Identification of a neutralizing epitope in the {beta}E–{beta}F loop of VP1 of equine rhinitis A virus, defined by a neutralization-resistant variant

Rachel A. Stevenson{dagger}, Jin-an Huang{ddagger}, Michael J. Studdert and Carol A. Hartley

Centre for Equine Virology, School of Veterinary Science, University of Melbourne, Parkville, VIC 3010, Australia

Correspondence
Carol A. Hartley
carolah{at}unimelb.edu.au


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 Glu->Lys change at position 658, which is located in the predicted {beta}E–{beta}F (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.

{dagger}Present address: Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia.

{ddagger}Present address: Pfizer, Animal Health R&D, 45 Poplar Road, Parkville, VIC 3052, Australia.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Equine rhinitis A virus (ERAV) is an important respiratory pathogen of horses and is classified with foot-and-mouth disease virus (FMDV), albeit as a separate cluster, in the genus Aphthovirus of the family Picornaviridae. ERAV shares many physico-chemical and biological properties with FMDV, and the genome structures and sequences of the two viruses are also similar (Li et al., 1996; Newman et al., 1973; Plummer, 1963; Studdert & Gleeson, 1978; Wutz et al., 1996).

As a picornavirus, ERAV has an icosahedral capsid made up of 60 copies each of four structural polypeptides termed VP1, VP2, VP3 and VP4 (Rueckert, 2001). The amino acid composition of the capsid proteins of many picornaviruses is typically highly variable as a result of selection pressure mediated by virus-neutralizing antibodies (Baranowski et al., 2001; Schiappacassi et al., 1995). Of the four capsid proteins, VP1 exhibits the most variability, particularly in the loops that project from the virion surface (Rueckert, 1996). However, in marked contrast to FMDV, poliovirus and human rhinoviruses (Stanway, 1990), the predicted amino acid sequence of ERAV P1 (the complete viral capsid protein-coding region), and of VP1 in particular, 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 {beta}E–{beta}F (EF) loop of VP1 and the {beta}A2–{alpha}Z loop of VP2, although some variation also occurs at the N terminus, and the {beta}C–{beta}D (CD) and the {beta}G–{beta}H (GH) loops of VP1 (Varrasso et al., 2001). Amino acid sequence variation may indicate that these regions are subject to antibody-mediated selection pressure.

A common approach to the characterization of neutralization epitopes in picornaviruses is the generation and sequencing of antibody-resistant variants (Usherwood & Nash, 1995). Such studies have shown that escape from neutralization by antibodies can be mediated by a single amino acid substitution (Lea et al., 1995) occurring within antibody-binding sites (Hughes, 1992; Hughes & Hughes, 1995). For ERAV strain 393/76 (ERAV.393/76), B-cell epitopes have been mapped to the loop regions of VP1 and VP3 using linear glutathione S-transferase (GST) fusion peptides (Stevenson et al., 2003). ERAV VP1 contains epitopes against which neutralizing antibody is directed (Warner et al., 2001), and a single highly conformational neutralization epitope of ERAV has recently been identified using neutralization-resistant monoclonal antibody (mAb)-selected variants. This epitope is thought to be formed when the C terminus of a VP1 molecule on one protomer approaches the EF loop of VP1 on the neighbouring protomer (Kriegshäuser et al., 2003). Given that ERAV VP1 has a somewhat different antigenicity against rabbit and horse ERAV.393/76 antisera (Stevenson et al., 2003), we attempted to define further epitopes involved in the neutralization of ERAV.393/76 in the natural host. In this study, escape mutants of ERAV.393/76 with increased resistance to neutralization by equine polyclonal antiserum (EPA) were selected and these viruses were characterized and compared with their neutralization-sensitive parent viruses.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus and cells.
Vero cells were grown in minimal essential medium (MEM; Gibco) as described previously (Warner et al., 2001). The ERAV isolates used in this study have been described previously (Li et al., 1996; Studdert & Gleeson, 1978; Wutz et al., 1996). Infected cells were maintained at 37 °C in MEM or Dulbecco's MEM (DMEM) as above except that the concentration of fetal bovine serum was reduced to 0·5 %. ERAV.393/76 virions were purified by a procedure described previously (Hartley et al., 2001). ERAV.PERV/62 virions were purified using PEG precipitation and sucrose cushions (Rueckert & Pallansch, 1981).

