PRIMA Full Proposal Template Table of Contents

PRIMA Full Proposal Template

Version 0.0 – 14 June 2018

PRIMA

Full Proposal Template

Table of Contents

1. Excellence 1

1.1 Objectives 1

1.2 Relation to call and topic 4

1.3 Concept and methodology 5

(a) Concept 5

(b) Methodology 5

1.4 Ambition 8

2. Impact 8

2.1 Expected impacts 8

2.2 Measures to maximise impact 9

(a) Dissemination and exploitation of results 9

(b) Communication activities 12

3. Implementation 13

3.1 Work plan 13

Table 3.1a: List of work packages 15

Table 3.1b: Work package description 15

Work package 1 15

Work package 2 18

Work package 3 20

Work package 4 22

Work package 5 23

Work package 6 25

Table 3.1c: List of deliverables 27

3.2 Management structure, milestones and procedures 29

3.2.1 Description of the organisational structure and the decision-making 29

Table 3.2a: List of milestones 30

3.2.2 Decision-making mechanisms in LAGMED 31

3.2.3 Innovation management 31

3.2.4 Critical risks for implementation 32

Table 3.2b: Critical risks for implementation 32



3.3 Consortium as a whole 34

3.3.1. Consortium’s expertise 34

3.3.2. Partners’ contributions for the project 35

3.3.3. Industrial/commercial involvement 41

3.3.4. Other countries and international organisations 41

3.4 Resources to be committed 41

Table 3.4a: Summary of staff effort 41

Table 3.4b: ‘Other direct cost’ items 41

4. Members of the consortium 43

4.1. Participants (applicants) 43

4.2. Third parties involved in the project (including use of third party resources) 59

5. Ethics and Security 60

5.1 Ethics 60

5.2 Security 61



Page 1 of 61

PRIMA

Full Proposal Template

Project proposal (Part II)

Title of Proposal Improvement of preventive actions to emerging LAGoviruses in the MEDiterranean basin:

development and optimisation of methodologies for pathogen detection and control

Acronym LAGMED

List of participants

Participant No *

PI name Organisation Country

1 (Coordinator) Joana Abrantes CIBIO/InBIO-UP Portugal

2 Partner 1 Esther Blanco INIA Spain

3 Partner 2 Carlos Rouco Universidad de Córdoba Spain

4 Partner 3 Ghislaine Le Gall-Reculé ANSES France

5 Partner 4 Stéphane Marchandeau ONCFS France

6 Partner 5 Stéphane Bertagnoli INRA-ENVT France

7 Partner 6 Lorenzo Capucci IZSLER Italy

8 Partner 7 Lilia Messadi ENMV de Sidi Thabet Tunisia

9 Partner 8 Hacina Ain Baziz ENSV d’Alger Algeria

1. Excellence

1.1 Objectives

The traditional epidemiologic triad model holds that clinical infectious diseases result from the

interaction of the pathogen, the host and the environment. The initial impact in the 1980s of rabbit

haemorrhagic disease (RHD) on wild and domestic rabbits slowly abated over time in rabbitries as a

consequence of the use of efficient vaccination campaigns and other control measures. However, in

2010, a new genotype named RHDV2 or RHDVb, and more recently GI.2, emerged in France with an

unknown origin, but linked to the European (Mediterranean) area. This new genotype that possibly

represents a new serotype, was detected both in wild and farm animals, including rabbits vaccinated

against RHDV GI.1 (former G1-G6) or naturally immunised, and showed the relevant impact of this

disease, particularly on the fragile equilibrium of the Mediterranean ecosystem. Indeed, RHD is still

one of the most (if not the most) devastating diseases of rabbits, with high mortality rates occurring

within a few days and with an outstanding ability of transmission at long distance.

mailto:[email protected]



Page 2 of 61

The main objective of this project is to increase interdisciplinary scientific and technical knowledge

on the epidemiological characteristics of RHD and its aetiological agent, the rabbit haemorrhagic

disease virus (RHDV). This will contribute to develop effective preventive actions, capable to reduce

the socio-economic impact of future outbreaks or of the emergence of new genotypes of unknown

origin. This is especially relevant for African countries of the Mediterranean basin where rabbits have

been promoted for poverty reduction programmes due to the “low investment and early benefits, and

subsistence on renewable resources for feeding, housing and general management” (Oseni&Lukefahr,

World Rabbit Science, 2014).

Therefore, the following objectives (O) and specific objectives (SO) were set:

O1. 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.

O2. 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

http://www.vetbionet.eu/



Page 3 of 61

insight 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.

O3. 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).

O4. 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.



Page 4 of 61

O5. 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.

1.2 Relation to call and topic

This proposal relates to the unique call of PRIMA Section 2, Thematic area 2 - Sustainable farming

systems under Med environmental constraints and in particular to the Topic 2.2: Preventing

emergence of animal and plant diseases.

In agreement with the general goals of PRIMA, this proposal aims to build research and innovation

capacities and develop knowledge for agro-food systems in the Mediterranean area. The main goal of

this project is to improve the control of RHD, one of the worst rabbit diseases, associated with severe

economic and production losses in the meat rabbit industry, as well as high mortalities of wild

animals. Indeed, the European rabbit plays a key ecological role in Mediterranean ecosystems, it is

one of the most important small game species in several European countries, and in African countries

small family rabbit units have been promoted in several programmes aiming at reducing poverty.

In order to achieve the main goal of the project, the consortium will rely on multidisciplinary research

(epidemiology, microbiology, genetics, virology, diagnostic, immunology, vaccinology), and critical

mass from relevant public actors (Academia and Governmental Research organisms) joined to advise

relevant private sectors (hunting federations, local meat rabbit producers and non-governmental

organisations). Thus, this project will improve research and innovation capacities of several sectors.

This project fits within the "Operational Objective 5/Pests and Pathogens in farming", since the

aim is to design and promote the adoption of novel approaches to reduce the impact of RHDV in

farming. Notwithstanding, this proposal focuses mainly on priority 3 "Preventing emergence of

animal and plant diseases". When a disease breaks out it is usually already too late to find and

implement effective solutions, and this is particularly the case of RHD due to the short incubation and

hyperacute-acute course. In this project we will obtain scientific knowledge on leporid-lagovirus

(host-pathogen) interaction to understand and foresee outbreaks, as those that occurred with the

widespread of the new GI.2. This will lead to the implementation of preventive actions by improving

biosecurity measures and prevention strategies, with the development of more sensitive and specific

diagnostic tools and more efficient vaccines to control the impact of future outbreaks caused by novel

lagoviruses.

This project is also related with priorities 2 and 4. Regarding priority 2 "Developing sustainable

and productive agro-systems", the Mediterranean ecosystem vulnerabilities will be taken into

account in this proposal by assessing disease extension, the characteristics of the strains circulating in

the field, and their direct impact on Mediterranean biodiversity and in rabbitries and/or smallholder

farms. Since the more efficient control of emerging diseases caused by lagoviruses can have a

favourable impact on the efficiency of smallholder rabbit producers, the achievements of the project

will be positively linked to poverty alleviation and rural development included in priority 4

"Developing farming systems able to generate income, to create employment and to contribute

to a balanced territorial development".



Page 5 of 61

1.3 Concept and methodology

(a) Concept

Rabbit haemorrhagic disease (RHD) is a serious and highly contagious viral disease of both domestic

and wild European rabbits (Oryctolagus cuniculus), caused by a calicivirus (genus Lagovirus). RHD

causes important economic losses in the rabbit industry, a relevant agricultural sector in the

Mediterranean basin. Furthermore, it has a significant negative ecological impact among wild rabbit

populations that have a key role in this hotspot of biodiversity. Through grazing and seed dispersal,

rabbits help to preserve plant species diversity, their latrines have positive effects on soil fertility, and

rabbits serve as prey for critically endangered predators (e.g. Iberian lynx and Spanish Imperial

Eagle). The overall concept underpinning this project is to contribute to the survival of rabbit populations by

increasing the knowledge on one of their major causes of mortality. In the light of the emergence of

new RHDV strains, such as GI.2 that affects young rabbits and rabbits vaccinated for classical RHDV

and shows an expanded host range (including some species of hares), the need of studying circulating

lagoviruses in wild animals, as potential origin of future outbreaks in farms/rabbitries, and developing

improved control measures, are of utmost importance.

Towards enhancement of the Mediterranean basin preparedness to control the disease, the exchange

of different expertise and scientific achievements is crucial, as well as the contribution from different

stakeholders. For this reason, the consortium includes expert teams covering the fields of virology,

epidemiology, veterinary medicine, immunology, evolutionary biology, molecular biology and

vaccinology. Furthermore, tackling a challenge as the control of a disease causing high impact in

different agricultural and ecological sectors requires the contribution of hunter associations, farmers

and non-governmental organisations promoting the conservation of the natural environment. Thus,

representatives of each sector will be involved in this project as operational groups, allowing the best

use of the different outcomes of the project, as well as their wide dissemination.

Regarding Technology Readiness Level (TRL), we expect to reach at the best a TRL6 level by

initiating some pre-clinical studies at the very end of the project (c.f. Section 1.4).

National and international research activities will be linked to this project, working synergistically to

fill in gaps on the knowledge of this disease/pathogen and to improve the development of innovative

products more rapidly and fully validated. This is the case, for example, of the European project

VetBioNet, in which partners 1 and 3 are collaborating in order to produce new diagnostic reagents

for the development of more robust immunoassays. In addition, the Coordinator is currently enrolled

in project “+Coelho” to control GI.2 in Portugal and that involves the detection and characterisation

of circulating strains in wild rabbit populations, the development and testing of adequate conservation

measures and knowledge transfer to stakeholders. Coordinator and partners 3-6 are involved in the

European project ANIHWA ECALEP. Aims of this project are: a) the study of the emergence and re-

emergence of pathogenic lagoviruses, notably by exploring the hypothesis of a species jump involving

introduction of a reservoir host species, and b) the search for the genetic determinisms of

pathogenicity. Partners 3 and 4 are currently involved in the project “Evolago15-17” that investigates

the genetic evolution of lagoviruses in French rabbit and hare wild populations, updates the diagnostic

tools and detects viral emergences.

Meetings and workshops with other existing related initiatives are planned in order to identify

synergies and possible areas of collaboration, having in mind the strategic agendas (e.g. WWF, FAO,

etc), the calls already launched and the gaps still not covered.

(b) Methodology

LAGMED consortium represents an international competence network that is dedicated to increase

the scientific and technical knowledge on the epidemiology of RHD and its aetiological agent RHDV.

To achieve the main objectives, the project comprises six work packages (WP) dedicated to three

different, but complementary areas: Research (WP1-4), Networking (WP5) and Management (WP6).



Page 6 of 61

WP1: Characterisation of the epidemiology of lagoviruses

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.

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).

WP2: Optimisation and validation of detection tools for preventing and controlling RHDV

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.

WP3: Mechanisms of leporids’ protection against GI.2

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



Page 7 of 61

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.

WP4: Definition of biosecurity measures, control and prevention strategy, and intervention

plans

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.

WP5: Communication, dissemination, and training

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.



Page 8 of 61

WP6: Project Management

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.

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.

1.4 Ambition

In this project, we expect to shed light on the epidemiological dynamics and modelling of an animal

disease (TRL1. Basic principles observed). The project should also have an impact on government

decisions of the countries involved in the partnership and although this is not a technological product

per se, the results will be used in a real setting of application by the government agencies responsible

for the biosecurity measures in the target countries corresponding to a high level of the TRL scale

(TRL6-7). We also expect to develop a new concept of RHD diagnosis, by developing more accurate,

sensitive and rapid diagnostic tools (TRL4. Technology validated in the lab). Notably, this is expected

to prevent new outbreaks and mitigate disease impact and mortality. A strategic part of this proposal

is the development of more efficient vaccines against GI.2 (TRL2. Technology concept formulated).

This new product might allow the implementation of new services in already existing companies,

increasing economic growth.

In addition, LAGMED will provide training opportunities to form scientists and technical staff with a

solid background in different fields of RHD research as epidemiology, animal experimentation,

virological and serological diagnosis, vaccine assessment or biosecurity and intervention plans.

2. Impact

2.1 Expected impacts

O1. Epidemiology and surveillance

In this objective, we will perform a characterisation of the epidemiology of lagoviruses in different

countries of the Mediterranean area. This will determine the current extent and impact of RHD, along

with the identification and distribution of susceptible species and their role in disease dissemination

and persistence, particularly as spillover agents for domestic animals. This information will be crucial

for the implementation of adequate biosecurity measures to contain and limit the spreading of

infection according to the region. The applicability of this knowledge to rabbitries/local farms will

increase rabbit production for domestic and industry markets and generate economic growth.

O2. Optimisation and validation of technical tools for better control of RHDV

One of the main goals of the topic in which this proposal fits into is the development of preventive

actions for better control of pathogens and mitigation of disease impact. With this objective we expect

to develop more effective, accurate, sensitive and rapid detection tools. Such tools will allow the



Page 9 of 61

application of biosecurity measures at early stages of an outbreak in order to contain the dispersion of

the disease and limit biodiversity and economic losses. As part of the preventive actions, we expect to

develop vaccines against GI.2 that will reduce transmission, but more importantly, control mortality.

O3. Analyses of RHDV-host interactions; induction and regulation of immune responses

Host-pathogen co-evolution leads to an arms race between host resistance and pathogen evasion

mechanisms. Since lagoviruses cannot be propagated in cell culture, identification of host resistance

mechanisms will have to rely on indirect techniques such as inoculation trials. With this objective we

expect to characterise the immune response triggered upon GI.2 infection and use this knowledge for

vaccine design.

O4. Biosecurity measures, control and prevention strategy

One of the major goals of the current proposal is to identify the most adequate and efficient

biosecurity measures and prevention strategies to control the disease. We expect to produce a manual

of procedures and develop a specific intervention plan to prevent and/or control RHD outbreaks in

order to minimise their negative impact both in the field and in rabbit production systems, particularly

in North Africa.

O5. Project Management, Mediterranean networking and Technology transfer

Scientific findings will be communicated to the scientific community, but also to national sanitary

authorities, breeders, environmental and hunting associations, central veterinary offices, the wildlife

management and conservation agencies, and academic institutions, empowering them with knowledge

to take the most adequate biosecurity and conservation measures. Training and mobility of young

researchers/post graduates/post doctorates, especially from African countries, will be promoted.

Ultimately, this training might lead to the implementation of new companies in African countries

dedicated, for example, to vaccine production, creating new employment opportunities.

2.2 Measures to maximise impact

(a) Dissemination and exploitation1 of results

Draft “Plan for the dissemination and exploitation of the project’s results”

LAGMED is fully committed in ensuring the maximum possible outreach of the project results’ and

interaction with public and private sectors. For that purpose, it is considered within work package 5

(WP5) “Communication, dissemination, and training”, with the aim to disseminate information both

to project partners, stakeholders, and to a broader audience. In order to better exploit the project’s

results, we will rely on previous existent collaborations between the different partners and

government, academia and industry, but collaboration with other sectors that might benefit from the

project’s results will also be sought. WP5 will ensure that there will be a comprehensive plan for

dissemination of results and activities from LAGMED both at the international and national level.

Dissemination activities will be coordinated by CIBIO/InBIO by the currently existing

Communication Coordinator in collaboration with the Communication Coordinators of the other

research centers. These activities will be designed considering the guidelines established in the book

“Science Communication in the World: Practices, Theories and Trends”, such as defining key

messages; establishing target audiences; selecting the appropriate ways of communication; adequate

information to the intended outlets; building of good relationships with the media; evaluating results;

maximising the exposure of messages; and appointing useful European Commission and other

external resources.

1 See participant portal FAQ on how to address dissemination and exploitation in Horizon 2020



Page 10 of 61

During the project

Dissemination activities will identify (i) the scientific results with high innovation potential, (ii) the

definition of further research and/or training needs, (iii) the limitations for the use of knowledge

(ethics, costs, standards), (iv) the potential commercial exploitation of the outcomes (applications,

tools, services, markets, etc.). The results will be evaluated and discussed in the annual meetings

involving all partners, and will be reflected in the Communication and Dissemination Plan (WP5),

which will be set up and updated during the project. Knowledge generated in the project will be

documented through the yearly reports and policy recommendations, especially in the form of the

project deliverables and through papers in international peer-reviewed journals. It will be also

disseminated via conferences, workshops, educational materials, interchange of scientific events

either organised by or with the participation of the consortium partners.

Project partners will also be encouraged to engage in public activities and education actions. This will

include the participation in science fairs and school events focusing on engaging public opinion

towards science, and to this project in particular.

Regional and national exhibitions and fairs are the main events used by the agri-food sector to interact

within the sector and with the society. Therefore, participation in such events will be encouraged in

order to present the project and its potentialities of R&D&I to the sector. The Knowledge

Dissemination Officers might further auscultate the sector regarding the main challenges and the

possibility of establishing partnerships to test or develop products or services originating from this

project.

LAGMED will also take advantage of other communication channels for dissemination actions in line

with the project, such as:

- The creation of a website integrated into the existing research centers’ main websites for the

diffusion of the information generated in the project and to facilitate the transfer of knowledge and

know-how. This website will target a wide audience and will explain the project, its aims and updates;

- Press releases, videos, interviews, articles in magazines on new achievements by the partners’

Communication offices, using national and international news and media outlets;

- Creation of brochures and flyers about the disease, contingency measures, etc;

- Organisation of conferences, workshops and advancing training focusing on epidemiology,

immunology, virology, molecular biology tools for diagnostic, prevention and control, etc. These

events are particularly relevant for (PhD) students and researchers participating in this project or

studying similar themes elsewhere;

- Project participants will be encouraged to participate and present research results at seminars,

national forums, international conferences and science-policy meetings.

We will always give high visibility to EU’s support and funding. A mention to the PRIMA program

will be included in every presentation and publication elaborated by the partners. We will review the

progress of the project against the deliverables and milestones established. This will provide a “living

set of metrics” to track project progress. In particular we will focus on R&D&I indicators that will

reflect the social and economic impact of the project. Indicators for measuring the dissemination

activity will be based on the number of scientific and technological publications, number of

publications addressing the general public (e.g. websites, TV appearances, press releases), number of

publications addressing decision makers or public authorities, number of PhDs generated in

collaboration with the project, and number of people trained. The website of LAGMED will include

links to other social networks (e.g. Facebook, Twitter, etc).

After the project

Exploitation of results will be managed as outlined in the Communication and Dissemination plan.

CIBIO/InBIO will ensure a coherent strategy, which will take into account each partner’s interests and

role. Special attention will be paid to find synergies between academic and business interests, aiming

to promote the hybridization of elements from academia, industry and government. For example, by

creating/providing new services (MAbs, ELISA, diagnostic tools) for the industry, or new policy

recommendations for the Government. It is expected that members of the consortium pursue the

Communication and Dissemination plan even after the project conclusion. The exploitation of results

will focus on innovative ways of applying gathered knowledge to face societal challenges related to



Page 11 of 61

food security and sustainable agriculture and forestry. Therefore, the following actions are planned to

occur after the project completion:

- Calibrate and maintain the developed Communication and Dissemination plan to pursue the

dissemination of the results while also highlighting the application of these results as used by the

industry, government agencies or NGOs to promote sustainable development;

- Use established international network of collaborations to apply to international competitive

funding, namely to future framework programmes, ERC, Marie-Curie grants, etc.

