Detection of vesicular stomatitis virus (VSV) RNA in the central nervous system of infected mice by in situ hybridization

Summary Using in situ hybridization with a cloned DNA probe specific for the VSV G protein, viral RNA was detected and localized in CNS tissue of mice infected i.c. with either wild or ts G 31 VSV mutant. In both cases, brain and spinal cord neurons were the only cells seen to contain viral RNA. Virus-positive neurons were observed enclosed in spongious areas induced by the ts VSV mutant. These results suggest that the VSV shows a strong tropism for the neuronal cell and indicate that the vacuole formation might be associated with the expression of the VSV G protein gene in infected neurons.     

Virulent and avirulent strains of Semliki Forest virus show similar cell tropism for the murine central nervous system but differ in the severity and rate of induction of cytolytic damage

The pathogenicity of the avirulent, demyelinating A7 strain of Semliki Forest virus (SFV) and the virulent SFV4 strain (derived from an infectious clone) for the central nervous system of adult BALB/c mice following intranasal infection was compared. The techniques used included immunocytochemistry using anti-SFV antibody and antibodies to cell markers, in situ hybridization (ISH) using a biotinylated cDNA probe specific for SFV, and immunocytochemistry/ISH double labelling. Whereas SFV4 was lethal at 4 days post-infection, A7-infected mice appeared normal at all times. Neuronal necrosis in the pyriform cortex was present in both infections, but developed sooner and was more severe iollowing miection with SFV4 than with A7. Intact neurons and putative oligodendrocytes contained viral RNA and virus-specific antigen in SFV4 infected mice; viral RNA but not virus-specific antigen was detected in similar cells in A7-infected mice. These results confirm that SFV4 and A7 share similar cell tropisms for the murine central nervous system, but differ in the severity and rate of development of cytolytic damage. Intranasal infection is an efficient monitoring system for studies of the molecular basis of pathogenicity of SFV infection in mice.     

Simultaneous in situ detection of viral antigens and RNA in brain tissues from a patient with herpes simplex encephalitis by immunocytochemistry and in situ hybridization

In order to elucidate the correlation between herpes simplex virus (HSV-1) and the central nervous system tissue, we performed the simultaneous detection of viral antigens and RNA in the brain tissue sections from a patient with herpes simplex virus (HSV) encephalitis using immunocytochemistry and in situ hybridization. In the present study the hybridization protocol reported by Brahic M et al. in 1984 were applied for the simultaneous detection of viral RNA and antigens with a few modification. The sections were first immunocytochemically stained to detect HSV-1 antigens by ABC method, and then hybridized with 3H-labelled HSV-1 cDNA probe for the detection of RNA after the acetylation of slides for the prevention of nonspecific bindings of isotope to slides. In the present study, viral antigens were immunocytochemically stained to brown-colored deposits located in the cytoplasm and nucleus whereas viral RNA were detected as the accumulation of many silver grains over the nuclei or cytoplasm. In this case the light microscopic findings in a part of temporal lobe showed multiple areas of necrosis mainly involving the gray matter and a few inflammatory changes such as perivascular cell cuffings. HSV-1 infected Vero cells as positive control demonstrated both antigens and RNA as shown in Fig.1 a. However, no hybridization signals and color deposits were observed in uninfected Vero cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

A rat model of human T lymphocyte virus type I (HTLV-I) infection: in situ detection of HTLV-I provirus DNA in microglia/macrophages in affected spinal cords of rats with HTLV-I-induced chronic progressive myeloneuropathy

