title: Cloning of the genomes of equine herpesvirus type 1 (EHV-1) strains KyA and racL11 as bacterial artificial chromosomes (BAC).
abstract: The genome of equine herpesvirus type 1 (EHV-1) strain RacL11, a highly virulent isolate obtained from an aborted foal, and that of the modified live vaccine strain KyA, were cloned as bacterial artificial chromosomes (BAC) in Eseherichia coli. Mini F plasmid sequences were inserted into the viral genomes by homologous recombination instead of the gene 71 (EUS4) open reading frame after co-transfection of viral DNA and recombinant plasmid pdelta71-pHA2 into RK13 cells. After isolation of recombinant viruses by three rounds of plaque purification, viral DNA was isolated from RK13 cells infected with RacL11 or KyA virus mutants expressing the green fluorescent protein (GFP), and electroporated into Escherichia coli DH10B cells. Several bacterial colonies were shown to contain high-molecular weight BAC DNA with a restriction enzyme fragment pattern indicative of the presence of full-length RacL11 or KyA genomes. Two selected BAC clones were analysed by restriction enzyme analysis and Southern blotting, and were eventually termed pRacLI I and pKyA. respectively. Upon transfection of pRacL11 or pKyA DNA into RK13 cells, GFP-expressing fluorescing virus plaques could be identified from day 1 after transfection. Infectivity after transfection of pRacL11 or pKyA could be readily propagated on RK13 or equine cells, indicating that infectious full-length DNA clones of strains RacL11 and KyA were successfully cloned in Escherichia coli as BACs. The glycoprotein 2-negative progeny reconstituted from pRacL11 and pKyA (L11deltagp2 and KyAdeltagp2) exhibited different growth properties. Whereas both L11deltagp2 and KyAdeltagp2 extracellular titres were reduced by 15- to 32-fold, plaque diameters were only markedly (50%) reduced in the case of KyAdeltagp2.
authors.affiliation: Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, Insel Riems, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Rudolph
authors.forename: D J
authors.initials: DJ
authors.lastname: O'Callaghan
authors.forename: N
authors.initials: N
authors.lastname: Osterrieder
mesh_terms: D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D016679:Genome, Viral; D006566:Herpesviridae Infections / Q000662:veterinary / Q000821:virology; D004861:Herpesvirus 1, Equid / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity; D006734:Horse Diseases / Q000821:virology; D006736:Horses; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000662:veterinary; D014162:Transfection / Q000662:veterinary; D014613:Vaccines, Attenuated / Q000235:genetics; D014774:Virulence
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D014613:Vaccines, Attenuated