Mechanisms of escape of visna lentiviruses from immunological control.
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Animals↗
Biomedical subjects
O Narayan
Publications and source records attributed to O Narayan.
Lentivirus-induced arthritis. Chronic disease caused by a covert pathogen.
Lentiviruses are exogenous, nononcogenic retroviruses that cause persistent infections in monocytes and macrophages. Among the clinical manifestations of such infections in sheep and goats is a slowly progressive arthritis, primarily involving the carpal joints of adult animals. Initially, clinical disease begins as synovitis; this progresses to involve surrounding connective tissues and osseous structures as inflammation increases and progresses. Inflammatory cells include macrophages, lymphocytes, and plasma cells. Macrophages and monocytes are the only cells that are infected with virus and in the inflamed joint may represent 50 per cent of the cells in the synovial fluid. However, only a small number of these cells are infected. Cytotoxic T lymphocytes predominate over helper T lymphocytes in the synovial fluid. The role of these cells in the pathogenesis of disease is not currently known. The pathogenesis of disease caused by these lentiviruses is related to the infection of the monocyte-macrophage cells in the animal. This event, along with virus-specific factors, render the host incapable of eliminating the virus. Despite persistence of the virus, viral replication is maintained at a tightly restricted level at all times in the infected animal. This is achieved by factors that regulate the maturation of monocytes. Basically, the infection is latent in monocytes and its precursors and becomes more productive as the cells mature. The maturing infected macrophage presents a portion of the viral proteins that it synthesizes on its cell surface in close association with class II major histocompatibility complex antigens. Lymphocytes react with this cell and produce interferon. This lymphokine induces further expression of Ia antigens. This feedback loop of Ia expression, together with persistence of the virus, maintains macrophages in a constant state of antigen presentation. This forms the trigger for the inflammatory condition that eventually leads to the degenerative lesions seen in the joint.
Animals↗
Cytomegalovirus but not human T lymphotropic virus type III/lymphadenopathy associated virus detected by in situ hybridisation in retinal lesions in patients with the acquired immune deficiency syndrome.
Paraffin sections of retinal tissue from five patients who died from the acquired immune deficiency syndrome (AIDS) and retinopathy were examined by in situ hybridisation experiments with deoxyribonucleic acid (DNA) labelled with sulphur-35 of lentivirus, human T lymphotropic virus type III/lymphadenopathy associated virus (HTLV-III/LAV), and cytomegalovirus. HTLV-III/LAV ribonucleic acid (RNA) was not detected in any of the tissue sections. Cytomegalovirus RNA was identified, however, in three of the five patients. Retinopathy induced by cytomegalovirus may thus be one of the many syndromes potentiated by the immunosuppression caused by HTLV-III/LAV.
Acquired Immunodeficiency Syndrome↗
Tropism of sheep lentiviruses for monocytes: susceptibility to infection and virus gene expression increase during maturation of monocytes to macrophages.
Visna lentiviruses have a natural tropism for cells of the macrophage lineage of sheep and goats, but virus replication in these cells in vivo is restricted so that only small quantities of virus are produced. One restricting factor suggested in previous studies is that virus replication is dependent on the maturity of the cells: the more mature the cell, the less restrictive the replication of the virus. Since monocytes in peripheral blood are precursors of macrophages, we investigated the effect of cell maturation on virus replication under limited control conditions in vitro by inoculating blood leukocytes with virus and retarding the maturation of monocytes to macrophages during cultivation in serum-free medium. Using enzyme markers that identified the cells in their resting monocytic stage (peroxidase) and mature macrophage stage (acid phosphatase) along with quantitative in situ hybridization and immunocytochemistry with viral reagents to trace the efficiency of virus replication, we correlated virus replication with cell maturation. Only a few monocytes were susceptible to infection, and virus replication did not extend beyond a low level of transcription of viral RNA. In the acid phosphatase-positive, maturing macrophage, susceptibility of the cells to infection was increased and virus replication was greatly amplified to the level of translation of viral polypeptides. However, virus maturation was delayed by 3 days until further cell maturation had occurred. Thus, the entire life cycle of the virus, from its attachment to the target cell to its maturation in the cell, was dependent on the level of maturation/differentiation of the monocytic cell.
Animals↗
Sequence homology between cloned caprine arthritis encephalitis virus and visna virus, two neurotropic lentiviruses.
