Ultrastructural effects of herpes simplex virus type 2 infection of rat dorsal root ganglia in culture.

The ultrastructural consequences of herpes simplex virus type 2 (strain R-2) infection of organotypic cultures of embryonic rat dorsal root ganglia were studied. The intial consequences (dilation of endoplasmic reticulum and/or Golgi apparatus and distortion of mitochondrial structure) occurred in the cytoplasm. These effects were followed by several nuclear changes including loss of nucleoplasm, and margination of chromatin. Extensive nuclear membrane proliferation was accompanied by the viral maturation process. Two previously unreported observations were made. First, productive virus replications occurred in glial cells, as well as in neurons. Mature, enveloped virus was produced by nuclear budding and envelopment in the cytoplasm in both cell types. Second, neurons were observed to participate in polykaryocyte formation with other neurons and with glial cells. These polykaryocytes were usually composed of only three or four cells. Neuronal-glial polykaryocytes were more prevalent than neuronal-neuronal polykaryocytes. In general, however, the ultrastructural changes in neurons and glial cells in culture were consistent with previously reported changes occurring in nervous tissue of experimental animals suffering from acute herpes simplex virus infections. Therefore, the utilization of this in vitro system to further study the pathogenesis of acute herpetic infections of sensory ganglia appears to be reasonable.     

Ultrastructure of herpes simplex virus infection of the nervous system of mice

Summary The nervous system and small intestine of mice infected with herpes simplex virus were examined by electron microscopy from the viewpoint of virus-host interaction.The host cells examined included the neuron, astrocyte, oligodendrocyte, and Schwann cell. The susceptibility of the latter was not less than that of the neuron. The endothelial cell, perineural fibrocyte and smooth muscle cell were also host cells. Replication of herpes virus in the nervous system was proven to be identical to that occurringin vitro; initial reproduction of nucleocapsids in the nucleus and subsequent maturation at the nuclear membrane with envelope formation, followed by discharge into the cytoplasmic reticular cavities and finally release from the host cell. Inconsistency in the distribution of virus particles and viral antigen was chiefly concerned with the host cell nucleus and the glial cytoplasm.Herpes virions, though few, were identified in the axons of peripheral nerves, and in the periaxonal space of myelinated fibres in the brain and the nerve ganglia. Virions were present in tiny vesicles in the perikarya or as naked particles. In the distal parts of peripheral nerve, there was marked dissociation in the amount of virions between Schwann cells and the axon. The significance of the endoneural space and the axon in the neural speread of infection is discussed briefly.     

Spontaneous human herpes virus type 1 infection in a chinchilla (Chinchilla lanigera f. dom.)

A 1-year-old male chinchilla with a 2-week history of conjunctivitis suffered subsequently from neurological signs comprising seizures, disorientation, recumbency and apathy. After 3 weeks of progressive central nervous disease the animal was killed in view of the poor prognosis. A non-suppurative meningitis and polioencephalitis with neuronal necrosis and intranuclear inclusion bodies were observed at necropsy and by light microscopy. The brain stem and cerebral cortices were most severely affected. Both eyes displayed ulcerative keratitis, uveitis, retinitis and retinal degeneration, and optical neuritis. Additionally, a purulent rhinitis with focal erosions, epithelial degeneration and intranuclear inclusion bodies was present. Ultrastructurally, herpes virus particles were detected in neurons of the brain. Immunohistochemistry with antisera specific for human herpes virus types 1 and 2 resulted in viral antigen labeling in neurons, glial cells and in neuronal processes. Viral antigen was found in the rhinencephalon, cerebral cortices, hippocampus, numerous nuclei of the brain stem, single foci in the cerebellum, and in a solitary erosive lesion of the right nasal vestibulum. Viral antigen was not detected in the eyes. The virus was isolated from the CNS, and nucleic acid sequence analysis of the glycoprotein B and the DNA polymerase revealed a sequence homology with human herpes virus type 1 of 99% and 100%, respectively. The clinical signs, the distribution of the lesions and the viral antigen suggest a primary ocular infection with subsequent spread to the CNS. Chinchillas are susceptible to human herpes virus 1 and may play a role as a temporary reservoir for human infections.     

