Spread of herpes simplex virus type 1 in the central nervous system during experimentally reactivated encephalitis

Because many of the features of reactivated herpes simplex virus type 1 (HSV-1) central nervous systems (CNS) infections in vivo are incompletely understood, we used an animal model to study the development of the morphological, ultrastructural, radiological and immunological changes which occurred during acute and experimentally reactivated diseases. Rabbits were intranasally inoculated with HSV-1, and their latent trigeminal ganglionic and CNS infections were reactivated by intravenous injection of cyclophosphamide and dexamethasone. Technetium brain scans were performed to localize areas of blood-brain barrier breakdown, and cerebrospinal fluid (CSF) was analysed for IgG content by radial immunodiffusion assays. Nervous system tissues were studied by in situ hybridization and by immunofluorescent, light and electron microscopic techniques. Diffuse uptake of technetium was observed as HSV-1 spread transsynaptically into the brain during the acute phase of infection, and viral antigens and nucleic acids were detected in both the CNS olfactory and trigeminal systems. During latency, viral RNA was detected in the nuclei of neurons within the CNS olfactory cerebral and entorhinal cortices, indicating that HSV-1 became latent within the same CNS structures that were involved during the acute phase of infection. Following drug-induced reactivation, the brain scans revealed a more focal breakdown of the blood-brain barrier, and both neurons and neuronal processes in the entorhinal and olfactory cortices contained viral nucleic acids which correlated with the ultrastructural presence of HSV-1 virions. During the reactivated phase of infection a marked increase in the CSF IgG index occurred without an increase in the CSF: serum albumen ratio indicating a prompt intrathecal response in infected rabbits as compared to controls. To some extent, the CSF IgG index reflected the degree of histopathological damage.     

Expression of nectin-1 in normal and herpes simplex virus type 1-infected murine brain.

Nectin-1 is an adherens junction protein that serves as an entry receptor for neurotropic herpes simplex virus (HSV). The expression of nectin-1 in the central nervous system (CNS) has not been well defined. Furthermore, it is not known whether HSV infection has an effect on nectin-1 expression in the brain. To better understand nectin-1 expression in normal and HSV-infected brain, the authors used immunohistochemistry to characterize the expression of nectin-1 in brain tissue of uninfected adult mice and mice infected with HSV-1. In the CNS of untreated and mock-infected mice, virtually all neurons, ependymal cells, choroid plexus epithelial cells, meningothelial cells, and vascular endothelial cells expressed nectin-1. Many oligodendrocytes, astrocytes, and vascular smooth muscle cells also demonstrated nectin-1 expression, but a minority of these cells did not stain for nectin-1. Brain tissue derived from mice euthanized 5 to 8 days after intracerebral inoculation of HSV-1 showed inflammation and widespread expression of HSV-1 proteins in neurons. In HSV-1-infected brains, many inflammatory cells showed nectin-1 expression and neuronal nectin-1 staining showed a wider variation in signal strength than that detected in uninfected tissues. Many neurons showing nuclear fragmentation consistent with the morphologic appearance of apoptosis showed little or no evidence of nectin-1 expression, whereas occasional neurons stained more intensely positive for nectin-1 than those in uninfected brain tissue. These findings confirm and extend previous observations of nectin-1 expression in the nervous system and suggest that HSV-1 infection leads to changes in nectin-1 expression in the CNS, which may contribute to HSV-induced pathology and dissemination.     

Coronavirus infects and causes demyelination in primate central nervous system.

Two species of primates, Owl and African green monkeys, were inoculated intracerebrally with either the neurotropic mouse hepatitis virus JHM or the putative multiple sclerosis brain coronavirus isolate SD. These viruses caused an acute to subacute panencephalitis and/or demyelination in the infected animals. The course of pathogenesis and sites of detected viral RNA and antigen was dependent both on animal species and virus strain but the results clearly showed that these viruses replicated and disseminated in the central nervous system (CNS) of these primates. This study suggests that human CNS may be susceptible to coronavirus infection.     

