Monoclonal anti-I-A antibody reverses chronic paralysis and demyelination in Theiler's virus-infected mice: critical importance of timing of treatment

Susceptibility to demyelination caused by the WW isolate of Theiler's murine encephalomyelitis viruses is linked to class II genes of the major histocompatibility complex. SJL/J (H-2s) mice, expressing only I-As class II gene products of the major histocompatibility complex, are highly susceptible to Theiler's murine encephalomyelitis virus infection with the WW virus isolate, with chronic paralysis and severe inflammation and demyelination in the central nervous system. The effect of in vivo administration of anti-I-As monoclonal antibodies on Theiler's murine encephalomyelitis virus infection was observed. SJL/J mice were treated in various protocols pre- or postinfection. Anti-I-As monoclonal antibody reversed chronic paralysis and reduced inflammation and demyelination when given after the establishment of persistent infection. The effect was long lasting, but clinical signs, inflammation, and demyelination recurred 2 months after treatment ceased. Anti-I-As antibodies had no effect on viral titers within the central nervous system. The timing of the administration of monoclonal antibodies was critical. Administration of anti-I-As before the establishment of the persistent infection resulted in fatal encephalitis.     

Neurotropic viral infections leading to epilepsy: focus on Theiler's murine encephalomyelitis virus

Neurotropic viruses cause viral encephalitis and are associated with the development of seizures/epilepsy. The first infection-driven animal model for epilepsy, the Theiler's murine encephalomyelitis virus-induced seizure model is described herein. Intracerebral infection of C57BL/6 mice with Theiler's murine encephalomyelitis virus induces acute seizures from which the animals recover. However, once the virus is cleared, a significant portion of the animals that experienced acute seizures later develop epilepsy. Components of the innate immune response to viral infection, including IL-6 and complement component 3, have been implicated in the development of acute seizures. Multiple mechanisms, including neuronal cell destruction and cytokine activation, play a role in the development of acute seizures. Future studies targeting the innate immune response will lead to new therapies for seizures/epilepsy.     

Pathogens of house mice on arid Boullanger Island and subantarctic Macquarie Island, Australia

Studies on island populations of house mice (Mus domesticus) and their viruses reveal insights into viral persistence in isolated communities. We surveyed the ectoparasites, endoparasites, and antiviral antibodies for 11 murine viruses and two bacteria of house mice inhabiting two islands off Australia. House mice on Boullanger Island were seropositive to two viruses, murine cytomegalovirus and epizootic diarrhea of infant mice. On subantarctic Macquarie Island, house mice were seropositive for five viruses: murine cytomegalovirus, lymphocytic choriomeningitis virus, mouse parvovirus, epizootic diarrhea of infant mice, and Theiler's murine encephalomyelitis virus. The diversity of antiviral antibodies was lower among populations of house mice on islands than those inhabiting mainland Australia. The decreased diversity of viruses in island populations of house mice may be a function of which agent the founder mice transfer to the island and related to the low densities which the host population may periodically reach over time.     

Theiler's virus infection in nude mice: viral RNA in vascular endothelial cells

Infection of athymic (nu/nu) mice with Theiler's murine encephalomyelitis virus results in an acute encephalitis which resembles poliomyelitis. Immunohistochemistry and in situ hybridization were used to delineate the presence of viral proteins and RNA in the nervous systems of nude mice infected with the Daniels strain of Theiler's virus. This system permits the analysis of a viral infection in the absence of an effective immune response. By immunohistochemistry, viral antigen was found in the processes and cell bodies of neurons and glial cells. Besides the presence of viral antigen in these cell types, by in situ hybridization, Theiler's virus RNA was also found in cells associated with vascular endothelium in the brains and spinal cords of these infected mice. Theiler's virus RNA-positive endothelial cells were observed not only near the primary lesions but also away from demonstrable lesions in normal-appearing regions in the central nervous system. Earlier work had suggested an intra-axonal dissemination for this virus (M. C. Dal Canto and H. L. Lipton, Am. J. Pathol. 106:20-29, 1982). Our findings are consistent with this model but also suggest an additional mechanism for virus spread within the central nervous system, i.e., by infecting vascular cells and crossing the blood-brain barrier. Lastly, after Theiler's murine encephalomyelitis virus infection, not only glial cells but also endothelial cells express major histocompatibility complex class II (la) antigen on their surface (M. Rodriguez, M. L. Pierce, and E. A. Howie, J. Immunol. 138:3438-3442, 1987). Our demonstration of Theiler's virus-infected endotheliumlike cells may explain interactions of virus products in stimulating antigen presentation.     

