ABSTRACT
Tracking human immunodeficiency virus-type 1 (HIV-1) infection at the cellular level in tissue reservoirs provides opportunities to better understand the pathogenesis of infection and to rationally design and monitor therapy. A quantitative technique was developed to determine viral burden in two important cellular compartments in lymphoid tissues. Image analysis and in situ hybridization were combined to show that in the presymptomatic stages of infection there is a large, relatively stable pool of virions on the surfaces of follicular dendritic cells and a smaller pool of productively infected cells. Despite evidence of constraints on HIV-1 replication in the infected cell population in lymphoid tissues, estimates of the numbers of these cells and the virus they could produce are consistent with the quantities of virus that have been detected in the bloodstream. The cellular sources of virus production and storage in lymphoid tissues can now be studied with this approach over the course of infection and treatment.
Subject(s)
Dendritic Cells/virology , HIV Infections/virology , HIV-1/physiology , Leukocytes, Mononuclear/virology , Lymphoid Tissue/virology , Viral Load , Adult , Antisense Elements (Genetics) , Autoradiography , CD4 Lymphocyte Count , HIV Infections/drug therapy , HIV Infections/immunology , Humans , Image Processing, Computer-Assisted , In Situ Hybridization , Lymph Nodes/virology , Palatine Tonsil/virology , RNA Probes , RNA, Viral/analysis , RNA, Viral/blood , Sensitivity and Specificity , Spleen/virologyABSTRACT
In lymphoid tissue, where human immunodeficiency virus-type 1 (HIV-1) is produced and stored, three-drug treatment with viral protease and reverse transcriptase inhibitors markedly reduced viral burden. This was shown by in situ hybridization and computerized quantitative analysis of serial tonsil biopsies from previously untreated adults. The frequency of productive mononuclear cells (MNCs) initially diminished with a half-life of about 1 day. Surprisingly, the amount of HIV-1 RNA in virus trapped on follicular dendritic cells (FDCs) decreased almost as quickly. After 24 weeks, MNCs with very few copies of HIV-1 RNA per cell were still detectable, as was proviral DNA; however, the amount of FDC-associated virus decreased by >/=3.4 log units. Thus, 6 months of potent therapy controlled active replication and cleared >99.9 percent of virus from the secondary lymphoid tissue reservoir.
Subject(s)
Anti-HIV Agents/therapeutic use , Dendritic Cells/virology , HIV Infections/drug therapy , HIV-1/drug effects , Leukocytes, Mononuclear/virology , Palatine Tonsil/virology , Adult , CD4-Positive T-Lymphocytes/virology , DNA, Viral/analysis , Dendritic Cells/cytology , Drug Therapy, Combination , HIV Infections/virology , HIV Protease Inhibitors/therapeutic use , HIV-1/isolation & purification , HIV-1/physiology , Humans , Image Processing, Computer-Assisted , In Situ Hybridization , Kinetics , Lamivudine/therapeutic use , Leukocytes, Mononuclear/cytology , Macrophages/virology , Proviruses/genetics , RNA, Viral/analysis , Reverse Transcriptase Inhibitors/therapeutic use , Ritonavir/therapeutic use , Viral Load , Virus Replication/drug effects , Zidovudine/therapeutic useABSTRACT
In sexual transmission of simian immunodeficiency virus, and early and later stages of human immunodeficiency virus-type 1 (HIV-1) infection, both viruses were found to replicate predominantly in CD4(+) T cells at the portal of entry and in lymphoid tissues. Infection was propagated not only in activated and proliferating T cells but also, surprisingly, in resting T cells. The infected proliferating cells correspond to the short-lived population that produces the bulk of HIV-1. Most of the HIV-1-infected resting T cells persisted after antiretroviral therapy. Latently and chronically infected cells that may be derived from this population pose challenges to eradicating infection and developing an effective vaccine.