To investigate the pathogenetic role of human T lymphocyte virus type I (HTLV-I) in central nervous system disease, a rat model for HTLV-I-associated myelopathy/tropical spastic paraparesis, designated as HAM rat disease, has been established. Wistar-King-Aptekman-Hokudai strain rats with induced HTLV-I infection develop a chronic progressive myeloneuropathy with paraparesis of hind limbs after an incubation period of 15 months. In the affected spinal cord in these rats, white matter degeneration, demyelination and vacuolar change with microglia/macrophage infiltration are present as are the provirus DNA and the virus mRNA. To identify infected cells in the affected lesions, we carried out in situ hybridization of amplified fragments of the provirus DNA by polymerase chain reaction on thin sections, plus immunohistochemistry on the same sections. The provirus DNA was localized in some microglia/macrophages in the spinal cord lesion. In addition, the HTLV-I provirus was clearly evident not only in ED-1-negative lymphoid cells but also in ED-1-positive macrophages from lymph nodes. These observations suggest that cells of microglia/macrophage lineage may be one of dominant viral reservoirs in the spinal cords and lymph nodes in HAM rat disease. These infected microglia/macrophages may relate to cause the myeloneuropathy through neurotoxic cytokine synthesis. Received: 14 January 1998 / Revised, accepted: 30 July 1998     

Borna disease virus replicates in astrocytes, Schwann cells and ependymal cells in persistently infected rats: location of viral genomic and messenger RNAs by in situ hybridization

Borna disease (BD) is an immune-mediated neurological disease caused by infection of the nervous system with a negative strand RNA virus, Borna disease virus (BDV). The host range for BDV is broad and extends from birds to primates. A BDV-like agent may cause disease in humans. Until recently, BDV-infected neural cells could only be identified immunocytochemically using serum from BDV-infected animals. The advent of BDV cDNA clones allowed definition of the relationship between viral nucleic acids and viral proteins in vivo. In situ hybridization with strand-specific RNA probes from a BDV cDNA clone, pAF4, identified BDV genomic RNA and BDV mRNAs in neurons, astrocytes, Schwann cells and ependymal cells in an anatomic distribution consistent with that of BDV proteins. Genomic RNA was contained primarily within the nucleus, whereas mRNAs were found in both the nuclear and cytoplasmic compartments. Viral RNAs were demonstrated in neurons expressing BDV proteins and in glial cells by combined techniques of immunocytochemistry and in situ hybridization.     

Measles virus matrix protein gene expression in a subacute sclerosing panencephalitis patient brain and virus isolate demonstrated by cDNA hybridization and immunocytochemistry

Summary Subacute sclerosing panencephalitis (SSPE) is a rare, fatal disease of children caused by a persistent measles virus infection of the central nervous system. A defect in synthesis of measles virus matrix (M) protein may be a factor in virus persistence in the brain. This study details attempts to detect expression of M protein in the brain of an SSPE patient, in the cell-associated virus isolated from this brain, and in brains of ferrets inoculated with the isolate. In situ hybridization with a tritiated cloned cDNA probe was used to search for RNA encoding M protein. Immunostaining with monospecific antiserum and the avidin-biotin-peroxidase technique was done to locate the polypeptide. The data obtained indicate that although nucleotide sequences coding for M protein were detected in the patient and ferret brains, expression of M protein in these tissues could not be detected. In the cultured SSPE virus isolate, the results were the same until the infected cells were examined by electron microscopy and a very limited expression of M protein was revealed. This suggests either diminished synthesis and/or rapid degradation of M protein in this cell-associated virus strain.This paper is dedicated to the late Dr. George A. Jervis, first Director of the Institute for Basic Research, Physician and Scientist, for his humanity and many contributions to the mentally retarded and developmentally disabled.     