- Benefit from the contacts established along the project development to foster new collaborations

with industry partners, while further strengthening ongoing relationships with the development of

innovative and tailored tools and applications for the partners’ needs;

- Take advantage of partnerships established throughout the project with private partners to promote

economic development, especially in African countries, by improving productivity and

competitiveness in the agricultural sector (OECD (2012), Agricultural Policies for Poverty Reduction,

OECD Publishing; https://read.oecd-ilibrary.org/agriculture-and-food/agricultural-policies-for-

poverty-reduction_9789264112902-en#page9).

Management of research data

LAGMED aims to participate in the Open Research Data Pilot in Horizon 2020, following the

procedures and approaches set in the “Guidelines on Data Management in Horizon 2020”

(http://ec.europa.eu/research/participants/data/ref/h2020/grants_manual/hi/oa_pilot/h2020-hi-oa-data-

mgt_en.pdf), and thereby adopting the best EU practice in terms of data research management. Also,

the project will adopt the best practices in research data management, following the “OECD

Principles and Guidelines for Access to Research Data from Public Funding”

(http://www.oecd.org/sti/sci-tech/38500813.pdf). This includes making our data FAIR (findable,

accessible, interoperable and reusable) and, as part of that, the preparation of a detailed Data

Management Plan (DMP) within the first six months of the project. The DMP will detail what data the

project will generate, how it will be exploited and made accessible for verification and reuse, and how

it will be curated and preserved. Briefly, the following aspects and principles will be considered:

i. LAGMED will contribute to generate large amounts of primary research data, including DNA

sequences from RHDV and the European rabbit, serology data, etc.

ii. All data collected and generated by LAGMED will be subject to validation and quality control by

the databases curators, following standardised procedures.

iii. All data sets will be documented with detailed metadata, including information on both field

procedures (e.g. locality, date and methods of sampling, and names of collectors) and lab procedures

(e.g. equipment used, procedures adopted, and names of analysts).

iv. The project will use Darwin Core (http://rs.tdwg.org/dwc/) as the main body of standards to

document biological diversity data.

v. LAGMED will deposit data in specialised research repositories and take measures to make it

possible for third parties to access, mine, exploit, reproduce and disseminate – free of charge for any

user – the data (and associated metadata) needed to validate the results presented in scientific

publications as soon as possible, and all the other data following the conditions and deadlines

established in the DMP.

vi. DNA sequence data will be deposited in GenBank (http://www.ncbi.nlm.nih.gov/genbank).

Knowledge management and protection

We will implement a Knowledge Management System (KMS), including an intranet communication

platform for sharing of knowledge, materials, methods and results among partners to ensure project

fluency. Each partner’s leader will guarantee the communication between and within participants of

the project. The KMS infrastructure will guarantee accessibility to all partners as well as security,

since the platform will be protected against illicit uses (through a firewall, and secure communication

lines). In the intranet, there will also be information relevant for the management and networking,

including meeting minutes, templates and guidelines for internal reports, progress reports and

deliverables.

All results and reports generated will be freely available to other researchers and stakeholders after

project completion, as long as it does not interfere with yet unpublished results. All produced

https://read.oecd-ilibrary.org/agriculture-and-food/agricultural-policies-for-poverty-reduction_9789264112902-en#page9

https://read.oecd-ilibrary.org/agriculture-and-food/agricultural-policies-for-poverty-reduction_9789264112902-en#page9

http://ec.europa.eu/research/participants/data/ref/h2020/grants_manual/hi/oa_pilot/h2020-hi-oa-data-mgt_en.pdf

http://ec.europa.eu/research/participants/data/ref/h2020/grants_manual/hi/oa_pilot/h2020-hi-oa-data-mgt_en.pdf

http://www.oecd.org/sti/sci-tech/38500813.pdf

http://rs.tdwg.org/dwc/



Page 12 of 61

scientific papers will be published according to the “gold” model of open access publications,

meaning that papers will only be submitted to open access journals or hybrid journals that allow the

open access publication through the payment of fees.

Management of Intellectual Property Rights (IPR) will be in accordance with Annex H of the H2020

Grant Agreement (GA) and regulated by the terms based upon the DESCA 2020 Model Consortium

Agreement, using the Module GOV LP for small projects. The main principles for the management of

intellectual property (IP) are as follows:

- Results shall be openly disseminated in parallel with appropriate protection of IP to encourage

adoption of the Project’s scientific outputs. In event of publication, reasonable notification and

deferral procedures to ensure IP protection in accordance with the standard GA will be followed;

- Background IP shall remain the property of the party bringing it to the Project. Access rights to

Background IP shall be granted on the following basis: 1) for Project use (‘for implementation’) a

non-exclusive, royalty-free license for that purpose only; 2) for exploitation (‘for use’) a non-

exclusive, non-sub-licensable license to Background IP on fair and reasonable terms shall be given

from the owner of the Background to another party that needs it to exploit its own Foreground IP;

- Foreground IP (results of the project) shall be owned by the party creating it. If more than one party

is involved, such ownership shall be joint, in the respective proportions and the assumption shall be

that either party can exploit / ‘use’ the joint IP provided that it gives the other notice and the other

party does not object;

- Access rights to Foreground IP shall be granted as follows: on a royalty-free basis to each other

party for the sole purpose of the Project. For exploitation, where a party needs another’s Foreground

to exploit its own Foreground, then a non-exclusive, non-sub-licensable license on royalty-free terms

shall be granted;

- Standard provisions for Access Rights for any identified Affiliated Entities and IP ownership and

Access Rights terms for withdrawing and joining parties shall be in accordance with standard H2020

terms incorporated within the DESCA model agreement.

(b) Communication activities 2 3

LAGMED will also develop a special focus on communicating research outputs to a range of

intermediate and end users through an iterative and multi-directional process involving relevant

stakeholders for the planning, implementation, monitoring and evaluation of the project results and

processes. Therefore, the following activities are proposed:

- Contacts with potential end users will be established for the production of monoclonal antibodies,

the vaccine and other outputs of the projects considered relevant for the economic sector.

- Attendance at conferences and meetings will be helpful in establishing partnerships with industry

companies interested in developing and/or producing these deliverables;

- Attendance to fairs and other events where potential users are present. The potential stakeholders

will be industry companies in need of biodiversity sustainability, governmental decision-makers and

NGOs. These institutions represent the main potential end users of approaches and tools developed

under LAGMED;

- Organisation of thematic Workshops and Conferences to strengthen the research community and

industry partners at a regional level;

- Public presentation of the main results on the application of tools and research by project partners,

public or private; these events aim to further attract collaborations from the private sector while

demonstrating the value of the products developed (vaccines, diagnostic tools) as a cost-efficient

approach to address conservation needs.

The Communication activities will be further detailed by the Communication and Dissemination Plan,

which will be prepared within the first six months of the project, and reviewed and updated at month

18.

2See participant portal FAQ on how to address communication activities in Horizon 2020

3 For further guidance on communicating EU research and innovation for project participants, please refer to the

H2020 Online Manual on the Participant Portal.



Page 13 of 61

3. Implementation

3.1 Work plan — Work packages, deliverables

LAGMED is structured around six interconnected work packages (WPs) and tasks, which are

designed to improve RHD control and minimise its economical and ecological impact in the

Mediterranean basin. Indeed, the European rabbit is an important meat source, as well as one of the

major small game species in several European countries. Emergence of GI.2, that affects young and

adult rabbits and hare species, reinforces the need of continuous surveillance, the development of

highly reliable diagnostic tools, the search for more adequate prophylactic therapeutics and the

existence of robust intervention plans for prevention and control of the disease. The management,

coordination and supervision of actions and activities of the project are assured by WP6 (Project

management), which aims to build good work relationships between the partners’ institutions. WP5

(Communication, dissemination and training) has also a critical importance in project organisation

and will be conducted throughout the project lifetime to assure that LAGMED is actively

disseminated into the target audiences. Moreover, this work package will upgrade the research

capacity and capability of all partners, in particular those from African countries, by expanding the

human potential and fostering a critical mass of researchers with interdisciplinary expertise. Work

packages 1 to 4 are strongly interconnected being directed towards the project’s main goal. Work

package 1 will focus on disease surveillance and epidemiology in the Mediterranean basin by

monitoring leporid populations and characterising circulating lagoviruses. The collected information

will be used on WP2 to develop, optimise and validate highly sensitive, specific and low-cost

diagnostic tools. This will improve disease diagnosis that will contribute to efficiently control the

spread of the disease when facing an outbreak. On WP3, we will coordinate efforts to develop novel

vaccines that surpass current welfare and biosecurity concerns associated with the existing ones.

Knowledge gathered from WP1, 2 and 3 will be used for WP4 which will allow setting up and testing

biosecurity measures and defining intervention plans. For a schematic representation of work package

flow, see Figure a. The timeline of the project is represented in Figure b. For work packages

descriptions and task details, please refer to Tables 3.1a and b. The main deliverables are provided in

Table 3.1c.

Figure a: Pert diagram depicting the organisational structure of LAGMED work packages (WP).



Page 14 of 61

Figure b: Timeline of the work packages, tasks and main deliverables of the LAGMED project. The timing of the deliverables is represented by an x.

Project title: Improvement of preventive actions to emerging LAGoviruses in the MEDiterranean basin: development and optimisation of methologies for pathogen detection and control

Acronym: LAGMED

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

WP1: Characterisation of the epidemiology of lagoviruses

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

1.2. Genotyping of viral isolates detected in the collected tissue samples x

1.3. Analyses of the serological status of leporid populations x

WP2: Optimisation and validation of detection tools for preventing and controlling RHDV

2.1. Validation of optimised diagnostic assays for detection of virus x

2.2. Development and validation of immunassays to monitor serological antibody response in the Mediterranean basin x

2.3. Geographical identif ication of circulating lagoviruses x

WP3: Mechanisms of leporids' protection against GI.2

3.1. Characterisation of innate immune response x

3.2. Characterisation of adaptive immune response x

3.3. Eff icacy of novel vaccines for protection of rabbits against RHDV variants circulating in the Mediterranean basin x x

WP4: Definition of biosecurity measures, control and prevention strategy, and intervention plans

4.1. Biosecurity measures and rabbit management x

4.2. Intervention plans x

WP5: Communication, dissemination and training

5.1. Communication and dissemination x x x x x

5.2. Training of scientists and technicians x x x

WP6: Project management

6.1. Scientif ic, administrative and financial management x x x x x

6.2. Management of intellectual property rights and technology transfer activities x

6.3. Database and collection of data x

1st report 2nd report final report

Year 1 Year 2 Year 3



Page 15 of 61

Table 3.1a: List of work packages

Work

package

No

Work Package

Title

Lead

Participant

No

Lead

Participant

Short Name

Person-

Months

Start

Month

End

Month

1

Characterisation

of the

epidemiology of

lagoviruses

1 CIBIO/InBIO 79.9 1 32

2

Optimisation

and validation

of detection tools

for preventing

and controlling

lagoviruses

4 ANSES 48.6 5 36

3

Mechanisms of

leporids’

protection

against

lagoviruses

2 INIA 53.9 10 36

4

Definition of

biosecurity

measures,

control and

prevention

strategy, and

intervention

plans

7 IZSLER 29.4 4 32

5

Communication,

dissemination

and training

1 CIBIO/InBIO 36.6 1 36

6 Project

management 1 CIBIO/InBIO 22.4 1 36

270.8

Table 3.1b: Work package description

Work

package

number

1 Lead beneficiary Coordinator – CIBIO/InBIO

Work

package

title

Characterisation of the epidemiology of lagoviruses

Participant

number 1 2 3 4 5 6 7 8 9

Short name

of

participant

CIBIO INIA UCO ANSES ONCFS INRA IZSLER ENMV ENSV

Person

months per

participant

11.2 3 20 4 0.5 4 4.5 7.7 25

Start

month 1 End month 32



Page 16 of 61

Objectives

RHD is one of the most devastating viral diseases of animals, responsible for more than 90% of

mortality within 2-3 days from infection and with an extreme ability of transmission also at long

distances. The risk of a new emergence of RHDV of unknown origin, linked to the European area, is

not negligible given that two different emergences have occurred in last 30 years. Therefore, the

main objective of WP1 is to increase the scientific and technical knowledge on the epidemiological

characteristics of the disease and its aetiological agent that can have a relevant impact on the

Mediterranean ecosystem. In order to address this concern, the approach will be to study the

distribution of susceptible species in Mediterranean area, to identify the presence of lagoviruses in

such populations and to determine the prevalence, extent and impact of the disease in the different

countries. This includes a characterisation of the circulating strains and the identification of

susceptible hosts, with an evaluation of their role in disease dissemination and persistence,

particularly as spill over agents for domestic animals.

Data gathered within this WP will provide the foundations for WP2, 3 and 4.

Description of work (where appropriate, broken down into tasks), lead partner and role of

participants

Task 1.1. Collection of rabbit/hare samples from the different geographical areas under study (M1-

M30).

Task Leader: Carlos Rouco

Partners involved: coordinator, partners 1-2, 4, 6-8

We intend to determine hotspots of outbreaks and identify susceptible leporid species (rabbits and

hares), particularly in North Africa. We will start by elaborating standardised protocols for sample

collection and storage. We will agree on the rules of the codes (for uniformisation and easy access)

and the information that should be registered for each sample (e.g. year, country, species, etc).

Samples (liver, spleen, duodenum, etc) will be collected from deceased leporids (rabbits and hares;

Task 1.2). Blood samples will be collected from live leporids either from rabbitries or from rabbits

trapped in the field to determine the population serostatus (Tasks 1.3); blood will be drawn from the

marginal ear/jugular vein. We will also take advantage of hunting activities to collect tissues and

blood (Task 1.2 and 1.3). Samples will be directly collected on site by some partners as well as

through existing collaborations with local administrations, hunting and farmers associations

(Federación Madrileña de Caza, ANPC, Fencaça, CNPC, etc), regional authorities (Centro de

Recuperación de Especies Silvestres Comunidad de Madrid, etc), producers associations/integrated

companies (e.g. Dr. Chakroun Chehid from Interprofessional Group of Poultry and Cuniculture

Products, Tunisia; Association des cuniculteurs algériens, Algeria), ongoing monitoring projects

(e.g. +Coelho in Portugal; SAGIR in France), or in collaboration with practitioners operating at

slaughterhouses. We expect to collect between 50-100 samples/year/country. We will also make an

inventory of already available samples (tissues and sera) at the consortium partners’ institutions. We

will choose representatives for the different Mediterranean countries epidemiological situation for

further analyses. In parallel, field surveys will be carried out in rabbit farms that had been

confronted with RHD to obtain background information on the management and sanitary programs

of the breeding. The surveys aim to gather information on the following: i) global information about

the breeding management (density, type of diet, origin of the rabbits, etc), ii) history of disease

problems (mortality rates, clinical signs, lesions, etc), iii) vaccination protocols and sanitary

measures, and iv) biosecurity measures. Information collected will be used for Tasks 4.1 and 4.2.

Task 1.2. Genotyping of viral isolates detected in the collected tissue samples (M4-M32).

Task Leader: Joana Abrantes

Partners involved: coordinator, partner 3, 5-8 Samples collected in Task 1.1 will be first screened by ELISA [partner 6], since this is a lowcost

assay with no interference from non-pathogenic lagoviruses. All the pathogenic lagovirus isolates

sent to IZSLER will be further tested by using a typing ELISA based on the use of approximately 45

specific MAbs. MAbs are all characterised in terms of quantity and specificity towards reference

strains. A string of results (antigenic profile) will be made available for each isolate where the MAb



Page 17 of 61

reactivity is classified as negative, low, medium, fully reactive. Comparison of the antigenic profiles

with the reference strains will provide a global picture of the pathogenic lagoviruses present in the

field in the years of the project. If during the epidemiological study non-pathogenic lagoviruses are

identified, a few of them will be selected based on their epidemiological or phylogenetical interest

and will be expressed as VLPs to study their profile. VLPs will be produced by partners 6 or 1.

ELISA positive samples will then be screened by conventional PCR [Coordinator, partners 3-7] for

genotyping, using conserved primers designed according to lagovirus genomes. These primers are

routinely used by different partners for sample screening, including partner 6 which is the OIE

Reference Laboratory for RHD. Extraction of RNA from liver samples and synthesis of cDNA will

be performed with commercially available kits according to manufacturers’ protocols. PCR-positive

samples will be sequenced with amplification primers to genotype the strains. Finally, relevant

genomes will be obtained by genome-walking or NGS as described elsewhere (Lopes et al. 2018; Le

Gall-Reculé, unpublished results) [coordinator, partners 3, and 5].

Recent studies have pinpointed a role of recombination in the evolution of GI.2. Therefore,

evolutionary analyses will be performed to determine recombination events (recombination

detection programs; phylogenetic reconstruction by Maximum-likelihood (ML) according to

detected recombination breakpoints) and possible origins of the outbreaks (ML trees; Network);

identification of virulence markers will be attempted by sequence comparison with moderately

pathogenic and non-pathogenic strains [Coordinator, partners 3 and 5]. We expect to obtain between 10-20 genomes/year, but these numbers might be adjusted based on

the genotyping PCR.

Task 1.3. Analyses of the serological status of leporid populations (M4-M30).

Task Leader: Lorenzo Capucci

Partners involved: coordinator, partners 1, 6

A serological surveillance will be conducted to monitor circulation of RHDV based on the titration

of specific antibodies. For this, rabbit and hare sera collected in Task 1.1 will be tested using

multiple ELISA methods [Partners 1 and 6]. First, the sera will be tested in two competition ELISAs

(cELISA) using GI.1 (RHDV) and GI.2 (RHDV2) as antigens, and by an indirect ELISA assay

using VLPs derived from those viruses. Combination of the two cELISAs allows establishing if a

serum is negative or positive and if positive to assign titer (cELISA are a semi-quantitative test) and

indicating if antibodies were induced by GI.1b-d/GI.1a or GI.2. In addition, a subset of sera will be

tested for IgM and/or IgA anti-GI.1/GI.2, with IgM as a marker of recent infection, and IgA, if IgM

negative, as a marker of reinfections. Since the circulation of non-pathogenic lagoviruses might

interfere with the conclusions drawn on RHD circulation, alternative methods developed by partner

6 within Task 1.2 will be also used. Altogether, this serological data will be of great help to obtain a

detailed epidemiological picture in terms of protection of the different geographic areas included in

the project.

We expect to test ~1500 field samples/year/country (Portugal, Spain), ~2000 samples from

rabbitries/year/country (Italy, Tunisia, Algeria) and ~500 samples already available from France.

Lopes, AM et al. (2018) Full genome sequences are key to disclose RHDV2 emergence in the

Macaronesian islands. Virus Genes 54:1-4.