HSV-1 brain infection by the olfactory nerve route and virus latency and reactivation may cause learning and behavioral deficiencies and violence in children and adults: A point of view

Two recent studies provided new evidence on the latency of HSV-1 DNA in 15.5% of olfactory bulbs and in 72.5% of trigeminal nerves from human corpses at forensic postmortems (1) and in 35% of 40 autopsied human brains (2). In the latter brains, latent HSV-1 DNA was found in the olfactory bulbs, amygdala, hippocampus, brain stem, and trigeminal ganglia. Although in these studies it is not known by which route HSV-1 entered the olfactory bulbs and brain, experimental studies in mice (3) revealed that injection of HSV-1 into the olfactory bulbs leads to virus migration into the brain amygdala and hippocampus via the olfactory nerve and locus coeruleus. If the olfactory ciliary nerve epithelium is the port of entry of HSV-1 into the olfactory bulbs and brain in humans as well, protection of the nose against HSV-1 infection may be needed to prevent virus latency in neurons in the amygdala and hippocampus (3). Infection of humans by HSV-1 was estimated to increase from 18.2% in the 0–20 year population group to 100% in persons older than 60 years (1), indicating that worldwide human populations at all ages are at risk of brain infection by the olfactory nerve route. In addition, both primary infection and reactivation of latent DNA in the brain may lead to damage of neurons in the brain involved in memory, learning, and behavior, as observed in infected, acyclovirtreated mice (3). The current introduction of a live apathogenic varicella-zoster virus (VZV) vaccine to immunize children against chickenpox (4) may suggest that the time is ripe for immunization of children and adults against HSV-1 infections, especially infections by the olfactory nerve route, to prevent potential brain damage.     

Spread of a neurotropic murine coronavirus into the CNS via the trigeminal and olfactory nerves

The route of entry into the central nervous system (CNS) of most neurtropic viruses has not been established. The coronavirus, mouse hepatitis virus strain JHM (MHV-JHM), causes acute encephalomyelitis and acute and chronic demyelinating diseases and is an important model system for virus-induced neurological disease. Suckling C57BL/6 mice infected intranasally with MHV-JHM develop either the acute encephalomyelitis or a late onset, symptomatic demyelinating encephalomyelitis, depending on whether they are nursed by unimmunized or immunized dams. Analysis by in situ hybridization was used to determine the route of entry of MHV-JHM into the CNS in these mice. At early times, viral RNA was detected only in the trigeminal and olfactory nerves and in their immediate connections in all mice. A few days later, MHV-JHM RNA was found throughout the brain in mice dying of the acute encephalomyelitis, but remained confined to the entry sites in mice which did not develop acute disease. These results suggest that MHV-JHM enters the CNS via an interneuronal route in all mice, but that the presence of maternal antibody prevents the dissemination of virus via extracellular fluid. In addition, MHV-JHM may establish low-level persistence in the trigeminal or olfactory nerve or in one of its connections in mice that do not develop acute encephalomyelitis.     

利用缺陷型单纯疱疹病毒载体在大鼠中枢神经系统…

本研究利用构建的Ｉ型单纯疱疹病毒（ＨＳＶ－１）扩增子质粒载体ｐＨＳＬ，在辅助病毒ＨＳＶ－１ｔｓＫ（许可温度３１℃）的辅助下，以首尾相连的连接全形成包装成复制缺陷型的ＨＳＶ－１假病毒颗粒，得到一种ＨＳＶ－１混合毒种ｄｖＨＳＬ。将ｄｖＨＳＬ接种体外培养的大鼠胚胎脊髓运动神经元和大脑皮质神经元后均可获得报告基因的表达；接种大鼠角膜后ｌａｃＺ基因三叉神经节中持续表达两个月；直接注射接种到大鼠前脑上质后，可     

Traffic of leukocytes in the central nervous system is associated with chemokine up-regulation in a severe model of herpes simplex encephalitis: An intravital microscopy study

Herpes simplex virus type 1 (HSV-1) is a human pathogen that may cause severe encephalitis. The development of experimental models of HSV-1 encephalitis is relevant for the comprehension of the immune mechanisms involved in this infection. C57BL/6 mice were inoculated intracranially with 10⁴ PFU of neurotropic HSV-1. All animals developed signs of encephalitis and died until day 6 post-infection (pi). Using intravital microscopy, we demonstrated increased leukocyte rolling and adhesion in the brain microvasculature of infected mice at days 1, 3 and 5 pi. The infection was followed by a significant increase in chemokine levels, including CCL2, CCL3, CCL5, CXCL1 and CXCL9. TNF-α also showed a significant increase at day 3 pi. Histological analyses demonstrated diffuse meningoencephalitis characterized mainly by mononuclear cell infiltrates. The present model of HSV-1 encephalitis exhibits high mortality in the very first days of infection. Accordingly, there were increased rolling and adhesion of leukocytes along the brain endothelium wall and a high expression of chemokines in the central nervous system. These results corroborate the role of chemokines in leukocyte recruitment following HSV-1 infection in the central nervous system.     

