A further step in the evolution of rabbit hemorrhagic disease virus: the appearance of the first consistent antigenic variant

Abstract

Rabbit hemorrhagic disease virus (RHDV) is a noncultivable calicivirus that infects rabbits (Oryctolagus cuniculus) and causes epidemics of an acute fatal hepatitis. In 1997 we identified two RHDV isolates from geographically distant Italian regions, which differed antigenically from the reference strain RHDV.Bs89. In fact, they were not reactive with mAb 1H8, that is able to protect rabbits from RHD and showed a low reactivity with the rabbit convalescent serum raised against RHDV.Bs89. Experimental infection of rabbits with either RHDV isolates confirmed their high pathogenicity and their peculiar antigenic profile; nevertheless, rabbits vaccinated with the current vaccine were protected against challenge infection with these isolates. Sequence comparison definitely demonstrated that the two isolates originated from the same RHDV variant and that the similarity of their structural protein (VP60) sequences with the RHDV.Bs89 is equal to 93%. This variant was named RHDVa since shows consistent genetic and antigenic differences from the wild-type RHDV. In particular, 44% of amino acid substitutions in RHDVa VP60 were located between amino acids 344 and 370, where the similarity with RHDV.Bs89 drops to 70%, suggesting that this region probably contains the epitope recognized by mAb 1H8. In addition, this paper presents preliminary data concerning the amino acids of VP60 involved in the hemagglutination site of the virus.

Introduction

Rabbit hemorrhagic disease (RHD) is a highly contagious and usually fatal hepatitis of adult rabbits (Oryctolagus cuniculus), first observed and described in China in 1984 (Liu et al., 1984, Marcato et al., 1991). From then on, it has appeared in Asia, Africa, Central America and Europe, causing the death of hundreds of millions of rabbits and heavy economic losses in countries including Italy, Spain and France, where rabbits are bred for commercial purposes. Recently, RHDV has been accidentally introduced into both Australia (Holden, 1995) and New Zealand (Thompson and Clark, 1997) following an investigation on its potential role as a biological control agent for wild rabbits.

RHD is caused by a hemagglutinating virus belonging to the family Caliciviridae which has so far not been propagated in vitro (Ohlinger et al., 1990, Parra and Prieto, 1990, Meyers et al., 1991, Wirblich et al., 1996). Caliciviruses are non-enveloped positive-strand RNA viruses with a diameter of approximately 35–40 nm. Vesicular exanthema of swine (VES) is the prototype of the family, which includes also human pathogens such as Norwalk virus (NV) and other animal pathogens such as feline calicivirus (FCV). The virions are composed of a single-strand of RNA approximately 7.5 kb in length and a single major capsid protein with a molecular mass of 60–74 kDa. Therefore, like other RNA viruses, calicivirus has a high genetic mutation rate and the concept of `quasispecies' can be applied to the members of the family (Holland et al., 1982, Gould et al., 1997, Radford et al., 1998). In addition, cryo-electron microscopy studies on NV, and more recently on RHDV, showed that the structural protein folds in two main compact domains which are held together by a third domain that acts as an hinge region (Venkataram Prasad et al., 1994, Thouvenin et al., 1997). Thus, the capsid of calicivirus is made by two concentric shells functionally different: the inner shell made by the N-terminal half of the VP60s (S domain) that contains and protects the genome and the outer shell made by the C-terminal half of the VP60s (P2 domain). This last domain contains two highly variable regions designated C and E; the latter also contains the main antigenic determinants (Neill, 1992, Seal et al., 1993). This structural organization seems to allow the two shells a consistent degree of independence. Proof of this is the identification in the livers and spleens of rabbits and hares which died, respectively, of `chronic' RHD and European brown hare syndrome (EBHS), of a stable sub-viral particle made only with the inner shell of the virion (Capucci et al., 1991, Granzow et al., 1996, Barbieri et al., 1997).

Following the appearance of RHD in Europe, a second calicivirus was discovered to be the agent of an RHD-like disease in hares, named EBHS, already observed before 1980, but for which the cause was still obscure (Lavazza and Vecchi, 1989, Capucci et al., 1991, Chasey et al., 1992). The amino acid sequences of the structural proteins (VP60) of RHDV and EBHSV show an overall similarity of 76%, lowering to approximately 50% in the first portion of the C-terminal half of the VP60, that it is thought to be exposed on the surface of the capsid (i.e the external shell) (Wirblich et al., 1994). Accordingly, antigenic, serological and cross-protection experiments classify the difference between the RHDV and EBHSV at a serotypic level. However, epidemiological data and cross-infection experiments in rabbits and hares, concluded that RHDV and EBHSV were two separate virus species (Capucci et al., 1991, Chasey et al., 1992, Lavazza et al., 1996).

Finally, a third calicivirus has been recently identified in rabbits and, after its partial characterization, has been preliminary named rabbit calicivirus (RCV) (Capucci et al., 1996b). RCV causes an unapparent infection in rabbits and it seems to replicate only in the intestine where it is present at a very low level. Antibodies cross-reactive with RHDV are easily detectable in sera taken some few days after an RCV infection and they protect the rabbits from the challenge with the pathogenic virus (Capucci et al., 1996b). Indeed, the presence of a nonpathogenic virus closely related to RHDV was already considered at the time of the first RHD seroepidemiological surveys, when specific RHDV antibodies were found in the serum from farm and laboratory rabbits where RHD was never noted (Rodák et al., 1990, Capucci et al., 1991). Furthermore, a retrospective serological survey noted the presence of RHDV antibodies in rabbit sera collected at least 6 years before the appearance of RHD (Rodák et al., 1990).

Therefore, in these last 15 years three related caliciviruses have been identified in lagomorphs, two of which, RHDV and EBHSV, clearly emerged very recently considering that they are the cause of devastating diseases never before observed.

In spite of the supposed RHDV variability, after 13 years of spreading around the globe within rabbit populations quite different in their immunological state and, at least in Europe, probably in competition with RCV, all the genetic and antigenic studies carried out so far testify to a stability of RHDV (Nowotny et al., 1997, Le Gall et al., 1998). This paper describes the identification and characterization of the first consistent genetic and antigenic variant of RHDV.