Deliverables (brief description and month of delivery)

D1.1.1. Field surveys (M8)

D1.1.2. Biobank of leporid and lagovirus samples (M30)

D1.2.1. Manuscript on the epidemiology of lagoviruses in the Mediterranean basin (M32)

D1.2.2. Manuscript on the virus phylodynamics in the Mediterranean basin (M32)

D1.3.1. Report on leporid populations serological status (M30)



Page 18 of 61

Work

package

number

2 Lead beneficiary Partner 3 – ANSES

Work

package title

Optimisation and validation of detection tools for preventing and controlling

RHDV

Participant

number 1 2 3 4 6 7 8

Short name

of participant CIBIO INIA UCO ANSES INRA IZSLER ENMV

Person

months per

participant

7.5 9 10 11.3 4 5.5 1.3

Start month 5 End month 36

Objectives

The main goal of WP2 is to deliver novel analytical tools and techniques that allow a timely

response to contain RHD. Indeed, such tools should allow the application of more adequate

biosecurity measures at early stages of an outbreak in order to contain the dispersion of the disease

and limit biodiversity and economic losses. Within the LAGMED consortium, this WP has as

technical objective to provide advances to both virological (RT-PCRs, ELISAs) and serological

(ELISAs) diagnosis of RHDV. For this, the following key issues will be addressed:

i) Design of more accurate, rapid and sensitive molecular diagnostic tools, capable of monitoring

the spread and the impact of lagoviruses.

ii) Standardisation of diagnostic assays, to increase the efficiency of detection and strain typing of

RHDV viruses circulating in the Mediterranean basin.

iii) Validation of the diagnostic sensitivity and specificity in comparison with assays considered

the gold standard for RHDV detection.


participants

Task 2.1. Validation of optimised diagnostic assays for detection of virus (M5-M32).

Task leader: Ghislaine Le Gall-Reculé/Joana Abrantes

Partners involved: coordinator, partners 2, 3, 5, 6

Since the emergence of GI.2, different research groups developed their own diagnostic methods for

its detection in each country (capture ELISA, RT-PCR, RT-PCR-sequencing, real-time RT-PCR).

In several countries, molecular studies showed that GI.2 quickly recombinated with GI.1b or GI.4

viruses that co-circulate in rabbit populations and that some have spread outside the country of

emergence. Therefore, there is a need to develop and validate sensitive tools that can detect and

discriminate the viral genotypes and/or the emergence of a novel recombinant virus. The genomes

obtained in WP1 will be used to design novel molecular diagnostic tools for a more accurate, rapid

and sensitive RHDV detection. Different approaches will be explored, including capture ELISA

(cELISA) to test liver samples, multiplex RT-PCR and real-time RT-PCRs [Coordinator, partners

2, 3, 5, 6]. Standardisation of the methods should to help to increase the efficiency of detection and

strain typing of RHD viruses circulating in the Mediterranean basin [linked to WP5 training

activities and technology transfer]. Sensitivity and specificity of the different methods will be

determined first by testing in viral samples with known number of viral copies. Testing of the

methods on serial dilutions of these samples will further allow us to determine the detection limit.

The selected method(s) will be validated in blind samples. Indeed, validation will be achieved in

samples from: i) live animals collected in experimental studies; ii) animals captured within rabbit

control operations [partner 2]; iii) wild rabbits collected in epidemiological surveys. The best

method should allow a rapid and sensitive diagnostic and should allow testing non-invasive

samples (blood, saliva, faeces) which might be very useful to forecast outbreaks and contain the

disease. Furthermore, they should allow monitoring the spread of lagoviruses, evaluating the



Page 19 of 61

impact on wild rabbit and hare populations, and updating the control measures for rabbit farms if

necessary. Finally, the selected method(s) shall be used to efficiently test environmental samples

from rabbit farms (e.g. wipers, airlock entries, air ducts, etc) after decontamination to guarantee

they are lagovirus-free.

Task 2.2. Development and validation of immunoassays to monitor serological antibody response

in the Mediterranean basin (M5-M32).

Task Leader: Esther Blanco

Partners involved:1, 3, 6, 7

A proficiency test will be done based on a panel of rabbit and hare sera representative of different

epidemiological situations. Panels of reference sera will be produced against specific RHDV

variants [partners 3, 6] and will be used for the validation of the different assays [partners 1, 6].

Serological immunoassays developed by partners 1 and 3 in collaboration with INGENASA

(SME), in the framework of VetBioNet project, will be validated in this task as diagnostic tools for

surveillance of lagoviruses currently circulating in the Mediterranean basin regions under study.

In order to detect antibodies anti-hare calicivirus (HaCV) in hares, new ELISA methods will be

developed based on MAbs anti-HaCV already produced towards HaCV expressed VLPs

baculovirus [partner 6]. Similarly, a new ELISA specific for European non-pathogenic strains

(GI.4; Le Pendu et al. 2017) will be developed, but in this case, new MAbs will be produced

against GI.4 VLPs already produced following the traditional methods at IZSLER [partner 6].

Diagnostic sensitivity and specificity will be evaluated also in relation to the ability of different

methods to distinguish antibodies specifically induced by different lagoviruses. Indeed, regardless

of the infecting lagovirus, in rabbits and hare sera there is always present a subset of “common”

antibodies that present cross-reactivity with all lagoviruses. These antibodies interfere at different

levels in ELISA tests in relation to the type of the methods used.

Task 2.3. Geographical identification of circulating lagoviruses (M8-M36).

Task Leader: Joana Abrantes/Lorenzo Capucci

Partners involved: coordinator, partners 2, 3, 5, 6

Unpublished observations suggest some antigenic variation among geographically distinct GI.2

isolates. Recently, in some countries, highly pathogenic GI.2 strains have been detected (Capucci

et al. 2017; Le Gall-Reculé, unpublished results). Therefore, the antigenic profile of a collection of

viral strains representing different Mediterranean epidemiological situations (see Task 1.3) will be

carried out using a panel of specific MAbs developed and characterised by partner 6 against GI.2

France 10-32 isolated in 2010 and GI.2 from north Italy in 2014 responsible to higher mortality

rates in rabbits.

Furthermore, on the basis of the data collected within Task1.3, the probability of finding new GI.2

subtypes (i.e. GI.2 with variation in the main antigenic determinants) is quite high. Therefore,

“new” GI.2 subtypes will be inoculated in rabbits in order to produce sufficient virus to be purified

and produce a new set of MAbs as described above.

From the viral genomes obtained in Task1.2, patterns of disease transmission based on Single

Nucleotide Polymorphism (SNPs) that distinguish different populations of GI.2 will be analysed.

The combination of SNPs and the antigenic profiles will constitute tools for the identification of

the source of future outbreaks and patterns of disease transmission. This information gains further

relevance in the context of rabbit production systems where rabbits are imported or translocated

with no sanitary control.

Cappuci, L et al. (2017) Increased pathogenicity in rabbit haemorrhagic disease virus type 2

(RHDV2). Vet Rec 180: 426.

Le Pendu, J et al. (2017) Proposal for a unified classification system and nomenclature of

lagoviruses. J Gen Virol. 98:1658-1666.


D2.1.1. Standardised sensitive molecular tools for lagoviruses diagnostic, including for non-

invasive samples (M32)



Page 20 of 61

D2.2.1. Immunoassays for lagoviruses serological surveillance (M32)

D2.3.1. Tools for geographical ID of lagovirus strains (M36)

Work package

number 3 Lead beneficiary Partner 1 – INIA

Work package

title Mechanisms of leporids’ protection against GI.2

Participant

number

1 2 4 6 7

Short name of

participant CIBIO INIA ANSES INRA IZSLER

Person months

per participant 6.9 15 17.5 13 1.5


Objectives

WP3 aims to advance the scientific and technical state-of-the-art of RHDV-host interactions by the

study of host immune responses, including innate and adaptive immunity, in animals infected with

lagoviruses. These studies will allow gaining insight on the mechanisms of immune response with a

relevant role in protection (or susceptibility) and, in particular, in heterologous protection. In this

regard, a further objective of this WP is the development and assessment of alternative vaccines to

those commercially available, which are based on inactivated infectious viruses obtained by

amplifying the RHDV directly in rabbits. Such novel vaccines will contribute to control the disease

in the Mediterranean area more efficiently (better adaptation to the specificity of rabbit production

in North Africa, easier administration, lower interaction with maternal antibodies for GI.2 vaccine,

and lower cost). In addition, manufacturing of these vaccines will better respect ethical

considerations.

This WP combines both basic and applied research and will contribute for WP4.

WP3 will take advantage of the unique reagents and technical resources (i.e. RHDV isolates, RHDV

mAbs, BSL2/3 laboratories and animal facilities, immunological equipment as FACS, ELISPOT

readers, Multimode microplate reader, NGS-Platform, etc) that are already available at the

LAGMED partners.


participants

Task 3.1. Innate immune response (M13-M36).

Task leader: Joana Abrantes

Partners involved: partners 1, 3, 5

Previous studies on the pathogenesis of RHD highlighted the importance of the innate immune

response in different outcomes of the RHDV infection. Indeed, adult animals usually die in less than

3 days, a too early timing to mount an antigen-specific response (Marques et al. 2010, 2012, 2014;

Teixeira et al 2012). VLPs have been shown to induce innate immune responses, probably because

they resemble infectious virus particles, which might have implications for vaccine design (e.g.

Neutra et al. 2006). Thus, naïve rabbits will be experimentally infected with VLPs or inactivated

virus particle of selected strains. Experimental immunisation will be conducted at partners 1, 3 or 5

in BSL2/3 animal facilities. The number of rabbits to be used will take into account the Principle of

the 3Rs following a statistical approach. Indeed, the number of rabbits will be calculated according

to a 5% significance level, a 90% statistical power and a 25% minimum effect of the treatment

taking into account the anticipated variation effect of 20. Animals will be monitored daily and will

be housed in cages appropriate to the species behavior, with food and water ad libitum. A

standardised template will be created for compiling information and results on the samples collected

on the experimental infections. Characterisation of the innate immune response will be achieved by



Page 21 of 61

tracing the profile of inflammatory biomarkers (ILs, IFN type I, TNF-alpha, etc). For this, serum

samples will be recovered periodically and the profile of the cytokines elicited early after infection

will be analysed with commercially available ELISA kits.

The number of rabbits and immunisation challenges will be dependent on final approval from the

respective Ethical Commitees. Nevertheless, we will use a minimum of VLPs from four-five strains

in at least 4 rabbits per strain/challenge.

Task 3.2. Adaptive immune response (M13-M36).

Task leader: Esther Blanco

Partners involved: coordinator, partners 1, 3, 6

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 T cell responses to homologous and heterologous RHDV capsid proteins,

following VLP and/or virus particle inactivated immunisation (from Task 3.1). Rabbits will be

monitored every 4h post-infection. Humane endpoints, well established for RHD, will be used to

avoid unnecessary suffering; when observed, animals will be sedated and euthanised. Experimental

immunisation will be conducted at Partner 1 and 3 BSL2/3 animal facilities. We will develop GI.2

specific methods to evaluate T-cell responses. For instance, the production of T cell effector

cytokines such as IFNγ (by ELISPOT) and the proliferation of RHDV specific T cells after in vitro

re-stimulation with VLPs or recombinant polypeptides of VP60 domains (S, P1 and P2) [Partner

1,3]. 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, IgA) elicited in immunised rabbits will be tested using different

type of ELISAs [Partners 1, 6].

Task 3.3. Efficacy of novel vaccines for protection of rabbits against RHDV circulating in the

Mediterranean basin (M11-M36).

Task Leader: Stéphane Bertagnoli

Partners involved: 1, 3, 5 and 6

Recombinant VLPs from RHDV of different genotypes (GI.1-4) and chimeric recombinant

myxoma-RHDV vaccines will be generated by partners 1 and 5, respectively. It should be noted that

an efficient recombinant myxoma–RHDV (GI.1) vaccine is already available on the market, but

cross protection with other genotypes is not documented so far. The experimental design and trials

will be performed at partner 3 and 5 facilities, in accordance with EU Guidelines for animal welfare,

and following the recommendations described in the specific monograph 2325 of the European

Pharmacopoeia. These procedures will be evaluated by ethical committees (c.f. section 5.1). Clinical

signs and mortality will be monitored after vaccination and after challenge. Blood samples,

biological fluids or faeces will be collected at day 0 (before vaccination) and at several time points

before challenge to determine the presence of antibodies against RHDV. For this purpose, different

types of ELISAs will be used, including isotype ELISAs for the detection of specific IgM, IgG and

IgA isotypes [Partner 6].

Before assessing the protection conferred by the vaccines developed, pilot experiments will be

carried out [Partners 1, 3 and 5] to evaluate the immunogenicity (using the assays described in the

Task 3.2) of vaccine formulations and protocols, using different adjuvants as Montanide, TRL

ligands (i.e MPL, Imiquimod, poly-ICLC, CpG-ODNs) or others, to define vaccine dose

concentration and vaccination schedule (one dose with or without boost). In the case of the

Myxoma-RHD vaccine, in vitro assays will be carried out previously to confirm that such adjuvants

do not interfere with the adequate replication of myxoma virus [Partner 5]. Furthermore, we will

explore the feasibility to immunise by an alternative route to the subcutaneous administration, as

intradermal, oral or ocular, depending on the adjuvant selected.

Marques, RM et al. (2010) Early acute depletion of lymphocytes in calicivirus-infected adult rabbits.

Vet Res Commun 34:659-668

Marques, RM et al. (2012) Early inflammatory response of young rabbits attending natural



Page 22 of 61

resistance to calicivirus (RHDV) infection. Vet Immunol Immunopathol 150:181-188

Marques, RM et al. (2014) Immunosuppression abrogates resistance of young rabbits to Rabbit

Haemorrhagic Disease (RHD). Vet Res 45:14

Neutra MR, Kozlowski PA. (2006) Mucosal vaccines: the promise and the challenge. Nat Rev

Immunol 6:148-158

Teixeira, L. et al. (2012) Regulatory T cells are decreased in acute RHDV lethal infection of adult

rabbits. Vet Immunol Immunopathol 148:343-347


D3.1.1. Manuscript on the innate immune response against lagoviruses (M36)

D3.2.1. Manuscript on the adaptive immune response against lagoviruses (M36)

D3.3.1. Vaccines formulation and delivery settings (M24)

D3.3.2. Efficacy of vaccines developed to protect against RHD and/or Myxoma (M36)

Work package

number 4 Lead beneficiary Partner 7 – IZSLER

Work package

title

Definition of biosecurity measures, control and prevention strategy, and

intervention plans

Participant

number 3 5 7 8 9

Short name of

participant UCO ONCFS IZSLER ENMV ENSV

Person months

per participant 7 1 5 1.4 15


Objectives

RHD is one of the most devastating animal diseases causing significant mortalities and transmitting at

long distances. While GI.2 associated mortalities seem to be lower than those caused by GI.1 strains,

it has the ability to kill other leporid species and rabbits younger than 2 months. However, at present,

there is still no cure for RHD. Therefore, disease surveillance, preventive measures and control are the

main strategies to reduce the incidence and minimise the impact of RHDV outbreaks. Disease

surveillance will be improved with the development of more accurate, rapid and sensitive diagnostic

tools (WP2), but there is still the need to set up and test biosecurity measures (e.g. novel vaccines,

WP3) to prevent and control occurring outbreaks, especially in African countries. Effective measures

contribute not only to the prevention, but also to the avoidance of emergency measures. The major

goals to be pursued within this WP are:

1) to identify the most adequate and efficient biosecurity measures and prevention strategies to

prevent the disease;

2) to define the strategies of intervention during an outbreak to reduce and limit the diffusion of the

disease.


participants

Task 4.1. Intervention plans (M4-M32).

Task Leader: Antonio Lavazza

Partners involved: 2, 4, 6-8

The intervention plan already set up and employed in European countries for controlling RHD

outbreaks will be reviewed and adapted to the local conditions of farms in North Africa. Results from

the field surveys conducted within Task1.1 will be used to better identify rabbit farms with RHD

problems and to obtain background information on the farm management.The foreseen measures of

biosecurity, as well as disinfection, cleaning, pest control will be properly tailored to local conditions

by adapting those already reported in the documents set up in EU countries. In particular,



Page 23 of 61

environmental, cultural and technical aspects related to the different areas of intervention will be

considered in the definition of the standards of intervention. Definition of biosecurity measures,

control and prevention strategy will be decided by consent through a meeting gathering partners 2, 4,

6, 7, 8. Information will be gathered in a Manual and will include:

1. detailed operations and measures that should be taken to prevent (biosecurity measures), control

(type of vaccine to be used and protocols of vaccination) and solve (extinction) an outbreak,

2. the type of laboratory tests (e.g frequency of virological test and serological checks of non-

vaccinated sentinels) that should be run to check the persistence of RHDV in the environment,

3. the management procedures to be used (delay slaughtering, carcass and manure removal, cleaning

and disinfection, vaccination, etc). The manual will be completed by a flux diagram indicating responsibilities and competence of

different people involved in the management of the outbreaks. The contents of the manual will be

shared and agreed with the policy makers and then illustrated and explained to all the stakeholders

(WP5).

Task 4.2. Biosecurity measures and rabbit management (M6-M32).

Task leader: Antonio Lavazza

Partners involved: 2, 4, 6-8

Investigation of the efficiency of defensive and offensive sanitary measures adapted to the

particularities of North African rabbitries, notably their small size (often hutches and farms not over

200 does). We will perform virus detection and serological characterisation of rabbits for rabbit

production systems, along with a systematic monitoring of virus circulation in the faeces, cages,

before and after cleaning disinfection (according to tools developed within WP2). Due to their likely

role in virus dissemination, we will perform virus detection in passive/indirect vectors.


D4.1.1. Manual of procedures (M12)

D4.1.2. Specific intervention plan to prevent and/or control RHD outbreaks in the field and in rabbit

production systems (M12)

D4.2.1. Report on the effectiveness of the defined procedures for disease outbreak management

(M32)

Work

package

number

5 Lead beneficiary Coordinator – CIBIO/InBIO

Work

package

title

Communication, dissemination and training

Participant

number 1 2 3 4 5 6 7 8 9

Short name

of

participant


Person

months per

participant

5.1 3 5 5.5 1 2 2.5 1.5 11


Objectives

The overall goal of WP5 is to ensure proper networking coordination, dissemination, exploitation and

technology transfer of results generated by the project to key stakeholders. Another objective of WP5

is to provide technician, graduate and post-graduate students training opportunities to acquire

experience on interdisciplinary knowledge on the RHD epidemiology and its etiological agent. This



Page 24 of 61

WP will also aims at organising a database to collect and render accessible the samples collected

and/or generated during the project as well as the data generated.


participants

The creation of research networks including multidisciplinary teams, established in different countries

is of great importance for the achievement of the objectives proposed in this project. Proper

coordination will be essential for efficient collaboration and improve the knowledge generated on

RHD. Thus, this WP will be supervised by Coordinator and will involve all partners. Communication

and dissemination (Task 5.1) will be supported by the existing CIBIO’s Knowledge Dissemination

Officer (KDO) in collaboration with existing management structures from the other partners. Training

will involve all partners (Task 5.2).

Task 5.1. Communication and dissemination (M1-M36)


Partners involved: Coordinator, partners 1-8

In this task, a report outlining the communication and dissemination strategy of the project will be

elaborated (c.f. Section 2.2). This report will be prepared within the first 6 months and updated at

month 18. In the report, we will identify the target audiences and the mechanisms of interaction. The

communication and dissemination strategy will include the organisation and participation in

international conferences and meetings/workshops for knowledge sharing, dissemination and

networking. Indeed, partners will participate in national and international meetings which will be used

as platforms to disseminate new knowledge, tools and techonologies generated within LAGMED to

the scientific community, either through oral presentations or posters. We will also define a

publication strategy of the outputs obtained within LAGMED in peer-reviewed journals. Particular

attention will be given to the mandatory Open Access policy requested in H2020 work programme.