Caprine arthritis encephalitis virus (CAEV) is an exogenous, nononcogenic retrovirus which causes neurological disease and crippling arthritis in goats. A complete CAEV genome was cloned from unintegrated viral DNA in two fragments of 9.4 and 0.4 kilobases in length, respectively. The biological activity of these clones was tested by ligation of the fragments followed by transfection onto goat synovial membrane cells; infectious virus was recovered. Cloned CAEV and visna virus, a related neurotropic virus of sheep, were compared by heteroduplex and molecular hybridization analyses. These data demonstrated that the greatest overall conservation of nucleotide sequences occurred in the gag and pol gene regions and two smaller regions, sor and the putative tat gene. The region of greatest divergence occurred in the env gene and, in particular, was localized primarily in the region coding for the glycosylated outer membrane protein. These findings and the recently demonstrated genetic relationship of visna virus, CAEV, and human T-cell lymphotropic virus type III, the etiologic agent of the acquired immune deficiency syndrome, may have important implications concerning the biological properties of these related viruses for human and veterinary medicine.
Animals↗
Neutralizing antibodies to visna lentivirus: mechanism of action and possible role in virus persistence.
Lentiviruses are nononcogenic retroviruses that cause persistent infections and slowly progressive diseases. Visna virus, a lentivirus of sheep, persists in cells of the macrophage lineage despite the presence of neutralizing antibodies in the animal. These antibodies are measured by prevention of virus replication in sheep fibroblast cell cultures. In this study we have compared the antiviral properties of the antibodies in sheep fibroblast and macrophage cell cultures, the latter being more relevant to infection in the animal. Using infectivity assays, binding of radiolabeled virus to cell membranes, cellular processing of labeled virus into acid-precipitable and acid-soluble components, and in situ hybridization of viral nucleic acid, we show that the antibodies prevented virus replication in both fibroblasts and macrophages. However, the site of neutralization differed between the two cell types. In fibroblasts, the site of virus neutralization was at the cell membrane, when the antibodies prevented virus attachment. In macrophages, virus incubated with the antibodies was phagocytized rapidly, followed by uncoating of the virions. However, virus RNA was not transcribed. Despite this ability of the antibodies to abort virus replication in macrophages, the kinetics of binding of the antibodies to the virus was much slower than the binding of virus to the macrophages. Therefore, persistent virus replication in immune sheep may be the result of virus spreading from macrophage to macrophage before the agent can be neutralized by antibodies in the plasma.
Adsorption↗
Restricted replication of lentiviruses. Visna viruses induce a unique interferon during interaction between lymphocytes and infected macrophages.
Lentivirus infections are characterized by a persistent, restricted type of virus replication in tissues. Using sheep and goat lentiviruses, whose target cells in vivo are macrophages, we explored virus-host cell interactions to determine whether an interferon (IFN) is produced during virus replication in vivo which causes restricted replication. We show that the lentiviruses were incapable of inducing IFN directly in any infected cell, including macrophages and lymphocytes. However, after infection with these viruses, sheep and goat macrophages acquired a factor that triggered IFN production by T lymphocytes. Only sheep/goat lentiviruses were capable of inducing the factor and, although these viruses replicated productively in various cell cultures of the natural host animal, only infected macrophages developed the IFN-inducing factor. The factor was produced continuously and was strictly cell associated, requiring direct contact with lymphocytes. The lymphocytes responded with a single, sudden release of IFN beginning 7 h after cocultivation and reaching peak values at 48 h, after which they ceased production and became refractory. IFN production was not immunologically specific and did not require histocompatibility between donors of the two cell types. The IFN is a nonglycosylated protein of molecular weight 54,000-64,000, and is stable to heat and acid treatments. These findings identify a unique IFN and a new method for virus induction of IFN. The novel two-stage process of induction provides a mechanism for local amplification and continuity of production of IFN in vivo. This is compatible with infection in the animal whose lentivirus-induced pathologic lesions consist of accumulations of lymphocytes and infected macrophages in target tissues.
Animals↗
Persistent expression of Ia antigen and viral genome in visna-maedi virus-induced inflammatory cells. Possible role of lentivirus-induced interferon.
In this study we investigated the pathogenesis of the lymphoproliferative response in the chronic-active visna maedi virus-induced inflammatory lesions. Viral RNA expression was confined to macrophages, but only in tissues showing inflammatory lesions. A persistent and high level of Ia antigen expression was seen in macrophage-like cells in the inflammatory lesions, and the amounts of viral RNA and Ia expression were closely correlated. A small subpopulation of macrophages contained both viral RNA and Ia antigen, and these were found in greatest number in the lung. In vitro experiments showed that a lentivirus-induced interferon (LV-IFN) could induce Ia antigens in normal sheep spleen and lymph node cells as well as in a transformed sheep macrophage cell line. Ia antigen expression in macrophages was transient in the absence of a continuing IFN stimulus and persisted for at least 2 wk in the presence of LV-IFN. LV-IFN also restricted viral replication in macrophages. It is suggested that LV-IFN induced by the inflammatory cells in visna-maedi lesions may induce Ia antigen expression in macrophages, thereby indirectly causing the lymphoproliferative response and restricted virus replication.
