Objectives

In order to increase interdisciplinary scientific and technical knowledge on the epidemiological characteristics of RHD and its aetiological agent, the rabbit haemorrhagic disease virus (RHDV), and contribute to the development of effective preventive actions, capable to reduce the socio-economic impact of future outbreaks or of the emergence of new genotypes of unknown origin, especially for African countries of the Mediterranean basin, the following objectives (O) and specific objectives (SO) were set:

Epidemiology and surveillance

SO1.1

Distribution of susceptible species (Portugal, Spain, France, Italy, Tunisia, Algeria) 

With this SO we intend to determine hotspots of outbreaks and identify susceptible leporid species (rabbits and hares), particularly in North Africa. This information will be obtained through ongoing collaborations or projects (e.g. synergy with WWF Spain, Dr. Ramón Perez-Ayala); ongoing monitoring projects (e.g. +Coelho in Portugal); hunting and farmers associations; regional authorities; producers associations/integrated companies (e.g. Dr. Chakroun Chehid from Interprofessional Group of Poultry and Cuniculture Products, Tunisia; Association des cuniculteurs algériens, Algeria).

SO1.2

Current extent of disease

Lagovirus diagnostic and identification in the geographic regions under study will be carried out using ELISA and PCR-based approaches on the samples collected within SO1.1. Additionally, serological surveillance will be conducted to monitor circulation of RHD and establish the percentage of exposed animals. Since the presence of non-pathogenic lagoviruses might interfere with the results and the conclusions drawn, new methods will be developed to distinguish between pathogenic and non- pathogenic lagoviruses positive serologies. These analyses will provide data on geographical variations regarding RHD prevalence, which could be relevant to adopt adequate control measures in the Mediterranean basin.

Optimisation and validation of technical tools for better prevention and control of RHDV

SO2.1

Improved lagovirus detection, genotyping and antigenic typing

Different research groups use different diagnostic tools to detect the presence of lagoviruses. In this SO we propose to standardise such procedures in order to increase the efficiency of detection and strain typing of circulating viruses. Patterns of disease transmission based on selected Single Nucleotide Polymorphisms (SNPs) that distinguish different populations of GI.2 will be also analysed. Moreover, in order to obtain the antigenic profile of the GI.2 isolates, we will use a panel of anti-GI.2 MAbs produced by partner 6, but new MAbs will also be produced due to antigenic variation between strains from different geographic areas. The knowledge generated will improve our understanding on viral emergence and spread, and will contribute to better control the disease.

SO2.2

Validation of serological methods for detection of antibodies against circulating RHDV strains

Serological immunoassays under development by partners 1 and 3 together with a SME (INGENASA) and funded by the EU project VetBioNet (www.vetbionet.eu) will be used to analyse sera samples collected in the Euro-Mediterranean region (O1). In this SO, we expect to cover a gap of VetBioNet project by assessing and validating the potential of novel assays in detecting current circulating lagovirus strains in the Mediterranean basin. The specificity and sensitivity of such assays will be estimated for viruses circulating in the different geographical regions. The establishment of new serological immunoassays and their validation will pass through a comparison with already available tests such as those described by OIE Manual and used as gold standard. Furthermore, to get insights into the antibody response elicited by different viral strains, isotype serology (IgG, IgM and IgA) will be also extended to other leporids, in particular, IgA detection in different hare species using new MAbs recently produced by partner 6.

Analyses of RHDV-host interactions; induction and regulation of immune responses

SO3.1

Innate immune response

Although it is known that the antibody response (humoral immunity) is important in the protection against RHD, other mechanisms of immunity might also play a relevant role. Among them, elevated innate immune competence has a relevant role in protection, in particular for the induction of cross- protection, as has been recently demonstrated in infections with an Australian non-pathogenic lagovirus strain. Innate immune response has been reported, among other possible explanations, as potentially relevant for the resistance to classical RHDV (GI.1) infection of young animals in contrast to adults. We propose to analyse parameters of the innate immune response induced by different GI.2 strains since this response has been shown to be relevant also for vaccine design. The serum profile of cytokines elicited after infection will be determined from blood samples recovered periodically from rabbits experimentally inoculated with GI.2 strains.

SO3.2

Adaptive immune response

Adaptive immunity is critical for resistance against RHDV. While most RHDV immunity studies have focused on humoral immune responses, very few studies have gathered information on cell- mediated immunity, especially T-cells. To gain insight on the adaptive immune response against GI.2 we will characterise the T-cell response to homologous and heterologous RHDV capsid proteins, following VLP and/or virus particle inactivated immunisation. We will evaluate the production of T- cell effector cytokines such as IFNγ (by ELISPOT) and proliferation of RHDV specific T-cells after in vitro re-stimulation with VLPs or recombinant polypeptides of VP60 domains (S, P1 and P2). Eventually, T-cell epitopes will be identified using overlapping synthetic peptides. On the other hand, and given the role of antibody response in protection, the kinetics of induction of serum and mucosal antibodies (IgM, IgG and IgA) elicited in immunised rabbits will be studied.

SO3.3

Development of vaccines

Current commercially available vaccines are based on inactivated infectious viruses. Due to the lack of a cell culture system for efficient virus propagation, such vaccines are obtained by amplifying the virus directly in the host species, i.e. in rabbits. The absence of heterologous protection (e.g. GI.1-4 vs. GI.2 and vice-versa) joined to welfare and biosecurity concerns, justify the search of new alternatives for vaccine production. We will explore the immunogenicity and protection conferred by different vaccine candidates (recombinant VLPs from RHDV of different genotypes (GI.1-4), against homologous and heterologous strains).

Biosecurity measures, control and prevention strategy

SO4.1

Definition of biosecurity measures, control and prevention strategy

To prevent the entry and the persistence of lagoviruses in rabbitries, we propose to test defensive and offensive sanitary measures; perform virus detection and serological characterisation of rabbits for rabbit production systems, along with a systematic monitoring of virus circulation in the faeces and cages, before and after cleaning disinfection; due to their role in virus dissemination, we will perform virus detection in passive/indirect vectors taking advantage of SO2.1.

SO4.2

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 ongoing outbreaks will be set up to be used particularly in African countries. The measures will be tailored to local conditions by adapting those already reported in the documents available for the EU countries.

Mediterranean networking activities and technology transfer

SO5.1

Meetings

In addition to the ordinary annual meeting with all the consortium participants, other meetings/workshops will be held in order to disseminate results and advise stakeholders, and identify synergies and gaps not covered by other projects from different areas.

SO5.2

Training

We expect to provide training on diagnosis and technology and disease management capabilities, and to promote the mobility of research workers (PhD students, technicians, etc) among the partners, particularly partners in North Africa. Training of stakeholders will be also sought.