Theiler's murine encephalomyelitis virus--characterization of newly isolated viruses from Japanese mouse colonies

The hemagglutinating-inhibition (HI) test was used to detect antibodies for Theiler's murine encephalomyelitis virus (TMEV), and the virus was isolated from sero-positive mice derived from colonies in Japan. HI antibody was detected in conventional mice (38.7%; 137/354) at titers ranging from 1:8 to 1:512, but it not in SPF mice (0/90). To isolate the virus, weanling mice inoculated intracerebrally with samples obtained from sero-positive mice were sacrificed and 10% brain homogenates were subcultured. New isolates designated as YOC and AB strains were obtained, and their physicochemical and biological properties were characterized. The results indicated that the new isolates were similar to Theiler's original (TO) strain according to the following observations of persistent paralysis of the hind limbs, resistance to ether treatment, a particles size of 10 approximately 50 nm in diameter, stability at pH 3, a density of 1.35 g/cm3 and three major and one minor viral proteins, (VPO; 38 Kd, VP 1; 33 Kd, VP2; 32Kd, VP3; 25 Kd). Immunoblotting analysis also showed that VP 2 of YOC and encephalomyocarditis virus of the Cardiovirus group, reacted strongly with the antisera against the viruses as well as with the GDVII strain. These results suggest that TMEV infection does exist in conventional mouse colonies in Japan, and that these viruses resemble the TO strain of TMEV.     

Prevalence of natural virus infections in laboratory mice and rats used in Canada

Results of serological tests carried out over a period of 6 years to detect the presence of antibodies against 14 indigenous viruses in mice and rats used in 32 Canadian institutions are reported. Close to 20,000 individual sera were tested by the complement fixation or the hemagglutination inhibition technics. In order of mouse colony prevalence the six most common viruses present were pneumonia virus of mice, mouse hepatitis virus, rat virus, minute virus of mice, Sendai, and Theiler's mouse encephalomyelitis viruses. The most common viruses present in rat colonies were minute virus of mice, K virus, coronaviruses (rat coronavirus or sialodacryoadenitis virus), rat virus, H-1, pneumonia virus of mice, Theiler's mouse encephalomyelitis viruses, Sendai, and reovirus 3.     

B-lymphocyte requirement for vaccine-mediated protection from Theiler's murine encephalomyelitis virus-induced central nervous system disease.

The role of humoral immunity in the protection of vaccinated SJL/J mice from central nervous system disease induced by the DA strain (DAV) of Theiler's murine encephalomyelitis virus was investigated in B-cell-deficient mice. Mice were depleted of B cells by treatment with a mouse monoclonal antibody specific for immunoglobulin M. DAV-vaccinated, B-cell-deficient mice failed to clear viral infection and were no longer protected from Theiler's murine encephalomyelitis virus-mediated central nervous system disease. CD4+ T cells are required in this model of protection to provide help for the development of an antiviral antibody response in the central nervous system.     

Complementation for replicative form DNA replication of a deletion mutant of H-1 by various parvoviruses.