Subject(s)
CD4-Positive T-Lymphocytes/virology , HIV Infections/transmission , HIV-1/physiology , Lymphocyte Activation , Simian Acquired Immunodeficiency Syndrome/transmission , Simian Immunodeficiency Virus/physiology , Animals , Anti-HIV Agents/therapeutic use , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , Cell Cycle , Cervix Uteri/virology , Epithelial Cells/virology , Female , HIV Infections/drug therapy , HIV Infections/virology , Lymph Nodes/virology , Macaca mulatta , RNA, Viral/analysis , Simian Acquired Immunodeficiency Syndrome/virology , Time Factors , Virus ReplicationABSTRACT
Detection of human immunodeficiency virus-type 1 (HIV-1) on only one or a few occasions in infants born to infected mothers has been interpreted to indicate that infection may be transient rather than persistent. Forty-two cases of suspected transient HIV-1 viremia among 1562 perinatally exposed seroreverting infants and one mother were reanalyzed. HIV-1 env sequences were not found in specimens from 20; in specimens from 6, somatic genetic analysis revealed that specimens were mistakenly attributed to an infant; and in specimens from 17, phylogenetic analysis failed to demonstrate the expected linkage between the infant's and the mother's virus. These findings argue that transient HIV-1 infection, if it exists, will only rarely be satisfactorily documented.
Subject(s)
HIV Infections/virology , HIV-1/genetics , HIV-1/isolation & purification , Specimen Handling , DNA, Viral/analysis , DNA, Viral/genetics , Diagnostic Errors , Equipment Contamination , Female , Genes, env , HIV Infections/immunology , HIV Infections/transmission , Humans , Infant , Infant, Newborn , Infectious Disease Transmission, Vertical , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , RNA, Viral/analysis , T-Lymphocytes, Cytotoxic/immunology , Viremia/virologyABSTRACT
A clone of about 1 kb has been isolated from a human brain cDNA library. The clone possesses a 151 amino acid open reading frame that exhibits 72% amino acid identity with the E2 ubiquitin-conjugating enzyme encoded by the RAD6 gene of Saccharomyces cerevisiae. A 90% amino acid identity was observed in a central sequence surrounding a cysteine, which most likely contributes the sulfhydryl group involved in the formation of the ubiquitin-E2 thiolester linkage. Northern hybridization analyses have identified a poly(A)-containing mRNA of about 1 kb encoding the E2-like sequence in human CEM lymphoblastoid and HeLa cells, Novikoff rat hepatoma cells and S49 mouse leukemia cells. Southern hybridization analyses indicate the presence of a single gene encoding this sequence in both human cell lines, but of two or more related genes in the rodent cell lines.
Subject(s)
Carrier Proteins/genetics , Ligases , Saccharomyces cerevisiae/genetics , Sequence Homology, Nucleic Acid , Ubiquitin-Conjugating Enzymes , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Carrier Proteins/chemistry , DNA Repair/physiology , HeLa Cells , Humans , Mice , Molecular Sequence Data , Rats , Tumor Cells, CulturedABSTRACT
The structural determinants of lipopolysaccharide required for the induction of tissue factor in human umbilical vein endothelial cells were studied. Intact lipid A was essential for the induction of tissue factor whereas the incomplete lipid A precursors lipid IVA and lipid X, as well as monophosphoryl lipid A and acyloxyacyl hydrolase-treated lipopolysaccharide, were unable to induce tissue factor and tissue factor specific mRNA. However, the lipid A precursor, lipid IVA, was able to inhibit LPS-mediated induction of tissue factor; structural determinants distal to lipid A were found to be required for maximal induction of tissue factor activity and tissue factor mRNA. The presence of serum in the assay was found to amplify but was not obligate for tissue factor induction by LPS.
