Viral infection and dissemination through the olfactory pathway and the limbic system by Theiler's virus.

Theiler's murine encephalomyelitis virus (TMEV) infection of mice can produce a biphasic disease of the central nervous system (CNS). Most susceptible strains of mice survive the acute infection and develop a chronic demyelinating disease. In this report, we analyzed the routes of spread of TMEV within the CNS of nude mice and target sites eventually infected in the CNS. Compared to the immunocompetent mouse, in which an antiviral immune response is mounted but virus persists, the nude mouse develops a severe encephalomyelitis due to the lack of functional T lymphocytes and provides a useful model for the study of viral dissemination. We demonstrated, by immunohistochemistry, the presence of viral antigen in defined regions of the CNS, corresponding to various structures of the limbic system. In addition, we found a different time course for viral spread using two different sites of intracerebral inoculation, ie, via the olfactory bulb or the cortex. Limbic structures were rapidly infected following olfactory bulb infection and then showed a decrease in viral load, presumably due to loss of target neurons. Using either route of infection, the virus was able to disseminate to similar regions. These results indicate that limbic structures and their connections are very important for the spread of TMEV in the brain. In the spinal cord, not only neuronal but hematogenous pathways were suspected to be involved in the dissemination of Theiler's virus.     

Infectious entry of equine herpesvirus-1 into host cells through different endocytic pathways

We investigated the mechanism by which equine herpesvirus-1 (EHV-1) enters primary cultured equine brain microvascular endothelial cells (EBMECs) and equine dermis (E. Derm) cells. EHV-1 colocalized with caveolin in EBMECs and the infection was greatly reduced by the expression of a dominant negative form of equine caveolin-1 (ecavY14F), suggesting that EHV-1 enters EBMECs via caveolar endocytosis. EHV-1 entry into E. Derm cells was significantly reduced by ATP depletion and treatments with lysosomotropic agents. Enveloped virions were detected from E. Derm cells by infectious virus recovery assay after viral internalization, suggesting that EHV-1 enters E. Derm cells via energy- and pH-dependent endocytosis. These results suggest that EHV-1 utilizes multiple endocytic pathways in different cell types to establish productive infection.     

Successful protection by amantadine hydrochloride against lethal encephalitis caused by a highly neurovirulent recombinant influenza A virus in mice

A mouse model system for a lethal encephalitis due to influenza has been established by stereotaxic microinjection with the recombinant R404BP strain of influenza A virus into the olfactory bulb of C57BL/6 mice. The virus infection spread selectively to neurons in nuclei of the broad areas of the brain parenchyma that have anatomical connections to the olfactory bulb, leading to apoptotic neurodegeneration. The inflammatory reaction at the extended stage of viral infection involved the vascular structures affected by induction of inducible nitric oxide synthase and protein nitration; those were related to the etiology of fatal brain edema. The intraperitoneal administration of amantadine inhibited the viral growth in the brain and saved mice from the lethal encephalitis. The severity of neuronal loss paralleled the time lag between the virus challenge and the start of amantadine treatment. Thus, early pharmacological intervention is essential to minimize neurological deficits due to influenza virus-induced neurodegeneration.     

Spread of Venezuelan equine encephalitis virus in mice olfactory tract

Summary Spread of Venezuelan equine encephalitis (VEE) virus and damage of the central nervous system (CNS) in mice infected by respiratory route was studied. Virus concentration in organs and blood, dose-effect relationships, and ultrastructural lesions in various tissues were examined in immune and normal mice. We showed, via three independent methods — characteristic curve investigations, tissue virus concentration dynamics, and ultrastructural methods — the spread of VEE virus through the olfactory tract into the brain of immune mice. From these experiments it was concluded that in case of respiratory challenge VEE virus can enter the CNS of normal mice by both vascular and olfactory pathways, while in immune mice the main route is olfactory.     

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.     

Experimental infection with equine herpesvirus 9 (EHV-9) in cats

The pathogenicity for cats of EHV-9, a new neurotropic equine herpesvirus, was assessed by intranasal inoculation with 10(6) plaque-forming units. Four cats killed 4, 5, 6 or 10 days after inoculation showed neurological signs consisting of hyper-excitability and aggressiveness, followed by tremors, occasional convulsions, and depression. Histologically, the cats showed severe encephalitis characterized by neuronal degeneration and loss, intranuclear inclusions, perivascular cuffing and gliosis in the cerebrum. A positive immunohistochemical reaction for EHV-9 antigen was seen in degenerating neuronal cells. The lesions extended from the olfactory bulb to the rhinencephalon and hippocampus. All cats had rhinitis, with or without intranuclear inclusion bodies in the nasal mucosa, and interstitial pneumonia. These findings indicate that the cat, like certain other species such as the goat, is susceptible to experimental infection with EHV-9, and may be at risk from natural infection.     