We established a persistent infection in L 929 cells with the DA strain of Theiler's murine encephalomyelitis virus. Our studies showed that only a small number of cells in the cultures contained infectious virus or viral antigen. A role for interferon in the maintenance of persistence was suggested. Viral isolates from the cultures were not temperature sensitive, nor did they contain viral capsid polypeptide mutations or defective interfering particles. T1 oligonucleotide maps showed evidence of mutation in two of three isolates.     

Mode of spread to and within the central nervous system after oral infection of neonatal mice with the DA strain of Theiler's murine encephalomyelitis virus.

Theiler's murine encephalomyelitis virus is a neurotropic enterovirus known to cause biphasic neural disease after intracerebral inoculation into adult mice. The present study characterizes a neonatal mouse model with a high disease incidence for the study of the acute phase of the pathogenesis of the DA strain of Theiler's murine encephalomyelitis virus after oral infection. The route of viral spread to and within the central nervous system (CNS) was determined by examining the kinetics of viral replication in various organs and by performing histopathological analysis. Viral antigen was detected widely in the neonatal CNS, mainly in the gray matter, and it was asymmetrical and multifocal in its distribution, with considerable variation in lesion distribution from animal to animal. Necrotizing lesions appeared to expand by direct extension from infected cells to their close neighbors, with a general disregard of neuroanatomical boundaries. The diencephalon showed particular susceptibility to viral infection. Other areas of the CNS, including the cerebellum and dentate gyrus of the hippocampus, were consistently spared. Neurons with axons extending peripherally to other organs or receiving direct input from the peripheral nervous system were not preferentially affected. The kinetics of viral replication in the liver, spleen, and CNS and the histopathological findings indicate that viral entry to the CNS is via a direct hematogenous route in orally infected neonatal mice and that the disease then progresses within the CNS mainly by direct extension from initial foci.     

Proteins induced in tissue culture by four isolates of Theiler's murine encephalomyelitis virus.

The proteins specified by four Theiler's murine encephalomyelitis virus isolates in infected BHK-21 cells were studied. Their processing, sensitivity to trypsin, and the changeover after viral infection from synthesis of cellular proteins to synthesis of viral proteins were determined by one- and two-dimensional gel electrophoreses. The molecular weights and isoelectric points of the structural and nonstructural proteins of DA and WW isolates, which represent the less virulent subgroup of Theiler's murine encephalomyelitis virus, and of GDVII and FA isolates, which represent the virulent subgroup, were found to be the same. The sensitivity of DA and GDVII isolates to trypsin, as purified virions, and in infected cell extracts was similar. The shut-off of cellular protein synthesis in cells infected with the same two isolates and the changeover to the synthesis of viral proteins appeared to have the same pattern. These findings are interesting since the two subgroups of Theiler's murine encephalomyelitis virus differ in their pathogenicity, intracellular development in infected BHK-21 cells, and RNA composition, as determined by RNase T1 fingerprinting analysis.     

Early infection of the central nervous system by the GDVII and DA strains of Theiler's virus.

The DA strain of Theiler's virus, a murine picornavirus, causes a persistent infection of glial cells of the white matter of the spinal cord, associated with chronic inflammation and primary demyelination. The GDVII strain causes an acute fatal grey matter encephalomyelitis. We characterized the target cells of GDVII and DA viruses 4 days following intracerebral inoculation, and we compared the levels of viral RNA within these cells. GDVII virus infected approximately 10 times more cells than DA virus. Whereas GDVII virus infected neurons exclusively, DA virus infected also astrocytes and possible macrophage-microglial cells. The levels of viral RNA in neurons infected with GDVII and DA viruses were of the same order. These results show that DA virus infects glial cells already at the beginning of the disease and that the more efficient spread of GDVII virus is probably not due to a higher level of RNA replication per cell.     