Animals↗
cis- and trans-acting transcriptional regulation of visna virus.
Visna virus is a pathogenic lentivirus of sheep that is related to human T-cell lymphotropic virus type III (HTLV-III), the probable etiologic agent of the acquired immune deficiency syndrome (AIDS). The transcriptional activity of visna virus promoter and enhancer sequences was studied by means of an assay based on the transient expression of the bacterial gene chloramphenicol acetyltransferase (CAT). The results suggest that the high level of expression of visna virus is due in part to cis-acting enhancer sequences that give the viral promoter a high level of transcriptional activity. In addition, the rate of transcription from the visna virus promoter situated in a plasmid expressing the CAT gene was much greater in infected than uninfected cells. This phenomenon of trans-acting transcriptional activation may involve either virally or cellularly encoded factors.
Acetyltransferases↗
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.
Base Sequence↗
The mammary gland as a target organ for infection with caprine arthritis-encephalitis virus.
A herd of 15 dairy goats with a previous history of neurological disease in kids was screened for caprine arthritis-encephalitis (CAE) virus infection. Virus was isolated from the PBL of all animals as well as milk from lactating does. All animals had antibody detected by ELISA and gel immuno-diffusion. Two animals from this herd were examined for responses to infection. One was an adult female with poor milk production and the other was a 3-month-old female kid which developed acute neurological disease. Both animals had a chronic mastitis compatible with a viral aetiology, and CAE virus was isolated from the mammary tissue of both. The mammary gland must thus be considered an important target organ for virus infection and dissemination.
Animal Diseases↗
A double labeling technique for performing immunocytochemistry and in situ hybridization in virus infected cell cultures and tissues.
This report describes a combined immunocytochemical and in situ hybridization procedure which allows visualization of cellular or viral antigens and viral RNA in the same cell. Cultures infected with visna or measles virus were fixed in periodate-lysine-paraformaldehyde-glutaraldehyde, stained by the avidin-biotin-peroxidase technique using antibodies to viral or cellular proteins and then incubated with radiolabeled specific DNA probes (in situ hybridization). The immunoperoxidase stain was preserved through the hybridization procedure. Nonspecific 'sticking' of probes over peroxidase stained cells was prevented by incorporation of 0.1% Triton X-100 into the hybridization solution and the post-hybridization washes. The in situ hybridization signal (silver grains/cell) on peroxidase-stained cells was reduced relative to hybridization with unstained cells. The double labeling technique was also applied to sections of paraffin-embedded tissues from a sheep infected with visna virus and mice infected with the HNT strain of measles virus. Visna virus RNA was detected in immunocytochemically identified macrophages in the synovium. A greater number of these cells had viral RNA than had viral protein. In measles virus-infected brains viral RNA was detected only in cells with viral protein. This technique provides a new approach to the study of viral pathogenesis by: identifying the types of cells which are infected in the host and identifying points of blockade in the virus life cycle during persistent infections.
Animals↗
Efficiency of in situ hybridization as a function of probe size and fixation technique.
In an attempt to improve fixation technique for viral RNA detection by in situ hybridization, we have quantitatively compared the hybridization signal obtained when measles virus or visna virus infected cell cultures were fixed with eight different fixatives and hybridized with 35S-labeled virus-complementary DNA probes of several size ranges. Small probes (mean length, 70 bases) gave higher signals than larger probes (mean lengths 140, 350, and 780 bases) with all fixatives. This increase in signal was minimal with acetic ethanol or formalin, but was dramatic with fixatives containing glutaraldehyde; with these fixatives the signals with small probes were 6.5- to 22-fold greater than with large probes. The highest signals were obtained with periodate-lysine-paraformaldehyde-glutaraldehyde (PLPG) fixed cells hybridized with small probes, and were 1.5- to 6.7-fold greater than those obtained with the commonly used fixative acetic ethanol. PLPG and other glutaraldehyde based fixatives also greatly improved the preservation of cellular morphology compared to acetic ethanol.
Acetates↗
Slow, persistent replication of lentiviruses: role of tissue macrophages and macrophage precursors in bone marrow.
