title: Enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system.
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.
authors.affiliation: Department of Physiology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Jayasri
authors.initials: J
authors.lastname: Das Sarma
authors.forename: Esther
authors.initials: E
authors.lastname: Scheen
authors.forename: Su-Hun
authors.initials: SH
authors.lastname: Seo
authors.forename: Michael
authors.initials: M
authors.lastname: Koval
authors.forename: Susan R
authors.initials: SR
authors.lastname: Weiss
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000032:analysis; D001921:Brain / Q000821:virology; D002415:Cats; D002490:Central Nervous System / Q000821:virology*; D018352:Coronavirus Infections / Q000821:virology*; D004195:Disease Models, Animal; D049452:Green Fluorescent Proteins; D007739:L Cells; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D006517:Murine hepatitis virus / Q000235:genetics* / Q000472:pathogenicity; D012333:RNA, Messenger / Q000096:biosynthesis; D016865:Reassortant Viruses; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D012692:Serial Passage; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000235:genetics; D014774:Virulence; D014779:Virus Replication
chemical_list: D000956:Antigens, Viral; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins