Mapping B-cell epitopes in equine rhinitis B viruses and identification of a neutralising site in the VP1 C-terminus

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Abstract

Erbovirus is a genus of the family Picornaviridae and equine rhinitis B virus (ERBV) is the sole species. Erboviruses infect horses causing acute respiratory disease and sub-clinical and persistent infections. Despite the high seroprevalence and worldwide distribution of these viruses, the pathogenesis and antigenic structure of the three ERBV serotypes (ERBV1, 2 and 3) is poorly understood. To characterise linear epitopes on ERBV structural proteins, a set of fusion proteins were expressed in Escherichia coli. These proteins were tested in Western blot and ELISA and reactive proteins were also used to identify neutralisation epitopes. VP1 contained serotype specific epitopes whereas VP2 was highly cross-reactive across the serotypes. The C-terminus of VP1 accounted for most of the reactivity of full-length VP1 and was also the location of a neutralising site in each serotype.

Introduction

Respiratory disease in horses is of major economic importance, particularly in the performance horse industry. One of the lesser-studied equine respiratory pathogens is equine rhinitis B virus (ERBV). Equine rhinitis B virus has been isolated from horses with clinical signs including fever to 41 °C for 1–3 days, nasal discharge, anorexia, oedema of the legs, lethargy, and swelling and abscessation of lymph nodes of the neck with pain on palpation (Fukunaga et al., 1983, Hofer et al., 1972, Mumford and Thomson, 1978). The ability of ERBV to establish persistent infection over an 18–24-month period (Burrows and Goodridge, 1978, Mumford and Thomson, 1978) and associated reports of subclinical infection with ERBV have suggested a role for these viruses in the exacerbation of infection with other respiratory viruses and with secondary bacterial infections (reviewed by Carman et al., 1997).

Initially, ERBV was classified as a Picornavirus with the equine rhinoviruses (Hofer et al., 1972, Steck et al., 1978). Analysis of genome sequence identity led to the reclassification of ERBV as the sole species in the genus Erbovirus, family Picornaviridae (Pringle, 1999). There are three known serotypes, ERBV1, ERBV2 and ERBV3 that vary in their acid stability (Horsington et al., 2011). All three serotypes have been isolated from horses world-wide and neutralising antibodies have been detected in 50–80% of horses tested and in all age groups (Black et al., 2007, Carman et al., 1997, Dunowska et al., 2002, Dynon et al., 2007, Fukunaga et al., 1981, Holmes et al., 1978, McCollum and Timoney, 1992, Mumford and Thomson, 1978, Rose et al., 1974, Steck et al., 1978, Wernery et al., 1998). Simultaneous infection with multiple serotypes has been reported and neutralising antibodies to two or all three serotypes in the one horse is common (Black et al., 2007, Dynon et al., 2007, Horsington et al., 2011).

Antibodies are the major effectors of protection against picornavirus infection, although cell-mediated immunity plays an essential role in the immune response to at least some of these viruses (Glezen et al., 1969, McCullough et al., 1992, Pay and Hingley, 1987, Rossi et al., 1991). While many neutralising epitopes in picornaviruses are conformational, linear epitopes are found in a number of viruses including Theiler's murine encephalomyelitis virus (TMEV) (Inoue et al., 1994, Kim et al., 1992), poliovirus (PV) (Blondel et al., 1983, Chow et al., 1985, Hoatlin et al., 1987), Enterovirus 71 (Foo et al., 2007), human rhinovirus (Skern et al., 1987) and foot and mouth disease virus (FMDV) (Giavedoni et al., 1991, Mateu et al., 1989, Strohmaier et al., 1982). The external capsid proteins, VP1, VP2 and VP3, share a wedge-shaped, eight-stranded, β-barrel structure. Differences exist in the size and conformation of the connecting loops between the strands and the extensions of the N- and C-termini, and antigenic sites appear limited to the surface exposed loops (reviewed by Mateu, 1995, Rueckert, 1985). In many picornaviruses the VP1 protein is the major site of antigenic epitopes and is the target for neutralising antibodies, although antigenic sites have also been detected in VP2 and VP3 (reviewed by Inoue et al., 1994, Mateu, 1995, Usherwood and Nash, 1995).

The aim of this study was to better understand the antigenic relationship between ERBV serotypes and identify potential antigens for a serotype-specific ERBV antibody detection ELISA. Full-length VP1 and VP2 and a selection of proteins representative of the surface exposed loops were expressed as fusion proteins in Escherichia coli and were used to map linear B-cell epitopes. The antigenicity of the fusion proteins was tested in Western blot and ELISA, using sera obtained from ERBV-immunised rats and from naturally ERBV-infected horses. Linear neutralising epitopes were identified using these fusion proteins in competitive inhibition of neutralisation assays and virus neutralisation assays with polyclonal rat sera prepared against specific proteins.

Section snippets

Viruses and sera

Viruses used in this study included the prototype ERBV1 and ERBV2 strains, ERBV1.1436/71 and ERBV2.313/75 (Black et al., 2005), and the Australian ERBV3 isolate, ERBV3.2225AS (Horsington et al., 2011).

Rat sera to specific proteins or whole virus were prepared by subcutaneous inoculation with 10 μg of purified protein in Freund's complete adjuvant followed by a second inoculation of 10 μg purified protein in Freund's incomplete adjuvant 14 days later. Production of protein or virus specific

Expression of ERBV capsid proteins

A total of 27 recombinant ERBV fusion proteins were expressed in E. coli and purified as either GST or hexa-histidine tagged proteins. The genetic sequences for each protein were amplified by PCR and cloned into either the pGEX-4T2 or pQE vector. Constructs were sequenced to confirm the protein coding sequences were correct and in-frame. Full-length VP1 and VP2, and the smaller loop proteins VP1-Nt, VP1-CD, VP1-EF, VP1-GH, VP1-HI, VP1-Ct and VP2-EF were expressed for each serotype (Fig. 1A and

Discussion

The surface-exposed regions of the capsid proteins of picornaviruses participate in receptor binding and antigenicity and contain the neutralising epitopes that distinguish serotypes. Of the three external capsid proteins, VP1 has the greatest sequence variation between serotypes and in many picornaviruses VP1 is the site of major neutralisation epitopes (reviewed by Inoue et al., 1994, Mateu, 1995, Usherwood and Nash, 1995). This study identified parallels in the antigenic structure of

Conclusion

A set of ERBV fusion proteins was tested for their reactivity to ERBV polyclonal rat sera and sera from naturally infected horses in Western blot and ELISA. The VP1-Ct was the most reactive protein in each serotype, accounting for most of the reactivity to full-length VP1. This region was the location of major B-cell epitopes in ERBV and the location of a major neutralising site in ERBV2. The VP1-Ct was also a neutralising epitope in ERBV1 and ERBV3, however, the results suggested other more

Acknowledgements

We thank Nino Ficorilli, Bob Geyer and Cynthia Brown for technical assistance. J.J.H. was the recipient of a Melbourne Research Scholarship. Other funding support was from the Special Virology Fund, Centre for Equine Virology, The University of Melbourne.

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