Up-regulation of mRNA for matrix metalloproteinases-9 and -14 in advanced lesions of demyelinating canine distemper leukoencephalitis

Matrix metalloproteinases (MMPs) comprise a family of proteolytic zinc- and calcium-dependent enzymes that are capable of disrupting the blood-brain barrier and mediating the destruction of extracellular matrix and myelin components. MMPs are also involved in facilitating leukocyte migration into inflammatory sites of the central nervous system. To determine the cellular localization and the amount of mRNA for MMP-9, MMP-14 and a tissue inhibitor of metalloproteinases (TIMP-1) in dogs with spontaneous demyelinating distemper encephalitis, formalin-fixed paraffin-embedded cerebella were investigated by in situ hybridization using specific digoxigenin-labeled RNA probes. Additionally, immunohistochemistry was performed to characterize the different types of plaques of demyelinating leukoencephalitis. Furthermore, virus antigen and mRNA were detected by immunohistochemistry and in situ hybridization. Healthy control dogs revealed a weak signal for mRNA for MMP-9, MMP-14, and TIMP-1 in various numbers of neurons, astrocytes, microglial cells and oligodendrocytes. In the cerebella of dogs with distemper, a strong increase of both number and staining intensity of MMP-9, MMP-14, and TIMP-1 mRNA-expressing cells, mainly in subacute inflammatory lesions and chronic plaques, was observed. The number of cells expressing mRNA for MMP-9 and MMP-14 increased about two- to threefold compared to TIMP-1 mRNA-expressing cells, whereas staining intensity of individual cells was similar. In early lesions, especially astrocytes and activated macrophages/microglial cells displayed a positive signal for MMPs and TIMP-1, whereas in older lesions activated microglia/macrophages and infiltrating lymphocytes represented the main source for MMP-9, MMP-14, and TIMP-1 mRNA synthesis as revealed by double-labeling techniques. In summary, the proportionally higher increase of MMP mRNA-expressing cells might indicate an MMP/TIMP imbalance as a cause for lesion initiation and progression in demyelinating canine distemper leukoencephalitis.     

Apoptosis in measles virus‐infected human central nervous system tissues

The extent of apoptotic cell death was examined in central nervous system (CNS) tissues from three cases of subacute sclerosing panencephalitis (SSPE). Apoptosis was demonstrated by in situ end‐labelling of DNA in formalin‐fixed, paraffin‐embedded tissue sections. Measles virus and cell types were labelled by immunohistochemistry and/or in situ hybridization. Furthermore, bcl‐2 expression in SSPE was examined by immunohistochemistry. All three cases exhibited varying degrees of apoptosis in all CNS areas studied. Brain tissue from a non‐neurological control case did not show any significant apoptosis. Characterization of cell types demonstrated neurons, oligodendrocytes, lymphocytes and microglia undergoing apoptosis. A linear relationship could not be established between virus burden and the extent of apoptosis in any particular area. Virus‐negative cells were observed which were undergoing apoptosis. Bcl‐2 immunoreactivity in SSPE was confined to the infiltrating cell population. These results suggest that apoptosis of various cell types may contribute to the neuropathogenesis of measles virus infection in the human CNS, either as a direct effect of viral infection or by cytokine‐mediated responses.     

In situ hybridization in neuropathology

In situ hybridization (ISH) allows the demonstration and localization of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in tissue sections, cells and chromosomes by utilizing a specific interaction with a labelled nucleotide probe of known composition. Although this technique has been employed for many years using radiolabelled probes, the recent development of non–isotopic labelling systems and the greatly increased availability of synthetic nucleic acids has allowed an enormous expansion in the potential applications of ISH. The technique is now applicable to unfixed and fixed tissues, including archival material. The use of enzyme–linked antibody techniques to detect labelled probes has greatly increased the sensitivity of non–isotopic ISH without a loss of specificity. The successful use of ISH demands careful selection of labelled probes, adequate tissue pretreatment to allow access of the probe, control of the stringency of probe binding and a sensitive reporter system, in addition to adequate controls. The accurate localization of nucleotides in the central nervous system (CNS) has many current research applications in the study of gene expression in multiple sclerosis and other inflammatory disorders, and a wide range of neurodegenerative disorders, viral infections and neoplasms. The technique is of diagnostic value in viral disorders, particularly where multiple infections occur. The combination of non–isotopic ISH with immuno–cytochemistry, electron microscopy and quantitative image analysis greatly increases its research potential, while the development of a related method, the in situ polymerase chain reaction, offers an additional opportunity for further enhancing the sensitivity of this technique.     

