Equine rhinitis A virus: structural proteins and immune response
Carol A. Hartley1,
Nino Ficorilli1,
Kemperly Dynon1,
Heidi E. Drummer2,
Jin-an Huang1 and
Michael J. Studdert1
Centre for Equine Virology, School of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia1
St Vincents Institute of Medical Research, 9 Princes Street, Fitzroy, Victoria 3065, Australia2
Author for correspondence: Carol Hartley. Fax +61 3 8344 7374. e-mail carolah{at}unimelb.edu.au
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Abstract
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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.
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Main text
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Equine rhinitis A virus (ERAV, previously known as equine rhinovirus type 1) infection of horses is characterized by fever (41±0·5 °C) and clinical signs that are generally considered to be confined to the respiratory tract, including nasal discharge, coughing, anorexia, pharyngitis and lymphadenitis (Plummer & Kerry, 1962
). Viraemia and persistent virus shedding from the pharyngeal region, urine and faeces also accompany infection (Plummer, 1963
; Plummer & Kerry, 1962
). ERAV is a newly classified member of the Aphthovirus genus of the Picornaviridae family (Pringle, 1999
) and is the only non-foot-and-mouth disease virus (FMDV) member of this genus. The homology of the nucleotide sequence of the ERAV genome with that of FMDV (Li et al., 1996
; Wutz et al., 1996
) and many physico-chemical properties of ERAV (Burrows, 1970
; Newman et al., 1973
, 1977
; Studdert & Gleeson, 1978
) are consistent with its reclassification. The production of viraemia and persistent virus shedding following ERAV infection are also more consistent with members of the Aphthovirus genus than with members of the Rhinovirus genus, in which ERAV was classified previously. Little is known about the antibody response of infected horses to this virus, with the exception that neutralizing antibody is elicited approximately 12 days after infection (Plummer & Kerry, 1962
) and that the prevalence of ERAV neutralizing antibodies varies according to the age of the horse, with maximum infection rates at about 50% in horses greater than 12 months of age (Studdert & Gleeson, 1978
). The aim of this study was first to identify the structural antigens of ERAV and then to use this information to characterize the antibody response to these proteins in experimentally and naturally ERAV-infected horses.
ERAV.393/76 (accession no. L43052) (Studdert & Gleeson, 1977
, 1978
) infected Vero cell culture lysate was clarified (10000 g, 15 min, 4 °C) and the virus in the supernatant was concentrated (100000 g, 2 h, 4 °C). The virus pellet was then resuspended in TNE buffer (0·01 M TrisHCl, pH 8·0, 0·1 M NaCl and 1 mM EDTA) containing 1% sarcosyl and 1% SDS and pelleted through a 10% sucrose cushion at 100000 g for 2 h at 4 °C. The resuspended virus was then purified through a 1545% (wt/vol) sucrose gradient at 80000 g for 4 h at 4 °C. The gradient was collected in 1 ml fractions. Fractions containing virus (as determined by SDSPAGE) were pooled before pelleting at 100000 g for 2 h at 4 °C and resuspended in TNE buffer. When separated on 1015% SDSPAGE under reducing conditions and stained with Coomassie brilliant blue, lanes containing purified ERAV.393/76 showed strong bands with molecular masses of approximately 26, 25 and 22 kDa and a minor band with a molecular mass of approximately 42 kDa (Fig. 1
). The predicted sizes of ERAV.393/76 P1 proteins, however, are 8, 25, 24 and 27 kDa for VP4, VP2, VP3 and VP1, respectively (Blom et al., 1996
; Li et al., 1996
; Wutz et al., 1996
). Although the molecular masses are within the correct range, the relative migration of these proteins did not correlate with the predicted sizes of each protein. To identify each band and the cleavage sites within the ERAV P1 polyprotein, the N-terminal amino acid sequence of the 26, 25 and 22 kDa bands was determined. As shown in Fig. 1
, the results established the order in which ERAV.393/76 capsid proteins separate as VP2, VP1 and VP3 by SDSPAGE. The predicted cleavage sites between VP2 and VP1 and VP1 and VP3 are similar to those used by FMDV 3C proteases. However, the autocatalytic cleavage site between VP4 and VP2 was shown to be a further 41 amino acids upstream from the predicted site. This has the effect of increasing the predicted size of VP2 and decreasing the predicted size of VP4 by approximately 4 kDa. The expected sizes for the capsid proteins are therefore 4, 29, 24 and 27 kDa for VP4, VP2, VP3 and VP1, respectively, and this now correlates well with the relative migration of the capsid protein bands from purified virus.

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Fig. 1. Coomassie brilliant blue-stained 1015% SDSPAGE showing the migration of purified ERAV.393/76 proteins. Capsid proteins VP2, VP1 and VP3 are indicated. The N-terminal amino acid sequences (actual) of the ERAV proteins in comparison to the picornavirus protease site prediction results are shown. Amino acid numbering is from the first residue of P1. The image was scanned on a Umax Astra 1220S desktop scanner using Adobe Photoshop 3.0 and annotated using Adobe Illustrator 6.0.