Murine viruses in an island population of introduced house mice and endemic short-tailed mice in Western Australia

House mice (Mus domesticus) were recently introduced to Thevenard Island, off the northwest coast of Western Australia. This island is also habitat for an endangered native rodent, the short-tailed mouse (Leggadina lakedownensis). Concerns have been raised that house mice may pose a threat to L. lakedownensis both through competition and as a source of infection. To assess the threat to L. lakedownensis posed by viral pathogens from M. domesticus, a serological survey was conducted from 1994 to 1996 of both species for evidence of infection with 14 common murine viruses (mouse hepatitis virus, murine cytomegalovirus, lymphocytic choriomeningitis virus, ectromelia virus, mouse adenovirus strains FL and K87, minute virus of mice, mouse parvovirus, reovirus type 3, Sendai virus, Theiler's mouse encephalomyelitis virus, polyoma virus, pneumonia virus of mice, and encephalomyocarditis virus) and Mycoplasma pulmonis. Despite previous evidence that populations of free-living M. domesticus from various locations on the Australian mainland were infected with up to eight viruses, M. domesticus on Thevenard Island were seropositive only to murine cytomegalovirus (MCMV). Antibodies to MCMV were detected in this species at all times of sampling, although seroprevalence varied. Infectious MCMV could be isolated in culture of salivary gland homogenates from seropositive mice. In contrast, L. lakedownensis on Thevenard Island showed no serological evidence of infection with MCMV, any of the other murine viruses, or M. Pulmonis, and no virus could be isolated in culture from salivary gland homogenates. Although MCMV replicated to high titers in experimentally infected inbred BALB/c laboratory mice as expected, it did not replicate in the target organs of experimentally inoculated L. lakedownensis, indicating that the strict host specificity of MCMV may prevent its infection of L. lakedownensis. These results suggest that native mice on Thevenard Island are not at risk of MCMV infection from introduced house mice, and raise interesting questions about the possible selective survival of MCMV in small isolated populations of M. domesticus.     

Role of sialyloligosaccharide binding in Theiler's virus persistence.

Theiler's murine encephalomyelitis viruses (TMEVs) belong to the Picornaviridae family and are divided into two groups, typified by strain GDVII virus and members of the TO (Theiler's original) group. The highly virulent GDVII group causes acute encephalitis in mice, while the TO group is less virulent and causes a chronic demyelinating disease which is associated with viral persistence in mice. This persistent central nervous system infection with demyelination resembles multiple sclerosis (MS) in humans and has thus become an important model for studying MS. It has been shown that some of the determinants associated with viral persistence are located on the capsid proteins of the TO group. Structural comparisons of two persistent strains (BeAn and DA) and a highly virulent strain (GDVII) showed that the most significant structural variations between these two groups of viruses are located on the sites that may influence virus binding to cellular receptors. Most animal viruses attach to specific cellular receptors that, in part, determine host range and tissue tropism. In this study, atomic models of TMEV chimeras were built with the known structures of GDVII, BeAn, and DA viruses. Comparisons among the known GDVII, BeAn, and DA structures as well as the predicted models for the TMEV chimeras suggested that a gap on the capsid surface next to the putative receptor binding site, composed of residues from VP1 and VP2, may be important in determining viral persistence by influencing virus attachment to cellular receptors, such as sialyloligosaccharides. Our results showed that sialyllactose, the first three sugar molecules of common oligosaccharides on the surface of mammalian cells, inhibits virus binding to the host cell and infection with the persistent BeAn virus but not the nonpersistent GDVII and chimera 39 viruses.     

Clonal expansion of infiltrating T cells in the spinal cords of SJL/J mice infected with Theiler's virus