Sera and antisera.
Experimental infection of horses with ERAV.393/76 has been described previously (Hartley et al., 2001). Post-infection antisera from horses C, G and S were used in this study. Sera from horse SM was obtained from a horse naturally infected with ERAV (Li et al., 1997). Rabbit hyperimmune antisera were prepared against whole, UV-inactivated ERAV.393/76 (Hartley et al., 2001). Rabbit hyperimmune antisera to whole, UV-inactivated ERAV.PERV/62 or the cloned EF loop region of VP1 of ERAV.393/76 were prepared by subcutaneous immunization of New Zealand White rabbits with 10 µg UV-inactivated ERAV.PERV/62 or 75 µg GST–EF fusion protein emulsified in complete Freund's adjuvant (Sigma). The rabbits received two further injections at 4 week intervals of UV-inactivated ERAV.PERV/62 or GST fusion protein in Freund's incomplete adjuvant (Sigma) and were bled 4 weeks after the final injection. Serum neutralization assays of test sera were carried out as described previously (Warner et al., 2001).

Depletion of GST-reactive antibodies from sera.
Sera were depleted of GST-reactive antibodies using a modification of the method of Crabb et al. (1992). Briefly, 25 µg GST diluted in double-strength reducing buffer was separated by SDS-PAGE and transferred to PVDF membranes. GST membrane strips were blocked to prevent non-specific binding by incubating for 1 h in PBS containing 5 % skimmed milk. Horse sera diluted 1 : 10 in 3 ml PBS containing 0·05 % Tween 20 (PBST) plus skimmed milk were added to each membrane and incubated for 3 h with constant rocking.

Isolation of neutralization-escape mutants.
To isolate neutralization-escape mutant viruses, 6x105 TCID50 of ERAV.393/76 was passaged in the presence of dilutions of EPA (1 : 250–1 : 4000 in DMEM) from horse S (Hartley et al., 2001) and incubated at 37 °C for 30 min. Virus/EPA mixtures were inoculated on to monolayer cultures of Vero cells in six-well plates and incubated at 37 °C. After 1 h, the inoculum was removed and the monolayers were washed once with DMEM. The DMEM was replaced and the cells were incubated for a further 72 h. Virus-infected supernatant was then harvested from the well containing the highest concentration of EPA in which CPE was visible on day 3 post-infection. Virus supernatant passaged in the presence of EPA and chosen for further passage was designated EPA+. In parallel experiments, ERAV.393/76 was also passaged in the absence of EPA (EPA). The selective cycle was performed 25 times in total.

Sequence analysis of the P1 region of virus isolates.
Viral RNA was extracted from virus-infected cell-culture supernatants using a QIAamp viral RNA mini kit (Qiagen), according to the manufacturer's instructions. Virus cDNA was synthesized using an ERAV-specific primer (5'-TTGCTCTCAACATCTCCAGC-3') and 100 U Superscript II RNase H reverse transcriptase (RT; Gibco BRL), according to the manufacturer's instructions. The resulting cDNA was used as a template in PCR to amplify the region between the leader peptide (L) and 2A, using two sets of overlapping ERAV-specific primers (Table 1) corresponding to nt –187 to 408 (reaction 1) and nt 391–798 (reaction 2) of P1 (Wutz et al., 1996). The amplified DNA was purified by gel extraction and sequenced at least three times using a Big-Dye Terminator sequencing kit (ABI Prism) according to the manufacturer's instructions, using overlapping ERAV-specific P1 primers (Table 1) and Perkin Elmer Sequenator model 377 machines.


