title: Switching species tropism: an effective way to manipulate the feline coronavirus genome.
abstract: Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPV Delta 7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPV Delta 7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.
authors.affiliation: Institute of Virology, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands.
authors.forename: Bert Jan
authors.initials: BJ
authors.lastname: Haijema
authors.forename: Haukeliene
authors.initials: H
authors.lastname: Volders
authors.forename: Peter J M
authors.initials: PJ
authors.lastname: Rottier
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002415:Cats; D002478:Cells, Cultured; D016765:Coronavirus, Feline / Q000235:genetics* / Q000472:pathogenicity; D016766:Feline Infectious Peritonitis / Q000401:mortality / Q000821:virology*; D005818:Genetic Engineering / Q000379:methods; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D014774:Virulence
chemical_list: D008562:Membrane Glycoproteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins