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Temperature-sensitive mutants of mouse hepatitis virus strain A59: isolation, characterization and neuropathogenic
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.
Insights
Researchers developed temperature-sensitive (ts) mouse hepatitis virus mutants to study viral RNA synthesis and neuropathogenesis. Most mutants were RNA-deficient, and all showed significant attenuation in mice, indicating potential for vaccine development.
Area of Science:
- Virology
- Molecular Biology
- Neuroscience
Background:
- Mouse hepatitis virus (MHV) is a significant pathogen.
- Temperature-sensitive (ts) mutants are valuable tools for studying viral replication and pathogenesis.
- Understanding MHV's genetic basis for virulence is crucial for disease control.
Purpose of the Study:
- To isolate and characterize temperature-sensitive mutants of mouse hepatitis virus strain A59.
- To investigate the role of viral RNA synthesis in MHV replication.
- To assess the neuropathogenic potential of MHV ts mutants in a mouse model.
Main Methods:
- Isolation of 5-fluorouracil-induced ts mutants from MHV-A59.
- Selection based on syncytia formation at restrictive and permissive temperatures.
- Analysis of viral RNA synthesis and protein expression.
- Assessment of neuropathogenicity through intracerebral inoculation in mice.
Main Results:
- Twenty ts mutants were isolated, with 16 being RNA-deficient (RNA-) and 4 RNA-positive (RNA+).
- No qualitative differences in viral RNA or proteins were observed among RNA+ mutants, except for an additional 46 kDa polypeptide in ts-209.
- All tested ts mutants exhibited significant attenuation in mice, with high doses causing no or transient disease.
- Histological changes in the central nervous system were observed in mice infected with specific ts mutants, despite a lack of clinical symptoms.
Conclusions:
- The study successfully generated and characterized MHV ts mutants, providing insights into viral RNA synthesis.
- The identified ts mutants are highly attenuated in vivo, suggesting their potential as vaccine candidates.
- Further investigation into the neurological effects of these attenuated mutants is warranted.