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Table 1. Primers used to sequence the P1 region of ERAV isolates

Nucleotide numbering is from the beginning of P1; negative numbers indicate pre-P1 sequence.

 
Cloning and expression of ERAV VP1 EF region fusion proteins.
The EF loop region of VP1 was amplified from the extracted RNA of ERAV.393/76, ERAV.PERV/62 and the 393/76 neutralization-escape mutant by RT-PCR using synthetic oligonucleotide primers. The positive-sense primer 5'-CGGGATCCGTTGGAGCACCAACCAAGAC-3' contained a BamHI cleavage site followed by nt 645–664 of ERAV P1 (Li et al., 1996; Wutz et al., 1996); the negative-sense primer for ERAV.393/76 and the 393/76 neutralization-escape mutant, 5'-ATAAGAATGCGGCCGCAGTGGGGGACATCCCTGCAA-3', as well as that for ERAV.PERV/62, 5'-ATAAGAATGCGGCCGCAGTGGGGGAAAGCCCTGCAA-3', contained a NotI cleavage site followed by nt 682–662 of ERAV P1 (Li et al., 1996; Wutz et al., 1996). The amplified product was cloned into pGEM-T (Promega) and subcloned into the BamHI and NotI restriction sites of expression vector pGEX-4T3 (Amersham-Pharmacia). Ligated products were transformed into Escherichia coli DH5{alpha} (Stratagene) and plasmid DNA from a positive clone was electroporated into E. coli JM109 (Invitrogen) for protein expression. Large-scale fusion-protein preparations were prepared as described previously (Warner et al., 2001).

Immunoblotting.
Immunoblotting of purified fusion proteins was carried out essentially as described by Warner et al. (2001), except that the membranes were incubated for 1 h at room temperature with either rabbit ERAV antiserum diluted 1 : 3000 or convalescent-phase horse sera that had been depleted of GST-reactive antibodies and diluted 1 : 200 in PBST/2·5 % skimmed milk.

ELISA.
ELISA of purified ERAV virions or bacterially expressed fusion proteins was carried out essentially as described previously (Stevenson et al., 2003), except that the bacterially expressed fusion proteins were absorbed on to the wells of Maxisorp (Nunc) ELISA plates at a concentration of 0·25 µg ml–1.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isolation of an ERAV.393/76 mutant virus
As the number of passages of ERAV.393/76 in the presence of EPA from horse S was increased, the virus caused CPE in progressively higher concentrations of EPA, from a 1 : 4000 dilution for passages 5–8 up to a 1 : 500 dilution for passages 22–25. To determine whether neutralization-resistant viruses had been selected, viruses from selected passage numbers were titrated in the presence and absence of EPA. When EPA+ and EPA viruses were compared in this assay, EPA+ viruses with increased resistance to neutralization compared with their EPA counterparts were obtained after 10 passages (Fig. 1). Although passage 20 and 25 EPA viruses also demonstrated a slight increase in resistance to neutralization, this was well below that of the EPA+ viruses. Viruses were passaged a further 15 times in the presence of EPA, but no further increase in resistance to neutralization was detected. Viruses at passage 15 were further characterized both as plaque-purified and as pooled (non-plaque-purified) populations. Individual EPA+ and EPA plaque-purified isolates were designated 1–6.



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Fig. 1. ERAV.393/76 EPA+ and EPA isolates from selected passage numbers titrated in the presence (filled bars) and absence (shaded bars) of 1 : 1000 EPA.