Subject(s)
Endothelium, Vascular/drug effects , Endotoxins/pharmacology , Lipid A/metabolism , Thromboplastin/biosynthesis , Blood Physiological Phenomena , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Glycolipids/pharmacology , Humans , Lipid A/analogs & derivatives , Lipid A/pharmacology , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/metabolism , Lipopolysaccharides/pharmacology , RNA, Messenger/biosynthesis , Salmonella typhimuriumABSTRACT
Visna virus and human immunodeficiency virus are prototypes of animal and human lentiviruses, respectively, that persist and are disseminated despite the host immune response because cells in the tissues and the bloodstream harbor viral genomes in a covert state. To facilitate identification of these latently infected cells, the polymerase chain reaction has been adapted to amplify viral DNA in fixed cells for detection by in situ hybridization. By using a multiple primer set that generates DNA segments with overlapping cohesive termini, visna virus DNA can be amplified, retained, and detected in infected cells with sensitivities that exceed those of existing methods by more than 2 orders of magnitude. This advance in single-cell technology should prove useful in diagnosing and gaining insight into the pathogenesis of viral infections and provide new opportunities to look for viruses in chronic diseases of unknown etiology.
Subject(s)
DNA, Viral/genetics , Genes, Viral , Retroviridae/genetics , Visna-maedi virus/genetics , Animals , Cells, Cultured , Choroid Plexus/microbiology , DNA, Viral/analysis , Nucleic Acid Hybridization , Polymerase Chain Reaction/methods , Retroviridae/isolation & purification , Sheep , Visna-maedi virus/isolation & purificationABSTRACT
Infection with Kaposi's sarcoma-associated herpesvirus (KSHV) is closely associated with Kaposi's sarcoma (KS) and primary effusion lymphoma, with viral genomes present in a latent state in the majority of tumor cells. Here we describe a cluster of latently expressed viral genes whose mRNAs are generated from a common promoter. Two mRNAs in this region encode the latency-associated nuclear antigen, the product of open reading frame 73 (ORF73). The larger RNA, of 5.8 kb, is an unspliced transcript that includes ORF72 and -71 at its 3' end; it initiates at nucleotides (nt) 127880 to 127886 from a promoter lacking recognizable TATA elements. A less abundant mRNA, of 5.4 kb, is a variant of this transcript, in which 336 nt of 5' noncoding information has been removed by RNA splicing. A third, more abundant RNA is generated from the same promoter region via splicing from the common splice donor at nt 127813 to an acceptor 5' to ORF72; this transcript is the presumed mRNA for ORF72, which encodes the viral cyclin D homolog. All three RNAs are 3' coterminal. In situ hybridization analysis with probes that can detect all three transcripts shows that the RNAs are detectable in a large fraction of BCBL-1 cells prior to lytic induction and in >70% of KS spindle cells in primary KS tumors. This confirms that these transcripts are indeed latent RNAs and suggests a role for their products in viral persistence and/or KSHV-associated proliferation.
Subject(s)
Genes, Viral , Herpesvirus 8, Human/genetics , Multigene Family , Alternative Splicing , Base Sequence , DNA Primers , DNA, Viral , Genes, Reporter , In Situ Hybridization , Open Reading Frames , Promoter Regions, Genetic , RNA, Messenger/genetics , Transcription, GeneticABSTRACT
Nucleotide sequence analysis of two molecular clones of transformation-defective avian sarcoma virus indicate that direct repeated sequences of 6 and 20 nucleotides are involved in the formation of the src deletions in these clones.
