Aggregation-associated loss of antigenicity observed for denatured virion protein 1 of Equine rhinitis A virus in an enzyme-linked immunosorbent assay (original) (raw)
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Several Recombinant Capsid Proteins of Equine Rhinitis A Virus Show Potential as Diagnostic Antigens
Clinical and Vaccine Immunology, 2005
. Despite these facts, knowledge of the prevalence and importance of ERAV infections remains limited, largely due to the absence of a simple, robust diagnostic assay. In this study, we compared the antigenicities of recombinant full-length and fragmented ERAV capsid proteins expressed in Escherichia coli by using sera from experimentally infected and naturally exposed horses. We found that, from the range of antigens tested, recombinant proteins encompassing the C-terminal region of VP1, full-length VP2, and the N-terminal region of VP2 reacted specifically with antibodies present in sera from each of the five experimentally infected horses examined. Antibodies to epitopes on VP2 (both native and recombinant forms) persisted longer postinfection (>105 days) than antibodies specific for epitopes on other fragments. Our data also suggest that B-cell epitopes within the C terminus of VP1 and N terminus of VP2 contribute to a large proportion of the total reactivity of recombinant VP1 and VP2, respectively. Importantly, the reactivity of these VP1 and VP2 recombinant proteins in enzyme-linked immunosorbent assays (ELISAs) correlated well with the results from a range of native antigen-based serological assays using sera from 12 field horses. This study provides promising candidates for development of a diagnostic ERAV ELISA.
Journal of General Virology, 2003
is an important respiratory pathogen of horses and is of additional interest because of its close relationship and common classification with foot-and-mouth disease virus (FMDV). As is the case with FMDV, the VP1 capsid protein of ERAV has been shown to be a target of neutralizing antibodies. In FMDV VP1, such antibodies commonly recognize linear epitopes present in the bG-bH loop region. To map linear B cell epitopes in ERAV VP1, overlapping fragments spanning its length were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. These fusion proteins were tested for reactivity with sera from ERAV-infected horses and with polyclonal sera from ERAV-immunized rabbits and mice. Regions at the N-and C-termini as well as the bE-bF and the bG-bH loop regions contained B cell epitopes that elicited antibodies in the natural host. GST fusion proteins of these regions also elicited antibodies following immunization of rabbits and mice, which, in general, strongly recognized native ERAV VP1 but which were non-neutralizing. It is concluded that the N-terminal region of ERAV VP1, in particular, contains strong B cell epitopes.
Equine rhinitis A virus: structural proteins and immune response
The Journal of general virology, 2001
Equine rhinitis A virus (ERAV) is a picornavirus that has been reclassified as a member of the Aphthovirus genus because of its resemblance to foot-and-mouth disease virus at the level of nucleotide sequence and overall genomic structure. The N-terminal amino acid sequence of three of the four capsid proteins of ERAV was determined and showed that the proteolytic cleavage sites within the precursor P1 polypeptide occur exactly as those predicted for an aphthovirus-like 3C protease, which generates the capsid proteins VP1 and VP3. However, the autocatalytic cleavage site between VP4 and VP2, which is independent of 3C protease cleavage, was different from that predicted previously. ERAV.393/76 antisera from horses and rabbits showed different reactivity to the viral structural proteins in both serum neutralization assays and Western blots. High neutralizing antibody titres appeared to correlate with strong reactivity to VP1 in Western blots.
2000
is an important respiratory pathogen of horses and is of additional interest because of its close relationship and common classification with foot-and-mouth disease virus (FMDV). As is the case with FMDV, the VP1 capsid protein of ERAV has been shown to be a target of neutralizing antibodies. In FMDV VP1, such antibodies commonly recognize linear epitopes present in the bG-bH loop region. To map linear B cell epitopes in ERAV VP1, overlapping fragments spanning its length were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. These fusion proteins were tested for reactivity with sera from ERAV-infected horses and with polyclonal sera from ERAV-immunized rabbits and mice. Regions at the N-and C-termini as well as the bE-bF and the bG-bH loop regions contained B cell epitopes that elicited antibodies in the natural host. GST fusion proteins of these regions also elicited antibodies following immunization of rabbits and mice, which, in general, strongly recognized native ERAV VP1 but which were non-neutralizing. It is concluded that the N-terminal region of ERAV VP1, in particular, contains strong B cell epitopes.