Intracerebral infection of susceptible mice with Theiler's murine encephalomyelitis virus results in immune-mediated inflammatory demyelination in the white matter and consequent clinical symptoms. This system has been utilized as an important virus model for human multiple sclerosis. Although the potential involvement of virus-specific Th cells has been studied extensively, very little is known about the nature of T cells infiltrating the CNS during viral infection and their role in the development of demyelinating disease. In this study, the clonal nature of T cells in the spinal cord during the disease course was analyzed using size spectratyping and sequencing of the TCR beta-chain CDR3 region. These studies clearly indicate that T cells are clonally expanded in the CNS after viral infection, although the overall TCR repertoire appears to be diverse. The clonal expansion appears to be Ag-driven in that it includes Th cells specific for known viral epitopes. Interestingly, such restricted accumulation of T cells was not detectable in the infiltrates of mice with proteolipid protein peptide-induced experimental autoimmune encephalomyelitis. The initial T cell repertoire (7-9 days postinfection) seems to be more diverse than that observed in the later stage (65 days) of virally induced demyelination, despite the more restricted utilization of Vbeta subfamilies. These results strongly suggest continuous stimulation and clonal expansion of virus-specific T cells in the CNS of Theiler's murine encephalomyelitis virus-infected mice during the entire course of demyelinating disease.     

Interleukin 22 is a candidate gene for Tmevp3, a locus controlling Theiler's virus-induced neurological diseases

After intracerebral inoculation, Theiler's virus induces in its natural host, the mouse, an acute encephalomyelitis followed, in susceptible animals, by chronic inflammation and primary demyelination. Susceptibility to demyelination among strains of laboratory mice is explained by the capacity of the immune system to control viral load during persistence. Also, differences of susceptibility to viral load between the susceptible SJL strain and the resistant B10.S strain are mainly due to two loci, Tmevp2 and Tmevp3, located close to the Ifng locus on chromosome 10. In this article, we show that the Tmevp3 locus controls both mortality during the acute encephalomyelitis and viral load during persistence. Most probably, two genes located in the Tmevp3 interval control these two different phenotypes with efficiencies that depend on the age of the mouse at inoculation. Il22, a member of the IL-10 cytokine family, is a candidate gene for the control of mortality during the acute encephalomyelitis.     

Receptors for Theiler's murine encephalomyelitis virus: characterization by using rabbit antiviral antiserum.

An immunological assay was developed to characterize the binding of Theiler's murine encephalomyelitis virus to BHK-21 cell receptors. After absorption of the virus and formaldehyde fixation, rabbit antibodies and Staphylococcus aureus protein A labeled with 125I formed a specific complex on the surfaces of the cells. The optimal multiplicity of infection in this system was 10 PFU per cell. The virus was internalized at 33 and 37 degrees C, but internalization did not take place at 25 or 4 degrees C. The binding was proportional to the number of cells and was significant within 30 s. Cell surface receptors were still active after fixation, and only intact viruses were bound, as demonstrated by the lack of binding of the purified, isolated virion proteins VP1, VP2, and VP3.     

Antibodies to bluetongue and epizootic hemorrhagic disease viruses in a barrier island white-tailed deer population.

From 1981 through 1989, serum samples from 855 white-tailed deer (Odocoileus virginianus) from Ossabaw Island, Georgia (USA), were tested for antibodies to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). During this period, prevalence of precipitating antibodies to BTV and EHDV as determined by agar gel immunodiffusion (AGID) tests decreased from 74% to 3% and from 34% to 1%, respectively. Antibodies were detected in serum samples from 0.5-yr-old deer only during 1981, 1982, and 1983, and with few exceptions, positive serological results after 1983 were restricted to older age classes. A decrease in prevalence of precipitating antibodies to BTV and EHDV in age classes exposed during 1981 indicates that AGID results from white-tailed deer populations underestimate the extent of previous exposure to these viruses. Serum neutralization test results from AGID-positive deer indicated that BTV 11 was the principal serotype responsible for infections during 1981. Since 1983, this serotype has been replaced by BTV 13; however, there has been a low level of transmission within the herd. Infection with EHDV 2 appeared most prevalent during 1982; as with BTV 13, there has been limited transmission in this high density deer population since 1983.     

Serologic survey of white-tailed deer on Anticosti Island, Quebec for bovine herpesvirus 1, bovine viral diarrhea, and parainfluenza 3.