Simultaneous in situ detection of JC virus antigens and RNA in progressive multifocal leukoencephalopathy (PML) by immunocytochemistry and in situ hybridization

We detected simultaneously JC virus (JCV) antigen and RNA in the frozen brain tissue from a patient with PML using immunocytochemistry and in situ hybridization. Histologically, the majority of JCV antigen positive cells were mainly located at the margin of demyelinated lesions. By simultaneous in situ detection of JCV antigen and RNA, the percentage of both JCV antigen and RNA positive cells in total number cells were 20 in the periplaque region and 2.3 in the central region of plaque respectively. On the contrary, only JCV RNA positive cells were detected 3.6% in the periplaque region and 4.6% in the central region of plaque, while there was not found any positive cells in apparently normal white matter. Moreover, JCV antigen in both antigen and RNA positive cells found in the periplaques was stained homogeneous and filled the whole swollen nucleus, whereas most of both antigen and RNA positive cells appeared in the center of plaques showed small amounts of antigen in the nucleus. From the results, it seems likely that most of the cells displaying only RNA detected in the periplaque were oligodendrocytes in the early stages of productive infection prior to viral protein synthesis. However, a small number of cells displaying only RNA in the center of plaques were likely to be oligodendrocytes and seemed to be in the nonproductive stages of a JCV infection, limited expression of viral protein, influenced by the host immune response including the infiltration of macrophages and humoral immunity.     

Reappraisal of progressive multifocal leukoencephalopathy due to simian virus 40

Several cases of progressive multifocal leukoencephalopathy (PML) have been associated with simian virus 40 (SV40), rather than with JC virus (JCV), the polyomavirus originally isolated from PML tissue. PML has, therefore, been defined as a demyelinating syndrome with possible multiple viral etiologies. Tissues from three of the cases thought to be associated with SV40 were available for reexamination. Monoclonal antibodies specific for SV40 capsid antigen VP1, virus-specific biotinylated DNA probes for in situ hybridization, and virus-specific primers in the polymerase chain reaction (PCR) were used. Macaque PML brain served as a positive control tissue for SV40 brain infection. Monoclonal antibodies to SV40 VP1 failed to recognize viral antigen in lesions from all three human PML cases. The biotinylated DNA probe, which reacted with SV40 in macaque PML, failed to detect SV40 in human PML. However, JCV could be detected by in situ hybridization with a JCV-specific DNA probe. Moreover, JCV DNA sequences were amplified by PCR from the human PML tissues, whereas SV40 DNA sequences were amplified only from the macaque brain. Thus, we could not confirm the original reports that the demyelinating agent in these three cases of PML was SV40, rather than JCV. We conclude that SV40 infection of the central nervous system need not be ruled out in the differential diagnosis of PML. Received: 1 December 1997 / Revised, accepted: 23 February 1998     

Search for virus nucleic acid sequences in postmortem human brain tissue using in situ hybridization technology with cloned probes: some solutions and results on progressive multifocal leukoencephalopathy and subacute sclerosing panencephalitis tissue