Central nervous system and genital infection with reactivation of latent herpes simplex virus type 2 in mice

To establish a reactivation model of genital and central nervous system infection, 3- to 12-week-old outbred or BALB/c mice were inoculated vaginally with the HG-52 strain of herpes simplex virus type 2 (HSV-2). Primary infection was confirmed by serially positive vaginal cultures. Mortality in 6- and 12-week old infected mice was about 20%. In survivors, clearance of infectious virus was confirmed in serially negative vaginal cultures. At 6 weeks, immunosuppression of survivors with cyclophosphamide and antilymphocyte serum was begun. Recurrent virus shedding, monitored by daily vaginal cultures, was detected in the majority of animals. All mice became moribund or died, usually during the third to fifth weeks of immunosuppression. Brains and spinal cords from which all sensory ganglia had been removed were homogenized and inoculated onto cultures. One or both central nervous system (CNS) samples were virus-positive in nearly half of these mice, and cell-free virus was isolated from most positive brain and cord supernatants tested. Three-fourths of mice had evidence of virus reactivation with immunosuppression, as indicated by vaginal or CNS isolations, and by failure to isolate virus by identical means in matched infected, non-immunosuppressed controls. Vaginal, spinal cord and brain isolates occurred independently of one another in many immunosuppressed mice, and could not be predicted from presence or absence of external genital lesions during primary infection. These experiments show that with immunosuppression, reactivations of latent HSV-2 infections in mice can be detected in the genital tract and CNS, and provide a model to study productive, recurrent CNS infection and disease.     

A thymidine kinase deficient HSV-2 strain causes acute keratitis and establishes trigeminal ganglionic latency,but poorly reactivates in vivo

The incidence of herpetic keratitis following in-tranasal or direct ocular infection with thymidine kinase-negative (TK^?) strains of herpes simplex virus (HSV)-2 has not been well studied, and the role of the TK gene in the establishment of latency and virus reactivation is controversial. To determine whether a TK^? strain of HSV-2 could establish trigeminal ganglionic latency and be reactivated in vivo to produce recurrent keratitis or nervous system infection, an animal model of acute and recurrent infection was utilized. Rabbits were infected by the intranasal or ocular routes, and latency was reactivated by immuno-suppression. Virus shedding in nasal and ocular secretions was monitored, and the eyes were examined for the presence of corneal epithelial lesions during acute and reactivated infections. Central nervous system (CNS) and trigeminal ganglionic tissues were assayed by histologic, virologic, and in situ hybridization techniques. All rabbits intranasally infected shed virus in both ocular and nasal secretions, whereas only 30% of rabbits infected in the eyes shed virus in nasal secretions. Virus was recovered from co-cultivation cultures, but not from cell-free ho-mogenates, of trigeminal ganglionic and CNS tissues from animals inoculated by both routes. The incidence of keratitis was much greater after direct ocular inoculation, although both routes of inoculation produced CNS and ganglionic inflammatory lesions. Keratitis healed in 92% of the animals infected by the ocular route by 26 days post infection. Of rabbits initially infected in the eyes and then subjected to drug-induced reactivation, only 30% shed virus, which was limited to a 24 hour period; there was no reappearance of epithelial keratitis, no animal became blind, and none died. In contrast, latently infected control rabbits uniformly reactivated. These studies show that this TK^? HSV-2 strain (i) replicates in the eye, (ii) is neuroinvasive but non-neurovirulent following intranasal and direct ocular infection; (iii) sheds in the eye more frequently and for longer periods after ocular than after intranasal inoculation; (iv) induces epithelial keratitis that usually heals spontaneously; (v) establishes latency in trigeminal ganglionic neurons, but no other ganglionic cells; and, (vi) reactivates in a small proportion of animals, but does not produce recurrent ocular lesions following drug-induced immunosuppres-sion. Thus, the TK gene appears directly involved in HSV latency and reactivation in vivo. © 1994 Wiley-Liss, Inc.     

Colonization of murine ganglia by a superinfecting strain of herpes simplex virus

We report on the colonization of murine trigeminal ganglia after sequential infection of mice by herpes simplex viruses (HSVs). In preliminary studies, we have established that whereas the HSV-1(F) strain efficiently colonizes ganglia when inoculated by either the ear or eye routes, the HSV-1 X HSV-2 recombinant C7D colonizes ganglia when inoculated by the eye route only. The experimental design consisted of inoculating the right eye with C7D on day 1 and with HSV-1(F) in both left and right eyes on day 26. Both right and left trigeminal ganglia were removed and analyzed independently for latent virus on day 52. Our studies indicate that HSV-1(F) viruses were recovered from all left trigeminal ganglia but from only a small number of right trigeminal ganglia. Some right trigeminal ganglia yielded no viruses, whereas others yielded both C7D and HSV-1(F) viruses identified on the basis of plaque morphology and restriction enzyme cleavage patterns of viral DNA. The results indicate that more than one virus may colonize the same ganglion and that trigeminal ganglia may be protected from colonization by a superinfecting virus by determinants acting at a local level in the absence of demonstrable virus.     