 
The neutralizing-antibody titre of a range of polyclonal ERAV antisera was determined against both the pooled and the plaque-purified isolates (Hartley et al., 2001). Pooled EPA+ virus populations demonstrated increased resistance to all polyclonal ERAV antisera tested when compared with the pooled EPA virus population (Table 2). In particular, horse C and horse SM antisera detected the greatest difference between isolates, with 5·5- and 8-fold increases, respectively. With horse S antiserum, used to select escape mutant viruses, there was a 3-fold increase in resistance to neutralization, the same as for rabbit polyclonal antiserum. Pooled EPA+ isolates showed only minor differences in neutralization titre compared with horse G antiserum. Both the plaque-purified EPA+ isolates and the pooled EPA+ virus population from which they were selected showed the same overall pattern of resistance to neutralization by the various polyclonal ERAV.393/76 antisera.


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Table 2. Serum neutralizing-antibody titres of various polyclonal ERAV antisera against pooled virus and individual purified plaques of EPA+ and EPA isolates

EPA from horse S was used to select variants. The neutralizing titre is the reciprocal of the highest dilution of serum that neutralized 100 TCID50 of each virus.

 
Viruses with increased resistance to neutralization share an amino acid change in the EF loop of VP1
The nucleotide sequence of P1 was determined for EPA+ and EPA isolates, and the sequences were aligned and compared with the published sequence of ERAV.393/76 (Li et al., 1996; Varrasso et al., 2001; Wutz et al., 1996). When EPA+ and EPA isolates were compared, it was found that both EPA+ and EPA+/1 had a G->A nucleotide substitution at position 1972 of P1 that was not present in either EPA/5 or EPA. This change resulted in an amino acid substitution at Glu-658->Lys in the predicted EF loop of VP1. An additional nucleotide substitution, from C to A at position 457 of P1, was also found in plaque-purified EPA+/1 but not in the pooled EPA+ virus population or the EPA viruses. This resulted in an amino acid substitution at Gln-152 of P1 to Lys in the BC loop of VP2. Passage 25 EPA+ and EPA were also sequenced and compared with passage 15 viruses. These sequences were identical to those of the equivalent passage 15 non-plaque-purified isolates, with EPA+ at passage 25 containing the single nucleotide and amino acid substitution within P1.

The amino acid substitution in VP1 is also present in naturally occurring ERAV isolates
The EPA+ and EPA isolates were compared in serum neutralization assays, using ERAV polyclonal sera raised in rabbits or in experimentally and naturally infected horses (Hartley et al., 2001), with 10 naturally occurring ERAV isolates whose P1 sequences had been determined previously (Li et al., 1996; Varrasso et al., 2001; Wutz et al., 1996). ERAV isolates 544/82, V1722/70 and PERV/62 also had increased resistance to neutralization by each of these polyclonal sera (Table 3). Other isolates, such as P346/75, showed increased resistance to some, but not all, of the polyclonal sera tested. Interestingly, like EPA+ isolates, PERV/62, 544/82 and V1722/70 also possess a Lys at amino acid position 658 of P1 (Fig. 2). Like ERAV.393/76 and EPA, all of the naturally occurring isolates possessed a Gln at position 152 in VP2 and not a Lys as seen in the plaque-purified EPA+/1 isolate.


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Table 3. Serum neutralizing-antibody titres of various polyclonal sera against ERAV isolates

EPA from horse S was used to select variants. The neutralizing titre is the reciprocal of the highest dilution of serum that neutralized 100 TCID50 of each virus. ND, Not determined.

 



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Fig. 2. Amino acid alignment of the P1 region of naturally occurring ERAV isolates compared with EPA+/1. Conservation of an amino acid among the isolates is indicated by an asterisk. Arrows ({downarrow}) indicate the location of the first amino acid for each of the four structural proteins. Bold lines indicate regions of proposed secondary structure ({alpha}-helices and {beta}-barrels) as determined by homology with picornaviruses of known three-dimensional structure (Wutz et al., 1996). The shaded box indicates the amino acid change in the EF loop region of VP1, which is seen in EPA+/1 compared with the naturally occurring isolates. Amino acid numbering is from the first amino acid of P1.

