Work Packages

Task 1.1

Collection of rabbit/hare samples from the different geographical areas under study.

Liver, spleen and duodenum samples will be collected from deceased leporids (rabbits and hares). When possible, blood samples will be collected to determine the population serostatus and to identify seropositive (resistant) leporids.

PARTNERSCIBIO/InBIO-UP, INIA

Task 1.2

Genotyping of viral isolates detected in the collected tissue samples.

A first screening by ELISA followed by traditional PCR, using conserved primers designed according to lagovirus genomes, and sequencing will be used to genotype the strains. Relevant genomes will be further obtained by genome-walking or NGS. Evolutionary analyses will be performed to determine recombination events, possible origin of the outbreaks, and virulence markers.

Task 1.3

Analyses of the serological status of leporid populations.

In order to monitor circulation of RHDV, titration of specific antibodies arising from natural infections will be monitored by OIE-prescribed immunoassays developed by partner 6 as well as by an indirect ELISA assay using GI.1 and GI.2 derived virus-like particles (VLPs) generated by partner 1 (Barcena et al. Veterinary Research, 2015; Rouco et al. Transboundary and Emerging Diseases, 2018).

Task 2.1

Validation of optimised diagnostic assays for detection of virus.

Genomes obtained in WP1 will be used to design more accurate, rapid and sensitive diagnostic tools, that will be further tested in non-invasive samples (blood, saliva, faeces). Validation of such tools in these samples might allow us to forecast outbreaks and contain the disease with the implementation of more adequate biosecurity measures.

Task 2.2

Development and validation of immunoassays to monitor serological antibody response in the Mediterranean basin.

The specificity and sensitivity of immunoassays developed under collaboration with INGENASA (SME) in the framework of VetBioNet project will be estimated as diagnostic tools for surveillance of lagoviruses currently circulating in the Mediterranean basin regions under study. Results will be also compared with available immunoassays used as gold standard. Depending on the results obtained, such assays will be adapted and/or optimised to be used for disease control.

Task 2.3

Geographical identification of circulating lagoviruses.

Furthermore, to get insight on the antibody response elicited by different circulating strains, the antigenic profile of a collection of viral strains representing different Mediterranean epidemiological situations will be carried out using a panel of specific MAbs developed and characterised by partner 6. Patterns of disease transmission will be determined based on Single Nucleotide Polymorphism (SNPs). We will combine information from both tools to identify the origin of ongoing outbreaks.

Task 3.1

Characterisation of innate immune response.

Characterisation of the innate immune responses will be achieved by determining serum profiles of innate immunity markers from experimentally infected animals. Experimental infections with caliciviruses will be conducted in BSL2/3 animal facilities already available. Partners will contribute by providing VLPs for in vivo inoculation, as well as by advising on the best procedures to carry out the infections. Serum profile of cytokines elicited early after infection will be analysed by commercially available ELISAs (ILs, IFN, etc). Data obtained will be useful for vaccine design (Task 3.3).

Task 3.2

Characterisation of adaptive immune response.

Some relevant aspects of adaptive immune response not studied yet will be addressed in this task. Specific cellular immune response elicited by VLPs and/or inactivated virus particles will be assessed by performing ELISPOT assays to detect IFNγ release by lymphocytes in ex-vivo assays. Likewise, specific T-cell proliferation will be measured by classical lymphoproliferative assays or CFSE methods using FACS. Potential T-cell epitopes will be able to be identified by in vitro recall, by stimulating with truncated versions of RHDV capsid protein and then with synthetic peptides. In addition, the specific induction of systemic and mucosal antibodies against RHDV will be analysed at different time post-immunisation in serum, nasal swabs and also milk. Antibodies will be isotyped (IgG, IgM and IgA) by ELISAs already available at OIE reference laboratory.

Task 3.3

Immunogenicity and protection conferred by different vaccine candidates (RHDV recombinant VLPs against homologous and heterologous challenges).

Recombinant VLPs from RHDV of different genotypes (GI.1-4) and chimeric recombinant myxo- RHDV vaccines will be generated by partners 1 and 5, respectively. Rabbits will be immunised with such constructions and humoral (antibody titration by ELISA) and cellular immune responses (Lymphoproliferation assay and IFNγ-ELISPOT) will be evaluated by testing different adjuvants. We will explore the administration of such vaccines by at least one of the alternative routes (subcutaneaous, intradermal, oral, and ocular). The vaccine strategies eliciting stronger immune responses will be assessed for protection capacity against homologous and heterologous challenges.

Task 4.1

Intervention plans

Based on the intervention plan already set up and employed in European countries for controlling RHD outbreaks, a specific intervention plan to prevent and/or control occurring outbreaks will be set up to be used particularly in African countries. The foreseen measures will be properly tailored to local conditions by adapting those already reported in the documents available for EU countries. A Manual will be prepared by including detailed operations and measures that should be taken to prevent (biosecurity measures), control and solve (extinction) an outbreak, including the type of necessary tests that should be run to check the persistence of RHDV in the environment and the management procedures to be used (delay slaughtering, carcass and manure removal, cleaning and disinfection, vaccination, etc).

Task 4.2

Biosecurity measures and rabbit management

Investigation of the efficiency of defensive and offensive sanitary measures, including disinfection, disinfestation, and biosecurity procedures, adapted to the particularities of North African rabbitries and search for passive/indirect vectors of lagoviruses.

Task 5.1

Communication and dissemination

In addition to the ordinary annual meeting with all the consortium participants, other meetings/workshops will be held in order to advise stakeholders, identify synergies and gaps not covered by other projects from different areas, and to disseminate results. Scientific findings will be further communicated in national and international meetings, and in peer-reviewed journals. Novel sequence data will be submitted to international nucleotide databases and will be available for the scientific community. National sanitary authorities, breeders, environmental and hunting associations will be informed by means of meetings, articles on generic and sector bulletins, technical and professional journals, and on relevant websites. We will liaise through existing contacts with the central veterinary offices, the wildlife management and conservation agencies, and academic institutions.

Task 5.2

Training of scientists and technicians

Workshops on diagnosis and technology and disease management will be promoted for training young researchers/post graduates/post doctorates, especially from African countries.

Task 6.1

Scientific, administrative and financial management.

We will set the management structure of the project for its successful implementation. This includes the management of the budget, and the supervision and quality control of the execution of the different WPs. The budget will be managed by the financial units of each partner and supervised by the coordinator. The coordinator will supervise the WPs’ progresses, and when necessary, conference calls with the other consortium partners will be undertaken to discuss proper adjustments.

Task 6.2

Management of intellectual property rights and technology transfer activities

Annual project reports will identify results and technologies that should be commercially protected. The “Consortium Agreement” will establish the rules for intellectual property rights. Partners’ technology transfer offices will provide advice and implement protection tools and will develop commercialisation strategies.

PARTNERSCIBIO/InBIO-UP

Task 6.3

Database and collection of data.

A database will be created for registering information on the samples collected or generated during the lifetime of the project and on the protocols developed/used. This database will be accessible to all partners of the consortium.