Novel sequence data will be submitted to international nucleotide databases and will be freely

available for the scientific community. One important aspect of the work is the interaction with the

media and the general public to ensure a continuous flow of information and an ability to reach a

broader audience. For this, we will create a website integrated into the existing research centers’ main

websites for the diffusion of the information generated in the project and to facilitate the transfer of

knowledge and know-how. We will further rely on existing contacts with the central veterinary

offices, the wildlife management and conservation agencies, academic institutions, national sanitary

authorities, breeders and hunting associations. Elaboration of newsletters, reports or technical sheets

are expected and will enable stakeholders to access to broader relevant information generated within

LAGMED.

Task 5.2. Training of scientists and technicians (M1-M36)



A relevant objective of LAGMED is to strengthen scientists and technicians training on diagnosis,

molecular epidemiology, biosecurity and disease management. For that, the mobility of staff, in

particular young researchers and technicians will be encouraged, enabling them through traineeships

to acquire skills on particular assays, use of equipment available on consortium research labs, and

other issues.

Expanding our training capabilities out of consortium by building partnerships with various

institutions (universities, NGOs, Professional associations) will be also important. This will be

achieved by the willingness of the consortium laboratories and research teams to receive external

researchers who need specific training in the target areas of this project.

We plan to offer in our training programme, in addition to specific experimental techniques,

workshops on diagnosis and technology and disease management. Workshops are expected to have

duration of 2 to 2.5 days, combining both theoretical and practical classes. The following workshops

are envisioned:

- Workshop 1: Diagnostic tools for RHD (following WP1+WP2 outputs)



Page 25 of 61

- Workshop 2: Biosecurity measures against RHDV (according to the manual produced on WP4)

- Workshop 3: Vaccination against GI.2 (according to WP3 results)

The coordinator will be responsible for Workshop 1 which will be held at CIBIO/InBIO facilities;

partners 2 and 6 will coordinate Workshop 2 which will be held at IZSLER facilities; partners 1, 3 and

5 will be responsible for Workshop 3 which might be sponsored by biotechnology companies.

Workshops might be converted as webinars that could be disseminated by internet and social media.

This teaching and training material will be useful to communicate research achievements of

LAGMED project to researchers but also to particular stakeholders and society.


D5.1.1. Communication and Dissemination Plan – version 1 (M6)

D5.1.2. Communication and Dissemination Plan – version 2 (M18)

D5.1.3. Media clippings of LAGMED impact (M12, 24, 36)

D5.1.4. Communications in workshops and conferences (M12, 24, 36)

D5.2.1. Reports on researchers exchange (M12, 24, 36)

D5.2.2. Reports of organisation of meetings, workshops and conferences (M12, 24, 36)

Work

package

number

6 Lead beneficiary Coordinator - CIBIO/InBIO

Work

package title Project management

Participant

number 1 2 3 4 5 6 7 8 9

Short name

of

participant


Person

months per

participant

4.3 3 9 2 1 1 1 0.2 0.9


Objectives

The overall management plan is aimed at evaluating the project progresses according to the work

plan. This will be measured against the milestones and deliverables estabilhed. In addition, at the

organisational level, it will optimize the infrastructural setup to support the project, with special

attention to financial, logistic, information and coordination issues. Finally, this WP will ensure that

Intellectual Property Rights of the results produced by the project are preserved and managed under

fair conditions between the partners.


participants

The management will be ensured by Dr. Joana Abrantes (CIBIO/InBIO) as scientific coordinator of

the project. CIBIO/InBIO will be in charge of the overall coordination and will act as the legal entity

responsible for all contractual arrangements and reporting to the EC. She will also keep each

participant updated of the information received from EU contact points.

Task 6.1. Scientific, administrative and financial management (M1-M36)



To ensure that the project is carried out according to the tasks’ description and deliverables, a kick-off

meeting (M1) and three annual meetings will be organised. The dates of these meetings will be

decided during the first meeting. During these meetings the project progress will be reviewed, the

potential problems will be discussed and solved. The coordinator will be responsible for the



Page 26 of 61

organisation and preparation of minutes, as well as for the collection of technical and financial reports

produced by each partner, to check them and submitting deliverables and periodic reports to the EU.

The Data Management Plan will be developed during the first six months of the project, and it will

detail what data the project will generate, how it will be exploited and made accessible for verification

and reuse, and how it will be curated and preserved.

Task 6.2. Management of intellectual property rights and technology transfer activities (M1-M36)



During annual project reports, the most economically valuable results and technologies which can be

protected in a perspective of future commercial exploitation will be identified. Appropriate measures

for protection will then be sought via each beneficiary's technology transfer/intellectual property (IP)

management office. The management of background and foreground intellectual property rights will

be regulated in detail through the "Consortium Agreement" following the guidelines of the EC.

With the techonology transfer offices of each partner, we will identify scientific findings worth of

being transferred for development and commercialization to third parties. The technology transfer

offices will then be responsible for providing advice on the forms of protection and on implementing

suitable protection tools for the IP generated in the project. They will also develop the

commercialisation strategies to private sector companies.

Intellectual Property management throughout the project and exploitation of project results will

follow the principles set out by the Code of Practice for universities and other public research

organisations concerning the management of intellectual property in knowledge transfer activities set

by the 2008/416 EC Recommendation of 10 April 2008 and the H2020 rules regarding intellectual

property set in Section 3 of the General Model Grant Agreement (version 5.0).

Task 6.3. Database and collection of data (M1-M35)

Leader: Joana Abrantes

Partners: Coordinator, partners 1-8

All the samples collected and/or generated by experimental infections in this project will be recorded

in easy standardized templates accessible to all LAGMED partners. Data generated by the project will

be added to such files, ensuring that they are consistently organised and are easily accessible to all

partners of the consortium. Common rules will be decided for the management of data generated by

the project (c.f. Section 2.2). Protocols, manuals and teaching materials will be compiled and stored

on the LAGMED database.


D6.1.1. Minutes of the kick-off meeting (M1)

D6.1.2. Data Management Plan (M6)

D6.1.3. Minutes of the first annual meeting (M14)

D6.1.4. Minutes of the second annual meeting (M26)

D6.1.5. Minutes of the final meeting (M34)

D6.2.1. Main protected results by the IP tools and main technology transfer achievements (M36)

D.6.3.1. LAGMED database (M2)



Page 27 of 61

Table 3.1c: List of deliverables4

Deliverable

(number)

Deliverable

name

Work

package

number

Short

name of

lead

participant

Type Dissemination

level

Delivery

date

(in

months)

D1.1.1 Field surveys WP1 CIBIO/InBIO DEM CO 8

D1.1.2

Biobank of

leporid and

lagovirus

samples

WP1 CIBIO/InBIO O PU 30

D1.2.1

Manuscript on

the epidemiology

of lagoviruses in

the

Mediterranean

basin

WP1 CIBIO/InBIO DEM PU 32

D1.2.2

Manuscript on

the virus

phylodynamics in

the

Mediterranean

basin

WP1 CIBIO/InBIO DEM PU 32

D1.3.1

Report on leporid

populations

serological status

WP1 CIBIO/InBIO R PU 30

D2.1.1

Standardised

sensitive

molecular tools

for lagoviruses

diagnostic,

including for

non-invasive

samples

WP2 ANSES DEM PU 32

D2.2.1

Immunoassays

for lagoviruses

serological

surveillance

WP2 ANSES DEM PU 32

D2.3.1

Tools for

geographical ID

of lagovirus

strains

WP2 ANSES DEM PU 36

D3.1.1

Manuscript on

the innate

immune response

against

lagoviruses

WP3 INIA DEM PU 36

D3.2.1

Manuscript on

the adaptive

immune response

WP3 INIA DEM PU 36

4 If your action is taking part in the Pilot on Open Research Data, you must include a data management plan as a

distinct deliverable within the first 6 months of the project. This deliverable will evolve during the lifetime of

the project in order to present the status of the project's reflections on data management. A template for such a

plan is available in the H2020 Online Manual on the Participant Portal.



Page 28 of 61

against

lagoviruses

D3.3.1

Vaccines

formulation and

delivery settings

WP3 INIA DEM CO 24

D3.3.2

Efficacy of

vaccines

developed to

protect against

RHD and/or

Myxoma viruses

WP3 INIA R CO 36

D4.1.1 Manual of

procedures WP4 IZSLER DEM PU 12

D4.1.2

Specific

intervention plan

to prevent and/or

control RHD

outbreaks in the

field and in rabbit

production

systems

WP4 IZSLER DEM PU 12

D4.2.1

Report on the

effectiveness of

the defined

procedures for

disease outbreak

management

WP4 IZSLER R PU 32

D5.1.1

Communication

and

Dissemination

Plan – version 1

WP5 CIBIO/InBIO R CO 6

D5.1.2

Communication

and

Dissemination

Plan – version 2

WP5 CIBIO/InBIO O PU 18

D5.1.3

Media clippings

of LAGMED

impact

WP5 CIBIO/InBIO DEC PU 12, 24, 36

D5.1.4

Communications

in workshops and

conferences

WP5 CIBIO/InBIO R PU 12, 24, 36

D5.2.1

Reports on

researchers

exchange

WP5 CIBIO/InBIO R CO 12, 24, 36

D5.2.2

Reports of

organisation of

meetings,

workshops and

conferences

WP5 CIBIO/InBIO O PU 12, 24, 36

D6.1.1 Minutes of the

kick-off meeting WP6 CIBIO/InBIO R CO 1

D6.1.2

Data

Management

Plan




Page 29 of 61

D6.1.3

Minutes of the

first annual

meeting


D6.1.4

Minutes of the

second annual

meeting


D6.1.5 Minutes of the

final meeting WP6 CIBIO/InBIO R CO 34

D6.2.1

Main protected

results by the IP

tools and main

technology

transfer

achievements


D.6.3.1 LAGMED

database WP6 CIBIO/InBIO O CO 2

3.2 Management structure, milestones and procedures

3.2.1 Description of the organisational structure and the decision-making

The organisational structure of the project is composed of five main bodies: the Coordinator, the

Governing Council, the External Advisory Board, the Stakeholder Platform and the Technology

transfer offices.

1. The Coordinator, Dr. Joana Abrantes (CIBIO): her primary role in the project is to act as

intermediary between the European Commission (EC) and the Consortium. She will supervise the

overall technical and scientific progress of LAGMED and will be responsible for ensuring smooth-

running of the project, submitting all required progress reports and deliverables and communicating

all information pertaining to the project to the EC. She will be responsible for work plan maintenance,

analysing the results, identifiying the problems and finding solutions with the consortium partners.

2. The Governing Council (GC) is the decision-making body of the project. Chaired by the project

Coordinator, it is composed of one representative per partner, each having one vote for decision-

making. The GC will be responsible for the strategic and political orientation of the project, and re-

orientation whenever necessary. The GC will analyse relevant information provided by the

Coordinator and will analyse the evolution of the context in which the project is carried out to ensure

the relevance of its activities with respect to external changes. Meetings of the GC will be held once

per year, unless the interest of a particular question requires an intermediate meeting, which could be

held by videoconference.

3. The External Advisory Board (EAB) provides an external point of view on how the project

should be conducted to bring maximum outcomes. This EAB will consist of up to three-four

individuals (e.g. representatives of rabbit meat producers, hunter associations, SME, International

experts on RHD emergence, NGOs as WWF/Adena, policy makers, etc). This group will give advice

on changes of the project in line with end-user priorities and on how to maximise the benefits for the

industry. They will review the progress of the project annually and will contribute to the

dissemination of the project results in their respective institutions.

4. The Stakeholder Platform is a list of preferential contacts that will be used to disseminate

information and to invite relevant stakeholders at events. This platform is not an advisory body (i.e.

FAO, OIE, hunter associations, WWF/Adena, INGENASA, rabbit meat producers, EMA, etc). This

group should include representatives from all players interested in the project results and will help to

disseminate information to relevant targets. Their role in the innovation management is quite relevant

since they provide the external view of the market and therefore can provide relevant information

about the products, processes or services that can be required as outputs.



Page 30 of 61

5. Technology transfer offices: these departments from institutions partners in LAGMED will give

advice on Intellectual property and technology transfer aspects, in order to facilitate rapid commercial

exploitation and improve project impact.

In order to keep track of progress made toward the deliverables set out in the project, the following

milestones have been identified (see Table 3.2a).

Table 3.2a: List of milestones

Milestone

number

Milestone

Name

Related

work

package(s)

Due date (in

month)

Means of

verification

MS1.1

Elaboration of standardised

protocols for sample collection,

coding and storage

WP1 2 Internal report

MS1.2

Biobank of leporid and

lagoviruses samples (collected or

archived)

WP1 12 List

MS1.3 Typing of circulating lagoviruses WP1 12 Internal report

MS1.4 Completion of field surveys WP1 8 Report

MS2.1 Selection of a panel of reference

samples for virological detection WP2 9 List

MS2.2 Selection of a panel reference of

sera for immunassays validation WP2 9 List

MS3.1 Selection of the RHDV strains

used for the challenges WP3 12 List

MS3.2 Key factors of the immune

response against lagoviruses WP3 36 Report

MS3.3 Achievement of recombinant

RHDV VLPs WP3 9 Report

MS3.4 Recombinant myxoma-RHD

viruses WP3 9 Report

MS3.5 Choice of relevant adjuvant WP3 12 Pilot

experiments

MS4.1 Consented biosecurity measures,

control and prevention strategies WP4 4 Report

MS4.2 Manual of procedures WP4 12 Report

MS5.1

Final version of the

Dissemination and

Communication Plan

WP5 6, 18 Internal report

MS5.2 Information on the LAGMED

webpage specifications WP5 3 Internal report

MS5.3

Information on personnel

exchange (dates, objectives, host

institution, country, etc)

WP5 6 Internal report

MS6.1 Kick-off meeting WP6 1 Internal report

MS6.2 First annual meeting WP6 12 Internal report

MS6.3 Second annual meeting WP6 24 Internal report

MS6.4 Final meeting WP6 33 Internal report

MS6.5

Selection of preferential contacts

(stakeholders) to disseminate

information

WP6 4 List



Page 31 of 61

3.2.2 Decision-making mechanisms in LAGMED

The Governance structure proposed for the project (Figure c) is appropriate given the complexity and

scale of the project, with a consortium of nine partners. The different main bodies included in the

organisational structure will cover different management issues. The decision-making mechanism

established will ensure that every partner is concerned with the project progress, and that the fair

decisions are taken in cooperation. In summary, these are the decision-making roles in LAGMED:

- Strategic management: GC will be in charge of the political and strategic orientation of the project as

well as the operational management of project activities. The coordinator will supervise the overall

technical and scientific progress and will take care of risk management.

- Administrative and risk management: the coordinator will keep the communication with the EC and

will send regular reports as requested by this organism. The WP management will be carried out by

the WP leaders, which will coordinate the activities as well as progress reporting.

- Innovation management: the stakeholder advisory board will provide input on the most relevant

results that could have interest for the sector. The Transfer technology offices will provide advice on

the management of Intellectual property and knowledge transfer.

Figure c: LAGMED Governance structure.

3.2.3 Innovation management

LAGMED activities include a wide range of innovations with interest for different sectors (industry,

meat producers, government sanitary authorities, enviromental organisations, etc). The Stakeholder

Platform jointly with the External Advisory Board will be essential bodies in the management

structure to foster innovation. They will provide advice in defining if more technological

developments are needed for the application of the new technologies deriving from LAGMED, or in

identifying potential industries with interest in the produced new technologies. It will then be the duty

of the owners of the results to exploit them, either internally or by looking for a third party interested

in the development of a product or service to be commercialised. A third component in the innovation

strategy will be the assistance of the Technology Transfer Offices of each institution partner in this

project. Indeed, they will propose measures to protect Intellectual property as well as appropriate

routes for exploitation.



Page 32 of 61

3.2.4 Critical risks for implementation

LAGMED presents some generic risks and uncertainties inherent to a project involving several

partners. Thus, a thorough risk analysis is needed to mitigate or remove the risks which threaten the

achievement of the project objectives before starting the project with periodical assessments. The

combination of available talent in the current proposal assures the existence of a critical mass able to

tackle challenging problems and offer innovative solutions. In this section, we present generic risks

associated with the project and suggest measures to be adopted for their mitigation. Additional risks

are described in Table 3.2b, which includes the level of risk, the related WP and the contingency plan

foreseen in the project.

A major risk is the delicate economic and geopolitical situation of the African partners that might

compromise the accomplishment of some of the goals of LAGMED. In order to minimise this risk, we

have opted not to include them as task leaders, but they will play a major role in the implementation

of WP4. Indeed, the project has been designed to promote and consolidate collaborations with these

African countries so as to improve their research capacity on RHDV. At the same time, collaboration

with the African teams would allow us to upgrade our knowledge on the epidemiology of RHDV in

Africa and to test and implement biosecurity measures to contain the disease in distinct

epidemiological scenarios. Overall, the success of the project will not be at risk even if sample

collection is not performed in Africa. Indeed, RHD is part of the list of the OIE notifiable diseases and

IZSLER (partner 6) is the reference laboratory to where samples should be sent for testing, including

those from African countries. Regarding the testing and implementation of the biosecurity measures,

this might be achieved by searching for equivalent epidemiological scenarios among the European

partners and attempt extrapolation. In case attendance to workshops for training is compromised,

workshops might be converted as webinars that will be shared with the African partners.

Another major issue is the failure to accomplish the milestones identified for each task, undermining

the completion of the work package. In this case, the tasks should be assessed for feasibility of the

planned results and the activity redirected to try to achieve the deliverables proposed. Intermediate

milestones and specifications have been designed to minimize the risk of delay or failure. The

experimental character of the project limits the risk of arriving at solutions that are not suitable in real

environments, hence assuring that the developed protocols and tools can be reliably used by third

parties. Handling intellectual property might also represent a challenge. A consortium agreement will

be signed before the start of the project and the Technology transfer offices will provide advice on

how to prepare such documents to facilitate the exchange of material preserving the property rights of

each partner. Finally, there might be difficulties in assuring long-term exploitation of the results for

commercial purposes or policymaking. Early-on engagement of potential end-users and stakeholders

and open access option for the data generated within LAGMED will ensure an exhaustive use of the

results, especially beyond the project lifetime. Moreover, most of the partners have an extensive

record of knowledge transfer to key decision-makers both in the private and public sector.

