The AIDS lentivirus connection.
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Gene Expression Regulation↗
Biomedical subjects
A T Haase
Publications and source records attributed to A T Haase.
Speculations on the role of transmissible agents in the pathogenesis of Alzheimer's disease.
Unconventional agents and conventional viruses provide model systems to investigate the pathogenesis of Alzheimer's disease (AD). The essay which follows examines the hypothetical role of herpes simplex in AD and presents some generally applicable experimental approaches to detecting genes in brain tissues. The concluding section, on parallels between AD and diseases of the brain caused by unconventional viruses, defines strategies for isolating genes related to pathology.
Aged↗
The pathogenesis of slow virus infections: molecular analyses.
Slow infections raise some novel issues in understanding the pathogenesis of viral diseases that have been increasingly addressed at the molecular level with in situ hybridization. These developments are reviewed as the framework for discussion of the realized and potential impact of these investigations for major neurological afflictions of humans and for AIDS.
Acquired Immunodeficiency Syndrome↗
Analysis of viral infections by in situ hybridization.
In situ hybridization provides a versatile means of analysis of the life cycles of viruses in single cells. This kind of analysis in "real life" situations has provided considerable insight into the spread of viruses, mechanisms of tissue damage by viruses, and virus-host cell interactions in chronic diseases. In this article I describe refinements in technology underlining these advances, especially developments that have made the technique such a sensitive and quantitative one. I also describe a method for combined macroscopic and microscopic in situ hybridization, new assays for the simultaneous detection of genes and gene products in a single cell, and a double-label in situ hybridization technique. These methods have already proved useful in analyzing the molecular ecology of viral infections, and should find wide application to problems of genetic regulation in many other systems.
Chronic Disease↗
Synthesis in cell culture of the gapped linear duplex DNA of the slow virus visna.
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).
Animals↗
Combined macroscopic and microscopic detection of viral genes in tissues.
A hybridization technique has been devised for detecting and quantitating viral genes in tissues that combines macroscopic and microscopic analyses in the same section. The method is based on dual labeling virus-specific probes with 125I and 35S to generate signals that can be detected both with X-ray films and nuclear track emulsions. The regions of increased hybridization evident in the X-ray film serve as a guide to the portion of the section that warrants microscopic examination. Detection of viral RNA in tissues with visna virus and viral DNA with hepatitis B virus are illustrated, and potential applications of this technique in virology and other disciplines are discussed.
Animals↗
Detection of two viral genomes in single cells by double-label hybridization in situ and color microradioautography.
Double labeling and color microradioautography were used in a new method of hybridization in situ to identify different genes in individual cells. The method is based on the unequal penetration of 3H and 35S into two layers of nuclear track emulsion separated by a thin barrier film. Hybridization of a 35S-labeled probe specific for one kind of gene results in silver grains over cells in both layers of emulsion; a 3H-labeled probe for a second gene provides grains only in the first layer of emulsion. Silver grains are converted to magenta-colored grains in the first layer and to cyan-colored grains in the second to facilitate enumeration of grains in each layer. This technique should be widely applicable in analyses of differential gene expression in single cells or in discrete populations of cells.
Autoradiography↗
Quantitative analysis of visna virus replication in vivo.
Visna virus is the prototype of the lentivirus subfamily, a group of nontransforming retroviruses that cause slow infections in sheep and goats. In nature, virus is acquired primarily by the respiratory route and subsequently spreads to several organ systems. These viruses persist for years in their hosts despite a vigorous immune response because of a block in virus gene expression. This report continues the analysis of persistence in vivo, and specifically examines a gene dosage hypothesis that has been advanced as an explanation for the decrease in transcription and virus production in the cells in infected animals. For this analysis a new pulmonary model has been developed that, in conjunction with quantitative in situ hybridization, provides an opportunity to examine in animals the molecular events that occur in the course of the viral life cycle. We establish the feasibility of such a longitudinal analysis in vivo, document restriction in gene expression in alveolar macrophages and provide evidence that this restriction cannot be accounted for simply by gene dosage. The approach illustrated with visna should be of general applicability to other dynamic and molecular investigations of virus infection.
Animals↗
Detection of visna virus antigens and RNA in glial cells in foci of demyelination.
Visna is a slow virus infection of sheep in which the characteristic pathological change is demyelination in foci of inflammation. The latter is thought to be the result of an immunopathological process directed against cellular and antigenic targets that have been difficult to define because of restricted viral gene expression. A new simultaneous detection assay is used to demonstrate viral RNA in cells identified unambiguously as oligodendrocytes and astrocytes. These cells were found in inflammatory foci. With a new strain of virus that causes a rapid form of visna in Icelandic sheep, viral antigens were demonstrated in cells in the inflammatory lesions. These findings are consistent with the postulated immunopathological mechanism of demyelination: cells that maintain intact myelin sheaths in the central nervous system are destroyed by the inflammatory response to viral antigens expressed in these cells.
Animals↗
Natural history of restricted synthesis and expression of measles virus genes in subacute sclerosing panencephalitis.
