Leishmania promastigotes evade interleukin 12 (IL-12) induction by macrophages and stimulate a broad range of cytokines from CD4+ T cells during initiation of infection.

Leishmania major are intramacrophage parasites whose eradication requires the induction of T helper 1 (Th1) effector cells capable of activating macrophages to a microbicidal state. Interleukin 12 (IL-12) has been recently identified as a macrophage-derived cytokine capable of mediating Th1 effector cell development, and of markedly enhancing interferon gamma (IFN-gamma) production by T cells and natural killer cells. Infection of macrophages in vitro by promastigotes of L. major caused no induction of IL-12 p40 transcripts, whereas stimulation using heat-killed Listeria or bacterial lipopolysaccharide induced readily detectable IL-12 mRNA. Using a competitor construct to quantitate a number of transcripts, a kinetic analysis of cytokine induction during the first few days of infection by L. major was performed. All strains of mice examined, including susceptible BALB/c and resistant C57BL/6, B10.D2, and C3H/HeN, had the appearance of a CD4+ population in the draining lymph nodes that contained transcripts for IL-2, IL-4, and IFN-gamma (and in some cases, IL-10) that peaked 4 d after infection. In resistant mice, the transcripts for IL-2, IL-4, and IL-10 were subsequently downregulated, whereas in susceptible BALB/c mice, these transcripts were only slightly decreased, and IL-4 continued to be reexpressed at high levels. IL-12 transcripts were first detected in vivo by 7 d after infection, consistent with induction by intracellular amastigotes. Challenge of macrophages in vitro confirmed that amastigotes, in contrast to promastigotes, induced IL-12 p40 mRNA. Reexamination of the cytokine mRNA at 4 d revealed expression of IL-13 in all strains analyzed, suggesting that IL-2 and IL-13 may mediate the IL-12-independent production of IFN-gamma during the first days after infection. Leishmania have evolved to avoid inducing IL-12 from host macrophages during transmission from the insect vector, and cause a striking induction of mRNAs for IL-2, IL-4, IL-10, and IL-13 in CD4+ T cells. Each of these activities may favor survival of the organism.

Slow persistent infection caused by visna virus: role of host restriction.

Proviral DNA has been demonstrated by in situ hybridization in foci of cells of a lamb infected with the RNA slow virus visna. A few of these cells also contain the major virion structural antigen p30. This restriction in virus gene expression in the infected animal provides a mechanism for persistence of virus in this chronic infection.

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.

Proteinase-resistant prion protein accumulation in Syrian hamster brain correlates with regional pathology and scrapie infectivity.

Multiple lines of evidence indicate that PrPSc, found only in scrapie, is a necessary component of the infectious scrapie agent. Equally compelling is the evidence that its accumulation in the brain causes the neuropathology characteristic of scrapie. We measured the regional concentration of PrPSc in nine brain regions throughout the course of scrapie in the Syrian hamster following intrathalamic inoculation of prions. PrPSc was compared to the regional concentration of glial fibrillary acidic protein, a measure of reactive astrocytic gliosis. PrPSc was detected first in the thalamus 14 to 21 days postinoculation and next in the septum at 28 days. Initiation of PrPSc synthesis and accumulation in the thalamus was attributable to the inoculum and in the septum to ventricular spread of de novo synthesized PrPSc. The timing and pattern of PrPSc accumulation in all other brain regions suggested transmission along neuroanatomic pathways. Reactive astrocytic gliosis followed PrPSc accumulation in each region by 1 to 2 weeks. Brain PrPSc, determined by summing the concentrations in each brain region, correlated well with scrapie infectivity titers throughout the course of infection (correlation coefficient = 0.975; slope of linear regression line = 1.136). Our results support the hypothesis that PrPSc participates in both the etiology and pathogenesis of prion diseases.

Serological definition of the lentivirus group of retroviruses.

The major polypeptides of visna viruses and other lentiviruses have been isolated and shown to be closely related if not identical in radioimmunoassays. By this criterion the lentiviruses form a distinct group of retroviruses unrelated to spuma viruses, mammalian and avian retroviruses that cause tumors, and unclassified retroviruses of cattle and horses. Two sera obtained from goats immunized with Mason-Pfizer monkey virus or squirrel monkey virus reacted with visna p30. Additional data suggest that this reaction represents infection of goats with a lentivirus or a new retrovirus closely related to the lentiviruses.

Gene expression in visna virus infection in sheep.

Visna is a slow degenerative disease of the central nervous system (CNS) of sheep caused by a nontumorigenic retrovirus. During the course of this disease, visna virus establishes a persistent infection of the CNS, lung and haematopoietic system, despite a specific humoral and cellular immune response. We have studied visna virus life cycle at the single-cell level in choroid plexus of experimentally infected animals, using a very sensitive and quantitative in situ hybridization assay. We report here that although proviral DNA is synthesized in significant amounts, its expression is blocked at the transcriptional level. This restriction of proviral DNA transcription offers an explanation for the slowness of the disease and the persistence of the infection.

