Janice E. Clements

Slow virus replication: The role of macrophages in the persistence and expression of visna viruses of sheep and goats

Lentiviruses of sheep and goats cause slowly progressive diseases of the central nervous system (visna), lungs (maedi) and joints (arthritis) in their natural hosts. However, the virus target cell(s) in these diseases are still unknown. In this report, using laboratory-adapted Icelandic visna virus and several field strains recently obtained from sheep and goats with natural disease in the U.S.A., we show that macrophages became persistently infected when inoculated in culture. Furthermore, macrophages were an invariable source of virus from experimentally and naturally infected animals. Virus-producing macrophages developed minimal cytopathic changes and virus assembly occurred mainly intracellularly, accumulating in cytoplasmic vacuoles. In contrast to macrophages, sheep choroid plexus fibroblasts developed syncytial cytopathic changes after inoculation and virus maturation occurred at the cell surfaces. Replication of the Icelandic virus was highly productive in this system but that of the field viruses was very inefficient. In some cases these agents failed to replicate in the fibroblasts and no cytopathic effect occurred. This block in the field virus replication was, however, overcome when infected nonproducer fibroblasts were co-cultivated with macrophages. In these cases, virus production with attendant cytopathic effect in the fibroblasts required the continuous presence of macrophages because the cells reverted to a non-productive state when separated from macrophages and became productive again when subcultures were added to new macrophages. The roles of the macrophage as a virus target cell and virus inducer in the virus-macrophage-fibroblast interactions are discussed with inferences to the well-known phenomenon of restricted virus replication in infected animals and the immunopathological aspects of the diseases.

Biochemical Characterization of the Virus Causing Leukoencephalitis and Arthritis in Goats

Goat leukoencephalitis-arthritis virus (GLV) has the density of a retrovirus in sucrose and contains an endogenous RNA-dependent DNA polymerase (reverse transcriptase). The virion reverse transcriptase utilizes the synthetic RNA template poly(rA). (dT)12 but not the synthetic DNA template poly(dA). (dT)12. A high mol. wt. RNA similar in size to visna virus RNA was isolated from 3H-uridine-labelled virions. The major structural protein of GLV has the same mol. wt. as that of visna virus. From these data the GLV appears to be a retrovirus.

Human Immunodeficiency Virus Infection of Human Astrocytes Disrupts Blood–Brain Barrier Integrity by a Gap Junction-Dependent Mechanism

HIV infection of the CNS is an early event after primary infection, resulting in neurological complications in a significant number of individuals despite antiretroviral therapy (ART). The main cells infected with HIV within the CNS are macrophages/microglia and a small fraction of astrocytes. The role of these few infected astrocytes in the pathogenesis of neuroAIDS has not been examined extensively. Here, we demonstrate that few HIV-infected astrocytes (4.7 ± 2.8% in vitro and 8.2 ± 3.9% in vivo) compromise blood–brain barrier (BBB) integrity. This BBB disruption is due to endothelial apoptosis, misguided astrocyte end feet, and dysregulation of lipoxygenase/cyclooxygenase, BKCa channels, and ATP receptor activation within astrocytes. All of these alterations in BBB integrity induced by a few HIV-infected astrocytes were gap junction dependent, as blocking these channels protected the BBB from HIV-infected astrocyte-mediated compromise. We also demonstrated apoptosis in vivo of BBB cells in contact with infected astrocytes using brain tissue sections from simian immunodeficiency virus-infected macaques as a model of neuroAIDS, suggesting an important role for these few infected astrocytes in the CNS damage seen with HIV infection. Our findings describe a novel mechanism of bystander BBB toxicity mediated by low numbers of HIV-infected astrocytes and amplified by gap junctions. This mechanism of toxicity contributes to understanding how CNS damage is spread even in the current ART era and how minimal or controlled HIV infection still results in cognitive impairment in a large population of infected individuals.

Sustained antibody-dependent cell-mediated cytotoxicity (ADCC) in SIV-infected macaques correlates with delayed progression to AIDS

Although several in vitro lines of evidence support the potential power of antibody-dependent cell-mediated cytotoxicity (ADCC) in controlling HIV infection, the role of ADCC in the pathogenesis of HIV infection in vivo remains uncertain. There are few studies to date that longitudinally determine the plasma ADCC activity in HIV-infected subjects. We sought to establish an SIV/macaque model to perform such a longitudinal study. In the rhesus macaque cohort studied here, three of five macaques (designated Group 1) maintained higher plasma ADCC activity for at least 1 year after inoculation with SIV/17E-Br. The ADCC activity of the two remaining macaques (Group 2) fell 12 weeks after inoculation. There were also differences in longitudinal measurements of anti-SIV envelope IgG titers and CD4 counts. Group 1 macaques maintained higher antienvelope IgG titers and higher CD4(+) T cell numbers as late as 60 weeks postinoculation, while Group 2 macaques had significantly lower titers at 1 year postinoculation and lower CD4(+) T cell counts by 30 weeks postinoculation. Our study shows a correlation between humoral response, ADCC activity, and disease progression (as measured by CD4(+) T cell counts). In these animals, ADCC activity is associated with delayed progression to AIDS. Further studies are underway to determine if ADCC is a protective immune response in SIV infection or if ADCC is a marker of intact cellular and humoral immune responses.