Sequence Conservation and Antigenic Variation of the Structural Proteins of Equine Rhinitis A Virus
Journal of Virology, 2001
The nucleotide and deduced amino acid sequences of the P1 region of the genomes of 10 independent equine rhinitis A virus (ERAV) isolates were determined and found to be very closely related. A panel of seven monoclonal antibodies to the prototype virus ERAV.393/76 that bound to nonneutralization epitopes conserved among all 10 isolates was raised. In serum neutralization assays, rabbit polyclonal sera and sera from naturally and experimentally infected horses reacted in a consistent and discriminating manner with the 10 isolates, which indicated the existence of variation in the neutralization epitopes of these viruses.
Journal of Veterinary Diagnostic Investigation, 2013
The objective of the present study was to validate a previously described competitive enzyme-linked immunosorbent assay (cELISA) to detect antibody to Equine arteritis virus (EAV) based on GP5-specific nonneutralizing monoclonal antibody (mAb) 17B7 9 using the World Organization for Animal Health (OIE)-recommended protocol, which includes the following 5 in-house analyses. 1) The assay was calibrated with the OIE-designated reference serum panel for EAV; 2) repeatability was evaluated within and between assay runs; 3) analytical specificity was evaluated using sera specific to related viruses; 4) analytical sensitivity was evaluated with sera from horses vaccinated with an EAV modified live virus (MLV) vaccine; and 5) the duration of cELISA antibody detection following EAV vaccination was determined. The positive cELISA cutoff of ≥35% inhibition (%I) was confirmed by receiver operating characteristic plot analysis. Analytical sensitivity of the cELISA was comparable to the serum neutralization (SN) assay in that it detected EAV-specific antibody as early as 8 days postvaccination. The duration of EAV-specific antibody detected by cELISA was over 5 years after the last vaccination. This cELISA could detect EAV-specific antibody in serum samples collected from horses infected with various EAV strains. In the field trial performed by American Association of Veterinary Laboratory Diagnosticians-accredited state laboratories and OIE laboratory, the diagnostic specificity of the cELISA was 99.5% and the diagnostic sensitivity was 98.2%. The data using various serum panels also had consistently significant positive correlation between SN titers and cELISA %I results. The results further confirm that the EAV antibody cELISA is a reliable, simple alternative to the SN assay for detecting EAVspecific antibodies in equine sera.
Development of a peptide ELISA for the diagnosis of Equine arteritis virus
Journal of Virological Methods, 2014
A peptide-based indirect ELISA was developed to detect antibodies against Equine 20 arteritis virus (EAV). Two peptides for epitope C of protein GP5 and fragment E of protein 21 M were designed, synthesized, purified and used as antigens either alone or combined. 22
Journal of General Virology, 2004
Equine rhinitis A virus strain 393/76 (ERAV.393/76) was passaged in the presence of post-infection ERAV.393/76 equine polyclonal antiserum (EPA). Viruses with increased resistance to neutralization by EPA were obtained after 15 passages. Compared with the parent virus, five plaque-purified, neutralization-resistant mutant viruses, in addition to the non-plaque-purified viruses that were examined, had a GluRLys change at position 658, which is located in the predicted bE-bF (EF) loop of VP1. Rabbit antiserum was prepared against the isolated EF loop of ERAV.393/76 VP1 expressed as a fusion protein with glutathione S-transferase. This antiserum bound to purified ERAV.393/76 in Western blots, but not to the neutralization-resistant mutant virus or to ERAV.PERV/62, a naturally occurring ERAV strain that has a Lys residue at position 658. These results suggest that the EF loop of VP1 is involved in a neutralization epitope of ERAV.