Table 3.2b: Critical risks for implementation

Description of risk (indicate level

of likelihood: Low/Medium/High)

Work package(s)

involved

Proposed risk-mitigation

Measures

Medium - high risk: the success of

this task will depend on the number

of samples sent from the field to

partners involved in laboratory

testing. This is particularly true for

samples coming from the south of

the Mediterranean area. The main

factors that can impair the activity

are 1) the reduced presence or

absence of lagoviruses in these

1

The training proposed in WP5 should

mitigate the lack of knowledge in

sample collection and storage, specially

for countries from the south of the

Mediterranean area



Page 33 of 61

areas, 2) unability to collect samples

due to lack of training, and 3) faible

collaboration between partners and

hunters, industry, etc

Low risk: the development and

validation of diagnostic methods is

partially dependent on sample

collection from all the geographic

areas involved in the project

2

Objectives could be reached with

archival samples, specially from OIE

reference lab collection, and/or with

samples coming from fewer participants

High risk: the development of a

vaccine might be a long-term

objective and depends on several

factors such as: unforeseen failures

in experimental procedures; the use

of a sufficient number of rabbits in

inoculation trials; the use of the best

immunogenic VLP or inactivated

virus particles; the choice of

adjuvant; the collaboration with

specialised companies for vaccine

manufacturing and production; etc

3

An appropriate statistical approach will

ensure robust results in terms of rabbit

numbers to be used; literature review on

immunogenic properties of older RHDV

strains and of adjuvants might shed light

on the best vaccine formulation;

previous experience of the partners

involved in the experimental procedures;

previous collaborations with specialiased

companies from other projects might

assure vaccine manufacturing and

production

Low - medium risk: resistance from

potential investors for investing in

novel diagnostic tools and vaccines

3

Most of rabbit meat producers are not

aware of the added value of novel tools

and prophylactic measures for their

activity. Partners of LAGMED have a

vast recognised international research

experience, which will contribute in

building trust with investors

Medium risk: the measures of

biosecurity, control and intervention

can be effectively verified during

outbreaks of the disease that can not

obviously be predicted. The

validation of the Manual, especially

for the post-outbreak intervention

strategy, could depend from the

number of situations in which it will

be used

4

A network with the stakeholders will be

set up in order to be aware of any

possible outbreak of disease, thus

increasing the number of potential cases

for testing the vailidity of the set up

measures

Low risk: slow and fruitless

communication channels between

project participants and stakeholders

5

The already established KDO of project

participants will provide support to

communication activities and will act as

liason agents to mitigate the lack of

openness by the stakeholders

Low risk: difficulties on the project

management and work package

coordination

6

The project coordinator will ensure that

project milestones are achieved, and

corrective actions will be undertaken if

and whenever appropriate. The existing

links of trust and cooperation among all

partners and the activation of

management procedures as soon as

problems first arise will reduce this class

of risks. The External Advisory Board

shall provide further advice for

addressing unforeseen challenges. This



Page 34 of 61

will be complemented by the periodic

project meetings and reporting from WP

leaders, followed by a tight budget

control

3.3 Consortium as a whole

3.3.1. Consortium’s expertise

Partners of the consortium have a long experience of project management, which assures that all the

tasks will be successfully completed in due time, and along the lines established in the work packages.

Also, the research staff of the research institutions involved in the proposal has a strong track record

of scientific achievements (c.f. Section 4), including the management of large national, EU, and

industry sponsored projects. The staff to be hired by LAGMED will reinforce these capacities, further

contributing to the success of the project. Researcher administrative staff from these instutions will

help in the preparation of the Communication, Dissemination and Exploitation plans and strategies

(WP5), in the development of the Research Program (WP1-4), and in the management of

administrative and financial issues (WP6).

In order to achieve LAGMED’s objectives, we sought and established collaborations to form a

multidisciplinary team. The teams involved can fill-in gaps in the know-how and networking

capacities presently available and each partner’s expertise will be a valuable contribution for the

consortium:

Joana Abrantes (coordinator) is an assistant researcher at CIBIO/InBIO’s group Immunity and

Emerging Diseases. In the last 12 years, she has been studying the immune system of leporids and the

emergence, evolution and epidemiology of RHDV. She developed a genome walking strategy to

obtain RHDV full genomes and demonstrated the importance of recombination for RHDV evolution.

Her expertise on virus evolution and epidemiology, along with her interest on the European rabbit

immune system, are keys for her leadership of LAGMED. Ana Margarida Lopes is a post-doc

researcher at CIBIO/InBIO’s group Immunity and Emerging Diseases. In the last 10 years, she has

been working on lagoviruses, with particular focus on the attachment factors used by these viruses to

initiate their infectious process. She is also interested in virus epidemiology and evolution and she has

experience on lagoviruses molecular detection tools and serology.

Esther Blanco (partner 1) is a research scientist in VACUVET, at CISA from INIA, with more than

20 years of experience in the development of new vaccine strategies and immunoassays for virus

detection. Juan Bárcena is a research scientist in the same unit with more than 25 years of experience

in different aspects of research in virology. He has worked in new vaccine approaches against RHDV,

including a live transmissible recombinant vaccine against RHDV and myxoma virus for wildlife use.

They are a recognised reference in the field of research on VLP-based vaccines for veterinary use.

Carlos Rouco (partner 2) is a wildlife field ecologist researcher at University of Cordoba with

sixteen years of research experience in ecology, epidemiology and wildlife management of small and

medium size mammal populations. His research has particularly focused on the improvement of

habitat management measures for favouring wild rabbits’ conservation in the wild, and on the

epidemiolology of myxomatosis and RHD by trying to understand how population dynamics affect

epidemics.

Ghislaine Le Gall-Reculé (partner 3) is a virology researcher at Anses and has twenty years of

experience in the study of the molecular evolution, epidemiology and control of caliciviruses in

rabbits and hares. She detected and described GI.2 for the first time, performed its characterisation

and followed its spreading throughout France. She currently follows its genetic evolution in French

rabbit populations and contributes to the epidemiology studies aiming the control of the disease in

rabbit breedings.



Page 35 of 61

Stéphane Marchandeau (partner 4) is an agronomic engineer and he is responsible for wild rabbit

management research programs. His research focuses on the myxomatosis/wild rabbit and RHD/wild

rabbit models to understand their impact in wild populations and to improve large-scale management

of this game species.

Stéphane Bertagnoli (partner 5) is a professor of virology at ENVT, and member of UMR INRA-

ENVT IHAP (Interactions hosts-pathogens) virology team. He specifically studies viral infections of

rabbits (myxomatosis, RHDV, Hepatitis E), and has expertise in vaccinology (recombinant poxvirus

vaccines).

Lorenzo Capucci (partner 6) is the expert for the RHD Reference Laboratory of the World

Organisation for animal Health (OIE). He works on the main viral diseases of lagomorphs (RHDV,

European brown hare syndrome and myxomatosis), and has been developing specific immunological

diagnostic techniques for these viruses since 1990. Antonio Lavazza is head of the Electron

Microscopy Laboratory that is part of the Animal Health and Welfare Department (IZSLER). His

main areas of activity include lagomorphs’ viral diseases and epidemiology, rabbit welfare, farming

and pathology, farming biosecurity and hygiene, and wildlife disease monitoring programs.

Lilia Messadi (partner 7) is professor of microbiology and immunology at ENMV and member of

the Research Laboratory “Epidemiology of enzootic infections of herbivorous” (Tunisia). She works

with other colleagues on bacterial and viral diseases of animals. Faten Ben Chehida is an associate

professor with a PhD in virology of small ruminants (capripoxvirus vaccine, PCR, molecular

characterisation, cloning, Western blot, etc).

Hacina Ain Baziz (partner 8) is a professor and researcher on rabbit and poultry breeding at ENSV

(National Veterinary School of Algiers-Algeria) and has thirty-year experience in the research study

of rabbits and poultry nutrition. She has a vast knowledge on the rabbit industry in Algeria. Mouna

Abed is a veterinary doctor and assistant professor at ENSV. She has experience on virus genetic

evolution and has good skills on molecular biology, classical virology and viral sequencing

techniques.

3.3.2. Partners’ contributions for the project

Participation of each partner in the different tasks of the work packages was envisaged according to

the parterns’ expertise and is described below. Figure d presents an overview of the partners’

contributions for LAGMED.

Figure d: overview of the partners’ contributions in the different tasks of the work packages. Darker

colours indicate who will supervise the task, while white boxes indicate no role in the task. JA: Joana

Abrantes; EB: Esther Blanco; CR: Carlos Rouco; GLGR: Ghislaine Le Gall-Reculé; SM: Stéphane

Marchandeau; LC: Lorenzo Capucci; AL: Antonio Lavazza; LM: Lilia Messadi; HAB: Hacina Ain

Baziz.

1.1 1.2 1.3 2.1 2.2 2.3 3.1 3.2 3.3 4.1 4.2 5.1 5.2 6.1 6.2 6.3

1 Coordinator JA 1, 5, 6

2 Partner 1 EB 3

3 Partner 2 CR -

4 Partner 3 GLGR 2

5 Partner 4 SM -

6 Partner 5 SB -

7 Partner 6 LC/AL 4

8 Partner 7 LM -

9 Partner 8 HAB -

TasksParticipant

nr.Short name PI WP leader



Page 36 of 61

Coordinator (CIBIO/InBIO)

WP1

T1.1: established collaborations with national sanitary authorities as well as environmental and

hunting associations will be used to obtain samples from leporids found dead in the field and hunted

in Portugal.

T1.2: will conduct the genotyping and sequencing of complete genome sequences of Iberian GI.2

strains (Sanger sequencing or NGS) and evolutionary analyses of the genomes characterised within

the proposal.

T1.3: will contribute to the sero-epidemiological picture of GI.2 by testing samples from the Iberian

Peninsula.

WP2

T2.1: will contribute to the optimisation and validation of more sensitive virological tools for GI.2

diagnosis. Validation will be achieved in samples of infected wild rabbits collected within studies on

GI.2 epidemiology ongoing at CIBIO/InBIO.

T2.3: will analyse and identify SNPs on GI.2 strains to predict patterns of disease transmission.

WP3

T3.1: will perform the characterisation of the innate immune response from serum samples collected

at different time points from rabbits inoculated experimentally.

T3.2: will contribute for the characterisation of the T cell responses against GI.2.

WP5

T5.1: will supervise the communication&dissemination activities through CIBIO/InBIO’s Knowledge

Dissemination Officer (KDO) in contact with existing management structures from other partners;

dissemination of the main results in at least one manuscript as senior authors; will organise and

participate in national and international congresses.

T5.2: will supervise and promote training programmes, including the preparation of the teaching and

training materials and its dissemination; will organise workshop 1 “Diagnostic tools for RHD”.

WP6

T6.1: will held the kick-off meeting and will prepare the minutes of the meetings; will be responsible

for the collection of technical and financial reports and their communication to the EU.

T6.2: will supervise the identification of economically valuable results and technologies for protection

and the elaboration of the “Consortium Agreement”; will supervise the knowledge transfer activities

of CIBIO/InBIO’s KDO and will be in contact with existing management structures from other

partners.

T6.3: will be the curator of the data and protocols produced within LAGMED that will be made

available to the other LAGMED partners via an internal database.

Partner 1 (INIA)

T1.1: established collaborations with national sanitary authorities as well as environmental and

hunting associations will be used to obtain samples from leporids found dead in the field and hunted

in Spain.

T1.3: will contribute to this task testing sera samples of leporid populations and participating on the

coordination of the analyses and interpretation of the results.

WP2

T2.2 optimisation and validation of an ELISA based on VLPs for an efficient RHDV serosurveillance.

In addition, we will supervise and coordinate the development and optimisation of other assays for

antibody detection as well as the preparation of a panel of reference sera for proficiency test.

T2.3 will contribute by collaborating on the antigenic characterisation of the GI.2 isolates found in

Spain.

WP3

T3.1: part of the experimental immunisation of rabbits with VLPs will be performed in INIA animal

facilities, and part of the samples can be analysed by ELISA for antibody and/or cytokines titrations.

T3.2: will supervise and coordinate the experimental immunisation of rabbits for analyses of adaptive

immune respon. Part of such experiments will be performed in INIA animal facilities. Assays’

procedures for specific evaluation of T-cell response to RHDV will be optimised, as well as IFNg-

ELISPOT or classical lymphoproliferation assays with 3HT.



Page 37 of 61

T3.3: will contribute to experimental design of vaccine assessment, to the analyses of immune

response to RHDV before and after of challenge and to the obtaining of immunogens based on

recombinant VLPs.

WP5

T5.1: presentation of the results in national and international congresses as well as dissemination of

the main results in at least one manuscript. Data and information will be provided to contribute to the

LAGMED webpage creation.

T5.2: will collaborate on training programmes, participating in the exchange of PhD and technicians

and in the organisation of Workshop 3 “Vaccination against GI.2”.

WP6

T6.1: will participate to the LAGMED annual meetings and organise the second annual meeting.

T6.2: INIA technology transfer office will collaborate to the management of intellectual property

rights, giving advice on the most adequate procedures.

T6.3: will provide data to be included in the project database.

Partner 2 (UCO)

WP1

T1.1: collection of rabbit/hare samples, mainly liver, spleen and duodenum samples, and when

possible, blood, from leporids killed through regular hunting season from several different

geographical areas from Spain. Additionally, samples will also be collected from deceased leporids

found by the network of collaborators (e.g. WWF, hunting federations, farmers, etc.).

T1.2: analysis of the epidemiological status and its potential correlation with different biotic and

abiotic variables obtained from samples collected and variables measured during the implementation

of Task 1.1.

WP2

T2.1: will test diagnostic tools developed in this task in samples collected during rabbit control

operations carried out in game emergency zones, to assess its rapid and sensitive diagnosis.

WP4

T4.1: participation on the review of the intervention plans and on the writing of a Manual and

implementation of specific intervention plans to prevent and/or control occurring outbreaks,

particularly in cases affecting African countries.

T4.2: will evaluate the efficiency of defensive and offensive measures; will contribute for the search

of passive vectors of the disease.

WP5

T5.1: participation to the consortium annual meetings and co-organisation of one of them;

presentation of the results in at least one international and one national congress; supervision of a PhD

dissertation based on the analysis of data collected along the project.

T5.2: co-organisation of workshop 2 “Biosecurity measures against RHDV”, in order to communicate

scientific knowledge gathered during the project to transfer best practices to reduce the impact of

RHD in wild rabbit populations and rabbitries.

WP6

T6.1: will participate in the development of the Data Management Plan.

T6.2: will contribute to the elaboration of the “Consortium Agreement”.

T6.3: will contribute with data and protocols produced within LAGMED for the internal database.

Partner 3 (ANSES)

WP1

T1.2: will contribute to the genotyping and the achievement of the complete genome sequences of

relevant French lagovirus strains (Sanger sequencing or NGS) to establish the genetic relationships of

the characterised strains and to perform evolutionary analyses of the genomes.

WP2

T2.1: will contribute to optimising and validating a sensitive virological tool for GI.2 diagnosis; will

validate these tools in samples from live animals collected during experimental studies performed in

ANSES’ BSL2 animal facilities and from infected wild rabbits collected in the framework of an

ongoing study on the GI.2 epidemiology in France.



Page 38 of 61

T2.2: will perform animal experiments in Anses’ BSL2 animal facilities to obtain new specific rabbit

immunsera to optimise GI.2 immunoassays.


WP3

T3.1: a part of the experimental infections with VLPs and/or inactivated viruses will be conducted in

ANSES’ BSL2 animal facilities as well as the titration of viruses before their inactivation

(quantitative real-time RT-PCR method already available).

T3.2: most of the rabbit experimental infections with VLPs and/or inactive vaccines will be performed

in ANSES’ BSL2 animal facilities. The characterisation of the adaptive immune response will be

assessed by developing GI.2 specific methods using ELISPOT and FACS apparatus available in the

ANSES’ Laboratory.

T3.3: a part of the experimental infections with vaccine candidates (VLPs) and their adjuvants will be

performed in ANSES’ BSL2 animal facilities. The immune responses will be measured and the

protection conferred against GI.2 will be experimentally evaluated.

WP5

T5.1: presentation of the results in at least one international and one national congresses; submission

of at least one manuscript in one international peer-reviewed journal with the concerned co-authors;

submission of the obtained French GI.2 sequences into public databases.


training materials and its dissemination; will co-organise workshop 3 “Vaccination against GI.2”.

WP6

T6.1: participation to the consortium annual meetings and organisation of the third annual meeting;

will participate in the development of the Data Management Plan.



Partner 4 (ONCFS)

WP1

T1.1: will make available data collected in France on viral strains involved in epizootics caused by

lagoviruses in wildlife.

WP4

T4.1: will provide advice on the intervention plans based on his experience on wild rabbit

management.

T4.2: will provide advice on the biosecurity measures based on his experience on wild rabbit

management.

WP5

T5.1: will be involved in the elaboration of communication and dissemination materials.

T5.2: will contribute to the preparation of the teaching and training materials for workshop 2

“Biosecurity measures against RHDV”.

WP6

T6.1: participation to the consortium annual meetings.



Partner 5 (INRA-ENVT)

WP1

T1.2: will contribute to the genotyping and the achievement of the complete genome sequences of

French GI.2 strains (Sanger sequencing or NGS) to establish the genetic relationships of the

characterised strains.

WP2

T2.1: will contribute to optimising and validating a sensitive virological tool for GI.2 diagnosis in

samples from live animals collected during experimental studies.




Page 39 of 61

WP3

T3.1: a part of the experimental infections with VLPs and/or inactivated viruses will be performed in

INRA-ENVT BSL2 animal facilities, and part of the samples will be analysed at INRA-ENVT.

T3.3: construction of recombinant myxoma-RHDV viruses, in vitro studies on adjuvants, and part of

the experimental infections with some vaccine candidates and adjuvants will be performed in INRA-

ENVT facilities. The immune responses will be measured and the protection conferred against

homologous and heterologous challenges will be experimentally evaluated.

WP5

T5.1: presentation of the results in at least one international and one national congresses; submission

of at least one manuscript in one international peer-reviewed journal with the concerned co-authors;

submission of the obtained French GI.2 sequences into public databases.


training materials and dissemination; will co-organise workshop 3 “Vaccination against GI.2”.

WP6

T6.1: participation to the consortium annual meetings; will participate in the development of the Data

Management Plan.



Partner 6 (IZSLER)

WP1

T1.1: collection of rabbit and hare samples from the farm/field or from animals involved in

inoculation trials.

T1.2: diagnosis of RHD in rabbits and EBHS and RHD-like disease in hares based on ELISA and the

use of specific anti-lagovirus MAbs panel. The methods will be used for primary diagnosis with

immediate typing (negative or positive results, with differentiation of GI.1, GI.2 and GII.1 as

causative agents) on liver from rabbits and hares collected on the field.

T1.3: will supervise and coordinate the analyses of the serological status of the leporid populations

from the other countries.

WP2

T2.1: optimisation and validation of serology methods for virus detection (cELISA).

T2.2: optimisation and validation of an efficient test to monitor serological antibody response;

development of new MAbs or new ELISAs for novel lagoviruses.

T2.3: will characterise antigenic variation among geographically distinct GI.2 isolates to predict

patterns of disease transmission.

WP3

T3.2&T3.3: IZSLER has BSL3 animal facility where it should be possible to perform experimental

infections with pathogenic lagoviruses which had been often used for RHDV infections of rabbits,

especially for testing viral pathogenicity of GI.2 and other strains. Indeed, this is the main finality

reported in the National authorization for experimental use of rabbits.

WP4

T4.1&T4.2: since 1990 Dr. Lavazza is in direct contact with the field and with breeders and hunting

associations and has a vast experience in the management of rabbit farms (industrial and rural) and of

wild animals. Dr Lavazza will contribute to the construction of a network of relationships among

partners for the aspects of biosecurity measures, control and prevention strategy in farm rabbit

management.

WP5

T5.1: participation to the consortium annual meetings; presentation of the results in at least one

international and one national congress: dissemination of the main results in at least one manuscript as

senior authors.

T5.2: co-organisation of the workshop 2 “Biosecurity measures against RHDV”, in order to

communicate scientific knowledge gathered during the project to transfer best practices to reduce the

impact of RHD in wild rabbit populations and rabbitries.