Neurovirulence of varicella and the live attenuated varicella vaccine virus

Varicella-zoster virus (VZV) is a neurotropic herpesvirus, which can cause a variety of complications during varicella infections. These range from meningoencephalitis to polyneuritis to retinitis. After primary VZV infection, VZV enters the dorsal root ganglia in a latent state. Reactivation from latency leads to zoster. The velocity of VZV is 13 cm per day, as the virus travels from ganglion to skin. The live attenuated varicella vaccine virus is markedly less neurovirulent than the wild-type virus. Nevertheless, a few cases of herpes zoster due to the vaccine virus have been documented. Usually, herpes zoster occurs in the same arm as the vaccination, often 3 or more years after vaccination. Thus, herpes zoster in a vaccinee often represents a reactivation of vaccine virus that was carried to the cervical dorsal root ganglia from a site of local replication in the arm. Finally, the role of autophagy during VZV infection is discussed. Autophagosome formation is a prominent feature in the skin vesicles during both varicella and herpes zoster. Therefore, autophagy is one of the innate immune mechanisms associated with VZV infection in humans.     

Multifocal varicella-zoster virus leukoencephalitis temporally remote from herpes zoster

Two patients with cancer, one with Hodgkin's disease and the other with a granulosa cell tumor of the ovary, developed a progressive, eventually fatal infection of the central nervous system exhibiting multifocal symptoms and signs. Pathologically, gross abnormalities of the brain resembled those in progressive multifocal leukoencephalopathy (PML), with discrete and confluent plaque-like lesions concentrated in the white matter, particularly along the gray-white junction. Microscopically, pathological changes differed distinctly from those associated with PML; in addition to confluent foci of white matter injury characterized by early demyelination and subsequent necrosis, prominent Cowdry type A eosinophilic intranuclear inclusions were noted in oligodendrocytes, astrocytes, and neurons. By electron microscopy, intranuclear spherical particles consistent in size and appearance with herpesvirus nucleocapsids were found within the lesions. Immunoperoxidase studies detected varicella-zoster virus (VZV) antigens in infected cells, implicating this virus as the responsible agent despite a lapse of many months between the cutaneous herpes zoster and onset of cerebral symptoms in both patients.     

Herpes simplex encephalitis: an autopsy case with isolation of type 1 herpes simplex virus

Summary An adult case of herpes simplex encephalitis was studied after autopsy. Postmortem examination revealed necrotizing encephalitis associated with Cowdry type A intranuclear inclusion bodies in glial cells. Herper simplex virus type 1 was isolated from the removed brain. Herpes simplex virus antigens were detected diffusely in wide areas of the brain by immunofluorescent test and viral particles characteristic to herpes simplex virus were demonstrated by electron microscopy. There was an apparent discrepancy between severity of histological changes and distribution of virus antigen.     

Intranuclear inclusions in paramyxovirus-induced encephalitis: evidence for altered nuclear body differentiation

Summary Intranuclear inclusion bodies (INB) are frequently encountered in viral infections, where they are thought to be accumulations of viral particles. However, for RNA viruses replicating in the cytoplasm, this compartmentalization represents a paradox not consistent with the viral replication cycle. To define the basis for intranuclear paramyxoviral inclusion bodies in astrocytes, natural cases of canine distemper virus subacute encephalitis were examined by light and transmission electron microscopy, and by quantitative confocal immunofluorescene microscopy. Although INB were viral antigen positive, they were not composed of structurally recognizable paramyxoviral nucleocapsids. The structural basis for the INB was instead viral antigen-associated forms of nucleolar development known as nuclear bodies. Three variants of the light microscopic Cowdry type A INB were complex nuclear bodies, giant beaded nuclear bodies (sphaeridia), and nuclear body-associated granulofilamentous matrices. In the latter, the granulofilamentous matrix frequently filled the nucleus, resulting in a fourth morphological INB variant, and was associated with morphological evidence of nuclear degeneration. These findings suggest a novel mechanism of virus-induced cytopathology whereby intranuclear viral protein exerts deleterious effects upon nucleolar differentiation in infected cells and hence altered host cell RNA metabolism.Supported by The State of Ohio Canine Research Fund     

Varicella-zoster virus encephalitis in acquired immunodeficiency syndrome: Report of four cases

