State of the p53 gene in hepatocellular carcinomas of ground squirrels and woodchucks with past and ongoing infection with hepadnaviruses.

Infection with hepadnaviruses and exposure to dietary aflatoxin are considered major risk factors in the development of hepatocellular carcinoma (HCC) both in humans and in animals. Recently, a broad range of mutations in the p53 tumor suppressor gene has been reported in human HCCs, predominantly from hepatitis B virus carriers in areas with either high or low levels of exposure to dietary aflatoxin. To determine whether p53 mutations are common to HCCs of hosts infected with related hepadnaviruses with and without treatment with aflatoxin, we studied the occurrence of mutations in the p53 gene in HCCs of ground squirrels and woodchucks with history of infection with ground squirrel hepatitis virus (GSHV) and woodchuck hepatitis virus, respectively. Sequencing of wild type p53 genes from ground squirrels and woodchucks revealed remarkable homology between the two species with only a few amino acid differences in exons 4, 8, and 9. Using direct polymerase chain reaction sequencing, we analyzed the state of the p53 gene (exons 4-9) in 20 HCCs from ground squirrels (2 uninfected, 7 with past, and 11 with ongoing infection with GSHV) and in 11 HCCs from woodchucks persistently infected with woodchuck hepatitis virus. Five GSHV carrier and two uninfected ground squirrels received i.p. administration of aflatoxin B1. We detected only one mutation in the p53 gene of the tested animals. This mutation was located in codon 176 of exon 5 in the HCC of a GSHV-positive ground squirrel treated with aflatoxin. Mutation was caused by a G to T transversion in the second position of the codon, resulting in the replacement of cysteine with phenylalanine, and was accompanied by a tumor-specific loss of heterozygosity. p53 allelic amino acid variation with sequences coding for aspartic acid or asparagine was present in codon 61 in the variable region of exon 4 in both HCCs and nonneoplastic tissues of ground squirrels. In view of the considerably lower apparent rate of mutations in comparison to human HCCs, we suggest a less important role for aflatoxin in the induction of p53 mutations in HCCs of ground squirrels. Alternatively, etiological factors other than p53 mutations may be of greater significance in the development of HCC in ground squirrels and woodchucks.

Construction of adenoviral and retroviral vectors coexpressing the genes encoding the hepatitis B surface antigen and B7-1 protein.

Recombinant retroviral (re-Rv) and adenoviral (re-Ad) vectors for delivery of two foreign genes were constructed, using the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) which mediates initiation of cap-independent translation. The first gene encoded the hepatitis B surface antigen (HBsAg) and the second encoded human or murine B7-1 molecule, a cell surface protein which is a costimulator for T cell activation. The EMCV IRES sequence was placed between the first and second coding sequences to form a dicistronic DNA fragment. In Rv vectors, the dicistronic fragment was inserted between the 5' long terminal repeat (LTR) and an internal promoter for the neomycin (neo) gene, so that the transcription initiated from the 5' LTR would generate a dicistronic mRNA for the HBsAg and B7-1 molecules. For Ad vectors, the dicistronic fragment was inserted between a cytomegalovirus promoter and a polyA signal to form a transcription cassette. This transcription cassette was inserted into the early region 1 of Ad5 genome to form a replication-defective re-Ad vector, or into early region 3 to form replication-competent vectors. Human cell line A549 infected with the re-Rv vectors or with the re-Ad vectors synthesized and secreted HBsAg at comparable levels, while the B7-1 molecules were detected at the surface of the infected cells, indicating both foreign genes carried by the Rv and Ad vectors were expressed efficiently.

Preliminary studies of acyclovir in chronic hepatitis B.

Three patients with HBsAg-positive and DNA-polymerase-positive chronic hepatitis were treated with increasing dosages of intravenous acyclovir. A fall in DNA polymerase activity was seen with all courses of acyclovir but no dose-response relationship was evident. In only one patient did DNA polymerase fall to zero where it has remained for five months. Two out of 10 courses were associated with significant side effects with the highest dosages of acyclovir but these promptly resolved when the agent was stopped. Acyclovir's apparently partial and transient action suggests that it will not have a role in the treatment of chronic hepatitis B virus infection.

