FVB mice transgenic for the H-2Db gene become resistant to persistent infection by Theiler's virus.

The DA strain of Theiler's virus causes a persistent infection of the white matter of the spinal cord with chronic inflammation and primary demyelination. Inbred strains of mice differ greatly in their susceptibility to this disease. It has been shown that both viral persistence and demyelination are controlled mainly by a gene located in the H-2D region. This raised the possibility that the H-2D gene itself controls viral persistence, which in turn determines demyelination. In the present work we introduced the H-2Db gene of resistant C57BL/6 mice into the genome of susceptible H-2q FVB mice and showed that the FVB mice become resistant to persistence of the infection and did not develop inflammatory lesions.     

The Th1/Th2 balance does not account for the difference of susceptibility of mouse strains to Theiler's virus persistent infection.

Theiler's virus causes a persistent infection with demyelination that is studied as a model for multiple sclerosis. Inbred strains of mice differ in their susceptibility to viral persistence due to both H-2 and non-H-2 genes. A locus with a major effect on persistence has been mapped on chromosome 10, close to the Ifng locus, using a cross between susceptible SJL/J and resistant B10.S mice. We now confirm the existence of this locus using two lines of congenic mice bearing the B10.S Ifng locus on an SJL/J background, and we describe a deletion in the promoter of the Ifng gene of the SJL/J mouse. We studied the expression of IFN-gamma, IL-2, IL-10, and IL-12 in the brains of SJL/J mice, B10.S mice, and the two lines of congenic mice during the first 2 wk following inoculation. We found a greater expression of IFN-gamma and IL-2 mRNA in the brains of B10.S mice compared with those of SJL/J mice. Also, the ratio of IL-12 to IL-10 mRNA levels was higher in B10.S mice. However, the cytokine profiles were the same for the two lines of resistant congenic mice and for susceptible SJL/J mice. Therefore, the difference of Th1/Th2 balance between the B10.S and SJL/J mice is not due to the Ifng locus and does not account for the difference of susceptibility of these mice to persistent infection.     

Two loci, Tmevp2 and Tmevp3, located on the telomeric region of chromosome 10, control the persistence of Theiler's virus in the central nervous system of mice

Theiler's virus persistently infects the white matter of the spinal cord in susceptible strains of mice. This infection is associated with inflammation and primary demyelination and is studied as a model of multiple sclerosis. The H-2D gene is the major gene controlling viral persistence. However, the SJL/J strain is more susceptible than predicted by its H-2(s) haplotype. An (SJL/J x B10. S)F1 x B10.S backcross was analyzed, and one quantitative trait locus (QTL) was located in the telomeric region of chromosome 10 close to the Ifng locus. Another one was tentatively mapped to the telomeric region of chromosome 18, close to the Mbp locus. We now report the study of 14 congenic lines that carry different segments of these two chromosomes. Although the presence of a QTL on chromosome 18 was not confirmed, two loci controlling viral persistence were identified on chromosome 10 and named Tmevp2 and Tmevp3. Furthermore, the Ifng gene was excluded from the regions containing Tmevp2 and Tmevp3. Analysis of the mode of inheritance of Tmevp2 and Tmevp3 identified an effect of sex, with males being more infected than females.     

High Numbers of Viral RNA Copies in the Central Nervous System of Mice during Persistent Infection with Theiler's Virus

The low-neurovirulence Theiler's murine encephalomyelitis viruses (TMEV), such as BeAn virus, cause a persistent infection of the central nervous system (CNS) in susceptible mouse strains that results in inflammatory demyelination. The ability of TMEV to persist in the mouse CNS has traditionally been demonstrated by recovering infectious virus from the spinal cord. Results of infectivity assays led to the notion that TMEV persists at low levels. In the present study, we analyzed the copy number of TMEV genomes, plus- to minus-strand ratios, and full-length species in the spinal cords of infected mice and infected tissue culture cells by using Northern hybridization. Considering the low levels of infectious virus in the spinal cord, a surprisingly large number of viral genomes (mean of 3.0 x 10(9)) was detected in persistently infected mice. In the transition from the acute (approximately postinfection [p.i.] day 7) to the persistent (beginning on p.i. day 28) phase of infection, viral RNA copy numbers steadily increased, indicating that TMEV persistence involves active viral RNA replication. Further, BeAn viral genomes were full-length in size; i.e., no subgenomic species were detected and the ratio of BeAn virus plus- to minus-strand RNA indicated that viral RNA replication is unperturbed in the mouse spinal cord. Analysis of cultured macrophages and oligodendrocytes suggests that either of these cell types can potentially synthesize high numbers of viral RNA copies if infected in the spinal cord and therefore account for the heavy viral load. A scheme is presented for the direct isolation of both cell types directly from infected spinal cords for further viral analyses.     