Subject(s)
Avian Sarcoma Viruses/genetics , DNA, Viral/analysis , Genes, Viral , Repetitive Sequences, Nucleic Acid , Base Sequence , Cloning, MolecularABSTRACT
Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) encodes a G-protein-coupled receptor (GCR) homolog. This protein is a potent, constitutively active signalling molecule that can influence both proliferation and angiogenesis when ectopically expressed in fibroblasts in vitro. Here we have examined the expression of the KSHV GCR gene in virus-infected lymphoid cells and in KS tumors. Our results show that in both situations the gene is expressed primarily during lytic replication; its transcription is unaffected by inhibition of viral DNA synthesis, indicating that it is expressed in the early phases of the lytic program. The major transcript bearing GCR sequences is bicistronic, harboring coding sequences for another viral gene, K14, at its 5' end. Extensive searches for monocistronic GCR mRNAs using nuclease mapping and reverse transcription-PCR failed to detect such species. The 5' end of K14/GCR mRNA maps to nucleotide (nt) 127848, and its poly(A) addition site maps to nt 130546; a 149-nt intron is present in the K14/GCR intergenic region. These results suggest that the KSHV GCR is translated by unconventional mechanisms involving either translational reinitiation, internal ribosomal entry, or leaky ribosomal scanning. The restriction of GCR expression to the lytic cycle has important implications for the potential role(s) of the GCR in KS pathogenesis.
Subject(s)
Gene Expression , Herpesvirus 8, Human/genetics , Receptors, Chemokine/genetics , Sarcoma, Kaposi/virology , Viral Proteins/genetics , 5' Untranslated Regions , Alternative Splicing , Animals , Base Sequence , COS Cells , DNA, Viral , GTP-Binding Proteins/metabolism , Humans , In Situ Hybridization , Molecular Sequence Data , Promoter Regions, Genetic , RNA, Messenger , RNA, Viral , Sarcoma, Kaposi/etiology , Tumor Cells, CulturedABSTRACT
Maedi and visna are, respectively, the pulmonary and neurological manifestations of slowly progressive infections of sheep caused by retroviruses of the lentivirus subfamily. Lentivirus infections are also persistent infections in which host defenses are generally not successful in eliminating the infectious agent because of restricted viral gene expression in many infected cells. In this report, we describe a method for amplifying and detecting viral DNA in tissue sections which has made it possible to verify experimentally the postulated existence of this reservoir of latently infected cells, as well as to estimate the actual number of cells which harbor viral genomes in infected tissues. In the discussion, we present a simple mathematical model that relates this number to the rate at which inflammatory lesions develop. This model can account for both the slow progression of natural infections and for the rapid accumulation of inflammatory foci in the high dosage experimental system analysed in our studies.
Subject(s)
DNA, Viral/analysis , Lung/microbiology , Pneumonia, Progressive Interstitial, of Sheep/microbiology , Visna-maedi virus/isolation & purification , Animals , DNA, Viral/genetics , Disease Models, Animal , Gene Expression , Nucleic Acid Hybridization , Polymerase Chain Reaction , RNA, Viral/analysis , Sheep , Visna-maedi virus/genetics , Visna-maedi virus/physiologyABSTRACT
Visna virus is a nontransforming retrovirus that causes slow infections in animals and a rapidly progressive-lytic infection in cell culture. The results of an analysis of the synthesis of viral DNA in cell culture are reported. Region- and strand-specific probes cloned in M13 have been used to define the dynamics of DNA synthesis and the major nucleic acid species formed. It is shown that (i) within the first hours of infection, a full-length copy of the viral RNA genome is synthesized by reverse transcription, (ii) early in infection a major species of DNA is formed that extends from a site near the center of the molecule to the 3' end, (iii) somewhat later a second major species of plus-strand DNA is generated that extends from the 5' end to the middle of the genome. As a consequence, most viral DNA molecules consist of a full-length minus strand, and two plus strands separated by a gap or nick in the center of the molecule (J. D. Harris, J. V. Scott, B. Traynor, M. Brahic, L. Stowring, P. Ventura, A. T. Haase, and R. Peluso (1981). Virology 113, 573-583). The implications of this viral DNA structure for one unusual aspect of the lentivirus life cycle, the production of viral RNA, and virions from extrachromosomal DNA are discussed (J. D. Harris, H. Blum, J. Scott, B. Traynor, P. Ventura, and A. T. Haase (1984). Proc. Natl. Acad. Sci. USA 81, 7212-7215).