WP6




Page 40 of 61



Partner 7 (ENMV)

WP1

T1.1: will collect samples from rabbits in several areas of Tunisia for virus and antibodies’ detection.

T1.2: will perform conventional PCR to detect the virus from collected samples and sequencing to

study the phylogeny of the Tunisian strains.

WP2

T2.2: will use immunoassays developed by partner 6 in order to evaluate their use as easy tools for

surveillance of the lagoviruses circulating in Tunisia.

WP4

T4.1: will gather information on the epidemiology of RHDV in Tunisia for the adaptation of the

European intervention plan to the African countries.

T4.2: will test biosecurity measures and supervise their implementation by rabbit breeders.

WP5

T5.1: the results of the epidemiological studies will be presented in the partners’ annual meeting and

in national or international meetings; will also publish the main results in scientific journals and

disseminate scientific reports to the decision-makers of the Ministry of Agriculture, and breeders and

hunting associations.

T5.2: seminars and laboratory training will be organised for young scientists and technicians, to

disseminate detection techniques in order to improve the RHD surveillance program.




Partner 8 (ENSV)

WP1

T1.1: samples’ collection (tissues and blood); field survey to identify rabbit farms that have had

problems with RHD and to obtain background information on the management of rabbit breeding in

some areas in Algeria. This task will be performed with the collaboration of Association des

Cuniculteurs Algériens.

T1.2: will contribute to the first genetic characterisation of RHDV in Algeria by NGS sequencing

with the collaboration of partner 3.

WP4

T4.1: will gather information on the biosecurity measures and vaccination practices in Algerian rabbit

farms (no information exists on viral diseases affecting rabbit farms in Algeria and there are no

official vaccination protocols available to breeders) for the adaptation of the European intervention

plan to the African countries. This task will be conducted in parallel with Task 1.1 by using the

information obtained with the surveys.

T4.2: proposal of adequate and efficient biosecurity measures and prevention strategies for RHD

control taking into account the results from the field surveys; will test biosecurity measures and

supervise their implementation by rabbit breeders.

WP5

T5.1: participation to the consortium annual meetings; organisation of an RHD workshop for

veterinarian and breeders; presentation of the results in at least one international and one national

congresses; publication of the main results in one scientific journal; submission in nucleotide

databases of the Algerian sequences obtained.

T5.2: organisation of training seminars for young scientists, technicians, veterinarians and breeders

with the collaboration of l’Association des cuniculteurs Algériens.






Page 41 of 61

3.3.3. Industrial/commercial involvement

We have already established contact with different industrial companies which will be essential for

the future exploitation of the main results expected for this project. In particular, partners 1 and 3 are

already collaborating with INGENASA in the framework of another project (VetBioNet), for the

optimisation of immunoassays for RHDV serosurveillance. The already established collaboration with

this well-known company in the field of Veterinary diagnosis, and in particular of RHDV (they

produce a kit for RHDV detection), will be quite important to accelerate the marketing of the new

products developed and validated in this project.

Regarding vaccine development, two different companies have been identified as potential

commercial companies interested in the vaccine strategies proposed in LAGMED for a better

prevention of RHDV. We plan to include these companies in the list of Stakeholders as priority in the

dissemination of the results achieved in this project. Under confidentiality agreements, promising

results obtained in LAGMED will be shared with such industrial partners.

3.3.4. Other countries and international organisations

Not applicable.

3.4 Resources to be committed Table 3.4a: Summary of staff effort

WP1 WP2 WP3 WP4 WP5 WP6

Total

Person-

Months per

Participant

1/CIBIO-

InBIO 11.2 7.5 6.9 0 5.1 4.3 35

2/INIA 3 9 15 0 3 3 33

3/UCO 20 10 0 7 5 9 51

4/ANSES 4 11.3 17.5 0 5.5 2 40.3

5/ONCFS 0.5 0 0 1 1 1 3.5

6/INRA-

ENVT 4 4 13 0 2 1 24

7/IZSLER 4.5 5.5 1.5 5 2.5 1 20

8/ENMV 7.7 1.3 0 1.4 1.5 0.2 12

9/ENSV 25 0 0 15 11 0.9 60

Total Person

Months 79.9 48.6 53.9 29.4 36.6 22.4

Table 3.4b: ‘Other direct cost’ items (travel, equipment, other goods and services, large

research infrastructure)

Participant

Number 1/CIBIO-InBIO

Cost

(€) Justification

Travel 8260.40

Participation in scientific

meetings and meetings

with other partners

Equipment 0 n.a.

Other goods and Services 10250 Workshop organisation;

manuscript fees

Total 18510.40



Page 42 of 61

Participant

Number 2/INIA

Cost

(€) Justification

Travel 8000

Scientific meetings;

meetings with other

partners

Equipment 0 n.a.

Other goods and Services 0 n.a.

Total 8000

Participant

Number 3/UCO

Cost

(€) Justification

Travel 18000

Fieldwork and sampling

collection; scientific

meetings; meetings with

other partners

Equipment 4000

Field investigation

materials and gear;

freezer; sampling

collection material

Other goods and Services 5000 Sub-contracting;

workshop organisation

Total 27000

Participant

Number 4/ANSES

Cost

(€) Justification

Travel 8000

National and international

congresses; meetings with

the consortium partners,

including 3 abroad

Equipment 0 n.a.


Total 8000

Participant

Number 6/INRA-ENVT

Cost

(€) Justification

Travel 8600 Meetings with the other

consortium partners

Equipment 0 n.a.

Other goods and Services 2000 Scientific congresses

Total 10600

Participant

Number 7/IZSLER

Cost

(€) Justification

Travel 5000


meetings with other

partners; field



Page 43 of 61

investigations

Equipment 0 n.a.


Total 5000

Participant

Number 8/ENMV

Cost

(€) Justification

Travel 10000


meetings with other

partners; internships

Equipment 0 n.a.

Other goods and Services 8000

Workshop organisation;

publication fees; practical

training workshops

Total 18000

Participant

No 9/ENSV

Cost

(€) Justification

Travel 8000


meetings with other

partners

Equipment 40000

Field investigation

materials (FTA card,

cooler, syringes, dry

tubes, etc);

ultracentrifuge;

micropipettes; PCR

thermocycler

Other goods and Services 2000 Other costs for field

investigation

Total 50000

4. Members of the consortium

4.1. Participants (applicants)

Coordinator (Participant number 1)

CIBIO/InBIO

CIBIO/InBIO's mission is to develop world-class research in the area of biodiversity. Its research

groups, based in several universities and research institutes across Portugal and in 1 university in

Angola, focus their activity on biodiversity and ecology, evolutionary biology and applied ecology,

and integrate experts in complementary fields, such as molecular and population genetics,

phylogeography, population biology, immunogenetics, taxonomy, ecology, bioinformatics

andcomputational biology, landscape management and conservation.

Since 2009, CIBIO/InBIO has participated in ~300 projects, both as a coordinator and a partner,

funded by the Portuguese Science and Technology Foundation (FCT) and Horizon2020.

CIBIO/InBIO is involved in training programmes ranging from undergraduate to post-doctoral levels,

being the main host institution of the Doctoral Programme in Biodiversity, Genetics and Evolution

and the Biodiversity, Genetics and Evolution Masters Course, by the Faculty of Sciences, University

of Porto.



Page 44 of 61

The Centre has a focused Knowledge and Technology Transfer strategy, providing services to the

community through the autonomous facilities of the Centre for Studies and Projects in Applied

Ecology (GEPE) and the Centre for Molecular Analysis (CTM). It also has a group dedicated to

science communication and outreach for disseminating scientific results, raise biodiversity awareness

and promote scientific culture. Both the Knowledge and Technology Transfer and the Communication

and Dissemination officer will be crucial for Tasks 5.1 and 5.2 (WP5).

CIBIO/InBIO is fully equipped with state-of-the-art laboratories, including NGS facilities (HiSeq

1500 high throughput sequencer and cBot, and MiSeq desktop sequencer, both from Illumina). These

laboratories are equipped with PCR and quantitative real time-PCR machines, a spectrophotometer, a

sequencer (3130xl Genetic Analyzer, Applied Biosystems, for Sanger sequencing), a nanodrop,

microscopes, etc, allowing for several molecular biology techniques (e.g. molecular cloning, PCR,

RT-PCR, qRT-PCR, Western Blot, ELISA, electrophoresis, DNA and RNA extraction, sequencing,

immunohistochemistry). This equipment and infrastructures guarantee the execution of Tasks 1.2, 1.3

(WP1) and 3.1 (WP3).

CIBIO/InBIO has classrooms, conference rooms, and an auditorium with a capacity for 250 people,

fitted with cabins for translation, projection and sound engineering. There are 10 fully equipped

double bedrooms and a canteen with daily service. These facilities are relevant for Task 5.3 (WP5).

Curriculum vitae

Name: Joana

Surname: Abrantes

Gender: female

Date of birth: 14 January 1982

Nationality: Portuguese

Position:

Auxiliary researcher at CIBIO/InBIO in the group Immunity and Emerging Diseases

Education:

2004: Degree in Biology, Faculty of Sciences, University of Porto

2010: PhD in Biology, Faculty of Sciences, University of Porto

Professional experience:

19.12.2013-present: Auxiliary researcher at CIBIO/InBIO, University of Porto

01.04.2011-18.12.2013: Post-doc researcher at CIBIO/InBIO, University of Porto, Portugal and

INSERM/Université de Nantes

01.03.2011-31.03.2011: Research technician at CIBIO, University of Porto, Portugal

01.01.2007-31.12.2010: PhD Student at CIBIO, University of Porto, Portugal and INSERM,

Université de Nantes, France

01.12.2005-31.12.2006: Research technician at CIBIO, University of Porto, Portugal

01.01.2005-30.11.2005: Research technician at CBA, University of Lisbon, Portugal

Main research interests: host-pathogen co-evolution; viral diseases; virus evolution; virus

epidemiology; immunogenetics.

Publications: Sixty-four published papers in the fields of virology, veterinary and epidemiology (e.g.

PLoS Pathogens, JVI, EID, Veterinary Microbiology, etc), being first author in 11 and

senior/corresponding author in 10.

Supervisions: one PhD student (concluded in June 2016) and one MSc student (concluded in

December 2016); training and support to technicians, MSc and PhD students, and post-docs of the

group Immunity and Emerging Diseases.

Projects: PI or researcher in 10 research projects, funded through Portuguese FCT national and

international calls (e.g. FCT-ANR joint call), the Portuguese government, and also through European

Union calls (ANIWHA).

Peer-review activity: associate Editor of BMC Veterinary Research (Virology section) since 2015;

peer review activity in international journals: PLoS Pathogens; Emerging Infectious Diseases;

Transboundary and Emerging Diseases; Veterinary Research; Virus Research; Infection, Genetics and

Evolution; PLoS One; European Journal of Wildlife Research; Research in Veterinary Science;

Journal of Integrative Agriculture; Journal of Wildlife Diseases.

FELASA Category C Laboratory Animal Course concluded in 2016.



Page 45 of 61

Other: organised the “1st European/Australian RHDV workshop” in 2015 that was held at

CIBIO/InBIO; currently part of the organising committee of the “2018 TIBE: Host-parasite

interactions” that will be held in December 2018 at CIBIO/InBIO.

Invited speaker at the 16th Congress of the Polish Society of Experimental and Clinical Immunology

(2017), Warsaw, Poland, and member of an international PhD thesis jury at University Pablo de

Olavide, Seville, Spain (2018).

Her research has attracted national and international media coverage (e.g. National Geographic,

Portuguese TV programs like Biosfera) and she has been involved in dissemination of knowledge

through invited communications in seminars.

Curriculum vitae

Name: Ana Margarida

Surname: Lopes

Gender: female

Date of birth: 31 August 1988

Nationality: Portuguese

Position:

Post-doc researcher at CIBIO/InBIO in the group Immunity and Emerging Diseases

Education:

2009: Degree in Biology, Faculty of Sciences, University of Porto

2016: PhD in Biodiversity, Genetics and Evolution, Faculty of Sciences, University of Porto and

Faculty of Sciences, University of Lisbon


01.12.2016-present: Post-doc researcher at CIBIO/InBIO, University of Porto, Portugal, UMIB - Unit

for Multidisciplinary Research in Biomedicine, ICBAS, University of Porto, Portugal and ANSES,

Ploufragan, France

01.03.2016-30.11.2016: Research technician at CIBIO/InBIO, University of Porto, Portugal

01.03.2012-29.02.2016: PhD Student at CIBIO/InBIO, University of Porto, Portugal and INSERM,

Université de Nantes, France

15.06.2010-29.02.2012: Research technician at CIBIO/InBIO, University of Porto, Portugal

01.10.2008-24.02.2009: Internship within the scope of BSc in Biology at CIBIO/InBIO, University of

Porto

Main research interests: host-pathogen interactions using leporids – lagoviruses models; infectious

processes and resistance to viral diseases; immunogenetics; molecular biology; virus impact on the

host; virus evolution.

Publications: Twenty-four published papers in the fields of virology, epidemiology and evolution (e.g.

PLoS Pathogens, JVI, EID, JGV, etc), being first author in 9.

Supervisions: training and support to technicians, MSc and PhD students of the group Immunity and

Emerging Diseases.

Projects: researcher in 6 research projects, funded through Portuguese FCT national and international

calls (e.g. FCT-ANR joint call), the Portuguese government, and also through European Union calls

(ANIWHA).

Peer-review activity: peer review activity in international journals (e.g. Transboundary and Emerging

Diseases).

FELASA Category B Laboratory Animal Course concluded in 2013.

Other: in 2015 she helped in the organisation of the “1st European/Australian RHDV workshop” that

was held at CIBIO/InBIO; currently she is part of the organising committee of the “2018 TIBE: Host-

parasite interactions” that will be held in December 2018 at CIBIO/InBIO. Young Scientist Travel

Award in 2013 promoted by the European Society for Virology. Dissemination of knowledge in

several international scientific meetings.

Publications

1. Silvério D, Lopes AM, Melo-Ferreira J, Magalhães MJ, Monterroso P, Serronha A, Maio E, Alves

PC, Esteves PJ, Abrantes J (2018) Insights into the evolution of the new variant rabbit hemorrhagic



Page 46 of 61

disease virus (GI.2) and the identification of novel recombinant strains. Transboundary and Emerging

Diseases 65:983.

2. Monterroso P, Garrote G, Serronha A, Santos E, Delibes-Mateos M, Abrantes J, Pérez de Ayala R,

Silvestre F, Carvalho J, Vasco I, Lopes AM, Maio E, Magalhães MJ, Mills SL, Esteves PJ, Simón

AM, Alves PC (2016) Disease-mediated bottom-up regulation: An emergent virus affects a keystone

prey, and alters the dynamics of trophic webs. Scientific Reports 6:36072.

3. Lopes AM, Dalton KP, Magalhães MJ, Parra F, Esteves PJ, Holmes EC, Abrantes J (2015) Full

genomic analysis of new variant Rabbit Hemorrhagic Disease Virus (RHDV2) revealed multiple

recombination events. Journal of General Virology 96:1309.

4. Nyström K*, Abrantes J*, Lopes AM, Le Moullac-Vaidye B, Marchandeau S, Rocher J, Ruvoën-

Clouet N, Esteves PJ, Le Pendu J (2015) Neofunctionalization of the Sec1 α1,2fucosyltransferase

Paralogue in Leporids Contributes to Glycan Polymorphism and Resistance to Rabbit Hemorrhagic

Disease Virus. PLoS Pathogens 11:e1004759.

5. Abrantes J, van der Loo W, Le Pendu J, Esteves PJ. (2012) Rabbit haemorrhagic disease (RHD)

and rabbit haemorrhagic disease virus (RHDV): a review. Veterinary Research 43:12.

Projects

- +Coelho “Plano de acção para o controlo da doença hemorrágica viral dos coelhos” as Researcher,

CIBIO/InBIO-UP (ongoing);

- ANIHWA ERA-Net "Emergence of highly pathogenic CAliciviruses in LEPoridae through species

jumps involving reservoir host introduction" as Collaborator, consortium ANSES (France) + ONCFS

(France) + INSERM (France) + IZSLER (Italy) + SVA (Sweden) + ENVT (France) (ongoing);

- PTDC/BIA-ANM/3963/2012 “How does the European rabbit (Oryctolagus cuniculus) immune

system recognise the rabbit haemorrhagic disease virus (RHDV) from its non-pathogenic form RCV-

A1?” as Principal Investigator (PI), CIBIO/InBIO (concluded);

- FCT-ANR/BIA-BIC/0043/2012 “Evaluation of the virulence of RHDV (Rabbit Haemorrhagic

Disease Virus) and mechanisms of host resistance” as Researcher, CIBIO/InBIO (concluded);

- PTDC/CVT/108490/2008 "Host-pathogen interaction: the Rabbit Haemorrhagic Disease (RHD) and

the European Brown Hare Syndrome (EBHS)" as Researcher, CIBIO/InBIO (concluded).

Infrastructures

CIBIO/InBIO NGS facilities are equipped with HiSeq 1500 high throughput sequencer and cBot, and

MiSeq desktop sequencer (Illumina). Laboratories are equipped with PCR and quantitative real time-

PCR machines, a spectrophotometer, a sequencer (3130xl Genetic Analyzer, Applied Biosystems, for

Sanger sequencing), a nanodrop, microscopes, etc, allowing for molecular biology techniques (e.g.

molecular cloning, PCR, RT-PCR, qRT-PCR, Western Blot, ELISA, electrophoresis, DNA and RNA

extraction, sequencing, immunohistochemistry).

Partner 1 (Participant number 2)

INIA

INIA (National Institute for Agricultural and Food Research and Technology) is a public research

organisation of the the Science, Technology and Innovation Ministry. CISA (Animal Health Research

Centre) which belongs to INIA was created in 1993 to enhance the prevention and control of animal

infectious diseases relevant in animal health. This centre has BSL3 and BSL3+ laboratories

specialised mainly in the viral diseases of farm pigs, sheep, fish and game species as rabbit, partridge

and wild boar. In addition, BSL2 facilities include Epidemiology and Risk assessment unit, where the

most relevant animal diseases are analysed. The VACUVET unit is devoted to improve animal

disease control with the development of new vaccines and diagnosis assays. This expertise is

nationally and internationally recognised, and the unit has participated in several networks, projects

and agreements with other governamental institutions, companies and international organisations as

FAO, OIE, etc.

Curriculum vitae

Dr. Esther Blanco (female) has a degree in Pharmacy (Universidad Complutense de Madrid) and has a

background in veterinary virology, diagnosis and vaccinology. She completed her PhD (1999) on the



Page 47 of 61

identification and characterisation of Foot-and-Mouth Disease virus T-cell epitopes and their

application to the development of new vaccine strategies at INIA (Spain) and at Institute of Virology

and Immunoprophylaxis (Mittelhaüsern, Switzerland). As post-doctoral, E. Blanco worked three years

at Pig Improvement Company (PIC) as research coordinator, leading a PRRS project and later she

started to work as researcher funded by “Ramon y Cajal” program, on the development and validation

of immunoassays for detection of African Swine Fever Virus. Currently, she has a permanent position

as Research scientist in the unit of Vaccine Veterinary Development (VACUVET), at Animal Health

Research Centre (CISA) from INIA. E. Blanco has participated in more than 23 projects, being

Principal researcher in 6 National and 4 International projects. The vaccine development performed in

her group has led to the obtaining of two patents on FMDV peptidic vaccine, one of them

(PCT/EP2013/056843) signed by Virbac (France) for marketing in China. The potential impact of

such achievement was recognised in 2014 with the Prize "Isabel Minguez" to best Innovation in

Animal Health.

