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Comparative analysis of rabbit hemorrhagic disease virus (RHDV) and new RHDV2 virus antigenicity, using specific virus-like particles
© Bárcena et al. 2015
Received: 27 May 2015
Accepted: 12 August 2015
Published: 24 September 2015
In 2010 a new Lagovirus related to rabbit haemorrhagic disease virus (RHDV) emerged in France and has since rapidly spread throughout domestic and wild rabbit populations of several European countries. The new virus, termed RHDV2, exhibits distinctive genetic, antigenic and pathogenic features. Notably, RHDV2 kills rabbits previously vaccinated with RHDV vaccines. Here we report for the first time the generation and characterization of RHDV2-specific virus-like particles (VLPs). Our results further confirmed the differential antigenic properties exhibited by RHDV and RHDV2, highlighting the need of using RHDV2-specific diagnostic assays to monitor the spread of this new virus.
Introduction, methods and results
Rabbit haemorrhagic disease (RHD) is a highly infectious and fatal disease of the European rabbit (Oryctolagus cuniculus), (reviewed in ). The etiological agent, rabbit haemorrhagic disease virus (RHDV), belongs to the Lagovirus genus within the Caliciviridae family. This genus comprises several non-pathogenic rabbit caliciviruses, which are genetically related to but relatively distant from RHDV [2,3], and European brown hare syndrome virus (EBHSV). RHDV is highly contagious and usually fatal in adult rabbits, with a mortality range up to 80-100% . Susceptibility to the disease begins in the 5-6th week of life of rabbits and steadily increases up to the 8-9th week when they become fully susceptible. After it was reported in China in 1984, RHD spread rapidly around the world, being currently enzootic in wild rabbit populations in Europe, Australia and New Zealand. When it emerged, RHD dramatically reduced wild rabbit populations and was responsible for great economic losses in the rabbit industry worldwide . Subsequently, efficient inactivated commercial vaccines against RHDV were introduced in the early 1990s, providing a good coverage, since all circulating strains are classified within a single serotype. These vaccines enabled the control of RHD in rabbitries for the last 20 years. Likewise, ELISA methods developed in Italy for the veterinary diagnosis of RHD in domestic rabbits [4,5], were proven effective tools for monitoring RHDV field epidemiology, particularly in surveys conducted in Australia among wild rabbit populations [6,7].
RHDV is a non-enveloped icosahedral single-stranded positive-sense RNA virus. The virus capsid (~40 nm diameter) comprises 90 dimers of a single capsid subunit, the VP60 protein. RHDV as most caliciviruses cannot be grown in cell culture, a fact that has hampered the study of this group of viruses, as well as the development of control measures. A major breakthrough was the finding that expression of recombinant VP60 protein in insect cells results in the formation of virus-like particles (VLPs) that are morphologically and antigenically identical to infectious RHDV virions . RHDV VLPs have been shown to induce full protection of rabbits against a lethal challenge with RHDV [9,10]. These VLPs have also been used for the development of sensitive and reliable tests for detection of antibodies to RHDV [9,11].
The VP60 protein has three domains , an N-terminal arm (NTA), a shell (S) forming a scaffold which protects the viral RNA, and a flexible protruding domain (P) at the capsid surface, which contains determinants for virus-host receptor interactions and antigenic diversity [12,13]. The P domain can be further divided into P1 and P2 subdomains, with P2 subdomain located at the outermost surface-exposed region of the viral capsid.
In 2010 a new RHDV related virus with a distinctive pathogenic profile was identified in France [14,15] and has since rapidly spread throughout domestic and wild rabbit populations of Italy, Spain, Portugal Germany, United Kingdom and the Azores islands [14,16-21]. Unlike RHDV, the new lagovirus, termed RHDV2 or RHDVb in the literature, kills rabbit kits under 30 days of age, as well as rabbits that had been previously vaccinated against RHDV. Further studies have pointed out other distinctive features of RHDV2: it causes an average mortality of 20% in experimentally infected rabbits , which is consistently less than RHDV, and exhibits a broader host range, since it infects other lagomorphs like different hare species [17,22], causing an RHD-like disease. RHDV2 has specific genetic and antigenic profiles. It has been shown that several monoclonal antibodies (MAbs) against RHDV capsid protein fail to react with cognate RHDV2 virions [14,17].
The situation originated by the emergence of RHDV2 virus has raised concerns regarding the impact of the new disease among domestic and wild rabbit populations . Given the serious epizootic situation created, new inactivated vaccines against RHDV2 have been urgently developed and their use has been provisionally allowed in European Union member states. Recently, the World Organization for Animal Health (OIE) Reference Laboratory for RHD (IZSLER, Brescia, Italy), has developed a serological assay based on the use of specific anti-RHDV2 MAbs, rabbit immune serum and virus capsid antigen obtained from RHDV2-infected rabbit liver extracts .
Here we report for the first time the generation and characterisation of RHDV2-specific VLPs, and their use together with RHDV-specific VLPs, to conduct a comparative serological study involving sera from vaccinated and virus-infected rabbits. Our results further confirmed the differential antigenic properties exhibited by RHDV and RHDV2 virus capsids, highlighting the need for using specific serological assays as tools to monitor the epidemiology of this emergent virus, where virus-specific VLPs could represent a convenient alternative to classical virus antigen.