Transmission of lymphadenopathy-associated virus/human T lymphotropic virus type III in sexual partners. Seropositivity does not predict infectivity in all cases.

To investigate transmission of lymphadenopathy-associated virus (LAV)/human T lymphotropic virus type III (HTLV-III) in long-term sexual partners, and the relationship between lymphadenopathy-associated virus seropositivity and transmission, nine couples (five heterosexual and four homosexual) at increased risk for acquired immune deficiency syndrome (AIDS) were studied. In two heterosexual couples, transmission of lymphadenopathy-associated virus from a seropositive man at increased risk to his monogamous wife occurred. In one couple, the wife of a man with hemophilia had lymphadenopathy-associated virus antibody and decreased T helper cells; in the other couple, the wife of a bisexual intravenous drug-user had AIDS. Neither woman had a recognized AIDS risk except marriage to a seropositive man at increased risk. However, study of the other couples revealed that regular sexual contact with seropositive persons over long periods did not always lead to evidence of lymphadenopathy-associated virus infection. This study suggests that presence of lymphadenopathy-associated virus antibody does not always indicate a high degree of infectivity.

Efficacy of glove combinations in reducing cell culture infection after glove puncture with needles contaminated with human immunodeficiency virus type 1.

OBJECTIVE: To study the effect of various latex and treated glove combinations in reducing the frequency of human immunodeficiency virus (HIV) infection of tissue culture cells after puncture by surgical needles contaminated with infectious human immunodeficiency virus type 1 (HIV-1). DESIGN: One, two, or three layers of sterile latex glove material, or two latex layers with intermediate cotton or Kevlar (with or without the virucidal compound nonoxynol-9) were used to cover 24-well cell culture dishes containing MT2 cells in cell culture medium. Surgical needles wet with cell culture medium containing HIV-1 (HTLV IIIA strain) were passed through the glove materials into the culture medium in the wells of the culture dishes. The culture medium in each well was then assayed biweekly for HIV-1 p24 antigen as a test for infection of cells in the well. RESULTS: The rate of HIV-1 infection of cell cultures after glove puncture was greater than 90% with a single latex surgical glove barrier, 23% to 60% with double or triple layers of latex gloves, less than 8% with an intermediate cotton glove impregnated with 4% nonoxynol-9, 6% with an intermediate Kevlar glove, and 0% with an intermediate Kevlar glove impregnated with nonoxynol-9. CONCLUSIONS: An intermediate glove of Kevlar or of Kevlar or cotton impregnated with virucidal compound nonoxynol-9 between standard latex gloves may improve surgical glove safety, compared with latex gloves alone with respect to needlestick transmission of HIV-1. The experimental model used may permit rapid investigation of other glove systems as barriers to the transfer of infectious agents through gloves by needlestick.

Genetic variation among hepatitis B and related viruses.

Hepatitis B virus (HBV) of man has several characteristics that distinguish it from viruses of other groups. These include its ultrastructure, viral DNA size and structure, a virion DNA polymerase which repairs a single-stranded region in the viral DNA, liver tropism, character of persistent infection, and association with hepatitis and hepatocellular carcinoma. Recently three other viruses have been found in other animal species that appear to share these characteristics although the viruses are not identical. HBV, Woodchuck hepatitis virus (WHV), ground squirrel hepatitis virus (GSHV), and duck hepatitis virus (DHV) appear to be members of a new virus group that might be designated the Hepadna virus group. Genetic variation among hepatitis B viruses includes the antigenic variation in the surface antigen (HBsAg) which constitutes the known HBsAg subtypes. There is also frequent variation in DNA base sequence among HBVs isolated from different patients.

DNA and DNA polymerase in the core of the Dane particle of hepatitis B.

The core of the Dane particle was shown to contain a DNA polymerase and a circular double stranded DNA with a molecular weight of 1.6 X 10(6) daltons which served as the primer-template for the enzyme. The product of the DNA polymerase reaction was in a base paired form and was covalently attached to the circular DNA. Neither the circular DNA nor the attached DNA product of the enzyme reaction was attacked by the DNase or released from intact cores until the cores were disrupted with sodium dodecyl sulfate, suggesting that they are internal components of the core. The DNA polymerase is a specific marker for Dane particles and can be used to distinguish sera with high and low concentrations of Dane particles. The DNA polymerase reaction can also be used to radiolabel Dane particle cores for a specific and sensitive radioimmunoprecipitation assay for antibody against the hepatitis B core antigen (anti-HBc).