Dr. Juan Bárcena (male) has a degree in Molecular Biology (Universidad Autónoma de Madrid) and

has been working for more than 25 years in different aspects of research in virology. He carried out

his PhD at the Instituto de Salud Carlos III (Majadahonda, Madrid) working on the molecular biology

of influenza virus. In 1994 he joined the CISA-INIA, where he was part of several research groups

working in the development of new vaccination strategies against different animal health related

diseases. Remarkably, he has worked in different vaccine approaches against RHDV, including

vaccines based on RHDV VLPs, as well as the development of a live transmissible recombinant

vaccine against RHDV and myxoma virus for wildlife use, which was supported by an agreement

between INIA, the National Hunters Federation of Spain and Fundación Biodiversidad (biodiversity

fundation, Ministry of Enviornment). In the last 10 years he has been working in the development of

new vaccine strategies against relevant pathogens in animal health (i.e. influenza A virus, FMDV),

based on engineered VLPs derived from RHDV, as platforms for foreign antigen multimeric display.

Currently, he has a permanent position as research scientist in the unit of Vaccine Veterinary

Development (VACUVET), at Animal Health Research Centre (CISA) from INIA.

Publications

1. Barcena J, Verdaguer N, Roca R, Morales M, Angulo I, Risco C, Carrascosa JL, Torres JM,

Caston JR (2004) The coat protein of Rabbit hemorrhagic disease virus contains a molecular switch at

the N-terminal region facing the inner surface of the capsid. Virology 322:118.

2. Bárcena J, Blanco E (2013) “Design of novel vaccines based on virus-like particles or chimeric

virions”. Structure and Physics of Viruses: An integrated Textbook. Springer. Mauricio G. Mateu

(Ed). Subcell Biochem. 68:631.

3. Bárcena J, Guerra B, Angulo I, Gonzalez J, Valcarcel F, Mata CP, Caston JR, Blanco E, Alejo A

(2015) Comparative analysis of rabbit hemorrhagic disease virus (RHDV) and new RHDV2 virus

antigenicity, using specific virus-like particles. Veterinary Research 46:106.

4. Moreno N, Mena I, Angulo I, Gómez Y, Crisci E, Montoya M, Castón JR, Blanco E, Bárcena J

(2016) Rabbit hemorrhagic disease virus capsid, a versatile platform for foreign B-cell epitope display

inducing protective humoral immune responses. Scientific Reports 6: 31844.

5. Rouco C, Abrantes J, Serronha A, Lopes AM, Maio E, Magalhães MJ, Blanco E, Bárcena J,

Esteves PJ, Santos N, Alves PC, Monterroso P (2017) Epidemiology of RHDV2 (Lagovirus

europaeus/GI.2) in free-living wild European rabbits in Portugal. Transboundary and Emerging

Diseases 65: e373.

Projects

- H2020-INFRAIA 2016-2017, project VetBioNet «Veterinary Biocontained facility Network for

excellence in animal infectiology research and experimentation interest » (2017-2022)

- National Research project MINECO: AGL2016-76445-R.2017-2020 “Modulation of the immune

response elicited by viral pseudoparticles; towards better control strategies of livestock viral

diseases".

- National Research project CAM: S2013/ABI-2906-CM. 2014-2018. Technological platforms for the

development of control strategies for Animal Health. PLATESA.



Page 48 of 61

- National Research project MINECO: AGL2013-48923-C2-1-R.2014-2017. “New strategies of

vaccination against livestock viral diseases using modified pseudoviral particles or VLPs".

- EPIZONE, FP62004Food3A. “Network of Excellence for Epizootic Disease diagnosis and control”.

Infrastructures

CISA (INIA) is the major research institution in Spain equipped with Biological Security Level 3

(BSL3) facilities, devoted to research with exotic infectious diseases affecting large and small

livestock animals. It is classified as SSTI (Singular Scientific and Technological Infrastructure). This

facility has all the equipments required to accomplish the proposed tasks, as well as the compulsory

authorisations and licenses to work with animals and handle pathogenic viruses. These equipments

include Real Time Thermocyclers, ELISPOT reader, Fluostar Omega multimode microplate reader,

FACS Scalibur, FACSAria cell sorter, Cell harvester, Microbeta counter, Zeiss LSM 800 with

Airyscan Confocal microscope, and others.


University of Cordoba (UCO)

University of Cordoba (UCO), with almost 16000 students, is currently top-ranked for research

quality and has an international reputation for research excellence. The UCO based on research

published in international journals with more impact and visibility (I-UGR ranking 2014) is ranked as

the 1st and 5th Spanish university in agrigulture and biological science research, respectively. The

international reputation for research excellence of the UCO results from effective blending of

traditions of a long-established university with up-to-date research facilities. In particular, the research

profile of the Department of Zoology covers a wide range of topics and approaches, consists of a

multidisciplinary group of professional lecturers and researchers with degrees in Biological Sciences

and Veterinary Medicine. Have extensive experience in studies of zoology, applied ecology,

epidemiology and pest control among others, and in the training of young researchers, thus provides a

broad array of expertise that generates a rich working environment. This is enhanced by the fact that

the UCO has an extensive library and on-line access to a large number of international scientific

journals in the areas of ecology, veterinary, agriculture and wildlife management. In addition, the

UCO, offers wide teaching and graduate programs. Finally, in addition to the excellency of the UCO,

its location in Andalucia meets optimal conditions to develop the study proposed here.

Curriculum vitae

First Name: Carlos

Name: Rouco

Gender: male

Date of birth: 28 October 1977

Nationality: Spanish

Position: Tenure track lecturership and Researcher (Wildlife Management) at University of Cordoba,

Department of Zoology, Edificio Charles Darwin (C-1), Campus Universitario de Rabanales, carrtera

N IV, km 395A, 14071, Cordoba, Spain. Tel: 0034957218517, e-mail: [email protected]

Education:

1995-2000: Biological Science degree (University of Seville) 2003-2005: Master in Game Wildlife

Management (National Game Research Institute-University of Castilla-La Mancha) 2003-2008: PhD

in Wildlife Management (University of Castilla-La Mancha).


- June 2008- May 2010: Postdoctoral fellow in the Department of Biodiversity Conservartion at

Doñana Biological Station-CSIC.

- June 2010-March 2012: Contractor Researcher at Wildlife Ecology and Epidemiology Team,

Landcare Research New Zealand Ltd.

- April 2012-June 2015: Postdoctoral fellow at Wildlife Ecology and Management Team, Landcare

Research New Zealand Ltd.

- October 2015-present: Tenure track lecturership and Researcher (Wildlife Management) at

Department of Zoology, University of Cordoba.

- Supervision of 1 PhD.



Page 49 of 61

- Scientific activities: host - virus interactions; Epidemiology of viral infections of rabbits

(myxomatosis, RHDV), wildlife management.

Publications

1. Pacios I, Moreno S, Selman C, Rouco C (2018) Oxidative stress in wild European rabbits naturally

infected with myxoma virus and rabbit haemorrhagic disease virus. European Journal of Wildlife

Research (in press).

2. Rouco C, Abrantes J, Serronha A, Lopes AM, Maio E, Magalhães MJ, Esteves PJ, Santos N, Alves

PC, Monterroso P (2018) Epidemiology of the new variant of rabbit haemorrhagic disease in free

living wild European rabbits. Transboundary and Emerging Diseases 65:e373.

3. Villafuerte R, Ramírez E, Castro F, Parra F, Recuerda P, Cotilla I, Rouco C (2017) Large-scale

assessment of myxomatosis prevalence in European wild rabbits (Oryctolagus cuniculus) 60 years

after first outbreak in Spain. Research in Veterinary Science 114:281.

4. Rouco C, Moreno S, Santoro S (2016) A case of low success of blind vaccination campaigns

against myxomatosis and rabbit haemorrhagic disease on survival of adult European wild rabbits.

Preventive Veterinary Medicine 133:108.

5. Santoro S, Pacios I, Moreno S, Bertó-Moran A, Rouco C (2014) Multi-event capture-recapture

modeling of host-pathogen dynamics among European rabbit populations exposed to myxoma and

rabbit hemorrhagic disease viruses: common and heterogeneous patterns. Veterinary Research 45:39.

Projects

- Spanish Ministery of Agriculture, fisheries and Environment. “Preventing Agricultural damage by

European rabbits” (2018)

- Talent Hub-Andalusian Knowledge Agency, co-funded by the European Union’s Seventh

Framework Program, the Marie Skłodowska-Curie actions. “Rabbits on the road: how human

transportation infrastructures turn key species into pest” (2015-2017).

- Proyecto Intramural CSIC ‘Interaction between parasites and viral diseases with wild rabbit

population dynamic, application for management and recovery programs’ (2008-2010).

Infrastructures

The University of Cordoba (UCO) also has all of the necessary facilities including modern

laboratories and equipment. UCO also has a transfer office of research results

(http://www.uco.es/webuco/otri/). This will facilitate the transfer to the public and the industry of the

main outcomes resulting from the research proposed. Finally, being in Andalucia, the UCO is

optimally located for the development of the current proposal.


ANSES (GLGR)

ANSES (French Agency for Food, Environmental and Occupational Health & Safety) is a

governmental organisation. It has a network of eleven reference and research laboratories operating in

three main areas: animal health and welfare, food safety (chemical and microbiological) and plant

health. These laboratories are internationally recognised in their respective fields of expertise:

epidemiology, microbiology and antimicrobial resistance, toxins and physico-chemical contaminants.

Thanks to their work in providing expertise, epidemiological monitoring, alerts and scientific and

technical assistance, they play a vital role in understanding various threats and in collecting data from

the network of accredited laboratories. The Ploufragan-Plouzané laboratory specialises in the health of

farmed poultry, rabbits, pigs and fish. It contributes to improving animal welfare as well as the health

quality of foodstuffs of poultry, rabbit and pig origin. It is composed of three Experimental

departments, including the “Avian and rabbit breeding and experimental” department, and eight

Research units, including the “Epidemiology and welfare in poultry and rabbit farming” and

“Virology, immunology and parasitology in poultry and rabbits (VIPAC)” units dedicated to avian

and rabbit productions. VIPAC unit studies the emerging diseases and the agents responsible for

diseases with a major economic impact on avian and rabbit productions or on the immune potential of

animals. It develops tools and methods for diagnosis and prevention of diseases in theses animals.



Page 50 of 61

One of the four teams (head: Dr Ghislaine Le Gall-Reculé) is in charge of research and

scientific/technical studies on rabbit and hare caliciviroses (RHDV and EBHSV).

Curriculum vitae

Name: Ghislaine

Surname: Le Gall-Reculé

Gender: female

Date of birth: 4 March 1963

Nationality: French

Position: Senior researcher in virology at Anses, Ploufragan-Plouzané Laboratory, Research unit of

“Virology, immunology, and parasitology in poultry and rabbits” (VIPAC), BP 53, 22440 Ploufragan,

France. Tel: +33 2 96 01 62 98. E-mail: [email protected]

Education:

1986: Master degree in Biological Oceanology, University of Paris VI, France

1988: Graduate (Engineer), Superior Institute of Animal Productions (ISPA), University of Rennes I,

France

1991: PhD in Life Science, University of Paris VI, France


1987-1989: PhD student at IFREMER (French Research Institute for Exploitation of the Sea),

Research unit of Pathology of Marine Invertebrates, La Tremblade, France. Involved in research on

the Rickettsiales-like organisms of the Saint-Jacques scallop Pecten maximus.

1989-1998: Associate researcher at Anses, Research unit VIPAC, Ploufragan, France. Involved in

research studies on Muscovy duck viruses (reovirus and parvovirus): virology, molecular and

antigenic diagnosis, molecular characterisation, vaccination strategies (DNA and VLPs).

1998-present: Researcher at Anses, VIPAC, Ploufragan, France. In charge of research and

scientific/technical support on rabbit and hare caliciviroses (RHD and EBHS): virology, molecular

diagnosis, molecular characterisation, molecular epidemiological survey, phylogeny and viral

evolution, pathogenicity, in vivo experimental studies on rabbits.

1998-2002: also in charge of researches to identify a viral etiological agent in Rabbit Epizootic

Enterocolitis (REE) disease.

2002-2011: also involved as associate researcher in studies on Avian Influenza viruses: molecular

characterisation and vaccination strategies (DNA and VLPs).

Scientific collaborations with national research institutes: CNRS-University of Lyon, CRCINA-

INSERM-Nantes, INRA-ENVT, ONCFS, and international: CIBIO/InBIO-UP (Portugal), CISA-INIA

(Spain), CSIRO (Australia), EMAI (Australia), ISZLER (Italy), NVRI (Poland), SVA (Sweden).

Publications: 157 publications (39 with international referees) in national and international scientific

journals, scientific congresses and popularising journals.

Reviewer in international scientific peer-reviewed journals on virology or animal science (Analytical

biochemistry, Antiviral Research, Archives of Virology, Central European Journal of Biology,

European Journal of Wildlife Research, MEEGID Infection Genetics and Evolution, Journal of

Biotechnology, Research in Veterinary Science, The Veterinary Journal, Veterinary Research, Viral

Immunology, Virology, Virology Journal, Virus Gene, World Rabbit Science).

Publications

1. Le Gall-Reculé G, Lavazza A, Marchandeau S, Bertagnoli S, Zwingelstein F, Cavadini P, Martinelli

N, Lombardi G, Guérin J-L, Lemaitre E, Decors A, Boucher S, Le Normand B, Capucci L (2013)

Emergence of a new lagovirus related to Rabbit haemorrhagic disease virus. Veterinary Research

44:81.

2. Esteves PJ, Abrantes J, Bertagnoli S, Cavadini P, Gavier-Widén D, Guitton J-S, Lavazza A,

Lemaitre E, Letty J, Lopes AM, Neimanis AS, Ruvoën-Clouet N, Le Pendu J, Marchandeau

S, Le

Gall-Reculé G (2015) Emergence of pathogenicity in lagoviruses: evolution from pre-exiting

nonpathogenic strains or through a species jump? PLoS Pathogens. 11: e1005087.

3. Le Gall-Reculé G, Lemaitre E, Bertagnoli S, Hubert C, Top S, Decors A, Marchandeau S, Guitton

J-S (2017) Large-scale lagovirus outbreaks in European brown hares (Lepus europaeus) in France



Page 51 of 61

caused by RHDV2 strains spatially shared with rabbits (Oryctolagus cuniculus). Veterinary Research

48:70

4. Lopes AM, Breimana A, Loraa M, Le Moullac-Vaidyea B, Galaninad O, Nyströma K,

Marchandeau S, Le Gall-Reculé G, Strive T, Neimanis A, Bovind NV, Ruvoën-Clouet N, Esteves PJ,

Abrantes J, Le Pendu J (2018) Host specific glycans are correlated with susceptibility to infection by

lagoviruses, but not with their virulence. Journal of Virology. doi: 10.1128/JVI.01759-17.

5. Lemaitre E, Zwingelstein F, Marchandeau S, Le Gall-Reculé G (2018) First complete genome

sequence of a European non-pathogenic rabbit calicivirus (lagovirus GI.3). Archives of Virology. doi:

10.1007/s00705-018-3901-z.

Projects

Current transnational research projects:

- Animal Health and Welfare ERA-Net call 2, project ECALEP «Emergence of highly pathogenic

CAliciviruses in LEporidae through species jumps involving reservoir host introduction» (2015-2018)

- CoVetLab Join Research 2017, project «Full-genome sequencing of viruses of veterinary importance

- development of sample preparation schemes for NGS» (2017-2018)

- H2020-INFRAIA 2016-2017, project VetBioNet «Veterinary Biocontained facility Network for

excellence in animal infectiology research and experimentation interest» (2017-2022)

Current national research studies:

- Research study Anses/ONCFS, project EvoLago15-17 «Genotyping and sequencing of RHDV and

EBHSV strains circulating in the French populations of rabbits and hares between 2015 and 2017 »

(2015-2018).

- Research study Anses/DGAL/CLIPP, project Epi-RHDV2 «Control of the Rabbit haemorrhagic

disease due to RHDV2 by a deep knowledge of the disease epidemiology in rabbit breedings and

wildlife» (2018-2019).

Infrastructures

VIPAC unit is composed of a BSL2/BSL3 building fully equipped for the handling of pathogenic

animal viruses and for serological, virological (ultracentrifuge) and molecular biology analyses

(thermocyclers, real-time thermocyclers, droplet-PCR, a genetic analyser for Sanger sequencing). The

staff has access to the facilities available in Ploufragan-Plouzané laboratory including a next-gen

sequencing platform with computational and technical assistance, a flow cytometer, an Elispot-

Fluorospot, and BSL2/BSL3 animal facilities equipped for experimental infections on rabbits.


ONCFS

The "Office national de la chasse et de la faune sauvage" (National Hunting and Wildlife Agency-

ONCFS) is a public agency under the shared administration of the Ministry for the Ecological and

Inclusive Transition (MTES) and the Ministry of Agriculture and Food (MAA).

ONCFS ensures the development of the huntable wildlife stocks while respecting their biological

equilibrium. Among its missions, ONCFS carries out studies, applied research and experimentation

for the conservation, restoration and management of wildlife and their habitats both at national and

international levels.

Within the framework of the research carried out on Lagomorphs and in partnership with Anses,

ONCFS ensures a monitoring of pathogenic Lagoviruses circulating in wild lagomorph populations.

Curriculum vitae

Stéphane Marchandeau (male) received a degree as engineer in agronomy in 1986. Since 1993, he is

project leader in ONCFS (National Hunting and Wildlife Agency) in charge of the wild rabbit

management research programs. He is an international expert of the Ministry for the Ecological and

Inclusive Transition (MTES) in the fields of sustainable management of natural resources and

biodiversity. The main part of his research is devoted to the myxomatosis/wild rabbit and RHD/wild

rabbit models to understand the impact of the diseases in wild populations and to improve large-scale

management of this game species. He has developed partnerships with several partners of the



Page 52 of 61

consortium to isolate and describe non pathogenic strains of rabbit lagoviruses and on molecular

mechanism in rabbit/RHDV interaction. He has authored 52 scientific publications in peer-reviewed

journals, 49 publications in technical journals and 3 book chapters. He is reviewer in several

international peer-reviewed scientific journals in ecology, wildlife management, conservation biology

and epidemiology.

Publications




48:70.




44:81.

3. 4. Lopes AM, Breimana A, Loraa M, Le Moullac-Vaidyea B, Galaninad O, Nyström K,

Marchandeau S, Le Gall-Reculé G, Strive T, Neimanis A, Bovind NV, Ruvoën-Clouet N, Esteves PJ,

Abrantes J, Le Pendu J (2018) Host specific glycans are correlated with susceptibility to infection by

lagoviruses, but not with their virulence. Journal of Virology. doi: 10.1128/JVI.01759-17.

