Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter species.

BACKGROUND: In the autumn of 1992, a novel form of chronic, active hepatitis of unknown etiology was discovered in mice at the National Cancer Institute-Frederick Cancer Research and Development Center (NCI-FCRDC), Frederick, Md. A high incidence of hepatocellular tumors occurred in affected animals. The disease entity was originally identified in A/JCr mice that were untreated controls in a long-term toxicologic study. PURPOSE: Our original purpose was to determine the origin and etiology of the chronic hepatitis and to quantify its association with hepatocellular tumors in mice of low liver tumor incidence strains. After a helical microorganism was discovered in hepatic parenchyma of diseased mice, we undertook characterization of the organism and investigation of its relationship to the disease process. METHODS: Hepatic histopathology of many strains of mice and rats, as well as guinea pigs and Syrian hamsters, in our research and animal production facilities was reviewed. Steiner's modification of the Warthin-Starry stain and transmission electron microscopy were used to identify bacteria in the liver. We transmitted the hepatitis with liver suspensions from affected mice and by inoculation with bacterial cultures. Bacteria were cultivated on blood agar plates maintained under anaerobic or microaerophilic conditions and characterized morphologically, biochemically, and by 16S rRNA sequence. RESULTS: We report here the isolation of a new species of Helicobacter (provisionally designated Helicobacter hepaticus sp. nov.) that selectively and persistently colonizes the hepatic bile canaliculi of mice (and possibly the intrahepatic biliary system and large bowel), causing a morphologically distinctive pattern of chronic, active hepatitis and associated with a high incidence of hepatocellular neoplasms in infected animals. CONCLUSIONS: The novel Helicobacter is a likely candidate for the etiology of hepatocellular tumors in our mice. The Helicobacter-associated chronic active hepatitis represents a new model to study mechanisms of carcinogenesis by this genus of bacteria. IMPLICATIONS: Adenocarcinoma of the stomach, the second most prevalent of all human malignancies world-wide, is associated with infection at an early age with Helicobacter pylori. Infection leads to several distinctive forms of gastritis, including chronic atrophic gastritis, which is a precursor of adenocarcinoma. H. hepaticus infection in mice constitutes the only other parallel association between a persistent bacterial infection and tumor development known to exist naturally. Study of the H. hepaticus syndrome of chronic active hepatitis and liver tumors in mice may yield insights into the role of H. pylori in human stomach cancer and gastric lymphoma.

Partial purification of native HIV transmembrane protein gp41: generation of polyclonal and monoclonal antibodies.

We partially purified the human immunodeficiency virus (HIV) glycoprotein gp41 from infected H9 cells by immunoaffinity chromatography using a column containing the M25 monoclonal antibody (diMarzo-Veronese et al., 1985). A pH 11.5 buffer worked best for eluting the glycoprotein from this column. The eluted gp41 was used in a sensitive slot blot immunoassay to detect antibodies to HIV in human sera and to prepare rabbit polyclonal antibodies and the 41-1S mouse monoclonal antibody. These antibodies reacted with gp41 in immunoprecipitation and in Western blot assays, but did not neutralize HIV in a syncytium-forming microassay. A pH 2.5 buffer was found to be the most effective solution for eluting gp41 from a 41-1S monoclonal antibody column.

Identification of the human papillomavirus E2 protein in genital tract tissues.

A 27-kilodalton protein representing approximately 60% of the E2 open reading frame of human papillomavirus type 6 (HPV-6) was synthesized in a bacterial expression system. Affinity-purified polyclonal antibody to this protein detected the probable E2 gene product as a 50-kilodalton protein in most condylomas by Western blot (immunoblot) analysis. The E2-positive condylomas were associated with HPV-6, HPV-11, HPV-16, or unidentified HPVs.

Microinjection and expression of an infectious proviral clone and subgenomic envelope construct of a human immunodeficiency virus.