Hepadnaviruses and hepatocellular carcinoma (HCC).

Members of the hepadnavirus family share properties of virion structure, genome structure and replication, epidemiologic behavior, and pathogenic effects. Persistent infections with hepatitis B virus (HBV) in man, woodchuck hepatitis virus (WHV) in Marmota monax, ground squirrel hepatitis virus (GSHV) in Spermophilus beecheyi, and duck hepatitis B virus (DHBV) in domestic ducks of China are associated with development of hepatocellular carcinoma (HCC). Epidemiological evidence implicating hepadnavirus infection in HCC includes the observation that the geographic distributions of HBV infection and HCC are similar, that the incidence of HCC is much higher in hepadnavirus-infected than uninfected hosts, and that viral DNA sequences are integrated in the cellular DNA of most (e.g., 80 to 90%), but not all, hepadnavirus-associated HCC. Cirrhosis further increases the risk of HCC in HBV-infected humans. The precise role of hepadnaviruses in development of most HCC is unclear, although the finding of viral integrations within or near protooncogenes in a few cases suggests the possibility that these integrations may play a direct role in these HCC. However, in the great majority of HCC, viral integrations are in different cellular DNA sites in different HCC, integrations are not within domains of known protooncogenes, and integrations are not found in some 10 to 15% hepadnavirus-associated HCC, suggesting that persisting viral sequences are not directly involved in the development of these HCC as viral sequences are for tumors caused by viruses with oncogenes or viruses that act by a "promoter-insertion" mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of hepatitis B virus in the development of primary hepatocellular carcinoma: Part I.

Chronic infections with hepatitis B virus (HBV) of humans and animal hepadnavirus infections in their natural hosts are strongly associated with primary hepatocellular carcinoma (HCC). Although viral integrations are found in cells of many HCC, no general viral-specific hepatocarcinogenic mechanism for hepadnaviruses has been identified. In approximately one half of HCC in woodchuck hepatitis virus (WHV) infected woodchucks, viral integrations near the c-myc or N-myc genes have been reported which result in enhanced expression of the respective gene. Such host gene-specific insertional mutagenesis has not been found in HCC of other hepadnavirus infected hosts. Thus in humans, ground squirrels and ducks hepadnaviral integrations appear to be at different host chromosomal DNA sites in each HCC and few integrations have been found within or near any cellular gene. Other possible hepadnavirus-specific carcinogenic mechanisms that are being investigated include transactivation of cellular gene expression by an hepadnavirus gene product (e.g. the X-gene), and mutation of host genes by unknown hepadnavirus-specific mechanisms. It should be noted, however, that chronic hepadnavirus infection is associated with chronic necroinflammatory liver disease with hepatocellular necrosis and regeneration (sometimes leading to cirrhosis in humans), a pathological process that is common to numerous other risk factors for HCC. This suggests the possibility that this pathological process is hepatocarcinogenic irrespective of the inciting agent and the role of hepadnavirus infection is no different from that of other risk factors in causing chronic necroinflammatory liver disease.

DNA and DNA polymerase of a virus-like particle in hepatitis B.

One of the particulate forms bearing hepatitis B antigen in human blood, the 42 nm Dane particle, has been shown to contain a small circular-stranded DNA with a molecular weight round 1.6 X 10(6) daltons and a DNA polymerase. The circular DNA functions as a primer-template for the DNA polymerase. The circular DNA, the DNA polymerase and the DNA product of the enzyme reaction appear to be internal components of the 28 nm core of the Dane particle. Dane particle cores labeled with H3 in a DNA polymerase reaction have been used in a sensitive double antibody precipitation assay for the antibody against core.

Molecular events in the pathogenesis of hepadnavirus-associated hepatocellular carcinoma.