Kinetics and pathogenicity of oral infection by equine herpesvirus-9 in mice and suckling hamsters

The pathogenesis and kinetics of oral infection by equine herpesvirus (EHV)-9 were studied in mice and hamsters. After oral inoculation of 10(5) plaque-forming units (PFU) of virus, 1-week-old suckling hamsters showed varying severity of neurological disease from 72 hours post inoculation (hpi) and all of these animals had died by 96 hpi. Four-week-old ICR mice inoculated orally with 4 × 10(4)PFU of virus showed no clinical signs, but they developed erosive and ulcerative gastritis from 36 hpi. Varying degrees of encephalitis were seen in infected mice and hamsters, and the hamsters also developed myelitis by 96 hpi. Immunohistochemistry performed on whole body sections of suckling hamsters revealed the kinetics of spread of the virus to the central nervous system. EHV-9 antigen was detected initially in macrophages of the oral and lingual submucosa. At 36 hpi virus antigen was detected in the nerve fibres and pseudounipolar neurons of the trigeminal ganglion and at 96 hpi antigen was present in the myenteric plexuses of the intestine. Virus antigen was also detected in the liver, lungs and heart of affected animals. EHV-9 DNA was detected by polymerase chain reaction in the brain, blood and spinal cord of suckling hamsters at 36, 48 and 96 hpi. These findings show that EHV-9 may spread via the trigeminal nerve when mice and hamsters are inoculated orally with virus.     

Borna disease virus infection in racing horses with behavioral and movement disorders

Summary.  Borna disease virus (BDV) is a neurotropic agent with capacity to infect and cause neurological disease in a broad range of warmblooded hosts including horses, sheep, cattle, cats, and possibly also humans. The epidemiology of BDV is largely unknown. However, it is likely that subclinically infected animals may represent potential virus reservoirs. In two groups of Swedish racing horses, one clinically healthy and one consisting of horses with diffuse neurological signs, the BDV seroprevalence was 24.5% and 57.7%, respectively. BDV RNA was detected in peripheral blood mononuclear cells in 8 out of 28 (28.6%) investigated horses, the majority of the BDV RNA-positive horses belonging to the group with neurological signs. There was a close relationship between the Swedish equine BDV isolates and previously reported equine BDVs in Europe. Our results point to an association of BDV infection with atypical disease patterns in horses such as diffuse mental and gait disturbances. These findings may be of importance for the understanding of the epidemiology of BDV infections in animals and man. Accepted September 4, 1998 Received May 27, 1998     

Meningoencephalitis in Mice Infected with an Equine Herpesvirus 1 Strain KyA Recombinant Expressing Glycoprotein I and Glycoprotein E

One of the consequences of equine herpesvirus 1 (EHV-1) infection in the natural host is a neurological disease that can lead to paralysis. The pathology associated with EHV-1-induced neurological disease includes vasculitis of the small blood vessels within the central nervous system and subsequent damage to the surrounding neural tissue. In a previous study, an EHV-1 recombinant KyA virus (KgI/gE/75) was generated in which the sequences encoding glycoprotein I (gI) and glycoprotein E (gE) were repaired [Frampton et al. 2002 (Virus Research 90: 287-301)] using genes of the pathogenic EHV-1 strain 89c25. In contrast to the parental KyA virus that lacks gI and gE, the recombinant KgI/gE/75 was able to spread to the brains of CBA mice after intranasal infection. Infection resulted in a meningoencephalitis characterized by lymphocytic cuffing of small blood vessels within the brain, consistent with that observed in EHV-1-infected horses exhibiting neurological signs. KgI/gE/75 was able to elicit cytopathology in the lung prior to spread to the brain. However, like the attenuated KyA strain, KgI/gE/75 did not persist in the lung and was completely cleared from lung tissue by day 5 postinfection. We propose that gI and gE are neurovirulence factors for EHV-1, and that the CBA mouse model can be extended to study neurologic sequelae resulting after EHV-1 infection.     

