virus (WHV). The cell line, designated WH44KA, grows as an adhering monolayer with a doubling time of 36 hr in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The cells not only showed epithelial origin on light and electron microscopic examination but also possess biosynthetic markers of the latter, such as albumin and α‐fetoprotein, which were demonstrated in cultured cells. When they were transplanted into athymic nude mice, tumors developed at the site of inoculation. These tumors were shown to he hepatocellular carcinoma, similar in morphology to the original tumor from which the WH44KA cells were derived. Chromosome analysis revealed a chromosome number ranging from 31 to 126, with a modal number of 35. Integration of WHV DNA was shown by Southern blot analysis. However, WHV surface antigen was not demonstrated in the cultured cells or supernatant medium. The WH44KA cell line appears to be a useful in vitro model for the study of virus‐induced hepatocellular carcinoma.