In 1985 unusual mortality was observed among the 3- to 4-yr-old white-tailed deer (Odocoileus virginianus) on Anticosti Island, Québec (Canada). A viral pathogen was suspected to be the cause of the deaths. Thus, a serologic survey for bovine herpesvirus 1 (BHV-1), bovine viral diarrhea (BVD) virus and parainfluenza-3 (PI-3) virus was conducted. We examined 396 deer sera from 1985. Results indicated that the high mortality mainly afflicted 3- to 4-yr-old deer. In 1985, 57% of deer sampled were seropositive for viral neutralizing antibodies against BHV-1. Prevalences decreased over the next 2 yr of the survey. Prevalence of antibodies against PI-3 virus, determined by hemagglutination inhibition test, remained high (82% to 84%) for the 3 yr period. No deer were seropositive for neutralizing antibodies against BVD virus during the survey period. Analysis of antibodies against BHV-1 and PI-3 viruses according to sex, age and antibody titers revealed that an epizootic BHV-1 infection occurred in 1985; PI-3 infection appears to be enzootic in Anticosti deer.     

The leader protein of cardioviruses inhibits stress granule assembly

Stress granules (SG) are cytoplasmic aggregates of stalled translation preinitiation complexes that form in cells exposed to various environmental stresses. Here, we show that stress granules assemble in cells infected with Theiler's murine encephalomyelitis virus (TMEV) mutants carrying alterations in the leader (L) protein, but not in cells infected with wild-type TMEV. Stress granules also formed in STAT1-deficient cells, suggesting that SG formation was not a consequence of increased type I interferon (IFN) production when cells were infected with the mutant virus. Ectopic expression of the wild-type L protein was sufficient to inhibit stress granule formation induced by sodium arsenite or thapsigargin treatment. In conclusion, TMEV infection induces stress granule assembly, but this process is inhibited by the L protein. Unlike poliovirus-induced stress granules, TMEV-induced stress granules did not contain the nuclear protein Sam68 but contained polypyrimidine tract binding protein (PTB), an internal ribosome entry site (IRES)-interacting protein. Moreover, G3BP was not degraded and was found in SG after TMEV infection, suggesting that SG content could be virus specific. Despite the colocalization of PTB with SG and the known interaction of PTB with viral RNA, in situ hybridization and immunofluorescence assays failed to detect viral RNA trapped in infection-induced SG. Recombinant Theiler's viruses expressing the L protein of Saffold virus 2 (SAFV-2), a closely related human theilovirus, or the L protein of mengovirus, an encephalomyocarditis virus (EMCV) strain, also inhibited infection-induced stress granule assembly, suggesting that stress granule antagonism is a common feature of cardiovirus L proteins.     

Major linear antibody epitopes and capsid proteins differentially induce protective immunity against Theiler's virus-induced demyelinating disease.

Theiler's murine encephalomyelitis virus-induced immunologically mediated demyelinating disease (TMEV-IDD) in susceptible mice provides a relevant infectious model for multiple sclerosis. Previously, we have identified six major linear antibody epitopes on the viral capsid proteins. In this study, we utilized fusion proteins containing individual capsid proteins and synthetic peptides containing the linear antibody epitopes to determine the potential role of antibody response in the course of virus-induced demyelination. Preimmunization of susceptible mice with VPI and VP2 fusion proteins, but not VP3, resulted in the protection from subsequent development of TMEV-IDD. Mice free of clinical symptoms following preimmunizations with fusion proteins displayed high levels of antibodies to the capsid proteins corresponding to the immunogens. In contrast, the level of antibodies to a particular linear epitope, A1C (VP1(262-276)), capable of efficiently neutralizing virus in vitro increased with the progression of disease. Further immunization with synthetic peptides containing individual antibody epitopes indicated that antibodies to the epitopes are differentially effective in protecting from virus-induced demyelination. Taken together, these results suggest that antibodies to only certain linear epitopes are protective and such protection may be restricted during the early stages of viral infection.