Chronic hepadnavirus infection is associated with hepatocellular carcinoma (HCC) in natural hosts such as humans, woodchucks, and Beechey ground squirrels. Several possible oncogenic mechanisms have been identified, including a potential role of the hepadnavirus x (hbx) gene, which transactivates transcription regulated by certain cis-acting sequences, e.g. regulatory sequences of the hepatitis B virus (HBV) and heterologous regulatory sequences of other viruses and cellular genes. The oncogenic potential of hbx is suggested by the observation of HCCs in hbx transgenic mice, the oncogenic transformation of cells expressing hbx in culture, and the transactivation of oncogenes c-myc and c-jun by hbx. Cis-activation of cellular oncogenes N-myc and c-myc by viral promoter insertion has been a common finding in woodchuck hepatitis virus (WHV)-associated HCCs of woodchucks. No such cis-activation of any cellular gene has been shown in virus-associated HCCs of ground squirrels or humans. Amplification and overexpression of the c-myc gene has been a common finding in HCCs of ground squirrels, and is rare in woodchuck or human HCCs. Point mutations in the p53 gene and allelic deletion of p53 have been common findings in human HCCs, but have not been found in HCCs in woodchucks and have been found rarely in ground squirrels. How each of these genetic changes in the different hosts contributes to HCC remains to be determined, but apparently different changes in different HCCs of hepadnavirus-infected hosts suggest that several separate genetic events may contribute to the development of HCC. These events may differ in each host, and some may not result from a direct virus-specific mechanism. Chronic hepadnavirus infection is often associated with chronic necroinflammatory liver disease and cirrhosis, a pathologic process common to several other risk factors for HCC. This suggests that this pathologic process (necroinflammatory disease) may be hepatocarcinogenic regardless of the inciting agent. Thus hepadnavirus infection may play an important role in the development of HCC by causing chronic hepatitis and HCC with the same mechanisms by which other risk factors for HCC cause chronic necroinflammatory liver disease and HCC.

Ribonucleic acid polymerase activity in Sendai virions and nucleocapsid.

After dissociation of purified Sendai virus with the neutral detergent Nonidet P-40 and 2-mercaptoethanol, it catalyzed the incorporation of ribonucleoside triphosphates into an acid-insoluble product. The enzyme activity was associated with viral nucleocapsid as well as whole virions. The reaction product was ribonucleic acid (RNA) which annealed specifically with virion RNA. Sedimentation of the (3)H-RNA reaction product revealed two components, a 45S component with properties of double-stranded RNA and 4 to 6S component which appeared to be mostly single-stranded RNA.

Common evolutionary origin of hepatitis B virus and retroviruses.

Hepatitis B virus (HBV), although classified as a double-stranded DNA virus, has been shown recently to replicate by reverse transcription of an RNA intermediate. Also, the putative viral polymerase has been found to share amino acid homology with reverse transcriptase of retroviruses. Using computer-assisted DNA and protein sequence analyses, we examined the genomes of 13 hepadnavirus isolates (nine human, two duck, one woodchuck, and one ground squirrel) and found that other conserved regions of the hepadnavirus genome share homology to corresponding regions of the genomes of type C retroviruses and retrovirus-like endogenous human DNA elements. Specifically, the most highly conserved sequence of the HBV genome, positioned at or near the initiation site for first-strand HBV DNA synthesis, is homologous over 67 nucleotides to the U5 region, a comparable region in retrovirus long terminal repeats. Within a highly conserved (i.e., 90%) 16-nucleotide sequence a heptanucleotide sequence CCTTGGG is 97% homologous between 27 virus isolates. Also, we found that the highly conserved HBV core, or nucleocapsid, protein shares 41% homology over 98 amino acids with the carboxyl-terminal region of the p30 gag nucleocapsid protein of type C retroviruses. In both cases, as with the previously reported polymerase homology, HBV is most homologous to the murine leukemia/sarcoma retroviruses. Further analysis revealed additional similarities between hepadnavirus and retroviral genomes. Taken together, our results suggest that HBV and retroviruses have a common evolutionary origin, with HBV arising through a process of deletion from a retrovirus, or retrovirus-like, progenitor.

