title: Recombinant mouse hepatitis virus strain A59 from cloned, full-length cDNA replicates to high titers in vitro and is fully pathogenic in vivo.
abstract: Mouse hepatitis virus (MHV) is the prototype of group II coronaviruses and one of the most extensively studied coronaviruses. Here, we describe a reverse genetic system for MHV (strain A59) based upon the cloning of a full-length genomic cDNA in vaccinia virus. We show that the recombinant virus generated from cloned cDNA replicates to the same titers as the parental virus in cell culture ( approximately 10(9) PFU/ml), has the same plaque morphology, and produces the same amounts and proportions of genomic and subgenomic mRNAs in virus-infected cells. In a mouse model of neurological infection, the recombinant and parental viruses are equally virulent, they replicate to the same titers in brain and liver, and they induce similar patterns of acute hepatitis, acute meningoencephalitis, and chronic demyelination. We also describe improvements in the use of the coronavirus reverse genetic system based on vaccinia virus cloning vectors. These modifications facilitate (i) the mutagenesis of cloned cDNA by using vaccinia virus-mediated homologous recombination and (ii) the rescue of recombinant coronaviruses by using a stable nucleocapsid protein-expressing cell line for the electroporation of infectious full-length genomes. Thus, our system represents a versatile and universal tool to study all aspects of MHV molecular biology and pathogenesis. We expect this system to provide valuable insights into the replication of group II coronaviruses that may lead to the development of novel strategies against coronavirus infections, including the related severe acute respiratory syndrome coronavirus.
authors.affiliation: Research Department, Cantonal Hospital St. Gallen, 9007 St. Gallen, Switzerland.
authors.forename: Scott E
authors.initials: SE
authors.lastname: Coley
authors.forename: Ehud
authors.initials: E
authors.lastname: Lavi
authors.forename: Stanley G
authors.initials: SG
authors.lastname: Sawicki
authors.forename: Li
authors.initials: L
authors.lastname: Fu
authors.forename: Barbara
authors.initials: B
authors.lastname: Schelle
authors.forename: Nadja
authors.initials: N
authors.lastname: Karl
authors.forename: Stuart G
authors.initials: SG
authors.lastname: Siddell
authors.forename: Volker
authors.initials: V
authors.lastname: Thiel
mesh_terms: D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000473:pathology / Q000821:virology; D002460:Cell Line; D003001:Cloning, Molecular; D018352:Coronavirus Infections / Q000473:pathology / Q000821:virology; D004260:DNA Repair; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D003711:Demyelinating Diseases / Q000473:pathology; D005818:Genetic Engineering; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000473:pathology / Q000821:virology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology*; D011995:Recombination, Genetic; D014616:Vaccinia virus / Q000235:genetics; D014774:Virulence; D014779:Virus Replication
chemical_list: D018076:DNA, Complementary; D004279:DNA, Viral