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.     

Viral Load and a Locus on Chromosome 11 Affect the Late Clinical Disease Caused by Theiler’s Virus

Theiler's virus causes a persistent infection and a demyelinating disease of mice which is a model for multiple sclerosis. Susceptibility to viral persistence maps to several loci, including the interferon gamma locus. Inactivating the gene coding for the interferon gamma receptor makes 129/Sv mice susceptible to persistent infection and clinical disease, whereas inactivating the interferon gamma gene makes C57BL/6 mice susceptible to persistent infection but not to clinical disease. This difference in phenotype is due to the difference in genetic background. Clinical disease depends on high viral load and Tmevd5, a locus on chromosome 11. These results have consequences for the identification of viruses which might be implicated in multiple sclerosis.     

Theiler's murine encephalomyelitis virus (TMEV) subgroup strain-specific infection in neural and non-neural cell lines.

GDVII subgroup strains of Theiler's murine encephalomyelitis virus (TMEV) are highly virulent and produce acute polioencephalomyelitis in mice. Neither viral persistence nor demyelination is demonstrated in the few surviving mice. In contrast, DA subgroup strains are less virulent and establish a persistent central nervous system infection which results in demyelinating disease. We previously reported a subgroup-specific infection in a macrophage-like cell line, J774-1 cells; i.e., GDVII strain does not replicate in J774-1 cells, whereas the DA strain actively replicates in these cells. In addition, this subgroup-specific virus growth is shown to be related to the presence of L* protein, a 17 kDa protein translated out-of-frame of the viral polyprotein from an AUG located 13 nucleotides downstream from the polyprotein's AUG. The present paper demonstrated that this subgroup-specific infection is observed in murine monocyte/macrophage lineage cell lines, but not in other murine cell lines including neural cells. An RNase protection assay also suggested that L* protein-related virus growth is regulated at the step of viral RNA replication. As macrophages are reported to be the major cell harboring virus during the chronic demyelinating stage, the activity of L* protein with respect to virus growth in macrophages may be a key factor in clarifying the mechanism(s) of TMEV persistence, which is probably a trigger to spinal cord demyelination.     

Sialylation of the host receptor may modulate entry of demyelinating persistent Theiler's virus

Theiler's murine encephalomyelitis virus (TMEV) is a picornavirus of the Cardiovirus genus. Certain strains of TMEV may cause a chronic demyelinating disease, which is very similar to multiple sclerosis in humans, associated with a persistent viral infection in the mouse central nervous system (CNS). Other strains of TMEV only cause an acute infection without persistence in the CNS. It has been shown that sialic acid is a receptor moiety only for the persistent TMEV strains and not for the nonpersistent strains. We report the effect of sialylation on cell surface on entry and the complex structure of DA virus, a persistent TMEV, and the receptor moiety mimic, sialyllactose, refined to a resolution of 3.0 A. The ligand binds to a pocket on the viral surface, composed mainly of the amino acid residues from capsid protein VP2 puff B, in the vicinity of the VP1 loop and VP3 C terminus. The interaction of the receptor moiety with the persistent DA strain provides new understanding for the demyelinating persistent infection in the mouse CNS by TMEV.     