Equine herpesvirus 9 induced lethal encephalomyelitis in experimentally infected goats

Summary.  The pathogenicity of a new neurotropic equine herpesvirus 9 (EHV-9) formerly designated gazelle herpesvirus 1 was evaluated using the goat as a representative of domesticated ruminants. Two goats inoculated intranasally with EHV-9 showed salivation, teeth grinding and other neurological disorders on day 8 post inoculation. One goat died 30 min after the onset of clinical signs and the other was sacrificed 3 h after the sudden onset of teeth grinding and foamy salivation. EHV-9 was recovered from peripheral white blood cells, the olfactory bulbs and brain, nasal swabs, concha, and lungs. Neuropathological lesions were located in the olfactory bulbs, cerebrum, midbrain and medulla oblongata with degeneration and necrosis of neurons, rarefaction, perivascular infiltration of mononuclear cells, and nodal glial reaction. EHV-9 antigen was detected in neurons in the lesions. These findings indicated that EHV-9 is highly pathogenic with high neurotropism for goats. Accepted May 29, 2000 Received February 20, 2000     

Equine major histocompatibility complex class I molecules act as entry receptors that bind to equine herpesvirus-1 glycoprotein D

The endotheliotropism of equine herpesvirus-1 (EHV-1) leads to encephalomyelitis secondary to vasculitis and thrombosis in the infected horse central nervous system (CNS). To identify the host factors involved in EHV-1 infection of CNS endothelial cells, we performed functional cloning using an equine brain microvascular endothelial cell cDNA library. Exogenous expression of equine major histocompatibility complex (MHC) class I heavy chain genes conferred susceptibility to EHV-1 infection in mouse NIH3T3 cells, which are not naturally susceptible to EHV-1 infection. Equine MHC class I molecules bound to EHV-1 glycoprotein D (gD), and both anti-gD antibodies and a soluble form of gD blocked viral entry into NIH3T3 cells stably expressing the equine MHC class I heavy chain gene (3T3-A68 cells). Treatment with an anti-equine MHC class I monoclonal antibody blocked EHV-1 entry into 3T3-A68 cells, equine dermis (E. Derm) cells and equine brain microvascular endothelial cells. In addition, inhibition of cell surface expression of MHC class I molecules in E. Derm cells drastically reduced their susceptibility to EHV-1 infection. These results suggest that equine MHC class I is a functional gD receptor that plays a pivotal role in EHV-1 entry into equine cells.     

The nucleotide sequence of the glycoprotein G homologue of equine herpesvirus 3 (EHV3) indicates EHV3 is a distinct equid alphaherpesvirus

Summary.  EHV3 causes equine coital exanthema and has been classified as an alphaherpesvirus on the basis of its biological properties; however due to the absence of any sequence information the phylogenetic relationship has not previously been examined. The complete nucleotide sequence of the EHV3 glycoprotein G (gG) gene was determined and showed that this virus is most closely related to the alphaherpesviruses equine herpesviruses type 1 (EHV 1) and type 4 (EHV4). EHV3 gG contains conserved and variable regions which are homologous to those previously defined for EHV1 and EHV4 gG proteins. Consistent with EHV1 and EHV4 gG, the variable region of EHV3 gG was found to elicit a strong antibody response in experimentally and naturally infected horses and could be exploited for use as a diagnostic reagent. Accepted May 21, 1999 Received March 15, 1999     

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.     

Borna disease virus induces acute fatal neurological disorders in neonatal gerbils without virus- and immune-mediated cell destructions

Borna disease virus (BDV) is a noncytolytic, neurotropic RNA virus that is known to cause neurological disturbances in various animal species. Our previous experiment demonstrated that neonate gerbils develop an acute fatal neurological disease following infection with BDV, Virology 282, 65-76). The study suggested that BDV directly causes functional damage of neuronal cells resulting in the lethal disorder in neonatal gerbils. To extend this finding, we examined whether BDV can induce neurological diseases in the absence of virus- and immune-mediated cell destruction, by using cyclosporine A (CsA)-treated neonatal gerbils. Although CsA completely suppressed specific antibody production and brain inflammation in the infected gerbil brains, the fatal neurological disorder was not inhibited by the treatment. Furthermore, we demonstrated that CsA treatment significantly decreased brain levels of cytokines, except interleukin (IL)-1 beta, in the infected gerbils. These results suggested that BDV replication, as well as brain cytokines, at least IL-1 beta, rapidly induces fatal disturbances in gerbil brain. We demonstrate here that BDV exhibits a unique neuropathogenesis in neonatal gerbil that may be pathologically and immunologically different from those in two other established rodent models, rats and mice. With this novel rodent model of virus infection it should be possible not only to examine acute neurological disturbances without severe neuroanatomical and immunopathological alterations but also to analyze molecular and cellular damage by virus replication in the central nervous system.     