Scrapie prion proteins are synthesized in neurons.

Scrapie is a slow degenerative encephalopathy of animals caused by unusual infectious particles termed prions. A cDNA encoding the only apparent component of the prion, a protein designated PrP 27-30, has recently been cloned and sequenced. By measuring mRNA levels using in situ hybridization with the PrP cDNA, the authors found that prion proteins are synthesized almost exclusively within neurons. The levels of PrP mRNA varied among different types of neurons, but did not change during scrapie infection. A cDNA encoding glial fibrillary acidic protein (GFAP) was a positive control; GFAP mRNA was confined to astrocytes. Our finding of PrP mRNA in neurons may explain the degeneration and vacuolation that occurs in these cells during scrapie infection.

A Trojan Horse mechanism for the spread of visna virus in monocytes.

Visna virus is the prototype of the lentivirus subfamily of retroviruses that cause slow infections of sheep and goats. These viruses persist and can be isolated from blood and cerebrospinal fluid for years despite neutralizing antibody. In the studies reported here we have used quantitative in situ hybridization to analyze infected leukocytes. We show that (1) monocytes harbor the visna genome; and (2) virus gene expression is as constrained in this cell as it is in glial and epithelial cells. These results are in accord with a Trojan Horse mechanism of virus dissemination in an immunologically responsive host.

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.

Antigenic variation in visna virus.

Two antigenic variants of visna virus were isolated sequentially from a single sheep inoculated with a plaque-purified strain of virus designated 1514. The genetically stable variants, LV1-1 and LV1-4, are of two classes: LV1-1 is partially neutralized by antibody to the inoculum strain 1514, while LV1-4 is not neutralized by antibody to 1514. The genetic mechanism responsible for generating the antigenic variants was investigated by comparing the chymotryptic and tryptic maps of the envelope glycoprotein gp135 and core polypeptides (p30, p16, p14), and by comparing the pattern of large oligonucleotides produced by digestion of the RNAs by T1 ribonuclease. We show that only the peptide maps of gp135 differ among strains, that the number of peptide fragments altered is small and that gp135 is the polypeptide that elicits neutralizing antibody. The maps of the RNAs are identical. We conclude that mutation in the glycoprotein gene rather than recombination is more probably responsible for antigenic variation, and speculate on the special aspects of visna virus replication relevant to this phenomenon.

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.

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.

Molecular cloning of a human prion protein cDNA.

Creutzfeldt-Jakob disease (CJD) of humans and scrapie of animals are degenerative, transmissible neurologic diseases caused by prions. The only known macromolecules within prions are prion proteins (PrP). The cDNA encoding the hamster prion protein (PrP 27-30) has been cloned and sequenced (Oesch et al., 1985). Using that hamster PrP cDNA, we screened a human retina cDNA library and sequenced the cDNA clone with the longest hybridizing insert. This insert was found to contain a long open reading frame (ORF) encoding the human prion protein. Northern transfer analysis showed that a related poly(A)+RNA measuring approximately 2.5 kb is expressed in a variety of human neuroectodermal cell lines. Human PrP differed from hamster PrP at 27 of 253 amino acids and at 98 of 759 ORF nucleotides. Conservation of PrP amino acid sequence between hamster and human is nearly 90%, reflecting similar structural features and shared antigenicity of the two proteins (Bockman et al., 1985). The human PrP sequence contained a presumptive amino-terminal signal peptide of 22 amino acids, two hydrophobic segments of sufficient length to span membranes, and two possible sites for N-glycosylation. The conservation between the hamster and human prion proteins suggests that they may have an important role in cellular metabolism and may explain the similarities between scrapie and CJD.

Development of a non-selecting, non-perturbing method to study human brain tumor cell invasion in murine brain.

The infiltrative nature of glial and some meningeal neoplasms is responsible for the failure of surgical removal and high recurrence rate of these tumors. Modeling of this process in vitro and in vivo will lead to a better understanding of the pathophysiology of this process and identify targets for novel therapy directed towards this phenotype. We present the results of the development and refinement of two model systems of tumor invasion: one in vitro barrier assay using the basement membrane extract Matrigel, and one in vivo where molecular detection of tumor cells allows single cell discrimination by in situ hybridization histochemistry. These techniques have strong correlations which validate their utility as measures of nervous system tumor invasion.

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.

Measles virus genome in infections of the central nervous system.

The measles virus genome was traced in acute and chronic infections of the central nervous system in hamsters and humans. The extent of viral replication and gene expression was assessed by the techniques of in situ hybridization and immunofluorescence. Both replication and gene expression were restricted in chronically infected hamsters and in humans with subacute sclerosing panencephalitis. It is proposed that restriction plays an important role in persistence of measles virus and the slow evolution of disease in these and other slow infections.

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.