Four patients with acquired immunodeficiency syndrome, a 27-year-old female intravenous drug abuser and three males (two drug addicts aged 27 and 33 years and a 40-year-old homosexual) presented with a rapidly progressive encephalopathy. Two had generalized varicella-zoster virus skin infection, one had had a regressive thoracic zoster rash 7 months previously and one had no history of cutaneous eruption. Neuropathological examination revealed, in each case, multifocal necrotic changes with numerous, intranuclear Cowdry type A inclusion bodies in glial cells, endothelial cells, macrophages and neurons, within and around the lesions. These inclusion bodies were stained positively for varicella-zoster virus by immunocytochemistry and contained herpes virus nucleocapsids by electron microscopy. Molecular biology using the polymerase-chain-reaction method demonstrated viral genome. In one case, zoster-induced non-inflammatory vasculopathy involved medium sized leptomeningeal vessels and was associated with circumscribed areas of cortico-subcortical infarction. In another case, varicella-zoster virus encephalitis was associated with human immunodeficiency virus encephalitis and a secondary cerebral lymphoma. Multinucleated giant cells expressing human immunodeficiency virus proteins in their cytoplasm, were found in the lymphomatous deposits and in the varicella-zoster virus necrotic lesions. In these latter lesions, Cowdry type A inclusion bodies could be seen in the nuclei of some multinucleated giant cells confirming previous observations of MGCs co-infected by HIV and CMV, and supporting the hypothesis that DNA viruses interact with HIV, thus increasing its effect.     

Herpes virus hominis type 2 and intranuclear tubular structures in organized nervous tissue cultures

Summary Mature explants of mouse spinal cord and ganglia have been infected with Herpes virus hominis type 2. After 48 h, a high titer of virus was detected in the explant and typical herpes-like particles were seen by electron microscopy. They were more numerous in sensory neurons and Schwann cells than in the CNS possibly because of a more rapid propagation through the extracellular space in spinal ganglia. In the infected nuclei, besides the naked particles, numerous tubular formations were found, apparently resulting from virus infection and specific for herpes type 2. Their dimensions and fine structure are illustrated and compared to other microtubular formations.In the cytoplasm of theneurons, a dramatic disorganisation occurred and, in the Schwann cell, the plasma membrane underwent disruption. Consequently, the normal axon — Schwann cell intimate contact was lost.This work has been supported by a grant n⁰ 1120 from the Fonds de la Recherche Scientifique Médicale (Belgium), by a University grant for the Laboratory of neuroanatomy (Dr. O. Périer) and by a grant from the Fonds de la Recherche Fondamentale Collective.Chargée de Recherches au Fonds National de la Recherche Scientifique Belgium.     

Morphological and immunohistochemical studies of the central nervous system involvement in papovavirus K infection in mice

Summary The murine papovavirus K causes fatal pneumonia in infant mice, but an asymptomatic infection in older mice. In order to establish whether the virus affects the central nervous system in the course of systemic infection, we carried out morphological and immunohistochemical studies on the experimentally infected mice. BALB/c mice, less than 4 days of age, were inoculated with K virus either intraperitoneally or intracerebrally. When the animals were moribund, usually 10 days or so, after inoculation, their brains were removed and examined. Acutely infected mice showed only minor changes: intranuclear eosinophilic inclusions in very rare capillary endothelial cells of the brain. However, immunoperoxidase studies, using specific antibody to K virus, revealed that a number of brain cells had positive nuclear staining. These nuclei were distributed throughout the brain, without an apparent site of predilection. Double-immunostaining showed that virtually all cells whose nuclei were positive for viral antigen were endothelial, because their cytoplasm was positive for factor-VIII or vimentin. There were no nuclei positive for viral antigen in astrocytes, as determined by positive staining for glial fibrillary acidic protein or glutamine synthetase. By electron microscopy, clusters of K virus particles were found only in the nuclei of brain capillary endothelial cells. Although these endothelial cells showed degeneration of varying degree, their basement membranes remained relatively intact and there was no disorganization in the endfeet of contiguous astrocytes. Neurons and glial cells had normal ultrastructures. Therefore, this study has demonstrated that there is involvement of central nervous system during systemic K virus infection and that the infection involves predominantly brain capillary endothelial cells.Supported by Deutsche Forschungsgemeinschaft and Alexander von Humboldt FoundationDepartment of Neuropathology, Institute of Brain Research, University of Tokyo, Tokyo, Japan     