Subject(s)
DNA, Viral/biosynthesis , Visna-maedi virus/genetics , Animals , Centrifugation, Density Gradient , Choroid Plexus/microbiology , Cloning, Molecular , DNA/analysis , DNA Restriction Enzymes , Electrophoresis, Agar Gel , Nucleic Acid Hybridization , Protein Biosynthesis , SheepABSTRACT
In slow infections caused by scrapie and other unconventional agents, and in Alzheimer's disease (AD), the formation of neuritic plaques and the increase in astrocytes and astrocyte-specific protein, glial fibrillary acidic protein (GFAP), are pathological changes common to both conditions. With the rationale that these parallels imply convergent pathogenetic mechanisms, we identified a gene whose expression increases in both. We now report the results of a more extensive analysis of this gene and show that by sequence analysis it is highly homologous and likely identical to GFAP. GFAP mRNA accumulates late in the course of scrapie in subpial and periventricular astrocytes and in cells in foci in the hippocampus. The increased abundance of GFAP mRNA is accompanied by an increase in the corresponding protein. GFAP mRNA is localized by in situ hybridization to the cell body and processes of astrocytes. In AD, the latter pattern predominates, consistent with induction of GFAP mRNA in the sites of synthesis in glial processes in the neuritic plaque.
Subject(s)
Alzheimer Disease/pathology , Astrocytes/pathology , Genes, Viral , Glial Fibrillary Acidic Protein/genetics , RNA, Messenger/genetics , Scrapie/pathology , Alzheimer Disease/genetics , Animals , Base Sequence , Blotting, Northern , Cricetinae , Glial Fibrillary Acidic Protein/analysis , Humans , Mice , Molecular Sequence Data , Nucleic Acid Hybridization , RNA, Messenger/analysis , Scrapie/geneticsABSTRACT
BACKGROUND: Tumor angiogenesis is essential for solid tumor growth. Yet, the importance of any particular factor in neoplastic proliferation is poorly defined. This study examines the clinical significance of increased expression of one of the angiogenic factors, vascular endothelial growth factor (VEGF), in early stage ovarian carcinoma. METHODS: Tumor specimens from 68 patients with International Federation of Gynecology and Obstetrics Stage I and II ovarian carcinoma were evaluated for VEGF expression. Antisense and corresponding sense (control) RNA probes were transcribed from the pCRII construct (Invitrogen, San Diego, CA), which contained human VEGF cDNA. The antisense probe was designed to include a highly conserved region of the VEGF coding sequence and thus detect all known variants. After in situ hybridization, sections were assessed for overexpression of VEGF. RESULTS: Twenty-nine of the tumor samples overexpressed VEGF, whereas 39 specimens did not. In patients whose tumors demonstrated elevated VEGF expression, 25% were without evidence of disease recurrence at last follow-up. In contrast, 75% of the patients whose tumors did not overexpress VEGF were without evidence of disease at last follow-up (P < 0.001). Median disease free survival for the VEGF positive group was 22 months, compared with > 108 months for the VEGF negative group (P < 0.001). When borderline tumors were excluded from the survival analysis, median disease free survival for the VEGF positive group was 18 months, compared with >120 months for the VEGF negative group (P < 0.001). Other possible prognostic variables had minimal impact on survival; these included age, stage, grade, cytology, and tumor size (P > 0.05). Assignment to a high risk group, as defined by the Gynecologic Oncology Group of the National Cancer Institute, was somewhat predictive of a shorter relapse free interval (P = 0.056). In a multivariate analysis, however, only elevated VEGF expression was associated with poorer survival. CONCLUSIONS: In this analysis, patients with early stage ovarian carcinoma with increased VEGF expression had a poorer prognosis. Further study of VEGF may ultimately lead to identification of patients with high risk lesions whose tumor biology portends a worse prognosis and who therefore may benefit from aggressive adjuvant therapy.
