Transcript analysis of the equine herpesvirus 1 glycoprotein B gene homologue and its expression by a recombinant...

Transcript mapping of the equine herpesvirus 1 (EHV-1) glycoprotein B (gB) gene homologue by Northern blot, S1 nuclease and primer extension analyses indicated that two overlapping transcripts of 3.4 and 4.6 kb originated from the same strand and were transcribed from left to right between coordinates 0.40 and 0.43 of the EHV-1 genome. The 3.4 kb transcript encoded EHV-1 gB and the 5' RNA terminus was located approximately 30 bases downstream from a probable TATA element. The coding region of the gB gene homologue was reconstructed from two subclones using oligonucleotide mutagenesis and inserted into vaccinia virus by homologous recombination. Cells infected with the recombinant virus synthesized EHV-1 gB antigen, which was detectable in the cytoplasm and on the cell surface by immunofluorescence using an EHV-1 neutralizing horse serum and EHV-1 monoclonal antibodies. On Western blots, bands of 138K to 143K, 80K to 90K and 55K to 57K were identified in recombinant virus-infected cells, by both EHV-1 monoclonal antibodies and the polyclonal horse serum. These were similar in Mr to bands identified by these sera in EHV-1-infected cells. Mice vaccinated with the recombinant virus produced antibodies which recognized proteins of the same Mr as EHV-1 gB, on Western blots, but did not have in vitro neutralizing activity.