Central nervous system susceptibility to herpes simplex infection.

Four days after inoculation of herpes simplex virus (HSV) on the rabbit cornea, distinctive and reproducible lesions appear in the trigeminal root entry zone. These viral lesions, situated in the central nervous system (CNS) portion of the root, consist of severe myelin destruction accompanied by mononuclear cell infiltration and partial sparing of axons. Immunofluorescent study demonstrated abundant viral antigen, and by electron microscopy viral nucleocapsids were found to be numerous within astrocytes and were rarely found in other cell types. In contrast, the adjacent peripheral nervous system (PNS) tissue appears unaffected by the presence of virus. The mechanism for this marked difference in response of the central nervous system and the peripheral nervous system may depend upon the susceptibility of astrocytes to viral infection and replication. The selective nature of the lesion provides an easily reproducible model for further investigation of the response of nervous system tissue to HSV.     

Herpes simplex virus genomes in human nervous system tissue analyzed by polymerase chain reaction

Herpes simplex virus (HSV) is known to establish latency in human trigeminal ganglia. It has been speculated that the virus might also be present in latent fashion in normal human brain, where it might be responsible for conditions such as herpes simplex encephalitis, and less plausibly as a cause for multiple sclerosis or Alzheimer's disease. To test the possibility that HSV exists in normal human brain, we utilized the polymerase chain reaction to assess the frequency and distribution of HSV genomes in the nervous system tissues of patients dying of nonneurological causes. Nine samples were obtained in a systematic fashion from olfatory bulb, gyrus rectus, hippocampus amygdala, calcarine cortex, pons, medulla, cerebellum, and trigeminal ganglia from each of 40 individuals dying of nonneurological disease. HSV genomes were sought in each sample using primers from four regions of the HSV genome. The primers identified in 26 (65%) of 40 samples of trigeminal ganglia. From 30 patients seropositive to HSV, sequences were amplified from 23 (77%). HSV genomic sequences could be amplified and detected in 14 (35%) of 40 brains. The positive areas included medulla, olfactory bulbs, pons, gyrus rectus, amygdala, and hippocampus. The study has confirmed the previous demonstration of latent HSV in trigeminal ganglia in normal humans. The frequency of latent HSV in trigeminal ganglia is in general agreement with results obtained by explanation of ganglia. In addition HSV sequences could be amplified from a variety of other central nervous system structures, suggesting the presence of the virus in latent state in normal central nervous system. Wheter the virus is important in the pathogenesis of nervous system diseases is not answered by these studies. However, it appears that the virus, commonly latent in trigeminal ganglia, may be detectable with lesser frequency in normal brain.     

Targets of herpes simplex virus type 1 infection in a mouse corneal model

Summary In animal models, spread of herpes simplex virus type 1 (HSV-1) from epithelial replication sites to the peripheral and central nervous system is known from analysis of individually dissected tissues. To examine virus spread in undissociated tissues, corneas of adult mice were inoculated with HSV-1. After 1 to 13 days groups of mice were perfused with formalin, and decalcified blocks of head and neck were embedded in paraffin. At intervals, serial sections were screened for HSV antigen. On days 1 and 2, viral antigen was restricted to cornea and conjunctiva but by days 3 and 4 was also seen in autonomic ganglia and the trigeminal system. On day 6, HSV antigen reached its maximum extent; infected sites included the trigeminal complex (ganglion, root, peripheral ophthalmic and maxillary branches and spinal nucleus and tract), ehtmoid sinus and olfactory buld, visual system, and autonomic ganglia (ciliary, pterygopalatine and superior cervical). Antigen progressively diminished on days 8 and 10, and was not detected on day 13. This method demonstrates a broader range of infected tissues and suggests a more complex pattern of HSV spread than has been previously recognized. Virus appears to reach the intracranial compartment by four different neural routes. When effects of higher and lower corneal inoculation doses were compared, a lower dose resulted in lower peak HSV titers in trigeminal ganglion and brain stem and later virus appearance in these tissues. Thus, dose may influence the kinetics of HSV spread from the peripheral inoculation site to the CNS.Supported in part by U.S.U.H.S. grant, R07396. the opinions or assertions contained herein are the private views of the authors and should not be construed as official or necessarily reflecting the views of the Uniformed Services University of the Health Sciences or Department of Defense. There is no objection to its presentation and/or publication     

