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 62 (glycoprotein L; gL) and downstream of gene 63 (a homologue of the herpes simplex virus transcriptional activator ICP0). The recombinant lacZ62/63-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 lacZ 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 HindIII site corresponding to Ab4 position 109,048. The lacZ62/63-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 beta-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.
Csellner
H
H
School of Biological Sciences, Macquarie University, Sydney, Australia.
Walker
C
C
Love
D N
DN
Whalley
J M
JM
D000818:Animals; D019677:Artificial Gene Fusion; D002460:Cell Line; D006224:Cricetinae; D004274:DNA, Recombinant / Q000032:analysis; D020405:Dermis / Q000166:cytology; D004195:Disease Models, Animal; D005260:Female; D006566:Herpesviridae Infections / Q000235:genetics* / Q000662:veterinary; D004861:Herpesvirus 1, Equid / Q000235:genetics* / Q000472:pathogenicity; D006736:Horses; D007668:Kidney / Q000166:cytology; D007763:Lac Operon / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames / Q000235:genetics*; D011817:Rabbits; D012141:Respiratory Tract Infections / Q000662:veterinary / Q000821:virology*; D014779:Virus Replication / Q000235:genetics*
D004274:DNA, Recombinant