An infectious proviral clone of the human immunodeficiency virus (HIV) was microinjected into the cell nucleus in six cell lines derived from caprine, ovine, bovine, or human solid tissue to study the utility of this method in effecting viral gene expression in nonlymphoid cells. Immunofluorescence assays for HIV demonstrated viral gene expression in only 5% of cells (100-200 cells per line) 24-48 h after microinjection; however, no reverse transcriptase activity was detectable, presumably due to a low level of virus release in this limited number of cells. Therefore, to indirectly assess infectious virus release, microinjected cells were cocultured with human T4 antigen-positive lymphocytes (H9) sensitive to HIV infection. Syncytia formation, electron microscopy, reverse transcriptase activity, and radioimmunoassay for HIV p24 were used to monitor viral gene expression in cocultures. HIV was efficiently recovered by cocultivating H9 with microinjected cells 48 h after microinjection, regardless of the tissue type or species of origin. H9 syncytia were visualized in some cocultures as early as day 5 but were readily apparent in all experiments on days 7-10. Syncytia induction in H9 was the earliest and most reliable indicator of infectious virus release. A recombinant construct containing a subgenomic envelope gene derived from the proviral clone of HIV was microinjected into human glioblastoma cells. Twenty-four to 48 h after manipulation, 5-20% of microinjected cells were found by immunofluorescence assay to express low levels of a putative gp120. These results suggest a possible approach to producing virus-free HIV envelope antigens in mammalian cells and may be relevant to subunit vaccine development.

Serological responses in chimpanzees inoculated with human immunodeficiency virus glycoprotein (gp120) subunit vaccine.

The major envelope glycoprotein of a human immunodeficiency virus (HIV) has been purified and was utilized as a prototype vaccine in chimpanzees. The 120,000-dalton glycoprotein (gp120) was purified from membranes of human T-lymphotropic virus (HTLV)-IIIB-infected cells and the final preparation contained low levels to no detectable HTLV-IIIB core antigen (p24) and low levels of endotoxin. Chimpanzees inoculated with gp120 responded by developing antibodies that precipitated radiolabeled gp120 and neutralized in vitro infection of HTLV-IIIB. Antibodies to HTLV-IIIB p24 were not detected in the gp120-immunized chimpanzees. Peripheral blood leukocytes from the vaccinated animals were examined for T4+ and T8+ cells, and no decrease in the T4/T8 ratio was found, indicating that immunization with a ligand (gp120) that binds to T4 has no detectable adverse effect on the population of T4+ cells. The only current animal model that can be reproducibly infected with HIV is the chimpanzee. Immunization of chimpanzees with HIV proteins will provide an experimental system for testing the effectiveness of prototype vaccines for preventing HIV infection in vivo.

Identification of the human papillomavirus type 6b L1 open reading frame protein in condylomas and corresponding antibodies in human sera.

Genital warts (condylomata acuminata) are among the most frequent sexually transmitted infections. Human papillomavirus type 6 (HPV-6), which is etiologically related to a majority of these lesions, has not been propagated in tissue culture. We generated two forms of HPV-6 viral antigens: a chemically synthesized oligopeptide (referred to as the C-terminal synthetic peptide) corresponding to residues 482 to 495 of the 500-amino-acid-long L1 open reading frame (ORF), and a bacterially expressed 54-kilodalton (kDa) fusion protein containing the N-terminal 13 amino acids encoded by the lambda bacteriophage cII gene followed by one vector-insert junctional residue and 462 amino acids of the L1 ORF sequence (residues 39 to 500). The cII-L1 fusion protein was specifically recognized by an antipeptide serum directed against the N-terminal 13 amino acids derived from the cII gene, an antiserum raised against the C-terminal synthetic peptide, and a genus-specific serum prepared by immunization with disrupted viral capsids. The 54-kDa fusion protein was purified, and the sequence of its first 36 amino acids was determined and found to be as predicted by the DNA sequence. Both the genus-specific anticapsid serum and the antiserum raised against the fusion protein identified authentic L1 ORF proteins in HPV-1-induced (58 kDa) and HPV-6/11-induced (56 kDa) papillomas. The synthetic peptide antiserum recognized the 56- to 58-kDa protein in HPV-6-induced warts, but not in HPV-1- or HPV-11-infected specimens. Using the fusion protein as antigen in immunoassays, we were able to detect the corresponding antibodies in human sera.

Expression of the bovine leukemia virus X region in virus-infected cells.