Factors that contribute to the transneuronal spread of herpes simplex virus

In viral encephalitis and retinal necrosis, different herpes simplex virus (HSV) strains spread between neurons in the central nervous system (CNS) by distinctly different routes. The steps of viral infection and spread in a single neuron type and nearby glial cells in vivo have been determined for three different strains of HSV (F, H129, and McIntyre-B). The corneas of mice were inoculated with equivalent titers of the strains. Two to 5 days later, the animals were killed. The spread of viral proteins within trigeminal cells was examined using immuno- and electron microscopy and Western blots with anti-HSV polyclonal antiserum. McIntyre-B virus infection resulted in fewer labeled ganglion cells, possibly as a result of reduced viral production in the corneal epithelium or trigeminal ganglion cells. Although the McIntyre-B strain was at least as, if not more efficient, at retrograde transport than the other strains, the amount of McIntyre-B virus that was transported in the trigeminal roots in an anterograde direction was significantly less than the other strains. Uptake by ganglionic satellite cells was qualitatively similar for the three strains, but maturation and release of virus from satellite cells to other neurons were reduced in the McIntyre-B strain. These characteristics may account for the preferential retrograde transneuronal spread of McIntyre-B strain. J. Neurosci. Res. 49:485–496, 1997. © 1997 Wiley-Liss, Inc.     

Herpes simplex encephalitis: Immunohistological demonstration of spread of virus via olfactory and trigeminal pathways after infection of facial skin in mice

An immunohistological study of viral antigen (VA) in the brain was carried out in mice which had been infected with herpes simplex type 1 virus (HSV) in the skin of the face. In 77% of the mice with VA in the brain the olfactory system as well as the trigeminal system/brainstem was affected. The remaining 23% had VA in the trigeminal system/brainstem only. Eye swab cultures yielded HSV from all mice with VA in the olfactory system. The ease of access of virus infecting the face to the olfactory system shown in this model may have implications for human infections.     

Effect of Herpes simplex virus infection on the trigeminal jaw-opening reflex in guinea pigs.

Herpes simplex virus (HSV) infection induces numerous electrophysiological and microscopic changes in neurons in vitro. To investigate the effect of HSV infection on in vivo neuronal activity, we induced an acute, latent and reactivated HSV infection of the trigeminal ganglia of guinea pigs through orofacial HSV inoculation and studied its effect on the trigeminal jaw-opening reflex of anesthetized guinea pigs. During the acute viral infection period both the threshold for elicitation of the reflex, and the latency to the onset of the reflex response were increased. During the latent viral infection in the trigeminal ganglia, the jaw-opening reflexes in the viral infected animals were not different from those of non-infected control animals. However, reactivation of the latent viral infection in these animals resulated in increases in both the threshold and latency of the jaw-opening reflex. These changes were similar to those found in animals with the acute viral infection. These results indicate that acute or reactivated latent HSV infection of the nervous system results in functional changes in the reflex pathways involving the trigeminal gasserian ganglia and beurons harboring infectious HSV-1.     

Targets of infection in a herpes simplex-reactivation model

Summary In mice surviving intracerebral inoculation with herpes simplex virus type 2 (HSV-2), recurrent infection was induced using high-dose immunosuppressive treatment. Targets of reactivated infection were identified in serial sections of heads using immunoperoxidase methods to detect viral antigen. Peripheral targets were cranial sensory and autonomic ganglia, peripheral nerves, and many non-neural structures. Central neural tissues included retina, optic nerve and tract, lateral geniculate nucleus and superior colliculus. Brain stem nuclei containing antigen were chiefly those associated with the trigeminal, facial, glossopharyngeal or vagus nerves. A few white matter regions were also positive. This is the first study using antigen methods to show that the central nervous system can be an important target of recurrent HSV infection in an animal model. Patterns of antigen-containing structures suggest that neural connections are important determinants of sites of reactivated infection. Decalcification of bone permits study of antigen distribution in tissues retaining their anatomical relationships.     