Subject(s)
Carcinoma/pathology , Endothelial Growth Factors/analysis , Lymphokines/analysis , Neovascularization, Pathologic/pathology , Ovarian Neoplasms/pathology , Adult , Aged , Aged, 80 and over , Antisense Elements (Genetics) , Capillary Permeability , Disease-Free Survival , Female , Humans , Logistic Models , Middle Aged , Neoplasm Staging , Prognosis , RNA Probes , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
In the lymph nodes of individuals infected with human immunodeficiency virus (HIV), there is evidence that points to three kinds of virus-cell relationships. Virions may be associated with CD4+ lymphocytes that are actively producing virus or may be bound at the surfaces of follicular dendritic cells like other antigens. HIV is also harbored in CD4+ lymphocytes and monocytes/macrophages in a latent form as transcriptionally silenced provirus. To ultimately investigate in vivo these and other HIV-cell interactions that play such critical roles in the persistence of virus, immune dysregulation, and depletion, we have developed an in situ hybridization method that discriminates multiply spliced from singly or unspliced viral transcripts. In this report we describe the method and the results obtained with it in an analysis of the switch from latent to productive infection of chronically infected T lymphocytes in culture. We found with this single-cell technique that there are two subpopulations in the culture, a minor one of productively infected cells and a major one of latently infected cells in which only low levels of viral transcripts terminated close to the 5' end of the viral genome were detected. Shortly after activation of viral gene expression with phorbol ester, transcripts encoding Tat and Rev increase in abundancy in individual latently infected cells and this is followed by increases in and cytoplasmic export of singly or unspliced mRNAs encoding structural proteins. These studies provide insights into the regulation of HIV gene expression from a single-cell perspective and, from that perspective, transcript profiles of productively infected cells as a frame of reference for defining HIV-cell relationships in individual cells in tissue sections.
Subject(s)
Gene Expression Regulation, Viral , HIV/physiology , RNA, Viral/metabolism , Virus Activation , HIV/genetics , Humans , RNA, Messenger/analysis , Tumor Cells, Cultured , Virus LatencyABSTRACT
Herpesvirus gene expression can be classified into four distinct kinetic stages: latent, immediate early, early, and late. Here we characterize the kinetic class of a group of 16 Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 genes in a cultured primary effusion cell line and examine the expression of a subset of these genes in KS biopsies. Expression of two latent genes, LANA and vFLIP, was constitutive and was not induced by chemicals that induce the lytic cycle in primary effusion lymphoma (PEL) cell lines. An immediate-early gene, Rta (open reading frame 50 [ORF50]), was induced within 4 h of the addition of n-butyrate, and its 3.6-kb mRNA was resistant to inhibition by cycloheximide. Early genes, including K3 and K5 that are homologues of the "immediate-early" gene of bovine herpesvirus 4, K8 that is a positional homologue of Epstein-Barr virus BZLF1, vMIP II, vIL-6, and polyadenylated nuclear (PAN) RNA, appeared 8 to 13 h after chemical induction. A second group of early genes that were slightly delayed in their appearance included viral DHFR, thymidylate synthase, vMIP I, G protein-coupled receptor, K12, vBcl2, and a lytic transcript that overlapped LANA. The transcript of sVCA (ORF65), a late gene whose expression was abolished by Phosphonoacetic acid, an inhibitor of KSHV DNA replication, did not appear until 30 h after induction. Single-cell assays indicated that the induction of lytic cycle transcripts resulted from the recruitment of additional cells into the lytic cycle. In situ hybridization of KS biopsies showed that about 3% of spindle-shaped tumor cells expressed Rta, ORF K8, vIL-6, vMIP I, vBcl-2, PAN RNA, and sVCA. Our study shows that several KSHV-encoded homologues of cellular cytokines, chemokines, and antiapoptotic factors are expressed during the viral lytic cycle in PEL cell lines and in KS biopsies. The lytic cycle of KSHV, probably under the initial control of the KSHV/Rta gene, may directly contribute to tumor pathogenesis.