 
The E->K change in the EF loop of VP1 eliminates antibody binding to this region
To investigate further the binding of rabbit ERAV.393/76 antiserum, purified ERAV.393/76, ERAV.PERV/62 and EPA+ virions were immunoblotted with rabbit antisera prepared against ERAV.393/76 (Fig. 3). Rabbit ERAV.393/76 antiserum bound to the capsid proteins of all three isolates. To compare the difference in the EF loop of the VP1 protein of these isolates, the viruses were also probed with rabbit antiserum prepared against the EF loop of ERAV.393/76 linked to GST (Stevenson et al., 2003). While this antiserum bound to the VP1 of ERAV.393/76 in Western blots, it did not bind to any proteins of ERAV.PERV/62 or EPA+/1, which suggests that the E->K change in the EF loop of VP1 is sufficient to reduce or eliminate the binding of these EF loop-specific antibodies significantly.



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Fig. 3. The E->K change in the EF loop of VP1 reduces binding of EF loop-specific antibodies. Purified ERAV virions 393/76, PERV/62 and EPA+ were separated by 12·5 % SDS-PAGE under reducing conditions, transferred to PVDF membranes and probed with rabbit ERAV.393/76 antiserum (R{alpha}), rabbit PERV/62 antiserum or rabbit GST–EF antiserum diluted 1 : 1000, 1 : 1000 and 1 : 500, respectively. Bound antibody was detected with HRP-conjugated swine anti-rabbit IgG (1 : 1000; Dako) and developed as described previously (Warner et al., 2001).

 
The VP1 EF loop regions of both ERAV.PERV/62 and EPA+/1 were expressed in E. coli as fusion proteins ligated to the C terminus of GST. To investigate the antigenicity in the natural host of the epitopes contained within the fusion proteins, ERAV antisera from horses (S, G and C) experimentally infected with ERAV.393/76 and antiserum from a naturally infected horse (SM) (Hartley et al., 2001) were used to probe the GST–EF fusion proteins. Antisera from all horses recognized all three GST–EF fusion proteins, but the reactivity of each serum was clearly strongest against the GST–EF fusion protein prepared from ERAV.393/76 and weakest against the GST–EF fusion protein prepared from EPA+/1, despite equivalent loading of each protein (Fig. 4). These results suggested that the Glu-658->Lys change in the EF loop of VP1 results in a reduction in the binding of antibodies to this region.



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Fig. 4. Immunoblot of E. coli-expressed GST fusion proteins of the VP1 EF loop regions of ERAV isolates 393/76, PERV/62 (PERV) and EPA+/1 (EPA+). A 0·5 µg sample of each purified GST–EF fusion protein was separated by 12·5 % SDS-PAGE under reducing conditions, transferred to PVDF membranes and probed with horse pre-immune serum and sera from horses experimentally infected with ERAV.393/76 (horses S, G and C), as well as serum from a horse naturally infected with ERAV (horse SM), at dilutions of 1 : 200, and developed as described previously (Warner et al., 2001). Bound antibody was detected with HRP-conjugated goat anti-horse IgG. All horse antisera were depleted of GST-reactive antibodies prior to use.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, an escape mutant virus of ERAV.393/76 that had increased resistance to neutralization by polyclonal ERAV antisera was produced. The most consistent mutation seen in ERAV escape mutants was a change from Glu-658 to Lys in the EF loop region of VP1. Both this change and the Gln-152->Lys change in the BC loop of VP2 (observed in the plaque-purified escape mutant of ERAV.393/76) may play a role in altering resistance to neutralization by polyclonal antisera, but the consistent identification of the Glu-658->Lys change in all escape-mutant populations strongly suggests that this was responsible for the increased resistance to neutralization. An amino acid change from a small, negatively charged Glu to a larger, positively charged Lys residue represents a radical modification, and this change in the EF loop was correlated with an alteration to both the antigenicity and the immunogenicity of this region as a linear epitope. Furthermore, naturally occurring ERAV isolates that also possess a Lys at residue 658 in the predicted EF loop of VP1 also demonstrated increased resistance to neutralization by ERAV.393/76-specific antisera from both horses and rabbits. Taken together, these results suggest that the EF loop of ERAV.393/76 VP1 contributes to a neutralization epitope of this virus.