Multifocal CNS demyelination following peripheral inoculation with herpes simplex virus type 1

The peripheral inoculation of herpes simplex virus type 1 (HSV 1) in experimental animals induces central nervous system (CNS) demyelinating lesions, but the potential relevance of this model to multiple sclerosis is lessened by the unifocal nature of the lesion. In this study, inbred strains of mice were selected on the basis of varying resistance to mortality following lip inoculation with virus. A spectrum of CNS pathology was observed, ranging from focal collections of inflammatory cells at the trigeminal root entry zone in resistant strains (C57BL/6J), to unifocal demyelinating lesions in moderately resistant strains (BALB/cByJ), to multifocal demyelinating lesions throughout the brain in susceptible strains (A/J). Findings from viral titration studies of the CNS support a direct cytolytic effect of virus in the development of demyelinating lesions at the trigeminal root entry zone but cannot exclude an immune-mediated component. Furthermore, 50% tissue-culture-infective doses, immunofluorescence, and electron microscopic studies of primary cultures of oligodendrocytes, derived from the three strains of adult mice, identify differences in resistance to HSV 1 infection in vitro, suggesting that differences at this level may also contribute to the pathological appearance. Multifocal lesions in A/J mice were first observed when the infectious virus could no longer be isolated from the CNS and may be the result of an immune-mediated process "triggered" by the acute CNS infection in susceptible strains of mice.     

Observations on the distribution of canine distemper virus in the central nervous system of dogs with demyelinating encephalitis

Summary The distribution of canine distemper virus in the central nervous system was examined in 11 dogs with demyelinating encephalitis by the direct fluorescent antibody technique on paraffin sections of brain and spinal cord. In the grey matter there was a good correlation between the presence and severity of lesions and presence and amount of viral antigen. Large concentrations of virus were found in neurons and their processes. In most demyelinating lesions only small amounts of viral antigen were found, mostly located in astrocytes. The potential importance of the role of the astrocyte in demyelination in canine distemper virus infection is stressed.     

Herpes simplex virus type I (HSV I)-induced multifocal central nervous system (CNS) demyelination in mice.

Multifocal central nervous system (CNS) demyelination develops in the brains of SJL/J, PL/J, and A/J mice following lip inoculation with a specific strain of herpes simplex virus I (HSV I). The lesions in all three inbred strains of mice share similar characteristics including demyelination, relative preservation of axons, and a mononuclear cell (MNC) infiltrate. The lesions, developing during the early phase of demyelination, also appear sequentially in the CNS (trigeminal root entry zone of the brainstem greater than cerebellum greater than cerebral hemispheres) of all three strains of mice but differ in the time of their initial appearance following infection as well as their morphology. In SJL/J mice, new areas of demyelination are observed for only 24 days following lip inoculation with virus. Late stage multifocal CNS demyelination persists throughout 28 weeks postinoculation (pi) in PL/J mice while in A/J mice the development of new areas of demyelination are restricted to 8 weeks pi. Although mononuclear inflammatory cells are present in the new areas of demyelination in either PL/J or A/J mice, viral antigens are not detected in the CNS beyond 12 days pi. In contrast, in situ hybridization studies using 35S-cDNA HSV probes and performed beyond day 12 pi identify probe-positive cells central to a number of the multifocal CNS demyelinating lesions in A/J mice. Results from studies with inbred and congenic strains of mice indicate that the major histocompatibility complex (H-2) does not determine the development of multifocal CNS demyelination following lip inoculation with HSV I but does influence the morphological appearance of the lesions that do develop.     

Herpes simplex virus,type 1 invasion of the rabbit and mouse nervous systems revealed by in situ hybridization

Summary Using a³H-labelled virion DNA probe applied to tissue sections, we have previously identified the precise microscopic anatomical localtion of herpes simplex virus (HSV) during the acute and latent stages of infection of the mouse trigeminal ganglia and central nervous system (CNS). In the present investigation, we compared the mouse and the rabbit with respect to their ability to support acute and latent infections of trigeminal ganglionic and central nervous system neurons. We found that HSV-1, strain F, produced acute and latent infection of trigeminal ganglion cells in both mice and rabbits; however, lower levels of HSV-1 RNA were expressed in rabbit neurons as compared to mouse neurons, and many fewer neurons of the rabbit supported an acute infection than in the mouse. Studies of the trigeminal system within the CNS revealed that HSV-1 established latency more readily in the mouse than in the rabbit. The histopathology observed in acutely infected rabbit brain was less intense and less widespread than in mouse brain.Supported by the Medical Research Service of the Veterans Administration and by Institutional and Biomedical Research Support Funds awarded by the Dean of the Medical School, University of Utah     