Subject(s)
Genes, Viral , Herpesvirus 8, Human/genetics , Chemokines/genetics , Gene Expression , Genes, Immediate-Early , Humans , Kinetics , RNA, Viral/analysis , Sarcoma, Kaposi/virology , Tumor Cells, CulturedABSTRACT
BACKGROUND: Human herpesvirus 8 (HHV-8) infection has been linked to the development of Kaposi's sarcoma and to rare lymphoproliferative disorders. METHODS: We used molecular methods, serologic methods, in situ hybridization, and immunohistochemical analyses to study HHV-8 infection in association with nonmalignant illnesses in three patients after transplantation. RESULTS: Primary HHV-8 infections developed in two patients four months after each received a kidney from the same HHV-8-seropositive cadaveric donor. Seroconversion and viremia occurred coincidentally with disseminated Kaposi's sarcoma in one patient and with an acute syndrome of fever, splenomegaly, cytopenia, and marrow failure with plasmacytosis in the other patient. HHV-8 latent nuclear antigen was present in immature progenitor cells from the aplastic marrow of the latter patient. Identification of the highly variable K1 gene sequence of the HHV-8 genome in both the donor's peripheral-blood cells and the recipients' serum confirmed that transmission had occurred. HHV-8 viremia also occurred after autologous peripheral-blood stem-cell transplantation in an HHV-8-seropositive patient with non-Hodgkin's lymphoma. Reactivation of the infection was associated with the development of fever and marrow aplasia with plasmacytosis; there was no evidence of other infections. HHV-8 transcripts and latent nuclear antigen were expressed in the aplastic marrow but not in two normal marrow samples obtained before transplantation. CONCLUSIONS: Primary HHV-8 infection and reactivation of infection may be associated with nonneoplastic complications in immunosuppressed patients.
Subject(s)
Bone Marrow Diseases/etiology , Disease Transmission, Infectious , Hematopoietic Stem Cell Transplantation/adverse effects , Herpesviridae Infections/transmission , Herpesvirus 8, Human/isolation & purification , Kidney Transplantation/adverse effects , Sarcoma, Kaposi/etiology , Adult , Antibodies, Viral/blood , Blood Cell Count , Bone Marrow/virology , Bone Marrow Diseases/blood , Bone Marrow Diseases/virology , Fatal Outcome , Genome, Viral , Herpesviridae Infections/etiology , Herpesviridae Infections/virology , Herpesvirus 8, Human/genetics , Herpesvirus 8, Human/immunology , Humans , Immunocompromised Host , Male , Middle Aged , Sarcoma, Kaposi/virology , Viremia/etiology , Virus ActivationABSTRACT
The behavior of two genetically different molecular clones of visna virus KV1772-kv72/67 and LV1-1KS1 was compared in vivo and in vitro. On intracerebral inoculation, clone KV1772-kv72/67 induced a similar response in five sheep as has already been reported with neurovirulent derivates of visna virus. Virus was frequently isolated from blood, cerebrospinal fluid (CSF), and lymphoid organs and induced characteristic central nervous system (CNS) lesions. A strong humoral immune response was detected by ELISA, immunoblotting, and neutralization. Six sheep infected with clone LV1-1KS1 showed a completely different picture. No virus could be isolated from blood or CSF during 6 months of infection. At sacrifice all organs were virus-negative except the CNS of one sheep. None of the six sheep developed significant neutralizing antibodies and only low titer antibodies were detected by ELISA and immunoblotting. Minimal CNS lesions were present in one sheep. The molecular clones were also tested in sheep choroid plexus cells (SCP) and macrophages. In macrophages LV1-1KS1 replicated to a significantly lower titer but induced much more cell fusion than KV1772-kv72/67. The clones replicated equally well in SCP cells. Thus, these molecular clones of visna virus, which differ only by 1% in nucleotide sequence, showed a profound difference in replication and pathogenicity both in vitro and in vivo. These results can be used to map viral genetic determinants important for host-lentivirus interactions.
