Characterization of the major glycoproteins of equine herpesviruses 4 and 1 and asinine herpesvirus 3 using monoclonal antibodies

Abstract

A panel of 14 monoclonal antibodies (MAbs) was used to characterize the high abundance glycoproteins of equine herpesviruses 4 (EHV-4) and 1 (EHV-1), and asinine herpesvirus 3 (AHV-3). The specificities of the MAbs, which had been determined previously for strains of EHV-4 and -1 from the U.S.A., in general were confirmed by ELISA for Australian strains of these viruses. Of the 14 MAbs seven were EHV-4 and -1 type-common and cross-reacted with AHV-3. Of the five MAbs that were EHV-1 type-specific, four crossreacted with AHV-3, whereas neither of the EHV-4 type-specific MAbs reacted with AHV-3, providing further evidence for a closer evolutionary relationship between EHV-1 and AHV-3 than that between either of these viruses and EHV-4. By Western blot and immunoprecipitation analyses, the identity of the six major glycoproteins, gp2, gpl0, gpl3, gpl4, gpl8 and gp2 t/22a, of an Australian EHV-1 isolate was verified, and it was shown that AHV-3 had cross-reactive glycoproteins of very similar Mr to those of EHV-1; five homologous glycoproteins of EHV-4 were also identified. It was determined that the EHV-4 gpl3 homologue had a much reduced Mr (67K) when the virus was grown in a continuous cell line than when grown in equine foetal kidney cells (95K). It is suggested that altered glycosylation by the cell line is responsible for this change in M~. Those glycoproteins acting as major immunogens in the naturally infected host, at least in their ability to elicit antibody, were identified. It was found that gp2, gpl3, gpl4, gpl8 and a glycoprotein at 120K (EHV-1) or l l6K (EHV-4) were all important immunogens in mares following EHV-l-induced abortion, and in a specific pathogenfree foal experimentally infected with EHV-1 and later cross-challenged with EHV-4. Gp2, gpl4 and gpl8 were the major immunogens in the donkey in response to AHV-3 infection. The type specificity associated with these glycoproteins was also examined and it was found that although most if not all contain typespecific epitopes, gp2 and a glycoprotein at 120K, and to a lesser extent gpl3 and gpl8, were significantly type-specific in the serum from a mare following natural EHV-1 infection and abortion.

Comparative studies of the structural proteins and glycoproteins of equine herpesviruses 2 and 5

“…SDS-PAGE and RIP were performed as described previously (Crabb & Studdert, 1990). ELISA was performed as described by Crabb et al (1991) using EHV-2 and EHV-5 purified virus, diluted to 10 mg/ml, to coat wells.…”

Characterization of glycoprotein B of the gammaherpesvirus equine herpesvirus-2.

“…Preparation of $&S-labelled viral antigens was carried out as described previously (Crabb et al, 1991). To saturate protein G beads with MAb, 10 µl packed 50 % protein G Sepharose beads was incubated with 50 µl of a 1\50 dilution of ascitic fluid in RIP buffer (0n05 M Tris-HCl, pH 8n0, containing 1 mM EDTA, 0n15 M NaCl, 0n25 % w\v BSA and 1 % v\v Triton X-100) for 1 h at 4 mC with shaking.…”

“…ELISAs were performed as described previously (Crabb et al, 1991) except that 96-well plates were coated with EHV-2.86\67 virions at a concentration of 5 µg\ml and added to plates in a total volume of BGCA 100 µl and horseradish peroxidase-conjugated affinity-purified rabbit anti-mouse antibody (Dako) diluted 1 : 1000 was used.…”

Herpesvirus-Associated Neurological Disease in a Donkey

“…2 Asinine herpesvirus 3 is an alphaherpesvirus based on sequence analysis of glycoprotein G and serological cross-reactivity of AsHV-3 with antibodies to EHV-1 and -4 glycoproteins. 3,6,9 Asinine herpesviruses 4, 5, and 6 were designated as novel gammaherpesviruses based on sequences obtained from donkeys with interstitial pneumonia. 7,8 The latter viruses were highly similar over ,165 bp in the DNA polymerase gene and ,380 bp in the terminase gene and appeared to represent closely related gammaherpesviruses.…”