Herpes simplex virus infection in capsaicin-treated mice

Following inoculation into the snout herpes simplex virus (HSV) spread to neurons in mouse trigeminal ganglion and subsequently to the brain. Capsaicin treatment of neonatal mice, which causes a loss of unmyelinated sensory neurons, some of which contain substance P, reduced the mortality rate of HSV-infected mice. Moreover, a lower percentage of mice survived the infection with reactivatable virus. There was also an extensive infection of glial cells proximal to the transitional zone in the trigeminal root between the peripheral and central nervous system. Distal to this zone there was an accumulation of substance P immunoreactivity in centrally directed fibres. This amplified degenerative effect on central branches of the substance P containing sensory nerves by glial infection may contribute to the deafferentiation pain syndrome following HSV infection.     

The weakly virulent herpes simplex virus type 1 strain KOS-63 establishes peripheral and central nervous system latency following intranasal infection of rabbits, but poorly reactivates in vivo.

The virological, clinical and electrophysiological manifestations of acute and experimentally reactivated infections of the rabbit central nervous system (CNS) and trigeminal ganglia have been studied after intranasal infection with herpes simplex virus type 1 (strain KOS-63). All animals shed virus in nasal secretions during the acute phase of infection. Although no rabbits developed clinical signs during the acute phase of infection, mild electroencephalographic (EEG) abnormalities consistent with viral invasion of the CNS were seen. KOS-63 produced only occasional gross and histopathologic herpetic lesions of the CNS and was very rarely recovered from the brain. These results indicate that KOS-63 was poorly neuroinvasive and only mildly neurovirulent during the acute phase of infection. However, KOS-63 did establish latency within the CNS and trigeminal ganglia of infected rabbits as demonstrated by in situ hybridization and by recovery of virus from co-cultivation cultures, but not from cell-free homogenates of nervous tissue. Cyclophosphamide and dexamethasone injections were used to reactivate latent CNS and trigeminal ganglionic infections. Following injection of the drugs, no animal shed virus in nasal secretions or developed obvious clinical or EEG changes. However, KOS-63 was recovered from co-cultivation cultures of brain and trigeminal ganglia at greater frequency following drug injection than during latency. These results indicate that KOS-63 was only poorly susceptible to drug-induced reactivation. In vivo experiments confirmed that the apparent poor neuroinvasiveness and weak neurovirulence of KOS-63 was not due to viral temperature-sensitive defects, deficient production of viral thymidine kinase, or abnormal defects in viral DNA polymerase function.     

Herpes simplex encephalitis: A serological follow-up study

A solid-phase radioimmunoassay method was used for the detection of herpes simplex virus (HSV) immunoglobulin M (IgM), IgA, and IgG antibodies within the central nervous system in 11 adult patients with acute HSV encephalitis. Serial cerebrospinal fluid (CSF) and serum specimens were sampled during the observation periods, extending up to 43 months after onset.
The clinical diagnosis of HSV encephalitis was confirmed demonstrating virus or virus antigen in the central nervous system in four patients and with significant HSV antibodies in CSF in all the patients. In acute stage CSF HSV antibodies of a significant level were demonstrated in one of four samples taken on days 3–4 after onset, and in samples taken on days 6–8 in five of nine patients. CSF HSV antibodies of a significant and high level were detected in all samples taken from day 10 after onset.
Intrathecal production of HSV IgM and IgA antibodies lasted from 7 weeks to 43 months during the observation periods. All patients had persistent intrathecal production of HSV IgG antibodies as well as of oligoclonal IgG during the total observation periods up to 43 months.     

Peripheral nervous system demyelination with herpes simplex virus

Inoculation of the cornea or footpad with herpes simplex virus Type I (HSV) has been shown to produce subsequent encephalitis or myelitis respectively. Although Schwann cells become infected, there is no destruction or demyelination in the peripheral nervous system (PNS). Demyelination only occurs in the central nervous system. Previous studies have shown that the Schwann cells infected with HSV do not produce enveloped viral particles. The studies presented here demonstrate that microinjection of HSV into the sciatic nerve of mice causes focal mononuclear cell infiltration and demyelination seven days after injection. The Schwann cells in this model produced enveloped virus. These studies demonstrate that when HSV is introduced into the extracellular space of the PNS, demyelination occurs.     