Bovine leukemia virus, like its closest relatives the human T-cell leukemia virus types I and II, contains a 1.8-kilobase X region between the env gene and the 3' long terminal repeat. In this communication, we report the detection and characterization of a subgenomic mRNA from which this X region is presumably translated. This mRNA was produced by a complex splicing mechanism which resulted in juxtaposition of the 5' end of the env gene and the two overlapping X-region open reading frames. Translation of this mRNA could yield at least two distinct proteins depending on which initiation codon is used. Detection of the protein encoded by the BLV X-region long open reading frame has been reported (N. Sagata, J. Tsuzuku-Kawamura, M. Nagayoshi-Aida, F. Shimizu, K.-I. Imagawa, and Y. Ikawa, Proc. Natl. Acad. Sci. USA 82:7879-7883, 1985). Using synthetic peptide antisera, we detected a protein encoded by the short open reading frame in virus-infected cells. The protein migrated in sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 19,000. It is a nuclear phosphoprotein.

Purification of 120,000 dalton envelope glycoprotein from culture fluids of human immunodeficiency virus (HIV)-infected H9 cells.

The outer envelope glycoprotein, gp120, has been purified from large volumes (greater than 100 liters) of HTLV-IIIB-infected H9 cell culture fluids using immunoaffinity chromatography resins prepared from immunoglobulins of AIDS patients plasma. By using a single-step immunoaffinity purification, between 7 and 28 micrograms of gp120 was recovered from each liter of culture fluid which represented between 60 and 95% of the total envelope glycoprotein present in the fluid. Envelope glycoprotein in culture media was concentrated more than 10,000 times over the starting material. The water-soluble gp120, containing trace contaminating proteins, was purified to apparent homogeneity by preparative polyacrylamide gel electrophoresis (PAGE). Envelope glycoprotein purified from culture fluids was immunogenic in laboratory animals in both native and PAGE-purified forms and was reactive with AIDS patient sera in immunoassays.

Prospect for prevention of human immunodeficiency virus infection: purified 120-kDa envelope glycoprotein induces neutralizing antibody.

This study initiates an effort to develop a safe vaccine against the acquired immunodeficiency syndrome (AIDS) that is caused by infection with a retrovirus designated human immunodeficiency virus (HIV) [formerly human T-cell lymphotropic virus type III (HTLV-III)]. Other retrovirus models have shown that purified external glycoprotein subunits are immunogenic. The external envelope glycoprotein of HIV (gp120) has a molecular size of 120 kDa, is responsible for virus infectivity, and induces strong antibody response in humans. Purified HIV virus preparations contain relatively little gp120 so HIV-infected cells were used as the antigen source. The gp120 was localized on cell membranes and was solubilized with low levels of nonionic detergent. The glycoprotein was further purified by immunoaffinity chromatography over a resin prepared from IgGs isolated from patients. Homogeneity was achieved following extensive dialysis and polyacrylamide gel electrophoresis. The gp120 isolated from infected cells was shown to be structurally identical by peptide maps to virion gp120 and the amino-terminal amino acid sequence confirmed that the molecule was specified by the HIV genome. Goat, horse, and rhesus monkey (Macaca mulatta) immune sera to gp120 precipitated the homologous antigen and neutralized the in vitro infectivity of HIV. The induction of neutralizing antibody indicates that a gp120 subunit vaccine against HIV is theoretically possible.

Human T-cell lymphotropic virus type III shares sequence homology with a family of pathogenic lentiviruses.

The etiologic agent of the acquired immune deficiency syndrome, human T-cell lymphotropic virus type III (HTLV-III), has recently been shown to morphologically resemble and share sequence homology with visna virus, a pathogenic lentivirus. Molecular hybridization, heteroduplex mapping, and DNA sequence analyses were used to compare HTLV-III to other lentiviruses of domestic animals, including visna, caprine arthritis encephalitis, and equine infectious anemia viruses. Hybridization results showed that a substantial amount of sequence homology exists between each of these viruses and HTLV-III. In addition, a closer relationship was found between visna and caprine arthritis encephalitis viruses than for any of the other lentiviruses studied. These results, along with nucleotide and amino acid sequence comparisons, have been used in a comprehensive effort to derive a systematic relationship for lentiviruses and to provide further evidence for classifying HTLV-III with the Lentivirinae subfamily of retroviruses. This relationship predicts that similarities in biology and disease process can be expected between HTLV-III and other Lentivirinae members.

Detection and characterization of the protein encoded by the v-rel oncogene.

To identify the protein encoded by v-rel, the oncogene of reticuloendotheliosis virus (REV-T), antisera have been raised to three synthetic peptides derived from the translation of our previously published v-rel DNA sequence [R.M. Stephens, N.R. Rice, R.R. Hiebsch, H.R. Bose, Jr., and R.V. Gilden, Proc. Natl. Acad. Sci. USA 80, 6229-6233 (1983)]. Sera to all three peptides precipitate a 59,000 Da protein from REV-T-transformed chicken lymphoid cells. This protein is not detectable in uninfected chick embryo fibroblasts, and its observed size is in good agreement with the 56,000 Da predicted by the DNA sequence. We conclude that this protein is the v-rel product and designate it p59rel. To search for evidence of post-translational processing of this protein, cells were grown in the presence of glycosylation inhibitors. These resulted in no detectable difference in the size of p59rel. Nor was its size detectably altered during the course of a pulse-chase experiment. Growth of cells in the presence of [32P] orthophosphate, however, revealed that p59rel is a phosphoprotein. It is also closely associated with a protein kinase activity, for precipitation with one of the peptide antisera (but not the other two) resulted in strong kinase activity in the immune complex pellet. During this reaction, p59rel itself becomes phosphorylated. Kinase activity was retained in the immune complex following detergent and high salt washes, leaving open the possibility that p59rel is itself a kinase.

Heteroduplex mapping in the molecular analysis of the human T-cell leukemia (lymphotropic) viruses.

The human T-cell lymphotropic virus (HTLV) family includes members associated with T-cell cancers (HTLV-I and HTLV-II) as well as the etiological agent of the acquired immunodeficiency syndrome (HTLV-III). Molecular clones of these viruses were used in heteroduplex mapping experiments to study their structural and evolutionary relationships. The HTLV-I subgroup, despite some restriction enzyme site polymorphism, demonstrated a high degree of sequence conservation. Heteroduplexes of HTLV-I and HTLV-II demonstrated a significant amount of sequence homology, with the strongest region of conservation occurring in the 3'-most coding sequences, designated pX, and to a lesser, although substantial extent in the rest of the genome. Thus, the genomic organization of HTLV-II appears to be very similar to that of HTLV-I. All HTLV-III molecular clones appeared to be identical, with a single exception, which showed heterogeneity in the env gene region. In heteroduplexes between HTLV-I and HTLV-III, very little homology was observed, being confined to the gap/pol region. In contrast to the latter result, a striking amount of homology was detected between HTLV-III and a morphologically similar, pathogenic, nononcogenic lentivirus, visna virus. These data provide strong evidence for a close taxonomic and thus evolutionary relationship between HTLV-III and the lentivirus subfamily of retroviruses. A taxonomic tree, based on the genetic relatedness and biological properties of the HTLV family, is proposed.

The gag and pol genes of bovine leukemia virus: nucleotide sequence and analysis.

The DNA sequence of the gag and pol regions of a provirus cloned from a bovine tumor is presented. In order to confirm these results the sequence of portions of a second clone, derived from a virus-producing cell line, was also determined. The gag gene was found to consist of 1179 nucleotides, which probably encode only three proteins: an N-terminal protein of 109 amino acids, a major core protein (p24) of 215 amino acids, and a nucleic acid binding protein (p12) of 69 residues. An open reading frame, whose translated product showed clear homology to the avian and murine proteases, was found beginning immediately upstream of the 3' end of gag. Following this protease region, a third long open reading frame, encoding 852 amino acids, showed clear homology to both avian and murine pol genes. The mechanism of translation of the protease and pol gene products cannot be predicted with certainty. Like Moloney murine leukemia virus (M-MuLV), BLV has a termination signal at the 3' end of gag, but unlike M-MuLV the protease is in a different reading frame. Like Rous sarcoma virus (RSV), BLV has a termination signal at the 3' end of the protease region and the reverse transcriptase is in a different (i.e., the third) reading frame. Possible translation mechanisms are discussed. Finally, the BLV gag and pol gene products are highly related to those of the human T-cell leukemia virus (HTLV); relatedness varied from 37% amino acid identities within the N terminal gag protein to 54% within the nucleic acid binding protein. Highly significant homology with both murine and avian type-C proteins was found within p24, p12, and the putative protease, reverse transcriptase, and endonuclease. Based on this homology, the BLV-HTLV family of viruses appears about equally distantly related to murine and avian type-C viruses.

Sequence homology and morphologic similarity of HTLV-III and visna virus, a pathogenic lentivirus.