The results of the present study strongly support the findings of Kriegshäuser et al. (2003), who developed a panel of neutralizing mAbs to select mAb-escape mutants of ERAV.PERV/62. The predominant amino acid changes seen in these ERAV.PERV/62 variants were in VP1 at Lys-114 (position 650 of P1, within the EF loop) and Pro-240 and Thr-241 (positions 776 and 777 of P1, within the C terminus of VP1). Although these mutations mapped to distant regions of the VP1 linear sequence, each of the mutants was cross-resistant to each of the four mAbs used, suggesting that these amino acids contributed to a single dominant antigenic site in mice. Kriegshäuser et al. (2003) developed a three-dimensional model of ERAV protomers and pentamers based on the known structures of FMDV and mengovirus. This model suggests that the three amino acids form a conformational epitope in the ERAV pentamer, where the C terminus of VP1 of one protomer extends towards the EF loop of VP1 in the neighbouring protomer.

Since the EF loop expressed as a GST fusion protein does not induce production of neutralizing antibodies when inoculated into rabbits (Stevenson et al., 2003), it seems that the conformation of the epitope containing residue 658 is crucial. Amino acids that are distant in a linear sequence can come together to form a discontinuous epitope or, alternatively, amino acids may contribute to the conformation of a distant epitope via an allosteric mechanism. These results show that the EF loop of VP1 contributes to a neutralization epitope of ERAV in the natural host, as well as in mice (Kriegshäuser et al., 2003). Since the amino acid involved in increasing resistance to EPA is eight residues downstream of that identified by Kriegshäuser et al. (2003), these residues might be considered to form part of a neutralization site, rather than a single epitope. Other than for hepatitis A virus (HAV) (Ping & Lemon, 1992), the EF loop of VP1 of picornaviruses has not commonly been reported to contain antigenic sites. However, unlike HAV, ERAV contains a very long EF loop in VP1, which is more consistent with a role for this loop in the antigenic structure of ERAV. However, like HAV, the neutralizing epitopes of ERAV appear to be highly conformational (Kriegshäuser et al., 2003). This is supported by results from previous studies (Stevenson et al., 2003) where linear peptides of regions of the P1 capsid, while found to be immunogenic, did not elicit neutralizing antibodies. In FMDV, the EF loop does not contain or contribute to a known neutralization site; however, its major antigenic site lies in the relatively long GH loop, which is exposed on the surface of FMDV virions (Acharya et al., 1989; Berinstein et al., 1995; Jackson et al., 2000; Neff et al., 1998). Compared with FMDV, the GH loop of ERAV is much shorter and does not contain an RGD integrin-binding motif. In contrast, the EF loop of ERAV is much longer than that in FMDV and thus parts of this loop are more likely to be exposed on the surface of the virion.