ELECTRON MICROSCOPIC STUDY ON NUCLEAR CHANGES IN MULTIPLE SCLEROSIS BRAIN BIOPSIES

Since intranuclear and cytoplasmic inclusions are characteristic features of virus infections, including SSPE, a biopsy analysis was made from ten MS cases in order to find out those changes suggesting possible viral particles and other signs of infected cells. In three acute cases intranuclear inclusions were found in astrocytes and oligodendrocytes. The majority of these inclusions consisted of small granules about 200 Å in diameter and the total diameter of the inclusions varied from 0.1 to 0.3 microns. Finely granular intranuclear inclusions were also infrequently found. Furthermore nuclear envelope often displayed abnormalities in the same cells which contained nuclear inclusions. Intracytoplasmic inclusions or giant cells were not found nor were clear-cut virus particles. These findings show that nuclear inclusions are characteristic of some acute MS cases. Further studies are necessary in order to relate these inclusions to possible viral etiology of MS.     

Herpes zoster ophthalmicus and delayed contralateral hemiparesis caused by cerebral angiitis: Diagnosis and management approaches

Four patients with herpes zoster ophthalmicus and delayed contralateral hemiparesis are described, and their findings are compared with those in patients previously reported in the English language literature. The current patients evidenced multifocal ipsilateral cerebral angiitis by angiography and multifocal infarcts in the distribution of the ipsilateral middle cerebral artery by computed tomographic scanning. Cerebrospinal fluid showed mononuclear pleocytosis, positive oligoclonal bands, and an elevated immunoglobulin G index. Two patients were treated with corticosteroids and acyclovir, and 1 with corticosteroids alone, all without apparent response. Necrotizing angiitis ipsilateral to the herpes zoster ophthalmicus was demonstrated postmortem in 1 patient with multifocal cerebral infarction and progressive leukoencephalopathy. Neither herpes varicella zoster immunocytochemical reactivity nor viral inclusions were seen. The leukoencephalopathy associated with herpes varicella zoster either may be caused by cerebral angiitis or, as previously reported, may be a temporally remote manifestation of persistent herpes varicella zoster infection. The cerebral angiitis associated with herpes varicella zoster is histologically similar to granulomatous angiitis, and both may be related to herpes varicella zoster infection of the cerebral vasculature.     

Restricted replication of herpes simplex virus in satellite glial cell cultures clonally derived from adult mice.

To determine the possible influence of satellite glial cells on restricting the spread of herpes simplex virus in the peripheral nervous system, HSV replication was studied in clonally derived cultures of satellite glial cells from adult animals. Satellite cells were purified by exploiting their close anatomical association with primary sensory neurons. Dissociated neurons from dorsal root ganglia were micro-manipulated to remove all but one of the attached satellite cells and cultured in the presence of the mitogenic stimulators bovine pituitary extract and cholera toxin. Following a lag phase of 20-30 days some of the individual satellite cells began to proliferate. Initial cultures demonstrated bipolar morphology similar to cultured Schwann cells, some of which differentiated into large astrocytic whorl-like cells on subsequent passage. Immunocytochemical and molecular studies demonstrated that these cells, designated Sat.1, express glial fibrillary acidic protein, confirming their glial origin and by electron microscopy they were shown to be phagocytic. Under single step viral growth conditions Sat.1 cells were restrictive for HSV replication, producing in the order of 1000 times less infectious virus than Vero cells, a standard permissive cell line. These results suggest that satellite cells, which tightly encase sensory neurons, play a role in restricting interneural spread of HSV within the peripheral nervous system.     

Alphaherpesvirus DNA replication in dissociated human trigeminal ganglia

Analysis of the frequency and PCR-quantifiable abundance of herpes simplex virus type 1 (HSV-1) and varicella zoster virus (VZV) DNA in multiple biological replicates of cells from dissociated randomly distributed human trigeminal ganglia (TG) of four subjects revealed an increase in both parameters and in both viruses during 5 days of culture, with no further change by 10 days. Dissociated TG provides a platform to analyze initiation of latent virus DNA replication within 5 days of culture.