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To obtain ERAV.393/76-specific antisera, four horses (B, C, G and S) were inoculated intranasally with this virus. Horses B and C received 10 ml of ERAV-infected equine foetal kidney (EFK) cell culture lysate (passage 5; virus titre 106·5 TCID50/ml) intranasally, while horses G and S received 10 ml of cell lysate (passage 22, four times in EFK cells and 18 times in Vero cells; virus titre 107·9 TCID50/ml) that had been clarified at 10000 g for 20 min at 4 °C. Sera collected from these horses over a 105 day period were tested for the presence of ERAV.393/76-neutralizing antibodies (Studdert & Gleeson, 1978
). As shown in Fig. 2
, neutralizing antibody titres peaked approximately 3 weeks post-infection for each horse and the antibody titres of horses S and G decreased with time thereafter. These titres were comparable to the antibody titres in sera taken from naturally infected horses, where titres of between 800 and 8900 were reached 20 days after infection (Li et al., 1997
). In contrast to the horse sera, immunization of rabbits with purified, UV-inactivated ERAV.393/76 induced significantly lower levels of neutralizing antibody (Fig. 2
).

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Fig. 2. Virus-neutralizing (VN) antibody titres of sera taken from (A) horses B (open bars) and C (filled bars) and (B) horses G (open bars) and S (filled bars) at the indicated days after inoculation with ERAV.393/76. (C) VN antibody titres of hyperimmune rabbit sera prepared by subcutaneous inoculation of rabbits R3 (open bars) and R4 (filled bars) with three doses of 20 µg of purified UV-inactivated ERAV.393/76 emulsified in Freunds complete adjuvant on day 0 and then Freunds incomplete adjuvant on days 26 and 63. VN antibody titres are expressed as the reciprocal of the highest dilution of serum that neutralized 100 TCID50 of ERAV.393/76.
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The capsid proteins of FMDV contain five overlapping antigenic sites with an immunodominant neutralization epitope located in the receptor-binding GH loop of VP1 (Mateu, 1995; Mateu et al., 1995
). To investigate the immunoreactivity of the ERAV capsid proteins during infection, sera obtained from ERAV-immunized rabbits and from experimentally and naturally ERAV-infected horses were used in Western blots to probe purified ERAV.393/76. Despite the low levels of neutralizing antibody in the hyperimmune rabbit sera, high levels of antibody against VP1, VP2 and VP3 were present (Fig. 3
). In each case, sera from the experimentally infected horses showed strong reactivity to VP1 and some reactivity to VP3. VP2 did not elicit a strong immune response in experimentally infected horses and no antibody was detected to VP0, although such antibodies may not have been detected because the preparations of purified virus used in these Western blots contained very little VP0 (Fig. 2
and data not shown). Bands that correspond in size to VP4 were not seen in either Coomassie blue-stained SDSPAGE or Western blots. Sera from naturally infected horses showed variable reactivity in Western blots. While each serum sample tested showed some reactivity to VP1, serum from horse SM showed a much stronger reactivity to VP3. The reason for the poor reactivity of these sera to ERAV.393/76 proteins, despite the presence of neutralizing antibody to this virus, is not known, but may be explained, in part, by the variation between the infecting virus and the virus that was used for Western blot analysis.

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Fig. 3. Western blots of the structural proteins of ERAV.393/76 with equine and rabbit antisera. Blots were probed with rabbit sera taken from two rabbits (R3 and R4) prior to (day 0) and after (day 125) three immunizations with UV-inactivated purified ERAV.393/76. Blots were also probed with sera from ERAV.393/76-inoculated horses B, C, G and S taken at the indicated days post-inoculation or with plasma from naturally infected horses SM, PE and H (Li et al., 1997 ). Sera from the naturally infected horses showed VN antibody titres against ERAV.393/76 of 3200, 400 and 600, respectively. Primary antibodies, diluted 1:200 (horse) or 1:5000 (rabbit) in 2·5% skimmed milk in PBS containing 0·5% Tween 20, were used to probe PVDF membranes for 1 h at room temperature prior to washing six times for 5 min with PBS containing 0·5% Tween 20. Bound antibody was detected with either horseradish peroxidase (HRP)-conjugated rabbit anti-horse IgG (1:20000, Sigma) or by HRP-conjugated swine anti-rabbit IgG (1:1000, Dako). Blots were then washed and developed with ECL substrate (Amersham). Following autoradiography, images were scanned on a Umax Astra 1220S desktop scanner using Adobe Photoshop 3.0 and annotated using Adobe Illustrator 6.0.
