Enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system
- 1 January 2002
- journal article
- research article
- Published by Springer Science and Business Media LLC in Journal of NeuroVirology
- Vol. 8 (5), 381-391
- https://doi.org/10.1080/13550280260422686
Abstract
Targeted recombination was used to select mouse hepatitis virus isolates with stable and efficient expression of the gene encoding the enhanced green fluorescent protein (EGFP). The EGFP gene was inserted into the murine coronavirus genome in place of the nonessential gene 4. These viruses expressed the EGFP gene from an mRNA of slightly slower electrophoretic mobility than mRNA 4. EGFP protein was detected on a Western blot of infected cell lysates and EGFP activity (fluorescence) was visualized by microscopy in infected cells and in viral plaques. Expression of EGFP remained stable through at least six passages in tissue culture and during acute infection in the mouse central nervous system. These viruses replicated with similar kinetics and to similar final extents as wild-type virus both in tissue culture and in the mouse central nervous system (CNS). They caused encephalitis and demyelination in animals as wild-type virus; however, they were somewhat attenuated in virulence. Isogenic EGFP-expressing viruses that differ only in the spike gene and express either the spike gene of the highly neurovirulent MHV-4 strain or the more weakly neurovirulent MHV-A59 strain were compared; the difference in virulence and patterns of spread of viral antigen reflected the differences between parental viruses expressing each of these spike genes. Thus, EGFP-expressing viruses will be useful in the studies of murine coronavirus pathogenesis in mice.Keywords
This publication has 20 references indexed in Scilit:
- Demyelination Determinants Map to the Spike Glycoprotein Gene of Coronavirus Mouse Hepatitis VirusJournal of Virology, 2000
- Retargeting of Coronavirus by Substitution of the Spike Glycoprotein Ectodomain: Crossing the Host Cell Species BarrierJournal of Virology, 2000
- Altered Pathogenesis of a Mutant of the Murine Coronavirus MHV-A59 Is Associated with a Q159L Amino Acid Substitution in the Spike ProteinVirology, 1997
- FACS-optimized mutants of the green fluorescent protein (GFP)Gene, 1996
- Codon usage limitation in the expression of HIV-1 envelope glycoproteinCurrent Biology, 1996
- Dissociation of demyelination and viral clearance in congenitally immunodeficient mice infected with murine coronavirus JHMJournal of NeuroVirology, 1996
- Mouse hepatitis virus A59 increases steady-state levels of MHC mRNAs in primary glial cell cultures and in the murine central nervous systemMicrobial Pathogenesis, 1992
- Determinants of coronavirus MHV pathogenesis are localized to 3' portions of the genome as determined by ribonucleic acid-ribonucleic acid recombination.1990
- Experimental demyelination produced by the A59 strain of mouse hepatitis virusNeurology, 1984
- Pathogenic murine coronaviruses II. Characterization of virus-specific proteins of murine coronaviruses JHMV and A59VVirology, 1979