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.

Searching for Clues: Tracking the Pathogenesis of Human Immunodeficiency Virus Central Nervous System Disease by Use of an Accelerated, Consistent Simian Immunodeficiency Virus Macaque Model

An accelerated model of human immunodeficiency virus central nervous system disease was developed in which more than 90% of infected macaques develop typical simian immunodeficiency virus (SIV) encephalitis with neuronal dysfunction by postinoculation (pi) day 84. Infected macaques had replicating virus and microglial activation in the brain 10 days after inoculation; viral replication and microglial activation were suppressed at pi day 21. By pi day 56, viral recrudescence in the brain was detected in 2 of 6 infected macaques. CD4 cells were the predominant lymphocytes in the brain during acute and asymptomatic infection; cytotoxic T lymphocytes and NK cells predominated in macaques with encephalitis. Low levels of peripheral blood NK lytic activity at pi day 10, elevated cerebrospinal fluid (CSF) monocyte chemoattractant protein-1 after 28 days, and high CSF viral RNA after 42 days predicted SIV encephalitis. This model is ideal to track the viral, cellular, and immunologic changes in the brain during acute and asymptomatic infection and during viral recrudescence and SIV encephalitis.

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.

Suppressor of Cytokine Signaling 3 Inhibits Antiviral IFN-β Signaling To Enhance HIV-1 Replication in Macrophages

HIV-1 replication within macrophages of the CNS often results in cognitive and motor impairment, which is known as HIV-associated dementia (HAD) in its most severe form. IFN-β suppresses viral replication within these cells during early CNS infection, but the effect is transient. HIV-1 eventually overcomes this protective innate immune response to resume replication through an unknown mechanism, initiating the progression toward HAD. In this article, we show that Suppressor of Cytokine Signaling (SOCS)3, a molecular inhibitor of IFN signaling, may allow HIV-1 to evade innate immunity within the CNS. We found that SOCS3 is elevated in an in vivo SIV/macaque model of HAD and that the pattern of expression correlates with recurrence of viral replication and onset of CNS disease. In vitro, the HIV-1 regulatory protein transactivator of transcription induces SOCS3 in human and murine macrophages in a NF-κB–dependent manner. SOCS3 expression attenuates the response of macrophages to IFN-β at proximal levels of pathway activation and downstream antiviral gene expression and consequently overcomes the inhibitory effect of IFN-β on HIV-1 replication. These studies indicate that SOCS3 expression, induced by stimuli present in the HIV-1–infected brain, such as transactivator of transcription, inhibits antiviral IFN-β signaling to enhance HIV-1 replication in macrophages. This consequence of SOCS3 expression in vitro, supported by a correlation with increased viral load and onset of CNS disease in vivo, suggests that SOCS3 may allow HIV-1 to evade the protective innate immune response within the CNS, allowing the recurrence of viral replication and, ultimately, promoting progression toward HAD.

Borna disease virus: nature of the etiologic agent and significance of infection in man

This review presents data on the characterization of Borna disease virus (BDV) and its potential as a possible causative agent in humans. The isolation of: (i) BDV-specific cDNA clones that encode various BDV-specific proteins and (ii) partially purified virus particles led to the conclusion that the viral genome consists of negative-sense, single-stranded RNA. The organization of the BDV-specific RNA species appears to be a nested set of overlapping subgenomic RNA transcripts. Furthermore, evidence is presented that BDV can infect humans and may cause certain psychiatric and neurological disorders. This concept is supported by: (i) the finding of virus-specific antibodies in sera of patients with neuropsychiatric diseases and (ii) results obtained during attempts to isolate BDV or a BDV-related agent from the cerebrospinal fluid of seropositive patients.

Analysis of borna disease virus-specific RNAs in infected cells and tissues

Borna disease virus (BDV) is an infectious agent that causes profound disturbances in motor function and behaviour in a wide range of animal species and possibly humans. The infectious nature of BDV has long been established, but the aetiological agent has not been isolated or classified. Recently, we have reported the isolation of BDV-specific cDNA clones using subtractive libraries constructed from mRNA from infected material. Here we describe studies on one of these cDNA clones, B8, and confirm its specificity by in situ hybridization on sections of BDV-infected brain. The complete nucleotide sequence of BDV-specific clone B8 was determined. Oligonucleotides of positive and negative polarity synthesized from sequences from the 5 and 3 ends, as well as the central part, of clone B8 identified both positive- and negative-strand BDV-specific RNAs in infected rat brain. All B8 sequences used as oligonucleotide probes were found to be contained in the larger positive- and negative-strand RNAs. Thus, the structure of the BDV-specific RNAs appears to be a nested set of multiple, overlapping subgenomic positive- and negative-strand RNA transcripts.