Strains from Both Theiler's Virus Subgroups Encode a Determinant for ...

JOURNAL OF VIROLOGY, Dec. 1990, p. 6345-6348

Vol. 64, No. 12

0022-538X/90/126345-04$02.00/0 Copyright C) 1990, American Society for Microbiology

Strains from Both Theiler's Virus Subgroups Encode Determinant for Demyelination

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JIANLIN FU,1 MOSES RODRIGUEZ,2 AND RAYMOND P. ROOS'* Department of Neurology, University of Chicago Medical Center, 5841 South Maryland Avenue, Chicago, Illinois 60637,1 and Department of Neurology, Mayo Clinic, Rochester, Minnesota 559052 Received 20 July 1990/Accepted 18 September 1990

The GDVII strain and other members of the GDVII subgroup of Theiler's murine encephalomyelitis viruses (TMEV) cause an acute lethal neuronal infection in mice, whereas the DA strain and other members of the TO subgroup of TMEV cause a chronic demyelinating disease associated with a persistent virus infection. We used GDVII/DA chimeric infectious cDNAs to produce intratypic recombinant viruses in order to clarify reasons for the TMEV subgroup-specific difference in demyelinating activity. We found that both the GDVII and DA strains contain a genetic determinant(s) for demyelinating activity. No demyelination occurs following GDVII strain inoculation because this strain produces an early neuronal disease that kills mice before white matter disease and persistent infection can occur.

Theiler's murine encephalomyelitis viruses (TMEV) are picornaviruses, closely related to cardioviruses (8, 12), that cause enteric and neurological disease in mice. Although TMEV strains are 90% similar at the nucleotide level and 95% similar at the amino acid level (10, 12), they fall into two subgroups on the basis of their markedly different biological activities (5, 7). The GDVII strain and other members of the GDVII subgroup cause an acute, fatal polioencephalomyelitis. In contrast, the DA strain and other members of the TO subgroup produce a restricted, persistent infection with demyelination that begins approximately 3 weeks after inoculation and continues for the rest of the life of an infected mouse. The DA disease has special interest because it serves as an excellent experimental model for multiple sclerosis (a human demyelinating disease of unknown cause) (3, 4). A question under continuing study has been whether GDVII subgroup strains lack a determinant for demyelination that is present in TO subgroup strains or whether all TMEV strains possess a genetic determinant for demyelination but GDVII subgroup strains kill mice with an acute neuronal disease before demyelination can occur. In this study, we present results from experiments with infectious cDNAs that indicate that all TMEV strains encode a determinant(s) for demyelination. We have previously published details regarding the generation of full-length infectious pDAFL3 and pGDVIIFL2 cDNAs from TMEV parental strains as well as the production of intratypic chimeric cDNAs (1, 14). These recombinant virus studies identified the GDVII 1B-2C coding region as containing a determinant critical for neurovirulence, although GDVII sequences 5' to this region also contributed to the full neurovirulence phenotype (1). We now use chimeric cDNAs (Fig. 1) to delineate determinants important in the late demyelinating disease. The chimeric cDNAs were constructed so that a genome segment from the GDVII strain was substituted with a segment from the DA strain, or vice versa, with maintenance of the polyprotein reading frame. The chimeras are named by listing the strain that contributes the majority of the genome after the region of the strain that *

contributes the substituted segment. Junctions of the substituted segment were sequenced by a dideoxynucleotide method (11). Each of the cDNAs was transcribed in vitro and then transfected into L cells as previously described (14). The progeny virus was plaque purified, and a virus stock was prepared in BHK-21 cells. Generally, 30 ,ul of undiluted virus stock (which varied from 5 x 104 to 2.8 x 107 PFU/ml) was inoculated intracerebrally into 5 to 10 3-week-old SJL/J mice (Jackson Laboratory). Tenfold serial dilutions of virus were used for inoculations when infection with undiluted virus killed animals within the first month, e.g., with GDVIIFL2 and GDlB-2C/DAFL3 viruses. Forty-five days after inoculation, survivors infected with the lowest dilutions were sacrificed by intracardiac perfusion with phosphatebuffered 4% formaldehyde with 1.5% glutaraldehyde (pH 7.2) (13). The spinal cord from each mouse was processed to provide 15 to 20 1-,um-thick glycol methacrylate-embedded sections that were stained with modified Eriochrome stain with cresyl violet counterstain to detect demyelination and inflammation. Animals inoculated with virus produced from transfected pDAFL3 and pGDVIIFL2 in vitro-derived transcripts had disease indistinguishable from that seen with parental viruses (1, 14). DAFL3 virus did not cause death within the first 45 days of inoculation. Histopathological examination of DAFL3-inoculated animals at this time consistently showed demyelinating myelopathy. The spinal cords generally exhibited a meningeal infiltrate as well as perivascular cuffs located within the white matter. The white matter, especially the posterior and lateral columns, exhibited demyelination with preservation of axons (Fig. 2B). In contrast, animals inoculated with varied dilutions of GDVIIFL2 died within 1 month of infection with neuronal infection. Since the 50% lethal dose of GDVIIFL2 was 0.7 PFU (2), only animals receiving a very small amount of virus survived the acute neuronal disease. These survivors had no evidence of histopathological abnormalities in the central nervous system, perhaps reflecting the fact that they had not been infected by the small amount of virus used in the inoculation. The animals that were inoculated with undiluted stocks of several of the recombinant viruses (GD5'-lB/DAFL3 [Fig.

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VOL. 64, 1990

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FIG. 1. TMEV chimeric intratypic cDNAs and their demyelinating activity. DA segments are shown as the open areas of the genome, while GDVII segments are shown as the solid areas. The restriction sites used to generate the intratypic chimeric cDNAs are noted above the individual plasmid constructs. The position of the restriction sites is aligned with the TMEV coding area shown at the top of the figure, and the nucleotide position in the genome is shown at the bottom. The ability of the virus to produce demyelination at 45 days after inoculation is noted by a plus sign. No demyelination was seen after inoculation with GDVIIFL2; an asterisk is placed beside the minus sign to note that no infected animals actually survived for 45 days after inoculation (see text). FIG. 2. Spinal cord sections from SJL/J mice infected for 45 days with GDVII/DA recombinant viruses. Glycol methacrylate-embedded sections were stained with modified Eriochrome with cresyl violet counterstain. (A) Normal spinal cord from SJL/J mouse mock infected with diluent intracerebrally. The other panels show evidence of primary demyelination and perivascular inflammation in mice infected with DAFL3 (B), GD5'-lB/DAFL3 (C), GD1B-2C/DAFL3 (D), GD2C-3C/DAFL3 (E), and GDNC/DAFL3 (F).

2C], GDlB-2C/DAFL3 [Fig. 2D], GDNC/DAFL3 [Fig. 2F], GD2C-3C/DAFL3 [Fig. 2E], and GD3C-3'/DAFL3 [Fig. 3]) survived the first month, and some developed a progressive lower-extremity weakness. Histopathological examination consistently showed typical features of DA strain inflammatory demyelinating disease (Fig. 2). However, the extent of pathologic abnormalities varied between groups infected with different recombinant viruses, with the most severe disease observed in animals infected with GD3C-3'/DAFL3 virus (Fig. 3). In the case of GDlB-2C/DAFL3 virus, animals tended to die within a month over several different dilutions of the inoculum, i.e., although the 50% lethal dose was 102-7 PFU (1), there was a "jagged" survival curve. For this reason, 11 surviving SJL/J mice that had been inoculated with 7.5 x 101 to 7.5 x 103 PFU were studied histopathologically. Nine of these animals had evidence of the typical features of DA strain inflammatory demyelinating myelopathy; two of the five animals inoculated at the lowest dose of virus had no evidence of histopathological abnormalities. It was of interest to note that demyelination was seen after infection with this recombinant virus, even with a low dose of inoculum, suggesting that GDlB-2C/DAFL3 virus may actually have an enhanced ability to cause demyelination; this finding is presently under study. In a companion study, eight DBA/2 mice (Charles River Laboratories) were inoculated with GDlB-2C/DAFL3 virus at doses comparable to those used with SJL/J mice. DBA/2 mice are known to be susceptible to the late demyelinating disease, although they are less susceptible than SJL/J mice (6). There was no demyelination in the DBA/2 mice despite an observation period of 106 days. These differences be-

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tween the susceptibility of DBA/2 mice to demyelination following infection with GDlB-2C/DAFL3 and their susceptibility after DAFL3 infection suggest that there may be an especially strong dependence on host genetics for full expression of demyelinating activity with this particular recombinant. Our studies demonstrate that recombinant viruses that replace any part of the DA genome with the corresponding segment from the GDVII genome are still capable of producing demyelination in infected survivors. These findings indicate that the GDVII strain, as well as the DA strain, has a determinant(s) for demyelination. We presume that demyelination is not seen after GDVII strain infection because GDVII causes an acute, lethal neuronal disease in which every animal that is infected dies, i.e., 1 infectious unit equals 1 50% lethal dose (5). Our ability to produce attenuated recombinant viruses made the demyelinating capacity of the GDVII strain apparent. Our findings indicate that the demyelinating potential of a neurovirulent virus that ordinarily does not produce white matter disease may become apparent after attenuation; this observation is of potential concern with respect to vaccine production. This study suggests that at least one determinant for TMEV demyelination is present in all TMEV strains. However, it is likely that multiple demyelination determinants exist and that some of the determinants are unique to TO subgroup strains, considering the complexity of the demyelination process. Published studies indicate that amino acid residues 101 and 268 in VP1 are important in DA strain demyelinating activity (9, 15); site-directed mutagenesis of infectious TMEV cDNA clones is in progress to precisely identify these DA strain-specific sequences critical in demyelination. These studies may lead not only to a better understanding of TMEV demyelination but also of other demyelinating diseases, such as multiple sclerosis. We thank Lee Baksas for secretarial assistance and T. Bodwell, L. Rosenstein, and B. Shim for technical help. This research was supported by Public Health Service grants 5 P01 NS21442-06 and 5 P01 NS24575-03 from the National Institutes of Health and grant RG 1512-C-4 from the National Multiple Sclerosis Society to R.P.R. M.R. is supported by grants RG 1878-B-2 and RG 2174-A-3 from the National Multiple Sclerosis Society and by Public Health Service grant RO1-NS24180 from the National Institutes of Health.

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3. Lehrich, J. R., B. G. W. Arnason, and F. Hochberg. 1976.

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NOTES

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