Lentiviruses, as exemplified by visna virus of sheep, are nononcogenic retroviruses that cause slowly progressive diseases after prolonged periods of incubation. Earlier studies on visna have shown that the long incubation period of the disease is associated with constant production of minimal quantities of virus in tissues, whereas virus could be obtained by culturing monocytes and macrophages from explants of lymphatic tissues and inflamed organs. In this study the role of macrophages in lentivirus infection was explored using two sheep that were intrabronchially inoculated with virus. When sections of paraffin-embedded tissue, processed by a recently described technique which combines immunocytochemistry for the identification of macrophages and in situ hybridization for identification of viral nucleic acid, were examined, we found that virus replication is associated almost exclusively with infection in selected populations of macrophages in the interalveolar region of the alveoli, in inflammatory exudate cells in the lung, in lymph nodes, and in the spleen. Although large numbers of alveolar macrophages had viral RNA, few of these cells produced virus. While this minimally productive type of viral replication provides an explanation for the slow pace of the infection, restricted replication in terminally differentiated, short-lived macrophages does not explain persistent virus replication in the animal. With the discovery of clusters of infected macrophage precursors in the bone marrow, a mechanism for persistence was found. The macrophage precursor cells provide an important missing link in the virus-target-cell circuit and may be the reservoir of latently infected cells which perpetuate lentivirus infections in both animals and humans.
Animals↗
Chaos in a periodically forced Lorenz system.
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Journal Article↗
Susceptibility of skeletal muscle to Coxsackie A2 virus infection: effects of botulinum toxin and denervation.
Coxsackie A viruses can infect denervated but not innervated mature skeletal muscles. The role of synaptic transmission in preventing susceptibility to Coxsackievirus infection was studied by surgically denervating leg muscles of mice or injecting the muscles with botulinum toxin to block quantal release of acetylcholine. Control muscles were injected with heat-inactivated toxin. Subsequent injection of Coxsackie A2 virus resulted in extensive virus replication and tissue destruction in the denervated and botulinum toxin-treated muscles, while the control muscles showed only minimal changes. This suggests that the susceptibility of skeletal muscle to Coxsackievirus infection is regulated by synaptic transmission.
Animals↗
Lack of neutralizing antibodies to caprine arthritis-encephalitis lentivirus in persistently infected goats can be overcome by immunization with inactivated Mycobacterium tuberculosis.
The pathogenesis of the persistent progressive diseases of sheep (visna-maedi) and goats (arthritis-encephalitis) is dependent on continuous replication of the causative lentiviruses. One subgroup of these viruses, Icelandic visna virus, accomplishes this form of replication by undergoing antigenic mutation. Mutant viruses arising late in the infection escape neutralization by antibodies directed to the parental virus. In contrast, we show here that viruses obtained from persistently infected sheep and goats with natural disease in this country do not induce virus-neutralizing antibodies, although antibodies to virus core proteins were produced. The lack of neutralizing antibodies was not overcome by hyperimmunization of animals with concentrated preparations of live or inactivated virus. Thus, failure to produce these specific antibodies was not due to lack of sufficient antigen or interference with the immune response because of the ability of these viruses to infect macrophages. The hyporesponsive state, however, was overcome by immunization of animals with virus and large amounts of inactivated Mycobacterium tuberculosis. Induction of agglutinating and neutralizing antibodies by this method was probably due to a unique form of antigen processing by macrophages activated by M. tuberculosis. Neutralizing antibodies were produced for the first time against the caprine arthritis-encephalitis virus by this method. These antibodies have similar biological properties to those induced by Icelandic visna virus. They belong to the immunoglobulin G1 subclass, they are effective against a narrow range of caprine arthritis-encephalitis viruses, and they identify (for the first time) antigenic variants among these caprine agents.
Animal Diseases↗
Genetic variation among lentiviruses: homology between visna virus and caprine arthritis-encephalitis virus is confined to the 5' gag-pol region and a small portion of the env gene.
Visna virus of sheep and arthritis-encephalitis virus of goats are serologically related but genetically distinct retroviruses which cause slowly progressive diseases in their natural hosts. To localize homologous regions of the DNAs of these two viruses, we constructed a physical map of caprine arthritis-encephalitis virus DNA and aligned it with the viral RNA. Cloned probes of visna virus DNA were then used to localize regions of homology with the caprine arthritis-encephalitis virus DNA. These studies showed homology in the 5' region of the genome encompassing U5 and the gag and pol genes and also in a small region in the env gene. These findings correlate with biological data suggesting that the regions of the DNA which are homologous may be responsible for virus group characteristics such as the closely related virus core antigens. Regions which did not show homology such as large sections in the env gene may represent unique sequences which control highly strain-specific characteristics such as the neutralization antigen and specific cell tropisms.
Animals↗