The inability of the neutralization-resistant variants of ERAV to escape neutralization completely clearly suggests that another neutralization epitope(s) exists. Polyclonal sera contain multiple subclasses and high-affinity antibodies that are likely to recognize both linear and conformational epitopes. It is likely that multiple neutralizing antibodies with a range of specificities are present in EPA, and the mutation in the EPA+ viruses resulted in escape from neutralization by a subset of these. Although it was demonstrated that antisera raised against ERAV.393/76 showed a reduced ability to neutralize viruses containing Lys-658 in the EF loop, it was not known whether the reciprocal was also true. It was initially hypothesized that antisera raised to ERAV.PERV/62 or EPA+/1 might show a reduced ability to neutralize viruses containing Glu at this position. In contrast, however, rabbit ERAV.PERV/62 antiserum neutralized the escape mutants and each virus isolate to equivalent titres, regardless of the EF loop sequence. In Western blots, rabbit ERAV.PERV/62 antiserum did not bind to GST–EF fusion proteins of the EF loops of ERAV.PERV/62, ERAV.393/76 or its escape mutant. This suggests that in ERAV.PERV/62, the EF loop is not immunogenic in rabbits. Although it is not certain whether this is the case in the natural host, this further supports the idea that other ERAV neutralization epitopes exist. Consistent with results obtained with rabbit ERAV.393/76 antiserum, the rabbit ERAV.PERV/62 antiserum was shown in immunoblots to bind to GST fusion proteins of the N- and C-terminal regions of VP1. Neutralization epitopes may be contained within these regions of VP1 or, more likely, may be composed of several regions that combine to form a conformational antigenic site, as antisera prepared against the N- or C-terminal fusion proteins did not neutralize ERAV.393/76.


   ACKNOWLEDGEMENTS
 
We thank Nino Ficorilli and Cynthia Brown for their excellent technical assistance. This work was supported in part by Racing Victoria and a Special Virology Fund. R. A. S. is the recipient of a University of Melbourne Research Scholarship.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Acharya, R., Fry, E., Stuart, D., Fox, G., Rowlands, D. & Brown, F. (1989). The three-dimensional structure of foot-and-mouth disease virus at 2·9 Å resolution. Nature 337, 709–716.[CrossRef][Medline]

Baranowski, E., Ruiz-Jarabu, C. M., Lim, F. & Domingo, E. (2001). Foot-and-mouth disease virus lacking the VP1 G–H loop: the mutant spectrum uncovers interactions among antigenic sites for fitness gain. Virology 288, 192–202.[CrossRef][Medline]

Berinstein, A., Roivainen, M., Hovi, T., Mason, P. W. & Baxt, B. (1995). Antibodies to the vitronectin receptor (integrin {alpha}V{beta}3) inhibit binding and infection of foot-and-mouth disease virus to cultured cells. J Virol 69, 2664–2666.[Abstract]

Crabb, B. S., Nagesha, H. S. & Studdert, M. J. (1992). Identification of equine herpesvirus 4 glycoprotein G: a type-specific, secreted glycoprotein. Virology 190, 143–154.[Medline]

Hartley, C. A., Ficorilli, N., Dynon, K., Drummer, H. E., Huang, J. & Studdert, M. J. (2001). Equine rhinitis A virus: structural proteins and immune response. J Gen Virol 82, 1725–1728.[Abstract/Free Full Text]

Hughes, A. L. (1992). Positive selection and intrallelic recombination at the merozoite surface antigen-1 (MSA-1) locus of Plasmodium falciparum. Mol Biol Evol 9, 381–393.[Abstract]

Hughes, M. K. & Hughes, A. L. (1995). Natural selection on Plasmodium surface proteins. Mol Biochem Parasitol 71, 99–113.[CrossRef][Medline]

Jackson, T. J., Sheppard, D., Denyer, M., Blakemore, W. & King, A. M. Q. (2000). The epithelial integrin {alpha}v{beta}6 is a receptor for foot-and-mouth disease virus. J Virol 74, 4949–4956.[Abstract/Free Full Text]

Kriegshäuser, G., Wutz, G., Lea, S., Stuart, D., Skern, T. & Kuechler, E. (2003). Model of the equine rhinitis A virus capsid: identification of a major neutralizing immunogenic site. J Gen Virol 84, 2365–2373.[Abstract/Free Full Text]

Lea, S., Abu-Ghazaleh, R., Blakemore, W. & 7 other authors (1995). Structural comparison of two strains of foot-and-mouth disease virus subtype O1 and a laboratory antigenic variant G67. Structure 3, 571–580.[Medline]