Mode of entry of a neurotropic arbovirus into the central nervous system. Reinvestigation of an old controversy

The mechanism by which neurotropic arboviruses gain access to the central nervous system remains uncertain, although it is generally assumed that viremic infection results in growth across or passive diffusion through brain capillaries. In contrast to the natural reservoir hosts of these arboviruses, clinical hosts (e.g., horses, humans) have viremias of very brief duration and low magnitude. We investigated the question of neuroinvasion in 5- to 6-week-old Syrian hamsters infected with St. Louis encephalitis virus (strain TBH-28). This model shares with the human disease low or undetectable viremia and many clinical and pathoanatomical features. The mortality rate after intraperitoneal inoculation of a moderate viral dose was 88%. No viremia was detectable by a sensitive assay in 31% of the animals. In the remaining hamsters, the mean peak viremia was 1.0 log10 plaque-forming units/0.05 ml and the mean duration 1 to 2 days. There was no correlation between viremia and outcome of infection, length of incubation period, or brain virus titer. Tissue infectivity studies showed a rise in titer in the olfactory neuroepithelium on day 4 postinoculation, then in the olfactory bulbs (day 5 postinoculation), and finally in the remainder of the brain (day 6 postinoculation). Specific immunofluorescence was demonstrated in the bipolar neurons of the olfactory epithelium, their dendrites, and in axon bundles of the olfactory nerves in the submucosa. By electron microscopy, virus particles and associated tubular structures were demonstrated within dendrites, perikarya, and axons of olfactory neurons, and to a lesser extent in macrophages and Bowman's gland cells in the lamina propria. In cells of Bowman's glands large numbers of virions were sequestered within secretory granules. Virus was recovered from nasal washings on day 4 postinoculation. Similar findings were obtained in weanling mice inoculated intraperitoneally with another (mouse-virulent) St. Louis encephalitis viral strain (77V-12908). These data taken together indicate that the olfactory pathway is the principal route of viral entry into the central nervous system. After peripheral inoculation a low-level viremia results in infection of highly susceptible cells in the olfactory neuroepithelium, allowing centripetal axonal transport of virus to the olfactory bulb, whence spread is unimpeded throughout the neuropil of the central nervous system. Infection of Bowman's gland cells in the olfactory mucosa and shedding of virus in nasal mucus may be an adaptation for nonarthropod-borne transmission, a feature of many flaviviruses.     

West Nile virus infection modulates human brain microvascular endothelial cells tight junction proteins and cell adhesion molecules: Transmigration across the in vitro blood-brain barrier

Neurological complications such as inflammation, failure of the blood-brain barrier (BBB), and neuronal death contribute to the mortality and morbidity associated with WNV-induced meningitis. Compromised BBB indicates the ability of the virus to gain entry into the CNS via the BBB, however, the underlying mechanisms, and the specific cell types associated with WNV-CNS trafficking are not well understood. Brain microvascular endothelial cells, the main component of the BBB, represent a barrier to virus dissemination into the CNS and could play key role in WNV spread via hematogenous route. To investigate WNV entry into the CNS, we infected primary human brain microvascular endothelial (HBMVE) cells with the neurovirulent strain of WNV (NY99) and examined WNV replication kinetics together with the changes in the expressions of key tight junction proteins (TJP) and cell adhesion molecules (CAM). WNV infection of HBMVE cells was productive as analyzed by plaque assay and qRT-PCR, and did not induce cytopathic effect. Increased mRNA and protein expressions of TJP (claudin-1) and CAM (vascular cell adhesion molecule and E-selectin) were observed at days 2 and 3 after infection, respectively, which coincided with the peak in WNV replication. Further, using an in vitro BBB model comprised of HBMVE cells, we demonstrate that cell-free WNV can cross the BBB, without compromising the BBB integrity. These data suggest that infection of HBMVE cells can facilitate entry of cell-free virus into the CNS without disturbing the BBB, and increased CAM may assist in the trafficking of WNV-infected immune cells into the CNS, via ‘Trojan horse’ mechanism, thereby contributing to WNV dissemination in the CNS and associated pathology.     