The role of VlsE antigenic variation in the Lyme disease spirochete: persistence through a mechanism that differs from other pathogens

The linear plasmid, lp28-1, is required for persistent infection by the Lyme disease spirochete, Borrelia burgdorferi. This plasmid contains the vls antigenic variation locus, which has long been thought to be important for immune evasion. However, the role of the vls locus as a virulence factor during mammalian infection has not been clearly defined. We report the successful removal of the vls locus through telomere resolvase-mediated targeted deletion, and demonstrate the absolute requirement of this lp28-1 component for persistence in the mouse host. Moreover, successful infection of C3H/HeN mice with an lp28-1 plasmid in which the left portion was deleted excludes participation of other lp28-1 non-vls genes in spirochete virulence, persistence and the process of recombinational switching at vlsE. Data are also presented that cast doubt on an immune evasion mechanism whereby VlsE directly masks other surface antigens similar to what has been observed for several other pathogens that undergo recombinational antigenic variation.     

Minute virus of mice: antibody response, viral shedding, and persistence of viral DNA in multiple strains of mice

Minute virus of mice (MVM) is a major concern for laboratory animal facilities because it remains with considerably high prevalence despite strict barrier systems. The aim of this study was to elucidate potential risks associated with MVM infection by investigating the role of the genetic background on antibody production and persistence as well as viral shedding. Mice of various strains and stocks were inoculated oronasally with the immunosuppressive strain MVMi; in addition, natural infection was modeled through contact exposure. As determined by serology, seroconversion and serum levels of IgG differed considerably among strains and stocks, especially in the contact-exposed group. For example, C57BL/6J mice responded well to exposure in contrast to FVB/N, NMRI, ICR, and C3H/HeN mice. Titration studies indicated that the viral dose necessary to induce seroconversion was strain-dependent. Experiments to dissect the role of the major histocompatibility complex haplotype in the response to MVMi gave inconclusive results. To detect viral persistence, spleens and feces were analyzed by PCR at 16 wk after exposure, and the infectivity of PCR-positive spleens was investigated by IP and oronasal inoculation of naive mice. Although DNA was detected in the spleens of some mice, feces remained negative, and naive mice were not infected by inoculation. In addition, viral shedding declined rapidly after day 20 postinoculation. In summary, the data show that seroconversion and antibody response to MVMi infection depend on the genetic background of mice, with the infective dose being a critical factor. The role of viral DNA in chronically infected mice will require further elucidation.     

Contributions of CD8+ T cells and viral spread to demyelinating disease

Acute and chronic demyelination are hallmarks of CNS infection by the neurotropic JHM strain of mouse hepatitis virus. Although infectious virus is cleared by CD8+ T cells, both viral RNA and activated CD8+ T cells remain in the CNS during persistence potentially contributing to pathology. To dissociate immune from virus-mediated determinants initiating and maintaining demyelinating disease, mice were infected with two attenuated viral variants differing in a hypervariable region of the spike protein. Despite similar viral replication and tropism, one infection was marked by extensive demyelination and paralysis, whereas the other resulted in no clinical symptoms and minimal neuropathology. Mononuclear cells from either infected brain exhibited virus specific ex vivo cytolytic activity, which was rapidly lost during viral clearance. As revealed by class I tetramer technology the paralytic variant was superior in inducing specific CD8+ T cells during the acute disease. However, after infectious virus was cleared, twice as many virus-specific IFN-gamma-secreting CD8+ T cells were recovered from the brains of asymptomatic mice compared with mice undergoing demyelination, suggesting that IFN-gamma ameliorates rather than perpetuates JHM strain of mouse hepatitis virus-induced demyelination. The present data thus indicate that in immunocompetent mice, effector CD8+ T cells control infection without mediating either clinical disease or demyelination. In contrast, demyelination correlated with early and sustained infection of the spinal cord. Rapid viral spread, attributed to determinants within the spike protein and possibly perpetuated by suboptimal CD8+ T cell effector function, thus ultimately leads to the process of immune-mediated demyelination.     

L* Protein of the DA Strain of Theiler’s Murine Encephalomyelitis Virus Is Important for Virus Growth in a Murine Macrophage-Like Cell Line

Strain GDVII and other members of the GDVII subgroup of Theiler’s murine encephalomyelitis virus (TMEV) are highly virulent and cause acute polioencephalomyelitis in mice. Neither viral persistence nor demyelination is demonstrated in the few surviving mice. On the other hand, strain DA and other members of the TO subgroup of TMEV are less virulent and establish a persistent infection in the spinal cord, which results in a demyelinating disease. We previously reported that GDVII does not actively replicate in a murine macrophage-like cell line, J774-1, whereas DA strain productively infects these cells (M. Obuchi, Y. Ohara, T. Takegami, T. Murayama, H. Takada, and H. Iizuka, J. Virol. 71:729–733, 1997). In the present study, we used recombinant viruses between these strains of the two subgroups to demonstrate that the DA L coding region of DA strain is important for virus growth in J774-1 cells. Additional experiments with a mutant virus indicate that L* protein, which is synthesized out of frame with the polyprotein from an additional alternative initiation codon in the L coding region of TO subgroup strains, is a key determinant responsible for the cell-type-specific restriction of virus growth. L* protein may play a critical role in the DA-induced restricted demyelinating infection by allowing growth in macrophages, a major site for virus persistence.     

The shiverer mutation affects the persistence of Theiler's virus in the central nervous system.

Theiler's virus persists in the white matter of the spinal cord of genetically susceptible mice and causes primary demyelination. The virus persists in macrophages/microglial cells, but also in oligodendrocytes, the myelin-forming cells. Susceptibility/resistance to this chronic infection has been mapped to several loci including one tentatively located in the telomeric region of chromosome 18, close to the myelin basic protein locus (Mbp locus). To determine if the MBP gene influences viral persistence, we inoculated C3H mice bearing the shiverer mutation, a 20-kb deletion in the gene. Whereas control C3H mice were of intermediate susceptibility, C3H mice heterozygous for the mutation were very susceptible, and those homozygous for the mutation were completely resistant. This resistance was not immune mediated. Furthermore, C3H/101H mice homozygous for a point mutation in the gene coding for the proteolipid protein of myelin, the rumpshaker mutation, were resistant. These results strongly support the view that oligodendrocytes are a necessary viral target for the establishment of a persistent infection by Theiler's virus.     

Role of dendritic cells in differential susceptibility to viral demyelinating disease

Although persistent viral diseases are a global health concern, the mechanisms of differential susceptibility to such infections among individuals are unknown. Here, we report that differential interactions between dendritic cells (DCs) and virus are critical in determining resistance versus susceptibility in the Theiler murine encephalomyelitis virus-induced demyelinating disease model of multiple sclerosis. This virus induces a chronic demyelinating disease in susceptible mice, whereas the virus is completely cleared in resistant strains of mice. DCs from susceptible mice are more permissive to viral infection, resulting in severe deficiencies in development, expansion, and function, in contrast to DCs from resistant mice. Although protective prior to viral infection, higher levels of type I interferons (IFNs) and IFN-gamma produced by virus-infected DCs from susceptible mice further contribute to the differential inhibition of DC development and function. An increased DC number and/or acquired resistance of DCs to viral infection render susceptible mice resistant to viral persistence and disease progression. Thus, the differential permissiveness of DCs to infectious agents and its subsequent functional and developmental deficiencies determine the outcome of infection- associated diseases. Therefore, arming DCs against viral infection-induced functional decline may provide a useful intervention for chronic infection-associated diseases.     

Localization of the growth hormone gene to the distal half of mouse chromosome 11

A DNA fragment size variant for the growth hormone gene, Gh, has been identified among inbred strains of mice. The inbred strains SM/J and CAST/Ei carry the less frequent allele Ghb and 11 other strains carry the Gha allele. Segregation analysis of data from two crosses involving SM/J and NZB/BINJ and a cross involving BALB/cJ and CAST/Ei confirmed the assignment of Gh to mouse chromosome 11 and placed the locus 2.6 +/- 1.8 map units distal to Erba (avian erythroblastosis oncogene A), a position consistent with the assignment of the Gh locus to the q22-q24 region of chromosome 17 on the human map. Segregation analysis also refined the location of Sparc (secreted acidic cysteine-rich glycoprotein) on mouse chromosome 11 to a position 16.7 +/- 4.2 map units proximal to Evi-2 (ecotropic viral integration site 2).