Li, F., Browning, G. F., Studdert, M. J. & Crabb, B. S. (1996). Equine rhinovirus 1 is more closely related to foot-and-mouth disease virus than to other picornaviruses. Proc Natl Acad Sci U S A 93, 990–995.[Abstract/Free Full Text]

Li, F., Drummer, H. E., Ficorilli, N., Studdert, M. J. & Crabb, B. S. (1997). Identification of noncytopathic equine rhinovirus 1 as a cause of acute febrile respiratory disease in horses. J Clin Microbiol 35, 937–943.[Abstract]

Neff, S., Sá-Carvalho, D., Rieder, E., Mason, P., Blystone, S. D., Brown, E. J. & Baxt, B. (1998). Foot-and-mouth disease virus virulent for cattle utilizes the integrin {alpha}v{beta}3 as its receptor. J Virol 72, 3587–3594.[Abstract/Free Full Text]

Newman, J. F. E., Rowlands, D. J. & Brown, F. (1973). A physiochemical sub-grouping of the mammalian picornaviruses. J Gen Virol 18, 171–180.[Medline]

Ping, L. H. & Lemon, S. M. (1992). Antigenic structure of human hepatitis A virus defined by analysis of escape mutants selected against murine monoclonal antibodies. J Virol 66, 2208–2216.[Abstract]

Plummer, G. (1963). An equine respiratory enterovirus: some biological and physical properties. Arch Gesamte Virusforsch 12, 694–700.[Medline]

Rueckert, R. R. (1996). Picornaviridae: the viruses and their replication. In Fields Virology, 3rd edn, pp. 609–654. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott–Raven.

Rueckert, R. R. (2001). Picornaviridae: the viruses and their replication. In Fields Virology, 4th edn, pp. 685–722. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.

Rueckert, R. & Pallansch, M. A. (1981). Preparation and characterization of encephalomyocarditis (EMC) virus. Methods Enzymol 78, 315–327.[Medline]

Schiappacassi, M., Rojas, E., Carrillo, E. & Campos, R. (1995). Response of foot-and-mouth disease virus C3 Resende to immunological pressure exerted in vitro by antiviral polyclonal sera. Virus Res 36, 77–85.[CrossRef][Medline]

Stanway, G. (1990). Structure, function and evolution of picornaviruses. J Gen Virol 71, 2483–2510.[Medline]

Stevenson, R. A., Hartley, C. A., Huang, J., Studdert, M. J., Crabb, B. S. & Warner, S. (2003). Mapping epitopes in equine rhinitis A virus VP1 recognized by antibodies elicited in response to infection of the natural host. J Gen Virol 84, 1607–1612.[Abstract/Free Full Text]

Studdert, M. J. & Gleeson, L. J. (1978). Isolation and characterization of an equine rhinovirus. Zentralbl Veterinarmed B 25, 225–237.[Medline]

Usherwood, E. J. & Nash, A. A. (1995). Lymphocyte recognition of picornaviruses. J Gen Virol 76, 499–508.[Medline]

Varrasso, A., Drummer, H. E., Huang, J., Stevenson, R. A., Ficorilli, N., Studdert, M. J. & Hartley, C. A. (2001). Sequence conservation and antigenic variation of the structural proteins of equine rhinitis A virus. J Virol 75, 10550–10556.[Abstract/Free Full Text]

Warner, S., Hartley, C. A., Stevenson, R. A., Ficorilli, N., Varrasso, A., Studdert, M. J. & Crabb, B. S. (2001). Evidence that equine rhinitis A virus is a target of neutralizing antibodies and participates directly in receptor binding. J Virol 75, 9274–9281.[Abstract/Free Full Text]

Wutz, G., Auer, H., Nowotny, N., Grosse, B., Skern, T. & Kuechler, E. (1996). Equine rhinovirus serotypes 1 and 2: relationship to each other and to aphthoviruses and cardioviruses. J Gen Virol 77, 1719–1730.[Abstract]

Received 7 April 2004; accepted 28 May 2004.



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