Herpes simplex virus type 1 DNA sequences which direct spread of virus from cornea to central nervous system

The virulence of a herpes simplex virus (HSV) intertypic recombinant possessing HSV-1 DNA sequences from map units 0.31 to 0.44 and HSV-2 sequences from map units 0 to 0.30 and 0.45 to 1.0 were compared with the virulence of the two parental strains. Following ocular inoculation, both the intertypic recombinant and the HSV-1 parent replicated at the infection site and spread to the peripheral and central nervous system (CNS) to produce fatal encephalitis. The HSV-2 parent also replicated at the infection site but failed to progress to the CNS. However, when inoculated intracerebrally, the HSV-2 strain was as lethal as the HSV-1 parent. Furthermore, the HSV-2 strain could produce thymidine kinase at 37 and 39 degrees in levels comparable to the HSV-1 strain. The results indicate that transfer of the HSV-1 DNA sequences imparted to the recombinant virus the necessary genetic information to spread from the cornea into the central nervous system.     

Evidence that the gene for herpes simplex virus type 1 DNA polymerase accounts for the capacity of an intertypic recombinant to spread from eye to central nervous system

HSV-1(17) replicates 100-fold more efficiently than HSV-2(186) within trigeminal ganglia following ocular infection. In order to identify the nucleotide sequences responsible for the differences in the capacity of the two HSV strains to grow within the peripheral nervous system, an intertypic recombinant was generated by infecting neuroblastoma cells with HSV-2(186) and a HSV strain possessing nucleotide sequences from HSV-1(17). The genome of the intertypic recombinant was composed entirely of HSV-2(186) DNA except for 2.0 kb of HSV-1(17) DNA positioned between m.u. 0.413 and 0.426. Following corneal infection of mice, the intertypic recombinant grew to higher titers in both ocular tissues and trigeminal ganglia than did the HSV-2 parent. Most significantly, the intertypic recombinant could spread into the brain from the trigeminal ganglion and kill the host whereas mice inoculated with the HSV-2(186) parent survived infection. The 2.0 kb of HSV-1(17) DNA inserted into the genome of the intertypic recombinant encodes the 5' terminus of the HSV-1 gene for DNA polymerase. Thus, the results suggest that the difference in the capacity of two HSV strains to replicate within the trigeminal ganglion of its host and to spread into the brain is determined by nucleotide sequences within the gene for DNA polymerase.     

Herpes simplex virus type 1 DNA sequences which direct spread of virus from comes to central nervous system

The virulence of a herpes simplex virus (HSV) intertypic recombinant possessing HSV-1 DNA sequences from map units 0.31 to 0.44 and HSV-2 sequences from map units 0 to 0.30 and 0.45 to 1.0 were compared with the virulence of the two parental strains. Following ocular inoculation, both the intertypic recombinant and the HSV-1 parent replicated at the infection site and spread to the peripheral and central nervous system (CNS) to produce fatal encephalitis. The HSV-2 parent also replicated at the infection site but failed to progress to the CNS. However, when inoculated intracerebrally, the HSV-2 strain was as lethal as the HSV-1 parent. Furthermore, the HSV-2 strain could produce thymidine kinase at 37 and 39° in levels comparable to the HSV-1 strain. The results indicate that transfer of the HSV-1 DNA sequences imparted to the recombinant virus the necessary genetic information to spread from the cornea into the central nervous system.     

Herpes simplex virus-specific memory CD8+ T cells are selectively activated and retained in latently infected sensory ganglia

This study challenges the concept that herpes simplex virus type 1 (HSV-1) latency represents a silent infection that is ignored by the host immune system, and suggests antigen-directed retention of memory CD8(+) T cells. CD8(+) T cells specific for the immunodominant gB(498-505) HSV-1 epitope are selectively retained in the ophthalmic branch of the latently infected trigeminal ganglion, where they acquire and maintain an activation phenotype and the capacity to produce IFN-gamma. Some CD8(+) T cells showed TCR polarization to junctions with neurons. A gB(498-505) peptide-specific CD8(+) T cell clone can block HSV-1 reactivation from latency in ex vivo trigeminal ganglion cultures. We conclude that CD8(+) T cells provide active surveillance of HSV-1 gene expression in latently infected sensory neurons.     

