Linda Stowring

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.

Detection of Viral Nucleic Acids by in Situ Hybridization

Publisher Summary Methods to adapt hybridization techniques for preparations of cells and chromosomes were devised and introduced in 1969 and used successfully to localize highly reiterated or amplified genes. Improvements in both hybridization techniques and labeling of specific probes have extended the compass of in situ hybridization to detection of single genomes of viruses in cells and single genes in chromosomes. These developments herald a new era in the analysis of viral infections and the role of viruses in chronic diseases and present some exceptional opportunities to examine the expression of genes in individual cells in other disciplines, such as developmental biology. This chapter provides descriptions of methods, presents examples of the use of in situ hybridization in virology, and discusses some of the important issues of which solution will be greatly influenced by in situ hybridization. In in situ hybridization, a nucleic acid probe, labeled radioactively or with a reporter molecule, is annealed to suitably prepared cells or chromosomes, and the formation of hybrids is assessed by autoradiography, by fluorescence, or by histochemical means. The great increase in sensitivity achieved in current techniques is the result of the increased signal generated by contemporary probes and the increased efficiency of hybridization. This chapter reflects these improvements, first, in the preparation of probes and, second, in the treatment of cells and conditions for hybridization.

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.

Detection of visna virus antigens and RNA in glial cells in foci of demyefnation

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.

Visna DNA synthesis and the tempo of infection in vitro

Visna virus is the prototype of the subfamily of nontransforming retroviruses that cause slow infections in vivo, and lytic infections in vitro. In this paper we present the first quantitative single cell analysis of the synthesis of viral nucleic acids, using improved methods of in situ hybridization; we identify early visna virus DNA synthesis as the rate-limiting step in transcription, virus production, and cell death in vitro; and we show that by manipulating the extent of early DNA synthesis we can slow the tempo of infection in vitro from 3 days to 3 weeks.

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.

Visna virus DNA: Discovery of a novel gapped structure

Abstract We have analyzed the structure of visna virus DNA synthesized during the life cycle of this lytic virus in confluent cultures of sheep choroid plexus cells. Viral DNA is of predominantly two forms: A linear duplex of about 6 million daltons corresponding to a transcript of viral RNA of subunit size; and, a structure with a gap in the center comprised of a full-length minus strand, and two long plus strands that together are about 300–500 bases shorter than the minus strand. A physical map of the linear DNA derived with restriction enzymes was ordered with respect to viral RNA, and the DNA was shown to bear terminal repeats of 350–450 nucleotides. Two circular forms of visna DNA differing only by the length of one terminal repeat also were detected, but constituted a very minor population. We discuss the relationship of these observations with this prototype of the lentiviruses to those recorded for transforming retroviruses.

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.

Detection by Hybridization of Viral Infection of the Human Central Nervous System

Studies of animal models of demyelination and investigation of the epidemiology of multiple sclerosis (MS) both point to the possibility of virus infection as one inciting factor in the disease, but until recently there has been little evidence for the central prediction of this hypothesis, persistence of viruses in the human brain. Advances in technology, particularly in methods for detecting virus genes by hybridization, have now provided us with our first glimpse of an indigenous viral flora of the brain, and

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.