In order to obtain a useful and readily applicable in situ hybridization (ISH) protocol for progressive central nervous system (CNS) diseases of unknown etiology that are possibly due to persistent viral infection, known and well described diseases were studied, namely, progressive multifocal leukoencephalopathy (PML) and subacute sclerosing panencephalitis (SSPE). The procedures described were validated by confirming results obtained by other investigators using histology, immunocytochemistry, electron microscopy, and ISH. A number of frequently encountered problems of tissue preparation are addressed as well as techniques to reduce autoradiography exposure times. A multi-staged specific, sensitive, reliable, and valid procedure for detection of viral genomes, mRNA and proteins is approached. Formalin-fixed and paraffin-embedded (FFPE) brain material from six patients who died with PML and one patient who died from SSPE were studied using ISH with a tritium-labeled cloned JC virus DNA probe and a measles-cloned nucleocapsid (NC) gene cDNA probe, respectively. This report constitutes a methodological framework as well as a detailed neuropathological analysis of identified brain cell populations within which in situ hybridization was detected. In early PML lesions, swollen nuclei or oligodendrocytes were the predominant cells labeled, whereas older lesions revealed increased numbers of reactive and bizarre hypertrophic astrocytes hybridized at the outer periphery of the demyelinated lesions. The hybridization varied greatly in intensity in different cells. Intense hybridization was noted very rarely in microglial cells, including rod cells and rarely in venular pericytes, intravascular mononuclear cells, or in vascular endothelial cells. These results, considered together with previous findings, indicate that in PML the viral infection runs different courses in the various cells: in astrocytes the viral genome persists for a long time inducing pathological changes in some cells. In oligodendrocytes the infection rapidly lyses the cells. There was a good correlation between chromatic changes observable in routinely stained sections and virus presence. In addition, in situ hybridization using a measles-NP-cloned probe in white matter from FFPE SSPE brain is presented confirming earlier results in SSPE cryopreserved brain.     

Cerebral endothelial cell infection by measles virus in subacute sclerosing panencephalitis: ultrastructural and in situ hybridization evidence

Infection of vascular endothelium plays a central role in the pathogenesis of acute measles virus infection outside the central nervous system (CNS) but has not been described in the human CNS. An ultrastructural survey was made of blood vessels in five cases of subacute sclerosing panencephalitis (SSPE) to determine whether or not infection of cerebral vascular endothelium occurred in this persistent fatal CNS disease caused by measles virus. Morbillivirus nucleocapsids were found in a few endothelial cells in three necropsy cases but not in the limited tissue available from two biopsies. In a severe parenchymal lesion in one necropsied case, endothelial cells hybridized in situ with a biotinylated probe specific for the N genomic RNA of measles virus. It is concluded that human cerebral endothelium is susceptible to measles virus infection.     

In situ hybridization histochemistry with oxytocin synthetic oligonucleotide: strategy for making the probe and its application

A 25 mer synthetic oligonucleotide, complementary to a specific region of the oxytocin-neurophysin preprohormone messenger RNA (mRNA), was designed for its application to in situ hybridization histochemistry. The probe was 3′-end labeled with [³H] deoxycytidine triphosphate (dCTP) by using terminal deoxynucleotidyl transferase, and hybridization of the labeled probe to the mRNA in the rat hypothalamus was visualized autoradiographically. Hybridization products were specifically localized in the dorsal part of the supraoptic nucleus and the peripheral part of the paraventricular nucleus. Not only is the oligomer designed useful for distinguishing oxytocin from vasopressin gene expressing neurons, but also it is proving useful for studies of estrogen-progesterone effects on neurons in the paraventricular nucleus. Thus, these results indicate that in situ hybridization histochemistry with synthetic oligonucleotide can be a valuable approach to measuring gene expression in hypothalamic neuroendocrine cells.     

Evidence of human immunodeficiency virus type 1 infection of nestin-positive neural progenitors in archival pediatric brain tissue

Human immunodeficiency virus type 1 (HIV-1) central nervous system (CNS) infection in children is associated with impaired brain growth and neurodevelopmental delays. Neural progenitors are critical for neurogenesis. Human multipotential neural progenitors grown in culture are permissive for HIV-1 infection, but it is not known if infection of these cells occurs in vivo. Brain tissue from pre-highly active antiretroviral therapy (HAART) era pediatric acquired immunodeficiency syndrome (AIDS) patients was examined for evidence of HIV-1 infection of nestin-positive neural progenitors by in situ hybridization; or after laser microdissection harvest, DNA extraction, and polymerase chain reaction (PCR). HIV-1 or viral DNA was identified in nestin-positive cells in four of seven HIV-1-infected children, suggesting in vivo infection of neural progenitors.