Equine herpesvirus genomes: Heterogeneity of naturally occurring type 4 isolates and of a type 1 isolate after heterologous cell passage

Summary The restriction endonuclease DNA fingerprints of 20 low passage, epidemiologically unrelated isolates of equine herpesvirus 4 (equine rhinopneumonitis virus) showed considerable heterogeneity in certain fragments, the positions of which were assigned to quite restricted positions on the 141 kilobase (kb) genome. We note that the heterogeneity observed in the restriction endonuclease DNA fingerprints of EHV1 (equine abortion virus) and of pseudorabies virus also tend to map to these same restricted regions.The restriction endonuclease DNA fingerprints of an EHV1 strain was invariant using low (<1) multiplicity of infection during 20 passages in equine cells but when adapted to hamster cells developed an approximately 0.8 kb deletion in the unique short region of the genome between 8 and 11 passages.With 3 Figures     

High level expression of equine herpesvirus 1 glycoproteins D and H and their role in protection against virus challenge in the C3H (H-2K) murine model

N and C-terminal truncated forms of equine herpesvirus 1 (EHV 1) glycoproteins gD and gH were expressed in baculovirus resulting in the production of secreted recombinant proteins. A carboxy-terminal histidine tag was included on each of the genes for protein isolation by nickel affinity chromatography. Recombinant gD was recognized by three gD specific monoclonal antibodies, 20C4, 5H6 and F3132. F3132 is a conformationally dependent monoclonal antibody with virus neutralizing activity. Expression of gH was confirmed by reacting the protein with the gH peptide specific antiserum R319. The truncated gD gene was also expressed as a β-galactosidase fusion protein which was purified from E. coli by nickel affinity chromatography. C3H mice were inoculated with purified recombinant gD or gH or insect cells which had been infected with recombinant baculoviruses. Mice were subsequently challenged with EHV 1. Purified recombinant baculovirus gD provided the most protection and produced high levels of virus neutralizing antibodies. The gD fusion protein was less effective at protecting mice and insect cells infected with either of the recombinant baculoviruses or purified recombinant gH were poor at conferring protection. The results emphasize the importance of using purified proteins in vaccine formulations and of including EHV 1 gD as a component of a subunit vaccine.     

A Comparison of the Tissue Lesions Produced in Adult Hamsters by Two Strains of Avirulent Venezuelan Equine Encephalomyelitis Virus

Adult hamsters were infected with two avirulent strains of Venezuelan equine encephalomyelitis virus, TC-83 and the small plaque variant (SPV) of Fe 3-7c. Histologic examination showed that significant brain lesions were produced by both strains and affected primarily the olfactory bulbs, lateral olfactory tracts and the pyriform lobes of the cerebral cortex. The type of lesions produced by both strains were similar, but when the two groups were compared, more of the hamsters inoculated with SPV had lesions and they were of greater severity. Outstanding lesions observed were: vasculitis, lymphocytic perivascular cuffing and hemorrhage, demyelination of the lateral olfactory tracts and neuronal necrosis of the olfactory bulbs.     

The nucleotide sequence of asinine herpesvirus 3 glycoprotein G indicates that the donkey virus is closely related to equine herpesvirus 1

Summary The nucleotide sequence of the glycoprotein G (gG) homologue of asinine herpesvirus 3 (AHV3), a respiratory alphaherpesvirus of donkeys, was determined. The AHV3 gG gene consists of 1233 base pairs (bp) and codes for a predicted protein of 411 amino acids. This is identical in size to the equine herpesvirus 1 (EHV1) gG gene and 6 amino acids longer than the equine herpesvirus 4 (EHV4) gG gene. The predicted amino acid sequence of AHV3 gG has characteristics of a class 1 membrane protein. The amino acid sequence of AHV3 gG shows 92% and 60% identity to EHV1 gG and EHV4 gG respectively. Two regions within the gG amino acid sequences of EHV1 and EHV4 were previously defined, an N-terminal constant region and an immunodominant highly variable region located toward the C-terminus. In the corresponding constant region of AHV3 gG there was 96% and 75% amino acid identity with EHV1 and EHV4 gGs respectively. In the variable region, there was 73% and 24% identity respectively. Phylogenetic analyses using the gG nucleotide sequences indicated that AHV3 is much closer in evolutionary distance to EHV1 than either virus is to EHV4. These findings provide additional support for the view that AHV3, or another closely related virus, may be the progenitor of EHV1 and has adapted to horses in relatively recent times.