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Animal: RNA Viruses |
Centre for Equine Virology, School of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia1
St Vincent’s 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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). 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 Tris–HCl, 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 15–45% (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 SDS–PAGE) were pooled before pelleting at 100000 g for 2 h at 4 °C and resuspended in TNE buffer. When separated on 10–15% SDS–PAGE 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 SDS–PAGE. 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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). 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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). 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 SDS–PAGE 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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). 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 Pro–His–Gln 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 Pro–His–Gln 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 Pro–His–Gln 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 Freund’s 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’ (Arg–Gly–Asp) integrin-binding motif in the long G–H 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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Received 3 January 2001;
accepted 16 February 2001.
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