Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3

ARMANDO DAMIANI

Congreso; 10th International Equine Infectious Diseases Conference; 2016

Equine herpesvirus type 3 (EHV-3) is the causal agent of equinecoital exanthema, a disease characterized by pox-like lesions onthe penis of stallions and the vulva of mares. Although the com-plete genomic sequence of EHV-3 has been recently made avail-able, its genomic content remains poorly characterized and themolecular mechanisms of disease developmentnotyetelucidated.In an attempt to facilitate the genetic manipulation of EHV-3, wedescribe here the construction of a full-length infectious bacterialartificial chromosome (BAC) clone of EHV-3. Mini-F vector se-quences were inserted into the intergenic region between ORF19and ORF20 (the UL41 and UL40 homologs, respectively) of EHV-3strain C175 by homologous recombination in equine dermis cells(Nbl-6). DNA of the resulting recombinant virus was electro-porated into E. coli and a full-length EHV-3 BAC clone was recov-ered. The reconstituted virus obtained after transfection of theEHV-3 BAC into Nbl-6 cells showed growth propertiesin vitrothatwere indistinguishable from those of the parental virus. To assessthe feasibility of mutagenesis of the cloned EHV-3 genome, amutant virus with a deletion of the glycoprotein E gene wasgenerated using the Red recombination system in E. coli and itsinvitrogrowth properties evaluated. The cloning of EHV-3 as a BACwillsimplifyfuturestudiestoidentifytheroleofitscodinggenesinviral pathogenesis and host immune response