Downloaded from www.microbiologyresearch.org by IP: 54.159.126.221 On: Thu, 28 Jan 2016 09:00:06 Journal of General Virology (1998), 79, 1901–1909. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Antigenic structure of the capsid protein of rabbit haemorrhagic disease virus Jorge L. Martı nez-Torrecuadrada, 1 Elena Corte s, 1 Carmen Vela, 1 Jan P. M. Langeveld, 2 Rob H. Meloen, 2 Kristian Dalsgaard, 3 William D. O. Hamilton 4 and J. Ignacio Casal 1 1 Inmunologı a y Gene tica Aplicada SA (INGENASA), Hnos Garcia Noblejas 41, 28037 Madrid, Spain 2 ID-DLO, PO Box 65, NL-8200 Lelystad, The Netherlands 3 Danish Veterinary Institute for Virus Research, DK-4771 Kalvehave, Denmark 4 Axis Genetics plc, Babraham, Cambridge CB2 4AZ, UK Rabbit haemorrhagic disease virus (RHDV) causes an important disease in rabbits. The virus capsid is composed of a single 60 kDa protein. The capsid protein gene was cloned in Escherichia coli using the pET3 system, and the antigenic structure of RHDV VP60 was dissected using 11 monoclonal anti- bodies (MAbs) and 12 overlapping fragments of the protein expressed in E. coli. Two antigenic regions were found. Ten out of the 11 MAbs recognized different discontinuous epitopes in the most immunodominant region of the viral capsid. This Introduction Rabbit haemorrhagic disease (RHD) is a viral disease of the species Oryctolagus cuniculus and was first described by Liu et al. (1984). The disease, characterized by high mortality in adult rabbits (Liu et al., 1984 ; Ohlinger et al., 1989), is caused by rabbit haemorrhagic disease virus (RHDV), a member of the family Caliciviridae (Ohlinger et al., 1990). The family Caliciviridae comprises several groups of non- enveloped viruses which share many biological properties (for a review see Clarke & Lambden, 1997). Among these properties is the presence of cup-like depressions (calyces) in the surface of the virus. However, this property can no longer be regarded as a general feature of caliciviruses as many of them present an ill-defined surface morphology. Thus, antigenically well- characterized caliciviruses such as feline calicivirus (FCV) and human Norwalk virus (NV) present a fuzzy, non-distinctive surface morphology. In contrast, RHDV shows a classic ‘ Star of David ’ calicivirus morphology. The 3D structure of RHDV has not been determined. Analysis of the recombinant NV particles by electron cryo- Author for correspondence : Ignacio Casal. Fax 34 1 408 75 98. e-mail INGENASAalc.es domain was located between residues 31 and 250 of the VP60 N terminus. The other MAb revealed the presence of an antigenic site within 102 aa of the C terminus. This MAb did not recognize the major cleavage product of the full-length 60 kDa protein. These results indicate that, in contrast to other caliciviruses such as Norwalk virus (NV), the 36 kDa cleavage product probably forms the N-terminal region of VP60. However, as in NV, the cleavage region appears to be the most immunodominant region. microscopy and computer image processing techniques has shown that the capsid is composed of 90 dimers of the capsid protein that form a shell domain from which arch-like capsomers protrude (Prasad et al., 1994). Based on sequence similarities with other T 3 RNA viruses, it has been predicted that the N-terminal amino acids (aa) 1–250 form the shell domain and the remaining C-terminal residues make up the arches (Prasad et al., 1994). The RHDV genome consists of a single-stranded RNA molecule of positive polarity, approximately 75 kb in size. The genome organization shows one long ORF that represents 86 % of the viral genome, with the VPg located downstream of the 3C-like protease gene, in the 5 region (Meyers et al., 1991). The capsid protein of RHDV has an apparent molecular mass of 60 kDa. Expression of a cDNA that encodes RHDV VP60 in insect cells infected with a recombinant baculovirus results in spontaneous assembly of the protein into empty recombinant RHDV (rRHDV) virus-like particles (VLPs) (Laurent et al., 1994 ; Plana-Dura n et al., 1996). These VLPs are morpho- logically and antigenically similar to native RHDV particles. The immunogenicity of these particles is extremely high ; doses as low as 05 μg are able to fully protect rabbits by the systemic route. Since RHDV cannot be propagated in vitro, the availability of large amounts of rRHDV VLPs has facilitated 0001-5335 1998 SGM BJAB