AN EQUINE HERPESVIRUS-1 MUTANT WITH A LACZ INSERTION BETWEEN OPEN READING FRAMES 62 AND 63 IS REPLICATION COMPETENT AND CAUSES DISEASE IN THE MURINE RESPIRATORY MODEL

An equine herpesvirus 1 (EHV-1) mutant was constructed by inserting a lacZ expression cassette into the intergenic region upstream of gene 6 2 (glycoprotein L; gL) and downstream of gene 63 (a homologue of the h erpes simplex virus transcriptional activator ICP0). The recombinant l acZ62/63-EHV-1 had similar growth kinetics in cell culture to those of the parental wild type (wt) virus, with indistinguishable cytopathic effects and plaque morphology. Reverse transcriptase PCR confirmed tha t the lacZ insertion did not interfere with transcription of gL and im munoblot analysis indicated there was no modification to late gene exp ression as monitored by synthesis of EHV-1 glycoproteins C and D. The parental EHV-1 isolate HVS25A used here had almost identical nucleotid e sequence to that published for isolate Ab4, in a 1200 bp region surr ounding the insert, but lacked a HindIII site corresponding to Ab4 pos ition 109,048. The lacZ62/63-EHV-1 caused respiratory disease in BALB/ c mice with clinical signs, histopathology and virus titres in lungs t hroughout days 1-5 post infection similar to those induced by wt EHV-1 . X-gal staining for beta-galactosidase expression in murine lungs cle arly demonstrated EHV-1 infection in cells of the bronchiolar epitheli um and pulmonary parenchyma, with a peak of infection evident at day 2 post infection, when up to 50% of bronchioles demonstrated blue-stain ing and thus virus-infected epithelial cells. The construction of this replication competent virus carrying a reporter gene identifies a sit e for insertion of foreign genes and will facilitate studies on the pa thogenesis of EHV-1.