State of hepatitis B viral genomes in cirrhotic and hepatocellular carcinoma nodules.

DNA of individual cirrhotic nodules (CN) and hepatocellular carcinoma nodules (HCN) of three hepatitis B surface antigen positive autopsy cases with macronodular cirrhosis were analyzed by Southern blot and slot blot hybridization with a hepatitis B virus (HBV) DNA probe. Evidence of episomal or replicating viral DNA, viral DNA integration at the same cellular DNA site in many cells (clonal integration) and viral integration in different cellular DNA sites in many different cells (non-clonal integration) was found in different cirrhotic nodules of the same liver, indicating heterogeneity in the state of HBV in different cells and in different cirrhotic nodules within each infected liver. Episomal or replicating viral DNA forms were found in all cirrhotic nodules of one liver, in less than 10% of examined nodules of a second liver and in none of the third. Evidence of clonal viral integration was found in CN of all three livers and non-clonal integration in CN of the latter two. Cirrhotic nodules with apparent different integrations in many different cells (non-clonal integration) outnumbered those with the same integration site in many cells (clonal integration), and many cirrhotic nodules in those two livers had no detectable viral DNA. Cirrhotic nodules with a viral integration in the same cellular DNA site in many cells would appear to have been formed by clonal expansion of an original cell containing the viral integration, and cirrhotic nodules with different integrations in many different cells (non-clonal integration) may have been formed by recruitment of many different cells with different viral integrations or by clonal expansion of cells without HBV integrations and subsequent viral integrations occurring integration. In one liver, three different hepatocellular carcinoma nodules appeared to represent metastatic lesions because the clonal pattern of HBV integration was identical in each, and in another liver different HCN appeared to be of different clonal origin, i.e. to have arisen from different cells, because multiple viral integrations (i.e. multiple individual restriction fragments with HBV sequences) were each different in different HCN of that liver.

Hepatitis B virus DNA forms in nuclear and cytoplasmic fractions of infected human liver.

Human liver tissues obtained at autopsy from two patients chronically infected with hepatitis B virus (HBV) were found to contain several distinct species of HBV DNA. Southern blot analysis using a nick-translated HBV [32P]DNA probe identified specific DNA bands migrating at the positions expected for linear double-stranded DNA of 3.6 and 2.0 kb. These DNA bands were shown to represent relaxed circular and closed circular (supercoiled) HBV DNA, respectively. In addition to these distinct bands several minor bands as well as a heterogeneous population of HBV DNA molecules were present. When infected cell nuclei were isolated, and the nuclear and cytoplasmic nucleic acid separately analyzed, the nuclear fraction contained the 2.0-kb DNA species. This species was shown to be supercoiled 3.2-kb HBV DNA by electron microscopy, restriction endonuclease digestion, and thermal denaturation. The cytoplasmic fraction contained DNA forms similar to those found in virions isolated from plasma (i.e., migration in the position of linear double-stranded molecules of 3.6 and 3.2 kb) and no supercoiled DNA was detected. Particles isolated from the cytoplasmic fraction were able to incorporate dNTPs into viral DNA sequences. Southern blot analysis of the nucleic acid isolated from the particles revealed the presence of HBV DNA forms migrating in positions expected for 3.6- and 3.2-kb linear double-stranded molecules as well as a heterogeneous population of HBV molecules. The 3.6- and 3.2-kb species were identified as relaxed circular and double-stranded linear genome-length HBV DNA. Digestion of the viral nucleic acid with pancreatic ribonuclease increased the electrophoretic mobility of a portion of the heterogeneous HBV molecules and resulted in the appearance of a distinct 1.9-kb DNA band suggesting the same viral DNA was complexed with RNA. Experiments to be reported elsewhere showed this DNA species to be genome-length minus-strand HBV DNA which was released from DNA-RNA hybrid molecules by RNase digestion. Thus, supercoiled HBV DNA exists free in the nucleus of infected liver cells and cytoplasmic particles contain relaxed circular and linear HBV DNA as well as a heterogeneous population of HBV DNA and DNA-RNA hybrid molecules, and a DNA polymerase reaction in the particles results in incorporation of dNTP into DNA strands of these molecules.