title: Firefly luciferase as a marker for herpesvirus (pseudorabies virus) replication in vitro and in vivo.
abstract: Insertion of reporter genes into complex viral genomes and monitoring virus replication by detecting the corresponding protein products is increasingly used in pathogenesis studies. We present here the isolation and characterization of a recombinant neurotropic alpha-herpesvirus, pseudorabies virus (PrV), which stably carries the gene encoding firefly luciferase. To express the enzyme the complete open reading frame for luciferase was fused to the promoter and first seven codons of the non-essential glycoprotein gX gene of PrV. A recombinant PrV carrying the luciferase gene inserted into the gX gene and exhibiting strong luciferase activity after infection of cultured cells was further characterized. Kinetic analyses showed that luciferase activity was detectable as early as 90 min after infection. Luciferase expression could be monitored in cell extracts in a luminometer. For facilitating plaque isolation of luciferase recombinant viruses it was also visualized in situ on sensitive film. Kinetic experiments in mice proved the suitability of luciferase as an excellent marker for following herpesvirus spread in the animal. By way of luciferase detection we show that PrV invasion of the central nervous system after intranasal infection of mice occurred independently of replication in non-neural tissues such as lung or thymus. Furthermore, comparison of isogenic luciferase recombinant PrV strains carrying intact or deleted glycoprotein gI genes showed differences in the organotropism between these two viruses.
authors.affiliation: Federal Research Centre for Virus Diseases of Animals, Tübingen, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Kovács
authors.forename: T C
authors.initials: TC
authors.lastname: Mettenleiter
mesh_terms: D000818:Animals; D001483:Base Sequence; D001517:Coleoptera / Q000201:enzymology; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D005260:Female; D005814:Genes, Viral; D005819:Genetic Markers; D011558:Herpesvirus 1, Suid / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D007700:Kinetics; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011557:Pseudorabies / Q000209:etiology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics; D014779:Virus Replication / Q000235:genetics
chemical_list: D004279:DNA, Viral; D005819:Genetic Markers; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C042966:pseudorabies virus glycoproteins; D008156:Luciferases