Intracerebral inoculation of 4- to 6-week-old C57BL/6 mice with the A59 strain of mouse hepatitis virus (MHV), a murine coronavirus, produced biphasic disease. Acute hepatitis and mild meningoencephalitis were followed by subacute spastic paralysis with demyelinating lesions in the brain and spinal cord as determined by Epon-embedded toluidine-blue-stained sections and by electronmicroscopy. MHV-A59 was cultured by plaque assay from the blood, brain, spinal cord, and liver of infected mice during the acute phase, but not in the chronic stage. MHV-A59 antigen was detected by immunofluorescence (IF) until 3 months postinfection (PI). Serum anti-MHV-A59 antibodies were detected from 7 days to 5 months PI. The induction of demyelination by MHV-A59 provides a suitable system to study virus-induced demyelination further.

MATERIALS AND METHODSCells, Virus, and Virus Infection. HeLa, Vero, BHK21, CV-1, HEp-2, and human astrocyte cells were maintained in Eagle's minimal essential medium containing 10% fetal calf serum, 1% glutamine, and antibiotics. These cells were cultured in T75 flasks (Falcon) at 370C in 5% CO2 and passed twice weekly. Astrocytes derived from a human brain biopsy sample were positive for glial fibrillary acidic protein by immunofluorescent staining.

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Herpes simplex virus type 1 (HSV-1) is known to reside latently in the trigeminal ganglia of man. Reactivation of this virus causes skin lesions and may occasionally infect other tissues, including the brain. To determine whether the brain tissue of humans free of clinical signs of HSV-1 infection contains any trace of HSV-1, we examined the DNA from brain tissue by endonuclease digestion, separation of the fragments by gel electrophoresis, and hybridization with labeled HSV-1 DNA probes. Hybrid bands were detected autoradiographically in experiments using cloned and virion-purified fragments ofthe HSV-1 genome. HSV-1 DNA sequences were found in 6 of 11 human brain DNA samples tested. In some cases, these bands corresponded to the bands expected for the complete viral genome, whereas others contained bands representing only a part of the genome. In some cases, the terminal fragments could be found, suggesting that the DNA was in a linear, nonintegrated form.Four types of human herpesviruses can be identified: cytomegalovirus, Epstein-Barr virus, varicella zoster virus, and herpes simplex virus (types 1 and 2; HSV-1 and HSV-2, respectively). Ofthese, herpes simplex virus (HSV) has been studied most and, almost since its discovery, has been known by its ability to form latent infections (1). The genome of HSV-1 is a linear double-stranded DNA of 108 daltons (2) and consists of two unique segments bounded by inverted repeats (3). The G+C content ofthe genome is unusually high (67%; ref.2), and it is cut by restriction endonuclease BamHI (GIG-A-T-C-C) 40 times.There have been many reports that HSV is present in the trigeminal ganglia of animals (4, 5) including humans (6-8). Isolation of HSV from superior cervical and vagus ganglia of humans has also been reported (9). HSV sequences have been detected in human brain by in situ hybridization, although no virus could be isolated nor was any detected by immunofluorescence (10). Recently, Cabrera et aL (11) have shown that, in mice experimentally infected with HSV-1, infectious virus can be recovered from brain tissue ofonly 5% ofthe latently infected animals whereas, by reassociation kinetics analysis, HSV-1 DNA sequences can be detected in the brains of 30% of mice harboring latent HSV.Although some work has been done on the analysis of HSV-1 recovered from tissue, little has been done on the viral nucleic acid present in that tissue. Probably this is because only a small amount ofmaterial is present in latently infected tissue. Instead of the classical reassociation kinetics analysis, we have used restriction endonuclease digestion, gel electrophoresis, and Southern blotting (12) to produce filters that can be hybridized with 32P-labeled nick-translated DNA of the highest possible specific activity. The small amount of radioactivity in hybrids could be detected autoradiographically. This

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