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Distinct cleavage site preferences have been described for aphthovirus 3C protease in comparison to the protease site preferences described for the Enterovirus and Cardiovirus genera (Blom et al., 1996
). We have now shown that cleavage of the ERAV P1 polyprotein occurs exactly as that predicted for FMDV-like 3C proteases. The autocatalytic cleavage site identified between VP4 and VP2 of ERAV, however, was not predicted using this model. Considering the strong amino acid identity between the region predicted to be the ERAV VP4/VP2 autocatalytic cleavage site and that known to be the FMDV VP4/VP2 cleavage site (74LGTKLLA/DKKTEETTT88 for ERAV.393/76 and 80LFGALLA/DKKTEETT94 for FMDV.O1K) (Strohmaier, 1978
), the finding that the actual cleavage site for ERAV is 41 amino acids further upstream than predicted is surprising. The VP4/VP2 cleavage event is independent of 3C protease and for polioviruses is thought to occur with the concerted action of the conserved His residue at position 195 of VP2 (2195H), bound H2O molecules and RNA (Hindiyeh et al., 1999
). Less information is known about this cleavage event for FMDV. FMDV capsids have been shown to contain an homologous active site to support VP0 cleavage; however, it appears this event does not require RNA encapsidation (Curry et al., 1997
). ERAV contains the ProHisGln motif in VP2 (2200P, 2201H, 2202Q) and the penultimate Leu residue of VP4 (4038L), which are conserved residues across all members of the Picornavirus genus (the presence of the ProHisGln motif is not conserved, however, in hepatoviruses and Aichi virus). These conserved residues are thought to form the structural elements required for the putative active site of VP0 cleavage (Basavappa et al., 1994
; Curry et al., 1997
; Hindiyeh et al., 1999
). While the model of Blom et al. (1996)
predicts a conserved Glu at position 5 of VP2, this residue is not conserved in ERAV, which contains a Ser residue in this position (Li et al., 1996
; Wutz et al., 1996
). The relevance of VP2 position 5 to the autocatalytic cleavage event is not known. Despite the high degree of sequence identity in the predicted region, conformational differences between ERAV and FMDV VP0 might explain the difference in cleavage sites; cleavage may require the VP4/VP2 site be in close proximity to the ProHisGln motif of VP2.
ERAV.393/76 was first isolated from a nasal swab taken from a 4-year-old mare 4 days after the onset of acute respiratory illness, during which time temperatures as high as 41·4 °C were recorded (Studdert & Gleeson, 1977
, 1978
). Given the highly cell culture-adapted nature of ERAV.393/76, it was not known whether inoculation of this virus into horses would cause clinical signs of disease. Of the four horses inoculated, some clinical signs of illness (elevated body temperature) occurred in the two horses that received a higher dose of virus. It was from these horses (S and G) that ERAV was isolated from nasal swabs, urine and plasma, indicating that the virus had infected these horses (data not shown). When the antibody response to ERAV.393/76 in these horses was investigated, it was found that antibodies were primarily directed to VP1 and, to a lesser extent, VP3. Sera that showed the highest neutralizing antibody titres also showed the strongest reactivity to the viral proteins in Western blots, where intense binding to VP1 appeared to correlate with the much higher neutralizing antibody titres in these sera. The high neutralizing antibody titres of the horse sera and the strong immunogenic nature of VP1 in these horses may be consistent with the role of picornavirus VP1 proteins in antigenicity and receptor binding; however, in most picornaviruses, these sites are conformation-dependent epitopes, which would not necessarily be represented by the reactivity of the sera to denatured viral antigens in Western blots.
In contrast to the infected horses, rabbits received UV-inactivated purified virus emulsified in complete Freunds adjuvant. Although high levels of antibody to each of the capsid proteins were detected by Western blot analysis, these sera appeared to have a slightly less intense reactivity to VP1. Rabbits also produced low titres of neutralizing antibody after repeated (three) immunizations. ERAV VP1 does not contain an RGD (ArgGlyAsp) integrin-binding motif in the long GH loop analogous to FMDV (Li et al., 1996
; Logan et al., 1993
). Whether the neutralization epitopes of ERAV are continuous, as has been shown for site A of FMDV, or conformational, as for many other picornaviruses (for review see Mateu, 1995
), is not known. It is possible that emulsification of ERAV in adjuvant or UV inactivation resulted in some alteration to the structure of the neutralization epitopes and did not, therefore, provide as efficient targets for the production of neutralizing antibodies as the fully infectious virions given to the horses. Work is currently under way to identify the epitopes recognized by these sera and to determine which of these epitopes are targets for virus neutralization.
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Acknowledgments
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Project funding was from Racing Victoria, Rural Industries Research and Development Corporation and a Special Virology Fund. We thank Cynthia Brown for her excellent technical assistance.
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Received 3 January 2001;
accepted 16 February 2001.