Evidence that Equine Rhinitis A Virus VP1 Is a Target of Neutralizing Antibodies and Participates Directly in Receptor Binding

Abstract

Equine rhinitis A virus (ERAV) is a respiratory pathogen of horses and is classified as anAphthovirus, the only non-Foot-and-mouth disease virus (FMDV) member of this genus. In FMDV, virion protein 1 (VP1) is a major target of protective antibodies and is responsible for viral attachment to permissive cells via an RGD motif located in a distal surface loop. Although both viruses share considerable sequence identity, ERAV VP1 does not contain an RGD motif. To investigate antibody and receptor-binding properties of ERAV VP1, we have expressed full-length ERAV VP1 in Escherichia coli as a glutathione S-transferase (GST) fusion protein (GST-VP1). GST-VP1 reacted specifically with antibodies present in serum from a rabbit immunized with purified ERAV virions and also in convalescent-phase sera from horses experimentally infected with ERAV. An antiserum raised in rabbits to GST-VP1 reacted strongly with viral VP1 and effectively neutralized ERAV infection in vitro. Using a flow cytometry-based binding assay, we found that GST-VP1, but not other GST fusion proteins, bound to cell surface receptors. This binding was reduced in a dose-dependent manner by the addition of purified ERAV virions, demonstrating the specificity of this interaction. A separate cell-binding assay also implicated GST-VP1 in receptor binding. Importantly, anti-GST-VP1 antibodies inhibited the binding of ERAV virions to Vero cells, suggesting that these antibodies exert their neutralizing effect by blocking viral attachment. Thus ERAV VP1, like its counterpart in FMDV, appears to be both a target of protective antibodies and involved directly in receptor binding. This study reveals the potential of recombinant VP1 molecules to serve as vaccines and diagnostic reagents for the control of ERAV infections.Equine rhinitis A virus (ERAV), formerly known as equine rhinovirus 1, is a member of the Aphthovirus genus in the family Picornaviridae (26). This genus is otherwise comprised of the different serotypes of Foot-and-mouth disease virus (FMDV). In addition to considerable sequence identity (17, 34), ERAV and FMDV share a range of physicochemical and biological properties (14,23,24). ERAV infection of horses results in an acute febrile respiratory disease that is accompanied by viremia and persistent virus shedding in urine and feces (for a review, see reference 30). It has been shown to be responsible for relatively large outbreaks of acute respiratory illness in adult horse populations, although much remains to be learned about the epidemiology and pathogenesis of this pathogen (18). Such studies are complicated by the likelihood that many isolates are not cytopathic for in vitro-cultured cells (18). Despite being primarily an infectious agent of horses, ERAV is also pathogenic for a broad range of other animal species, including humans (24, 25). There is currently no vaccine to control ERAV infection, and only limited diagnostic tools are available.The genome of all picornaviruses is single-stranded, positive-sense RNA containing a single, long open...

Mapping epitopes in equine rhinitis A virus VP1 recognized by antibodies elicited in response to infection of the natural host

“…Amino acid variations that do occur among naturally occurring strains of ERAV locate mostly to the proposed bE-bF loop of VP1 and the bA2-aZ loop of VP2, although some variation also occurs at the N terminus and at the bC-bD and bG-bH loops of VP1 (Varrasso et al, 2001). Amino acid sequence variation in these regions suggests that the regions may contain epitopes that elicit neutralizing antibodies.…”

“…For example, the surface loops of VP1, VP2 and VP3 of poliovirus type 1 (PV-1) and human rhinovirus type 14 capsid structures protrude from the virion surface to form the receptor-binding canyon, as well as the major antigenic sites of these viruses. In comparison, the VP1 protein within the relatively smooth surface of FMDV particles contains a linear peptide, the bG-bH loop, which is both the dominant neutralization epitope and is also involved in receptor binding to various integrins via an RGD motif (Berinstein et al, 1995;Danen et al, 1995;Jackson et al, 2000;Mateu et al, 1995;Stanway, 1990;Strohmaier et al, 1982).In marked contrast to FMDV and human rhinoviruses, the predicted amino acid sequence of ERAV P1 and, in particular, VP1 has remained remarkably stable over time (Varrasso et al, 2001). Amino acid variations that do occur among naturally occurring strains of ERAV locate mostly to the proposed bE-bF loop of VP1 and the bA2-aZ loop of VP2, although some variation also occurs at the N terminus and at the bC-bD and bG-bH loops of VP1 (Varrasso et al, 2001).…”

“…Amino acid sequence variation in these regions suggests that the regions may contain epitopes that elicit neutralizing antibodies. We have shown that the ERAV capsid protein VP1 contains B cell epitopes that elicit neutralizing antibodies and has receptor-binding activity (Warner et al, 2001). It was anticipated, therefore, that the major antigenic sites for ERAV would be located within the surface structures of VP1.…”

“…In marked contrast to FMDV and human rhinoviruses, the predicted amino acid sequence of ERAV P1 and, in particular, VP1 has remained remarkably stable over time (Varrasso et al, 2001). Amino acid variations that do occur among naturally occurring strains of ERAV locate mostly to the proposed bE-bF loop of VP1 and the bA2-aZ loop of VP2, although some variation also occurs at the N terminus and at the bC-bD and bG-bH loops of VP1 (Varrasso et al, 2001).…”

“…3a), confirming the presence of authentic VP1 epitopes within the fusion proteins. As described by Warner et al (2001), rabbits immunized with the full-length recombinant VP1 (GST-VP1) produced neutralizing antibodies at a level comparable to rabbit ERAV antisera. Neutralizing antibodies were not detected in sera from rabbits immunized with any of the fusion proteins GST-NT, GST-EF, GST-GH or GST-CT (SN titres <10).…”