title: Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3 (EHV-3).
abstract: Equine herpesvirus type 3 (EHV-3) is the causal agent of equine coital exanthema, a disease characterized by pox-like lesions on the penis of stallions and the vulva of mares. Although the complete genomic sequence of EHV-3 has been recently made available, its genomic content remains poorly characterized and the molecular mechanisms of disease development not yet elucidated. In an attempt to facilitate genetic manipulation of EHV-3, we describe here the construction of a full-length infectious bacterial artificial chromosome (BAC) clone of EHV-3. Mini-F vector sequences were inserted into the intergenic region between ORF19 and ORF20 (UL41 and UL40, respectively) of EHV-3 strain C175 by homologous recombination in equine dermal cells (NBL-6). DNA of the resulting recombinant virus was electroporated into E. coli and a full-length EHV-3 BAC clone was recovered. Virus reconstituted after transfection of the EHV-3 BAC into NBL-6 cells showed growth properties in vitro that were indistinguishable from those of the parental virus. To assess the feasibility of mutagenesis of the cloned EHV-3 genome, recombinant viruses targeting the glycoprotein E (gE) gene were generated using Red recombination in E. coli and in vitro growth properties of the recombinant viruses were evaluated. We first repaired the gE (ORF74) coding region, since the parental virus used for BAC cloning specifies a truncated version of the gene, and then created gE-tagged and gE-null versions of the virus. Our results demonstrated that: (i) EHV-3 can be efficiently cloned as a BAC allowing easy manipulation of its genome; (ii) gE is dispensable for EHV-3 growth in vitro and is expressed as a product of approximately 110-kDa in infected cells; (iii) viruses having a deletion compromising gE expression or with a truncation of the cytoplasmic and transmembrane domains are significantly compromised with regard cell-to-cell spread. The cloning of EHV-3 as a BAC simplifies future studies to identify the role of its coding genes in viral pathogenesis and host immune responses.
authors.affiliation: Institut für Virologie, Zentrum für Infektionsmedizin Robert von Ostertag-Haus, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany; Laboratory Virology and Biotechnology Kyrgyz Research Veterinary Institute named after A. Duisheev, 60 Togolok Moldo, Bishkek, Kyrgyzstan.
authors.forename: Maksat
authors.initials: M
authors.lastname: Akhmedzhanov
authors.affiliation: Cátedra de Virología, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, Calle 60 y 118 S/N, 1900 La Plata, Argentina.
authors.forename: Mariela
authors.initials: M
authors.lastname: Scrochi
authors.affiliation: Instituto de Virología, CICVyA, INTA, Las Cabañas y Los Reseros S/N, Castelar 1712, Argentina.
authors.forename: Maria
authors.initials: M
authors.lastname: Barrandeguy
authors.affiliation: Instituto de Virología, CICVyA, INTA, Las Cabañas y Los Reseros S/N, Castelar 1712, Argentina.
authors.forename: Aldana
authors.initials: A
authors.lastname: Vissani
authors.affiliation: Institut für Virologie, Zentrum für Infektionsmedizin Robert von Ostertag-Haus, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany.
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Osterrieder
authors.affiliation: Institut für Virologie, Zentrum für Infektionsmedizin Robert von Ostertag-Haus, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany. Electronic address: amdamiani9@gmail.com.
authors.forename: Armando Mario
authors.initials: AM
authors.lastname: Damiani
mesh_terms: D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D015870:Gene Expression; D023061:Gene Order; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics; D016679:Genome, Viral; D004858:Herpesvirus 3, Equid / Q000235:genetics*; D016296:Mutagenesis; D016366:Open Reading Frames; D014162:Transfection; D010948:Viral Plaque Assay; D014779:Virus Replication
chemical_list: D004274:DNA, Recombinant
keywords: BAC; ECE; EHV-3; gE