A study was conducted of the genetic relation between human T-cell lymphotropic retroviruses and visna virus. The human T-cell lymphotropic viruses include those associated with T-cell malignancies (HTLV-I and HTLV-II) as well as the etiologic agent of the acquired immune deficiency syndrome (HTLV-III). Visna virus, a slowly replicating and pathogenic but nononcogenic retrovirus of sheep, is a member of the subfamily Lentivirinae. Results obtained by molecular hybridization and heteroduplex analysis indicated that a greater extent of nucleotide sequence homology exists between HTLV-III and visna virus than between HTLV-III and any of the other viruses. The homology observed under conditions of low stringency spanned the entire genome, but was strongest in the gag/pol region. The morphogenesis and fine structure of HTLV-III and visna virus also demonstrated striking similarities. The data provide strong evidence for a close taxonomic and thus evolutionary relation between HTLV-III and the Lentivirinae subfamily.

The nucleotide sequence of the env gene and post-env region of bovine leukemia virus.

The env gene of a bovine leukemia virus (BLV) tumor-derived proviral DNA clone has been located by comparison of the translated DNA sequence with amino acid sequence data on purified gp60 and p30env (A. M. Schultz, T. D. Copeland, and S. Oroszlan (1984) Virology 135, 417-427). There is a continuous open reading frame from the N terminus of gp60 for 1446 nucleotides; gp60 is predicted to contain 268 amino acids and p30env, 214. The predicted p30env shows structural features typical of type C viral transmembrane proteins. It is also clearly related to that of the human T-cell leukemia virus (HTLV), as predicted from the DNA sequence of Seiki et al. (M. Seiki, S. Hattori, Y. Hirayama, and M. Yoshida (1983) Proc. Natl. Acad. Sci. USA 80, 3618-3622) The two proteins show 36% identities in their amino acid sequence, in an alignment requiring six gaps. More distant relatedness is also seen between BLV p30env and both murine leukemia virus p15E and Rous sarcoma virus gp36. The gp60s of BLV and HTLV are more distantly related than their p30envs, but their homology is nonetheless statistically significant. Between the presumptive terminator of the env gene and the beginning of the 3'-long terminal repeat is a region of 1817 base pairs of unknown function. Just as in the HTLV post-envelope sequence, there are at least two reading frames which are open for a significant fraction of this region. In neither the tumor-derived clone nor a clone from a virus-producing cell line, however, is there a continuous open reading frame throughout the region. Comparison of the BLV and HTLV sequences within the post-envelope region revealed a very limited but possibly significant similarity.

Serological analysis of a subgroup of human T-lymphotropic retroviruses (HTLV-III) associated with AIDS.

The two main subgroups of the family of human T-lymphotropic retroviruses (HTLV) that have previously been characterized are known as HTLV-I and HTLV-II. Both are associated with certain human leukemias and lymphomas. Cell surface antigens (p61 and p65) encoded by HTLV-I are frequently recognized, at low titers, by antibodies in the serum of patients with acquired immunodeficiency syndrome (AIDS) or with signs or symptoms that precede AIDS (pre-AIDS). This suggests an involvement of HTLV in these disorders. Another subgroup of HTLV, designated HTLV-III, has now been isolated from many patients with AIDS and pre-AIDS. In the studies described in this report, virus-associated antigens in T-cell clones permanently producing HTLV-III were subjected to biochemical and immunological analyses. Antigens of HTLV-III, specifically detected by antibodies in serum from AIDS or pre-AIDS patients and revealed by the Western blot technique, are similar in size to those found in other subgroups of HTLV. They include at least three serologically unrelated antigenic groups, one of which is associated with group-specific antigens (p55 and P24) and another with envelope-related (p65) proteins, while the antigens in the third group are of unknown affiliation. The data show that HTLV-III is clearly distinguishable from HTLV-I and HTLV-II but is also significantly related to both viruses. HTLV-III is thus a true member of the HTLV family.

Simian AIDS: isolation of a type D retrovirus and transmission of the disease.

A type D retrovirus related to but distinct from Mason-Pfizer monkey virus was isolated in vitro from the blood of two rhesus monkeys (Macaca mulatta) with simian acquired immunodeficiency syndrome (SAIDS). Three juvenile rhesus monkeys that were injected intravenously with tissue culture fluids containing this virus developed SAIDS after 2 to 4 weeks.