Temperature-sensitive mutants of mouse hepatitis virus strain A59: Isolation, characterization and neuropathogenic properties

Abstract

Twenty 5-fluorouracil-induced temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 were isolated from 1284 virus clones. Mutants were preselected on the basis of their inability to induce syncytia in infected cells at the restrictive temperature (40 degrees) vs the permissive temperature (31 degrees). Of these mutants, only those with a relative plating efficiency 40 degrees/31 degrees of 3 x 10(-3) or smaller were kept. Virus yields at 40 degrees compared to 37 degrees and 31 degrees (leakiness) were determined. Most mutants (16) were RNA-, i.e., unable to synthesize virus-specific RNA at the restrictive temperature. The other four were RNA+. No qualitative differences were detected in the virus-specific RNAs in cells infected with RNA+ ts-mutants, both at 31 degrees and 40 degrees. Virus-specific proteins present in cells infected with ts-171 (RNA-) and the RNA+-mutants (ts-43, ts-201, ts-209, and ts-279) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates. No qualitative differences in the pattern of virus-specific cellular proteins were detected among the mutants except for an additional polypeptide of about 46,000 daltons in ts-209-infected cells. Finally, the neuropathogenic properties of eight of the mutants were investigated. Whereas 10(2) PFU of wild-type virus injected intracerebrally killed 50 to 100% of 4-week-old Balc/c mice within 1 week, the mutants were highly attenuated. A dose of 10(5) PFU lead to no or transient disease. However, 4 weeks after infection with ts-342, ts-43, or ts-201 obvious histological changes were observed in brain and spinal cord of clinically healthy mice.

Induction of Demyelination by a Temperature-sensitive Mutant of the Coronavirus MHV-A59 is Associated with Restriction of Viral Replication in the Brain

“…Intracerebral inoculation of certain ts mutants of MHV-A59 causes a subacute demyelinating disease in the CNS (Koolen et al, 1983). The present paper describes virological and neuropathological findings in mice infected with ts mutants of MHV-A59, including several that induce demyelination in the CNS.…”

“…In BALB/c mice, MHV-A59 causes, even at low dosage, an acute infection with high mortality (Koolen et al, 1983).…”

“…The isolation and characterization of the ts mutants of MHV-A59 and the preparation of virus stocks were described previously (Koolen et al, 1983), as was the isolation of revertants of ts-342 (van Berlo et al, 1986). The ratio of ts-342 plaque titres at 40 °C to those at 3 l °C was about 10-s, compared with ratios of 1.5 to 8 for the revertants or wild-type virus.…”

“…In rodents of the appropriate age and genetic background, intracerebral inoculation of a large dose of wild-type MHV causes an acute lethal disease (Knobler et al, 1981(Knobler et al, , 1982Wege et al, 1982). Low doses of wild-type virus or high doses of attenuated temperature-sensitive (ts) mutants of MHV strains JHM and A59 produce a chronic demyelinating disease in mice (Haspel et aL, 1978;Koolen et al, 1983;Lavi et al, 1984b;Weiner, 1973;Woyciechowska et al, 1984). Demyelination induced by MHV-JHM, which has been studied most extensively, is characterized by infection and lysis of oligodendrocytes, leaving axons well preserved (Sorensen et al, 1980).…”

Target and Effector Cell Fusion Accounts for B Lymphocyte-mediated Lysis of Mouse Hepatitis Virus-infected Cells

“…Target cells were infected by incubating cell monolayers with virus for 1.5 h at 37 °C followed by incubation for 16 h at 31 °C to delay cytopathic effects caused by the MHV(A59) virus (Koolen et al, 1983).…”