Subject(s)
Macrophages/virology , Visna-maedi virus/pathogenicity , Visna/virology , Animals , Antibodies, Viral/blood , Cell Line , Macrophages/cytology , Sheep , Visna/immunology , Visna/pathology , Visna-maedi virus/immunology , Visna-maedi virus/isolation & purificationABSTRACT
The recent discovery of DNA sequences of a new human herpesvirus in Kaposi's sarcoma (KS) has fueled speculation that this virus might cause KS. The mere presence, however, of a virus in a complex multicellular tumor like KS could just as well be construed as evidence of a passenger agent. We sought stronger evidence linking the KS-associated herpesvirus (KSHV) to tumor formation by using in situ hybridization to investigate the specificity, constancy, and timing of KSHV gene expression in KS tumor cells. Here we document expression of a 700-nucleotide viral RNA in every KS tumor examined, from the earliest histologically recognizable stage to advanced tumors in which the vast majority of identifiable spindle tumor cells contain this transcript. Two other KSHV RNAs were also detected in a smaller fraction of the tumor cells in all but the earliest lesion. These viral RNAs were expressed to relatively low levels in this subset; because one of these RNAs encodes a major viral capsid protein, these cells may be producing KSHV. We did not find these KSHV genes expressed in a variety of other tumors and proliferative processes, but we did detect viral gene expression in prostatic tissue, supporting a possible mechanism for sexual transmission of KSHV. The close relationship between KS and KSHV gene expression is consistent with the hypothesis that KSHV is directly involved in the etiology and pathogenesis of KS.
Subject(s)
AIDS-Related Opportunistic Infections/virology , Herpesvirus 8, Human/isolation & purification , RNA, Viral/analysis , Sarcoma, Kaposi/virology , AIDS-Related Opportunistic Infections/pathology , Gene Expression , Herpesvirus 8, Human/genetics , Humans , Sarcoma, Kaposi/pathology , Tongue/pathologyABSTRACT
Infectious molecular clones have been isolated from two maedi-visna virus (MVV) strains, one of which (KV1772kv72/67) is an antigenic escape mutant of the other (LV1-1KS1). To map the type-specific neutralization epitope, we constructed viruses containing chimeric envelope genes by using KV1772kv72/67 as a backbone and replacing various parts of the envelope gene with equivalent sequences from LV1-1KS1. The neutralization phenotype was found to map to a region in the envelope gene containing two deletions and four amino acid changes within 39 amino acids (positions 559 to 597 of Env). Serum obtained from a lamb infected with a chimeric virus, VR1, containing only the 39 amino acids from LV1-1KS1 in the KV1772kv72/67 backbone neutralized LV1-1KS1 but not KV1772kv72/67. The region in the envelope gene that we had thus shown to be involved in escape from neutralization was cloned into pGEX-3X expression vectors, and the resulting fusion peptides from both molecular clones were tested in immunoblots for reactivity with the KV1772kv72/67 and VR1 type-specific antisera. The type-specific KV1772kv72/67 antiserum reacted only with the fusion peptide from KV1772kv72/67 and not with that from LV1-1KS1, and the type-specific VR1 antiserum reacted only with the fusion peptide from LV1-1KS1 and not with that from KV1772kv72/67. Pepscan analysis showed that the region contained two linear epitopes, one of which was specific to each of the molecularly cloned viruses. This linear epitope was not bound by all type-specific neutralizing antisera, however, which indicates that it is not by itself the neutralization epitope but may be a part of it. These findings show that mutations within amino acids 559 to 597 in the envelope gene of MVV virus result in escape from neutralization. Furthermore, the region contains one or more parts of a discontinuous neutralization epitope.