Reactivity of anti-RHDV MAbs against the capsid proteins of RHDV, RHDV2 and EBHSV in indirect ELISA assays.
Epitope location (aa position within VP60 protein)
(aa 549-579, P1 subdomain)
NPISQVAP (aa 326-333, loop 2 at P2 subdomain)
PGNNAT (aa 305-310, loop 1 at P2 subdomain)
Using the same approach (with a dilution range starting from 1/30), we tested sera from rabbits vaccinated against RHDV (ARVILAP vaccine, Laboratorios Ovejero, Spain) or RHDV2 (NOVARVILAP vaccine, Laboratorios Ovejero, Spain or FILAVAC VHD VARIANT vaccine, Filavie, France) collected 21 days after vaccination (Figure 2B). All animals developed specific antibody responses which were consistently higher for the cognate than for the heterologous antigen. Taken together, the results obtained revealed a markedly different antigenicity between both viral capsids, which is consistent with the observed lack of efficient protection afforded by current commercial RHDV vaccines against RHDV2.
The available information regarding sequence alignment studies of lagovirus full-length capsid proteins indicate RHDV2 is phylogenetically distinct from all previously described members of the genus Lagovirus and forms a new genetic group . Amino acid identity among RHDV2 strains is close to 97%, whereas average identity is 89.2% when compared with RHDV strains [14,17]. When the sequence comparison is restricted to the 7 regions of the P domain that show the highest degree of genetic variation (regions V1 to V7 ), the identity between RHDV2 and RHDV strains drops to 60% . Moreover, the crystallographic resolution of the VP60 protruding P domains of RHDV  and RHDV2 , has enabled the analysis of structural differences between both viruses. Although their overall structures are similar, the P1 subdomain helices are slightly shifted and some P2 subdomain loops are oriented differently. Interestingly, amino acid changes between both viral capsid proteins tend to concentrate on the three extended loop regions located at the outer surface of the P2 subdomain . These results provide insights regarding the marked antigenic differences observed between both viruses, remarkably the lack of efficient protection against RHDV2 afforded by current RHDV inactivated vaccines, which has prompted the urgent development and provisional authorization of RHDV2-specific inactivated vaccines.
Epidemiological surveillance of RHD among domestic and wild rabbit populations is performed using serological assays based on the use of RHDV antigen obtained from infected-rabbit liver extracts and virus specific MAbs [4-6]. Commercial test kits based on RHDV VLPs are currently available and have been used in several studies [16,30]. In Australia, early seroepidemiologic surveys  evidenced the circulation of a non-pathogenic rabbit calicivirus (RCV-A1), which was subsequently identified and characterized . Serological assays enabling discrimination of both viruses, based on the use of virus specific VLPs, have been developed . In Europe, the rapid spread of the newly emergent virus RHDV2, which is apparently replacing classical RHDV strains among rabbit populations [14,18] and is capable of infecting lagomorphs from the Lepus genus [17,22], is a major cause of concern. The availability of virus specific reagents such as RHDV2 VLPs, would greatly ease monitorization of field evolution and circulation of RHDV2, as well as its interaction with RHDV when both viruses co-circulate in rabbit populations.
Here we report for the first time the generation and characterization of RHDV2-specific VLPs. Our results further confirmed the differential antigenic properties exhibited by RHDV and RHDV2 capsid proteins. Using sera from both, vaccinated and infected rabbits, it was shown that antibody responses were consistently higher for the cognate than for the heterologous antigen. Interestingly, the results obtained in the serological analysis of sera collected from an apparently healthy wild rabbit population from Central Spain, suggesting high prevalence of RHDV2, were in agreement with previous reports indicating rapid spread of the novel virus through the Iberian Peninsula [16,23].
The VLPs reported in this study might also be useful for the development of vaccines against RHDV2. Current provisionally authorised vaccines for RHDV2 are prepared from liver extracts of experimentally infected rabbits. Since RHDV2 induces consistently low mortality rates (20-30%) as compared to RHDV (close to 100%), this might represent an important drawback for vaccine production (i.e. lower yields of virus antigen per infected rabbit). Therefore, the development of new recombinant subunit vaccines for RHDV2 based on VLPs might provide greater benefit than the equivalent for RHDV.
In addition, this work enabled the characterization of our MAbs 2E7, 1G5 and 1C9, regarding their differential reactivity with three lagovirus capsid proteins (RHDV, RHDV2 and EBHSV). In summary, the RHDV2 VLPs and the MAbs reported in this study provide tools that might be useful for monitorization of virus circulation, development of control measures, as well as for research in different aspects of the biology of this relevant emergent virus.
We thank Yolanda Gómez for the technical assistance and David Ayensa for his collaboration providing sera samples from rabbits vaccinated with RHDV and RHDV2 vaccines. The authors are grateful to his Grace the Duke of Westminster for his support during the study as well as the staff of “La Garganta” and A. Sonia Olmeda and María Teresa Cutuli from the Veterinary Faculty of UCM for their assistance during sampling. This work was funded by grants AGL2013-48923-C2-1-R to EB and BFU2014-55475 to JRC (Spanish Ministry of Economy and Competitiveness) and P2013/ABI-2906 (PLATESA, Comunidad Autónoma de Madrid) to EB.
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