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 characterizedby pox-like lesions on the penis of stallions and the vulva of mares. Although the complete genomicsequence of EHV-3 has been recently made available, its genomic content remains poorly characterizedand the molecular mechanisms of disease development not yet elucidated. In an attempt to facilitategenetic manipulation of EHV-3, we describe here the construction of a full-length infectious bacterialartificial chromosome (BAC) clone of EHV-3. Mini-F vector sequences were inserted into the intergenicregion between ORF19 and ORF20 (UL41 and UL40, respectively) of EHV-3 strain C175 by homologousrecombination in equine dermal cells (NBL-6). DNA of the resulting recombinant virus was electroporatedinto E. coli and a full-length EHV-3 BAC clone was recovered. Virus reconstituted after transfection of theEHV-3 BAC into NBL-6 cells showed growth properties in vitro that were indistinguishable from thoseof the parental virus. To assess the feasibility of mutagenesis of the cloned EHV-3 genome, recombinantviruses targeting the glycoprotein E (gE) gene were generated using Red recombination in E. coli andin 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 dispensablefor 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 andtransmembrane domains are significantly compromised with regard cell-to-cell spread. The cloning ofEHV-3 as a BAC simplifies future studies to identify the role of its coding genes in viral pathogenesis andhost immune responses.