Herpes simplex virus type 1 induces filopodia in differentiated P19 neural cells to facilitate viral spread

Herpes simplex virus type-1 (HSV-1) is a neurotropic virus with significant potential as a viral vector for central nervous system (CNS) gene therapy. This study provides visual evidence that recombinant green fluorescent protein (GFP)-expressing HSV-1 travel down dendrites in differentiated P19 neuronal-like cells to efficiently reach the soma. The virus also promotes cytoskeletal rearrangements which facilitate viral spread in vitro, including often dramatic increases in dendritic filopodia. Viral movements, cell infection and filopodia induction were each reduced with the actin polymerization inhibitor cytochalasin D, suggesting the involvement of the actin cortex in these processes. The observation of neural cytoskeletal reorganization in response to HSV-1 may shed light on the mechanisms by which acute viral infection associated with herpes encephalitis produces cognitive deficits in patients.     

Poliovirus replication and spread in primary neuron cultures

While some neurotropic viruses cause rapid central nervous system (CNS) disease upon entry into the brain parenchyma, other viruses that are cytolytic in the periphery either result in little neuropathology or are associated with a protracted course of CNS disease consistent with persistent infection. One such virus, poliovirus (PV), is an extremely lytic RNA virus that requires the expression of CD155, the poliovirus receptor (PVR), for infection. To compare the kinetics of PV infection in neuronal and non-neuronal cell types, primary hippocampal neurons and fibroblasts were isolated from CD155+ transgenic embryos and infected with the Mahoney and Sabin strains of PV. Despite similar levels of infection in these ex vivo cultures, PV-infected neurons produced 100-fold fewer infectious particles as compared to fibroblasts throughout infection, and death of PV-infected neurons was delayed approximately 48 h. Spread in neurons occurred primarily by trans-synaptic transmission and was CD155-dependent. Together, these results demonstrate that the magnitude and speed with which PV replication, spread, and subsequent cell death occur in neurons is decreased as compared to non-neuronal cells, implicating cell-specific effects on replication that may then influence viral pathogenesis.     

Herpes simplex virus type 1, apolipoprotein E and Alzheimer' disease

Various infectious agents, and viruses in particular, have been proposed as potential causes of Alzheimer's disease. Herpes simplex virus type 1 (HSV-1) is one of the stronger candidates because it is neurotropic, ubiquitous in the general population and able to establish lifelong latency in the host. The body of evidence for the role of HSV-1 in Alzheimer's disease is contentious but centres around its presence in the regions of the brain affected by Alzheimer's disease. The fact that HSV-1 is also present in elderly patients without the disease suggests that the virus is not an independent cause of the condition. The incidence of Alzheimer's disease is highest in carriers of the apolipoprotein (APO) E-e4 allele who harbour HSV-1 DNA in the CNS, so it is possible that these agents are co-factors for the disease. However, studies investigating this have been small and limited by the need to access brain tissue from non-diseased APOE-e4 carriers. Human herpesvirus type 6 (HHV-6) is another virus investigated as a potential contributor to Alzheimer's disease. However, it is uncertain whether its presence is a cause or a consequence of the disease and it may be that HHV-6 merely exacerbates the potentially harmful effects of HSV-1 in APOE-e4 carriers. It is difficult to ascertain the role of an infectious agent in Alzheimer's disease due to the difficulty of establishing the timepoint at which the agent becomes involved. Further research into the possible link between herpesviruses and Alzheimer's disease is therefore required.     

Reactivation of herpes simplex type 1 in pneumococcal meningitis

BackgroundAcute bacterial meningitis (ABM) and herpes simplex type 1 (HSV-1) encephalitis are two rare but serious infections affecting the central nervous system (CNS). Concurrent bacterial and viral CNS infection has occasionally been reported.ObjectivesTo illustrate the possibility of intrathecal infection with both Streptococcus pneumonia and HSV-1 by presenting a case and to examine whether herpesvirus reactivation is common in ABM.Study designWe report a case diagnosed with HSV-1 reactivation in the cerebrospinal fluid (CSF) during treatment for pneumococcal ABM. A retrospective analysis of CSF samples from 21 patients with ABM was performed, with analysis of DNA from HSV-1 and four other neurotropic herpesviruses.ResultsAll 21CSF samples were negative for HSV-1, HSV-2, varicella zoster-virus, Epstein–Barr virus and human herpesvirus 6 DNA by PCR.ConclusionsAlthough herpesvirus infection does not seem to be a common phenomenon in ABM we suggest that HSV-1 reactivation could be kept in mind if patients with ABM show symptoms or signs compatible with encephalitis.