Localization of a neutralization site of Theiler''s murine encephalomyelitis viruses.

Theiler's murine encephalomyelitis viruses (TMEV) are picornaviruses that produce enteric and neurological diseases in mice. Subgroup TO strains of TMEV cause persistent infections with demyelination, while subgroup GDVII strains neither persist nor demyelinate. We produced neutralizing monoclonal antibodies (mAbs) to clarify the mechanisms of persistence and demyelination. Some of the neutralizing mAbs reacted with isolated VP1 on Western blots, while others were conformation specific. The neutralization site for the former TMEV mAbs was on the VP1 trypsin cleavage site of the intact virion. The neutralization site for the conformation-specific mAbs was distinct and was not affected by trypsin. Trypsin treatment of subgroup TO strains increased their infectivity for L cells, whereas the infectivity of subgroup GDVII strains was decreased by trypsin treatment. Subpopulations of virus in subgroup TO-infected tissue culture cells and in infected mouse brain homogenates contained VP1-cleaved virus; this VP1-cleaved virus gave rise to a large persistent fraction in neutralization tests when it was reacted with VP1-specific mAbs. These findings have implications regarding the pathogenesis of subgroup TO demyelinating disease. TMEV VP1 cleavage may be important for virus persistence because of disruption of a major neutralization epitope. The change in virus surface structure caused by VP1 cleavage may affect cell binding and lead to altered cytotropism. Immunocytes, which have been implicated in subgroup TO demyelination, may provide a source for proteases for VP1 cleavage.     

Trypsin-sensitive neutralization site on VP1 of Theiler''s murine encephalomyelitis viruses.

We generated Theiler's murine encephalomyelitis virus mutants resistant to several neutralizing monoclonal antibodies (MAbs) having their epitopes near a trypsin cleavage site of VP1. Neutralization and Western blot (immunoblot) studies suggest that two of the MAbs have identical epitopes that partly overlap the epitope of a third MAb. Sequencing of RNA of the mutants localized the epitopes to a site near the carboxyl end of VP1. The limited diversity of nucleotide changes seen in the mutants and the immunodominance of the site suggest that the carboxyl end of VP1 may have an important function.     

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.     

Theiler''s murine encephalomyelitis virus neutralization escape mutants have a change in disease phenotype.

DA strain of Theiler's murine encephalomyelitis virus produces a persistent demyelinating infection. We previously produced escape mutant viruses that are resistant to a neutralizing monoclonal antibody and have a mutation in VP1 amino acid residue 268 in a neutralization site (Y. Ohara, A. Senkowski, J. Fu, L. Klaman, J. Goodall, M. Toth, and R.P. Roos, J. Virol. 62:3527-3529, 1988). In contrast to wild-type DA strain, these escape mutants produce little if any demyelinating disease after inoculation into weanling mice.     

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.     

Neutralizing monoclonal antibodies to Theiler''s murine encephalomyelitis viruses.

Theiler's murine encephalomyelitis viruses (TMEV) are serologically related picornaviruses which cause both enteric and neurological disease in mice. The biological activities of TMEV vary between the two different TMEV subgroups (TO and GDVII) and with different passage histories of the same TMEV strain (e.g., mouse brain-passed versus tissue culture-passed DA strain of the TO subgroup). We raised neutralizing monoclonal antibodies (mAbs) against tissue culture-passed DA and GDVII strains of TMEV. We produced two mAbs against the DA strain which neutralized all members of the TO subgroup, but not the GDVII subgroup strains (GDVII and FA); these two DA mAbs reacted similarly with both mouse brain-passed DA and tissue culture-passed DA. Of six neutralizing GDVII mAbs, four reacted only to GDVII and FA, whereas two neutralized TO strains as well. These mAbs demonstrate the presence of TMEV group-specific as well as subgroup-specific neutralization and substantiate the division of TMEV into two distinct subgroups. On Western immunoblots one of the two DA mAbs reacted against isolated DA VP1, two GDVII mAbs (which were TMEV group specific) reacted against isolated GDVII VP1 and DA VP1, and the other DA mAb and four other GDVII mAbs required an intact virion conformation for reactivity. An analysis of the epitopes recognized by these mAbs may elucidate sites important in TMEV biological activities.     

The L-coding region of the DA strain of Theiler's murine encephalomyelitis virus causes dysfunction and death of myelin-synthesizing cells

The DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis virus (TMEV) induce an early transient subclinical neuronal disease followed by a chronic progressive inflammatory demyelination, with persistence of the virus in the central nervous system (CNS) for the life of the mouse. Although TMEV-induced demyelinating disease (TMEV-IDD) is thought to be immune mediated, there is also evidence that supports a role for the virus in directly inducing demyelination. In order to clarify the function of DA virus genes, we generated a transgenic mouse that had tamoxifen-inducible expression of the DA L-coding region in oligodendrocytes (and Schwann cells), a cell type in which the virus is known to persist. Tamoxifen-treated young transgenic mice usually developed an acute progressive fatal paralysis, with abnormalities of the oligodendrocytes and Schwann cells and demyelination, but without significant lymphocytic infiltration; later treatment led to transient weakness with demyelination and persistent expression of the recombined transgene. These findings demonstrate that a high level of expression of DA L can cause the death of myelin-synthesizing cells and death of the mouse, while a lower level of L expression (which can persist) can lead to cellular dysfunction with survival. The results suggest that expression of DA L plays an important role in the pathogenesis of TMEV-IDD. Virus-induced infection and death of oligodendrocytes may play a part in the demyelination of other diseases in which an immune-mediated mechanism has been stressed, including multiple sclerosis.     

Determinants of persistence and demyelination of the DA strain of Theiler''s virus are found only in the VP1 gene.

The DA strain of Theiler's virus persists in the central nervous systems of mice and causes chronic inflammation and demyelination. The GDVII strain, on the other hand, causes an acute encephalitis that kills the host in a matter of days. We constructed a series of recombinants between two infectious cDNA clones of the genomes of DA and GDVII viruses. Analysis of the phenotypes of the recombinant viruses yielded the following results. (i) Determinants of persistence and demyelination are found only in the VP1 capsid protein of DA virus. (ii) Whereas the VP1 capsid protein of DA virus is able to fully attenuate the neurovirulence of GDVII virus and to allow the chimeric virus to persist and demyelinate, the VP1 capsid protein of GDVII virus is unable to render DA virus neurovirulent. (iii) The mere attenuation of the neurovirulence of GDVII virus does not allow it to persist and demyelinate.     

Functional analysis of the internal translation initiation site of foot-and-mouth disease virus.

Mutagenesis of the large untranslated sequence at the 5' end of the genome of foot-and-mouth disease virus revealed that a region of approximately 450 nucleotides preceding the open reading frame of the viral polyprotein is involved in the regulation of translation initiation at two internal start sites. Variations in two domains of this region reduced the translation efficiency up to 10-fold, whereas an intermediate segment seemed to be less essential. A pyrimidine-rich sequence preceding the start codon was most sensitive in that conversion of single pyrimidine residues to purines decreased the translation efficiency strongly. The data are in agreement with a recently proposed general structural model for the internal ribosome entry site of the cardiovirusaphthovirus subgroup of picornaviruses (E. V. Pilipenko, V. M. Blinov, B. K. Chernov, T. M. Dmitrieva, and V. I. Agol, Nucleic Acids Res. 17:5701-5711, 1989). They suggest, however, that this model represents only a core structure for the internal entry of ribosomes and that foot-and-mouth disease virus and other members of the picornaviruses need additional regulatory RNA elements for efficient translation initiation.     

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.