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

Summary An equine herpesvirus 1 (EHV-1) mutant was constructed by inserting a lac Z expression cassette into the intergenic region upstream of gene 62 (glycoprotein L; gL) and downstream of gene 63 (a homologue of the herpes simplex virus transcriptional activator ICP0). The recombinant lac Z62/63-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 that the lac Z insertion did not interfere with transcription of gL and immunoblot analysis indicated there was no modification to late gene expression as monitored by synthesis of EHV-1 glycoproteins C and D. The parental EHV-1 isolate HVS25A used here had almost identical nucleotide sequence to that published for isolate Ab4, in a 1200 bp region surrounding the insert, but lacked a Hin dIII site corresponding to Ab4 position 109,048. The lac Z62/63-EHV-1 caused respiratory disease in BALB/c mice with clinical signs, histopathology and virus titres in lungs throughout days 1–5 post infection similar to those induced by wt EHV-1. X-gal staining for β-galactosidase expression in murine lungs clearly demonstrated EHV-1 infection in cells of the bronchiolar epithelium and pulmonary parenchyma, with a peak of infection evident at day 2 post infection, when up to 50% of bronchioles demonstrated blue-staining and thus virus-infected epithelial cells. The construction of this replication competent virus carrying a reporter gene identifies a site for insertion of foreign genes and will facilitate studies on the pathogenesis of EHV-1.