Centre for Equine Virology, School of Veterinary Science, University of Melbourne, Parkville, VIC 3010, Australia
Correspondence
Carol A. Hartley
carolah{at}unimelb.edu.au
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ABSTRACT |
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Present address: Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia.
Present address: Pfizer, Animal Health R&D, 45 Poplar Road, Parkville, VIC 3052, Australia.
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INTRODUCTION |
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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
E
F (EF) loop of VP1 and the
A2
Z loop of VP2, although some variation also occurs at the N terminus, and the
C
D (CD) and the
G
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.
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METHODS |
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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 GSTEF 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 : 2501 : 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 391798 (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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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 ml1.
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RESULTS |
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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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DISCUSSION |
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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 GSTEF 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.
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ACKNOWLEDGEMENTS |
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Received 7 April 2004;
accepted 28 May 2004.
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