Subacute sclerosing panencephalitis (SSPE) is a slow infection caused by measles virus in which several years separate recovery from typical acute measles and the development of a slowly progressive neurological disease. We have investigated replication of measles virus in brain tissue obtained after the onset of neurological disease and in the terminal phase. With a hybridization tomographic technique that combines in situ hybridization with macroradioautographic screening of large areas of tissue, we analyzed the spatial and temporal distribution of virus genes in vivo, using region- and strand-specific probes for the nucleocapsid and matrix genes. We show that early in the course of SSPE there is a global repression in the synthesis and expression of the genome. In the final stage of SSPE most infected cells still have depressed levels of plus- and minus-strand viral RNA and contain nucleocapsid protein but lack matrix protein. These findings provide further evidence for a unified view of slow infections of the nervous system, where the general constraints on virus gene expression provide an explanation for persistence of virus in the face of the host's immune response, and the slow evolution of pathological change. In the final phases of SSPE the more specific block in virus replication accounts for the cell-associated state of the virus and the difficulty in virus isolation.
Brain↗
Laboratory diagnosis of hepatitis B virus infection by nucleic acid hybridization analyses and immunohistologic detection of gene products.
The laboratory diagnosis of hepatitis B virus (HBV) infection is based on two types of analyses: (i) Immunochemical detection in serum or liver of viral antigens or their respective antibodies; (ii) Demonstration of HBV DNA in serum or liver by nucleic acid hybridization techniques (dot and transfer blots, in situ hybridization). These hybridization analyses have proven to be more sensitive than immunochemical assays for the detection of viral markers and have revealed HBV DNA in serum and liver of patients without serologic evidence of HBV infection. With appropriate controls, probes and hybridization conditions the hybridization assays are exquisitely specific. They are particularly useful in studying the molecular biology of the virus, its extrachromosomal replication or integration into the cellular genome and transformation of the infected hepatocytes. Further, recent studies have identified new target cells for HBV in liver (bile duct epithelial cells, endothelial cells, smooth muscle cells in blood vessel walls), spleen, bone marrow and white blood cells. While serological and immunohistochemical assays are still the basis of routine laboratory diagnosis of HBV infection, nucleic acid hybridization provides novel information useful in establishing the etiology of certain cases of viral hepatitis without serologic evidence for known viral agents (so-called non-A, non-B hepatitis), for demonstrating active viral replication in infected individuals, and for selecting and monitoring patients undergoing antiviral therapy.
DNA, Viral↗
Molecular pathogenesis of hepatitis B virus infection: simultaneous detection of viral DNA and antigens in paraffin-embedded liver sections.
Hepatitis B virus (HBV) DNA and viral antigens were simultaneously identified by immunohistochemical staining of formalin-fixed, paraffin-embedded liver sections followed by in situ hybridisation. In the developed radioautographs, silver grains indicate the location of viral DNA in the cell and the immunohistochemical stain marks sites of accumulation of viral antigen. In liver from a patient with chronic active hepatitis serologically positive for hepatitis B surface antigen and e antigen (HBsAg/HBeAg) viral nucleotide sequences, representing actively replicating DNA species, were demonstrated predominantly in the cytoplasm. Viral core antigen (HBcAg) was expressed in the liver cell nuclei. HBcAg was not detectable in most hepatocytes with high levels of viral replication. Conversely, most liver cells in which HBcAg was found did not contain replicating HBV. HBcAg and replicating viral DNA species were not detectable in hepatocytes undergoing pathological changes, such as ground glass cells. Because no pathological changes could be identified either in hepatocytes with high levels of HBV replication or expression of nuclear HBcAg, the liver cell damage in this patient with chronic hepatitis B was presumably induced by other mechanisms. The simultaneous observation of viral DNA, antigens, and pathological changes at the single cell level and their correlation with clinical findings should contribute to the understanding of the molecular mechanisms underlying HBV-induced liver cell injury.
Antigens, Viral↗
Detection of viral genes and their products in chronic neurological diseases.
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Animals↗
Asymmetric replication of hepatitis B virus DNA in human liver: demonstration of cytoplasmic minus-strand DNA by blot analyses and in situ hybridization.
In situ and blot hybridization techniques have been used with strand- and region-specific probes to characterize the forms of hepatitis B virus (HBV) DNA in the liver of a patient with chronic active hepatitis B. The hepatocytes contain a heterogeneous population of rapidly migrating DNA species in the 0.5-1.4 kb position that are localized predominantly in the cytoplasm and are of minus-strand polarity. The findings indicate that the replication is asymmetric, with separate pathways for plus- and minus-strand synthesis of HBV DNA; that viral DNA synthesis is initiated at a site near the nick in the minus strand of virion DNA; and that actively replicating forms of HBV DNA can be identified at the cellular level by in situ hybridization.
Cytoplasm↗
Simultaneous in situ detection of viral RNA and antigens.
We have combined in situ hybridization and immunocytochemistry to detect RNA and proteins in the same cell. We envision a wide range of applications for this method, and, as one example, we show that viral genomes and capsid antigens can be detected simultaneously in paraffin sections of the central nervous system of mice infected with Theiler's virus, the causative agent of a slow demyelinating disease.
Animals↗
Detection by hybridization of viral infection of the human central nervous system.
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Brain↗
Detection of hepatitis B virus DNA in hepatocytes, bile duct epithelium, and vascular elements by in situ hybridization.
A radiolabeled probe specific for hepatitis B virus (HBV) nucleotide sequences was hybridized in situ to liver tissue from three patients with chronic hepatitis B. The HBV genome was detected not only in infected hepatocytes but also in bile duct epithelial cells, endothelial cells, and smooth muscle cells. These findings extend the known host cell range for HBV, suggest new mechanisms of viral dissemination, and illustrate the usefulness of in situ hybridization in the study of pathogenesis of HBV infection.
Animals↗
Visna DNA synthesis and the tempo of infection in vitro.
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Animals↗