Early loss of astrocytes in herpes simplex virus-induced central nervous system demyelination

Immunohistochemistry was used to study herpes simplex virus type 1-induced central nervous system demyelination in the trigeminal root entry zone of mice inoculated with herpes simplex virus type 1 by the corneal route. There was no change in peripheral nervous system myelin as shown by immunostaining for P0 glycoprotein. Double immunoperoxidase staining for herpes simplex virus type 1 antigens and glial fibrillary acidic protein showed that most of the infected cells were astrocytes. Glial fibrillary acidic protein immunostaining was completely lost in the inferior medial portion of the trigeminal root entry zone at 6 days after herpes simplex virus type 1 inoculation, a time when central nervous system myelin was preserved as indicated by immunostaining for myelin basic protein. The pattern of glial fibrillary acidic protein staining did not change and herpes simplex virus type 1 antigens were no longer detected after day 8. There was a progressive loss of myelin basic protein staining within the area unstained by glial fibrillary acidic protein antisera on days 8 to 14. This pattern of astrocyte loss before central nervous system demyelination is strikingly different from the reactive astrocytosis seen in other demyelinating lesions, such as acute experimental allergic encephalomyelitis, progressive multifocal leukoencephalopathy, or acute multiple sclerosis. Herpes simplex virus type 1 infection in mice provides an unusual model of acute central nervous system demyelination preceded by a loss of astrocytes.     

Virus-induced demyelination in herpes simplex virus-infected mice

Herpes simplex virus (HSV) infection of the mouse trigeminal ganglia and the brain stem is associated with demyelination of axons in the central part of the trigeminal root and inflammatory cell infiltration and perivascular demyelination in the brain stem. Cyclophosphamide (CPA) treatment prior to or soon after HSV inoculation caused increased axonal spread of infective virus from the peripheral site of inoculation, more widespread and severe demyelination and increased mortality, suggesting that by CPA the virus invasion of the CNS was facilitated. A direct cytocidal effect of HSV on myelinating cells seemed one plausible explanation for the demyelination. Influence on demyelination at late stages of infection by cytotoxic immune reactions are not excluded by the results reported but seemed not to dominate the picture. Schwann cells from the peripheral part of the nerve root invaded demyelinated areas in the brain stem and remyelinated the axons.     

Herpes-simplex-related antigen in human demyelinative disease and encephalitis

Summary Using immunohistochemical methods optimized to detect herpes simplex virus type 2 (HSV-2) antigen, paraffin sections from human central nervous system tissues from 31 cases pathologically diagnosed as multiple sclerosis (MS), 34 cases of other neurological diseases, 4 adult cases of HSV encephalitis, and mouse brains infected with various HSV strains were examined. Two distinct patterns of immunoreactivity with HSV antisera were seen. In typical acute human and experimental encephalitis, antigen was readily detected using high dilutions of antisera to both HSV types –1 and –2, and was found nonselectiviely in both neurons and glia. Lesions were destructive, with necrosis of all neural cell types, and inflammation was a mixture of polymorphonuclear and mononuclear cells. By contrast, immunoreactivity in lesions in each of three MS cases and in one case of brain stem encephalitis was found only with HSV-2 antisera, and relatively high antiserum concentrations were required to detect it. Reactivity appeared to be largely restricted to glial cell nuclei within and near lesions that were selectively demyelinated. Only mononuclear inflammation was present. These experiments suggest that HSV-related antigen may be found in a broader spectrum of human CNS lesions than has previously been recognized, and that HSV or a related agent may be associated with a selective infection of glial cells and with CNS demyelination.     

SPONTANEOUS RECOVERY FROM THE ENCEPHALOMYELITIS IN MICE CAUSED BY STREET RABIES VIRUS

Recovery from rabies was studied in an experimental model. Young adult mice were inoculated in a hindlimb footpad with street rabies virus (fox salivary gland isolate). In a group of 62 mice, 97% developed clinical rabies with paresis of the extremities and spasticity, and 37% recovered with neurological sequelae. There was an acute inflammatory reaction in the brainstem and grey matter of the spinal cord, and degeneration of myelinated axons in the white matter of the cord and in the dorsal roots. Rabies virus antigen was found in the central nervous system of all mice examined between day 5 and 13, and also in trigeminal and dorsal root ganglia. Surviving mice had neutralizing antibodies in serum and brain tissue, and 90% survived an intracerebral challenge with the CVS strain of fixed rabies virus. Spontaneous recovery from rabies encephalomyelitis was demonstrated with evidence of viral replication and pathological changes in the central nervous system.