4. Nyström K, Le Gall-Reculé G, Grassi P, Abrantes J, Ruvoën-Clouet N, Le Moullac-Vaidye B,

Lopes AM, Esteves PJ, Strive T, Marchandeau S, Dell A, Haslam SM, Le Pendu J (2011) Histo-blood

group antigens act as attachment factors of rabbit hemorrhagic disease virus infection in a virus strain-

dependent manner. PLoS Pathogens 7: e1002188

5. Le Gall-Reculé G, Zwingelstein F, Fages M-F, Bertagnoli S, Gelfi J, Aubineau J, Roobrouck A,

Botti G, Lavazza A, Marchandeau S (2011) Characterisation of a non-pathogenic and non-protective

infectious rabbit lagovirus related to RHDV. Virology 410: 395.

Projects

Partner of the ECALEP project (Emergence of highly pathogenic CAliciviruses in LEporidae through

species jumps involving reservoir host introduction) supported by the ERA‑Net Anihwa (Animal

Health and Welfare), a coordination action funded under the European Commission’s ERA‑Net

scheme within the Seventh Framework Programme (Contract No. 291815).


INRA-ENVT

INRA is a large size research institute and a major actor in EU agronomic research. The joint research

unit UMR 1225 “Interactions Hôtes-Agents Pathogènes” (IHAP, Interactions hosts pathogens) brings

together scientists from the Animal Health Division of the French National Institute for Agriculture

(INRA) and from the National Veterinary School of Toulouse (ENVT). The UMR IHAP objectives

are to serve the field of veterinary sciences and agriculture by contributing to the control of infectious

diseases of livestock animals, and in turn to human health and food safety. The UMR INRA ENVT

IHAP is constituted by a core of 54 permanent staff, distributed in six research groups (Virology,

Bacteriology, Transmissible Spongiform Encephalopathy, Immunology, Parasitology and

Epidemiology). Over the last 10 years, the Unit acquired a strong experience in the management and

participation to EU funded/international research projects (over 15 projects - coordination of more

than 6 of them). Over the last 8 years, more than 200 peered-review articles have been published by

IHAP researchers. UMR INRA ENVT 1225 is located on the ENVT campus, and can use all the

facilities devoted to veterinary sciences and has a direct access to relevant clinical material. Beside the

laboratory equipment that is classically available in a virology laboratory (histology platform,

fluorescence and confocal microscopy, (real-time) PCR, cell analyser and sorter, ultracentrifugation),

the unit is full member of different technological platforms:

- ‘Structure Fédérative de Recherche Bio-Médicale de Toulouse’ (http://www.ifr-bmt.univ-tlse3.fr/),

which gathers a large number of research units involved in infectious disease study in Toulouse and

http://www.ifr-bmt.univ-tlse3.fr/



Page 53 of 61

provides access to its members to the most advanced technologies in cellular imaging, cell sorting,

and transgenesis.

- Genopole Toulouse platform, which provides to its members access and expertise to cutting edge

high throughput genomics and bioinformatics tools (http://www.genotoul.fr/index.php?id=43).

- proteomics platform in the “Institut de Pharmacologie et biologie structurale” in Toulouse As an

INRA unit, the IHAP has also dedicated access to the mass spectrometry platform located in Tours

Curriculum vitae

Name: Stéphane

Surname: Bertagnoli

Gender: male


Nationality: French

Position: Professor (Virology) at ENVT. Team co-Leader: Interactions Hôtes-Virus et Vaccinologie

(IHVV), UMR INRA-ENVT 1225 Interactions Hôtes-Agents pathogènes (IHAP), Ecole Nationale

Vétérinaire de Toulouse (ENVT), 23 chemin des Capelles, 31076 Toulouse Cedex 3, France Tel:

0561562590, e-mail: [email protected]

Education:

1984-1988: Veterinary medicine degree (Ecole Nationale Vétérinaire de Toulouse) 1991-1992:

Master in microbiology (Université de Toulouse) 1992-1996: PhD in virology (Université de

Toulouse); laboratory of molecular virology of UMR 960 INRA-ENVT (Toulouse, France, Dr A.

Milon; Construction of recombinant poxviruses to vaccinate against Myxomatosis and Rabbit

Hemorrhagic Disease).

2007: Habilitation à Diriger des Recherches (HDR) (Host-Myxoma virus interactions; poxvirus

vectors) (Université de Toulouse)


1997- August 2012: Associate professor in virology (ENVT)

From September 2012: Professor in virology (ENVT)

1997-2005: laboratory of molecular virology of UMR 960 INRA-ENVT

2005-present: Interactions Hôtes-Virus et Vaccinologie (IHVV), UMR INRA-ENVT 1225

Interactions Hôtes-Agents pathogènes (IHAP)

Supervision of 8 PhD.

Scientific activities: host - virus interactions; viral infections of rabbits (myxomatosis, RHDV,

Hepatitis E)

Publications

1. Bertagnoli S, Gelfi J, Le Gall G, Boilletot E, Vautherot J-F, Rasschaert D, Laurent S, Petit F,

Boucraut-Baralon C, Milon A (1996) Protection against myxomatosis and Rabbit Viral Hemorrhagic

disease with recombinant myxoma viruses expressing Rabbit Viral Hemorrhagic disease virus capsid

protein. Journal of Virology, 70: 5061.




44:81.

3. Esteves PJ, Abrantes J, Bertagnoli S, Cavadini P, Gavier-Widén D, Guitton J-S, Lavazza A,

Lemaitre E, Letty J, Lopes AM, Neimanis AS, Ruvoën-Clouet N, Le Pendu J, Marchandeau S, Le

Gall-Reculé G (2015) Emergence of pathogenicity in lagoviruses: evolution from pre-exiting

nonpathogenic strains or through a species jump? PLoS Pathogens 11:e1005087.




48:70.

5. Le Pendu J, Abrantes J, Bertagnoli S, Guitton JS, Le Gall-Reculé G, Lopes AM, Marchandeau S,

Alda F, Almeida T, Célio AP, Bárcena J, Burmakina G, Blanco E, Calvete C, Cavadini P, Cooke B,

Dalton K, Delibes Mateos M, Deptula W, Eden JS, Wang F, Ferreira CC, Ferreira P, Foronda P,

http://www.genotoul.fr/index.php?id=43



Page 54 of 61

Gonçalves D, Gavier-Widén D, Hall R, Hukowska-Szematowicz B, Kerr P, Kovaliski J, Lavazza A,

Mahar J, Malogolovkin A, Marques RM, Marques S, Martin-Alonso A, Monterroso P, Moreno S,

Mutze G, Neimanis A, Niedzwiedzka-Rystwej P, Peacock D, Parra F, Rocchi M, Rouco C, Ruvoën-

Clouet N, Silva E, Silvério D, Strive T, Thompson G, Tokarz-Deptula B, Esteves P. 2017. Proposal

for a unified classification system and nomenclature of lagoviruses. Journal of General Virology

98:1658.

Projects

- Animal Health and Welfare ERA-Net call 2, project ECALEP «Emergence of highly pathogenic

CAliciviruses in LEporidae through species jumps involving reservoir host introduction» (2015-2018)

- Research study Anses/DGAL/CLIPP, project Epi-RHDV2 «Control of the Rabbit haemorrhagic

disease due to RHDV2 by a deep knowledge of the disease epidemiology in rabbit breedings and

wildlife » (2018-2019).

Infrastructures

UMR IHAP has its own dedicated facilities: a biosafety level (BSL) 2 facility (over 650 m2); a state

to the art BSL3 (100 m2, built in 2011); a large capacity animal BSL3 facility (750 m2, built in 2008),

which is designed to host separately rodents, poultry, rabbits and large animals. The animal facility is

run by dedicated and fully qualified staff (7 people including a quality manager).


IZSLER

The Institute (IZSLER, www.izsler.it) is a governmental public Technical-Scientific Organisation

providing services as required by national and regional laws. At the present, IZSLER employs over

600 persons, 110 of them are veterinarians, biologists and chemists. IZSLER performs diagnosis for a

variety of viral infectious diseases of domestic animals as well as zoonotic and arthropod borne

diseases. Scientific research is an institutional duty. It is mostly aimed at the diagnosis and study of

animal transmissible diseases, at the hygiene of foodstuffs and to the technological development.

IZSLER acts as OIE reference laboratory for Foot and Mouth Disease, Swine Influenza Virus, Rabbit

Haemorrhagic Disease Virus, Myxomatosis and Swine Vesicular Disease.

IZSLER have extensive expertise and an international reputation in the production of monoclonal

antibodies and in the development of diagnostic immunoassays for antigen or antibody detection,

mainly based on ELISA.

Curriculum vitae

Dr Lorenzo Capucci (male) is the head of the Proteomic Structures at the Genomics Department at the

‘Instituto Zooprofilattico Sperimentale della Lombardia e dell’Emiglia Romagna’ (IZSLER), where

he has been responsible for approximately 15 research projects funded by the Italian Health Ministry.

He is the expert of the RHD Reference Laboratory of the World Organisation for animal Health (OIE)

since 1996. From 1982 to 1990, Dr Capucci worked at the Foot and Mouth diease virus (FMDV)

National Reference Laboratory. Now he works on the main viral diseases of lagomorphs (RHDV,

European brown hare syndrome and myxomatosis), and has been developing specific immunological

diagnostic techniques for these viruses since 1990. These methods have been used by Australian

Scientists since 1996. Dr. Capucci has been collaborating with Australian Scientists from CSIRO and

State Departments for over 20 years as senior advisor on RHDV epidemiology and antigenic

charaterisation. He has also been the responsible of the main Italian laboratory for rapid diagnosis of

Bovine Spongiform (BSE) and Scrapie since 2000. Dr Capucci is author/co-author of aproximatevely

60 articles on Scientific International peer-reviewed journal. He works as reviewer for several

Scientific Journals in the field of Veterinary Virology.

Dr. Antonio Lavazza (male) is pro-tempore Head of the Virology Unit of IZSLER. Responsible for

the NRL for viral diseases of lagomorphs, expert for the RHD Reference Laboratory of the World

Organisation for animal Health (OIE) for Rabbit Myxomatosis. He performs routine diagnosis based

on EM. His activities are particularly devoted to diagnosis of viral diseases of minor species,

companion and wild animals, surveillance and control of viral disease of livestock and wildlife, rabbit



Page 55 of 61

welfare, farming biosecurity and hygiene, classical diagnostic method for virology investigation,

vaccination and vaccines.

Publications

1. Capucci L, Cavadini P, Schiavitto M, Lombardi G, Lavazza A (2017) Increased pathogenicity in

rabbit haemorrhagic disease virus type 2 (RHDV2). Veterinary Record 180:426.

2. Velarde R, Cavadini P, Neimanis A, Cabezón O, Chiari M, Gaffuri A, Lavín S, Grilli G, Gavier-

Widén D, Lavazza A, Capucci L. Spillover events of infection of brown hares (Lepus europaeus) with

rabbit haemorrhagic disease type 2 virus (RHDV2) caused sporadic cases of an European Brown hare

syndrome like-disease in Italy and Spain. Transboundary and Emerging Diseases 64:1750-1761.

3. Lavazza A, Cavadini P, Barbieri I, Tizzani P, Pinheiro A, Abrantes J, Esteves PJ, Grilli G, Gioia E,

Zanoni MG, Meneguz PG, Guitton J-S, Marchandeau S, Chiari M, Capucci L (2015) Susceptibility of

the eastern cottontail (Sylvilagus floridanus) to European brown hare syndrome (EBHS) virus and

sporadic occurrence of EBHS-like disease suggests an active role for the lagomorph in the disease’s

epidemiology. Veterinary Research 46:13.

4. Le Gall-Reculé G, Lavazza A, Marchandeau S, Bertagnoli S, Zwingelstein F, Cavadini P,

Martinelli N, Lombardi G, Guérin J-L, Lemaitre E, Decors A, Boucher S, Le Normand B, Capucci L

(2013) Emergence of a new lagovirus related to Rabbit haemorrhagic disease virus. Veterinary

Research 44:81.

5. Puggioni G, Cavadini P, Maestrale C, Scivoli R, Botti G, Ligios C, Le Gall-Reculé G, Lavazza A,

Capucci L (2013) The new French 2010 variant of the rabbit hemorrhagic disease virus causes an

RHD-like disease in the Sardinian Cape hare (Lepus capensis mediterraneus). Veterinary Research

44:95.

Projects

- FP7 LAB ON SITE

- FP6 EPIZONE

- FP7 DISCONTOOLS

- EMIDA-ERANet program entitled APHAEA: “Harmonized Approaches in monitoring wildlife

Population Health, And Ecology and Abundance”. (2014)

- ANIHWA CE program entitled ECALEP (ID 49), “Emergence of highly pathogenic CAliciviruses

in LEporidae through species jumps involving reservoir host introduction. (2015)

Infrastructures

IZSLER is completely equipped for carrying out its work in the project. Infrastructure consists of

laboratories for cell culture, virology, histopatology, MAbs production, protein biochemistry,

molecular biology and immunology, virological and serological analysis, genomic typing and vaccine

production.

Furthermore, IZSLER is equipped with BSL3+ and high containment facilities for small and large

animal accommodation, suitable for conducting experiments with exotic and notifiable viral diseases.

Animal facilities include also BL2 and conventional units, a SPF-chicken unit (200SPF eggs/week)

and a small (5 sows) pig pathogen-free unit. Laboratories are equipped with incubators, air flow

cabinets, microscopes, including EM, centrifuges and ultracentrifuges, filtration and sterilization

equipment, cold storage rooms and liquid nitrogen tanks, thermocyclers, Real Time PCRs, automatic

nucleic acid extractors, sequencers, NGS and a flow cytometer.

A database for a “samples, reagents and material bank” (Biobank) was established for the record of

pathogens, tissue samples, pathological materials, sera, MAbs, cell cultures and reagents. A catalogue

is available on web (http://www.ibvr.org).

IZSLER has a library and training and conference rooms (up to 100 seats).


ENMV

The ENMV is a unique public hospital-university institution in Tunisia, created in 1974, under the

twofold supervision of the Ministry of Agriculture and the Ministry of Higher Education and

Scientific Research. Its role is to ensure (i) the graduation of veterinary students (6 years of study), (ii)



Page 56 of 61

the specialisation of veterinary doctors (4 years in several specialties); (iii) postgraduate training, and

(iv) scientific research through several research laboratories and several nationally or internationally

funded research projects. It benefits from collaborations with several regional institutions dedicated to

breeding or diagnosis and research.

The ENMV team (microbiology laboratory) is part of the research laboratory LR02AGR03 funded by

the Ministry of Higher Education and Scientific Research, entitled "Epidemiology of enzootic

infections of domestic herbivores" which deals with infectious diseases including vector-borne

diseases (anaplasmosis, ehrlichiosis, Q fever, theileriosis, babesiosis, etc). The microbiology

laboratory has participated in several national research projects as well as in a European project.

Research activities mainly target the detection of microbial pathogens by culture, the evaluation of the

prevalence of infections, the detection and molecular characterisation by PCR, ELISA tests, etc.

Members of the team have also experience in animal virology; collaborative activities include several

institutions and veterinary practioners in clinical and epidemiological studies.

Curriculum vitae

Name: Lilia

Surname: Messadi

Gender: female

Date of birth: 6 August 1957

Nationality: Tunisian

Position: Senior researcher in microbiology at the Ecole Nationale de Médecine Vétérinaire de Sidi

Thabet (ENMV), Tunisia

Education:

1980: graduation diploma in Veterinary Medicine (ENMV)

1981: certificate of Specialized Studies in Immunology (Institut Pasteur of Tunis)

1982: certificate of Specialized Studies in Parasitology (Faculty of Pharmacy of Monastir)

1983: certificate of Specialized Studies in Microbiology (Faculty of Medicine of Tunis)

1984: Doctorate in Veterinary Medicine (ENMV)

1989: master in Genetics and Molecular Biology (Faculty of Sciences of Tunis)


1984 to date: teacher-researcher in microbiology and immunology at the ENMV, in charge of

teaching, supervision of students in veterinary theses, masters (7), PhD (7), partner or PI of national

(5) or international research projects (cross-border cooperation program Italy-Sicily, IFS project).

1986-1997: Assistant professor in microbiology and immunology (ENMV)

1997- may 2003: Associate professor in microbiology and immunology (ENMV)

From june 2003: Professor in microbiology and immunology (ENMV)

From 1991: responsible of the laboratory of microbiology of the ENMV

1992 to date: Microbiology laboratory officer of the ENMV

Supervision of 4 PhD and 6 Master theses.

2013-to date: Chairman of the expert committee specialized in animal health within the National

Centre for Animal Health Surveillance

2015: Member of the national committee against antimicrobial resistance

2018: Member of the scientific council of the ENMV

Scientific collaborations with national research institutes: Institut de la Recherche Vétérinaire de

Tunisie (IRVT), Institut Pasteur de Tunisie (IPT), Groupement Interprofessionnel Cunicole et Avicole

(GIPAC), Anses Lyon (France), Veterinary deparment of Sassary (Italy), Istituto Zooprofilattico

Sperimentale della Sicilia (Italy), École nationale vétérinaire d’Alfort (France).

Publications: 25 publications in international scientific journals and scientific congresses.

Name: Faten

Surname: Ben Chehida

Gender: Female

Date of birth: 20 June 1985

Nationality: Tunisian



Page 57 of 61

Position: Assistant professor in microbiology and immunology at National school of veterinary

medicine of Sidi Thabet (ENMV)

Education:

2012: DVM, ENMV

2013: Certificate of Higher education in Avian pathology, School of veterinary medicine of Alfort,

Paris.

2017: PhD in virology-immunology, CIRAD Montpellier/Institute Pasteur of Tunisia/ Institute of

veterinary research of Tunisia

2017: Speciality degree in Microbiology/Immunology, ENMV

2013-2017: PhD student at CIRAD of Montpellier, unity of research CMAEE: Control of Exotic and

Emerging Animal Diseases/ Institute Pasteur of Tunisia (departement of veterinary virology)/ Institute

of veterinary research of Tunisia (department of virology)


2013-2017: Residence in microbiology and immunology at National school of veterinary medicine of

Sidi Thabet

July 2018: Assistant professor in microbiology-immunology at National school of veterinary medicine

of Sidi Thabet

Scientific collaborations with national (Institute Pasteur of Tunisia (IPT)/Institute of veterinary

research of Tunisia (IRVT)) and international research institutes (CIRAD Montpellier).

Publications

1. Ben Said M, Ben Asker A, Belkahia H, Ghribi R, Selmi R, Messadi L (2018) Genetic

characterization of Anaplasma marginale strains from Tunisia using single and multiple gene typing

reveals novel variants with an extensive genetic diversity. Ticks and Tick-borne Diseases 9:1275.

2. Belkahia H, Ben Said M, El Mabrouk N, Saidani M, Cherni C, Ben Hassen M, Bouattour A,

Messadi L (2017) Spatio-temporal variations and genetic diversity of Anaplasma spp. in cattle from

the North of Tunisia. Veterinay Microbiology 208:223-230.

3. Reusken CB, Messadi L, Feyisa A, Ularamu H, Godeke GJ, Danmarwa A, Dawo F, Jemli M,

Melaku S, Shamaki D, Woma Y, Wungak Y, Gebremedhin EZ, Zutt I, Bosch BJ, Haagmans BL,

Koopmans MP (2014) Geographic distribution of MERS coronavirus among dromedary camels,

Africa. Emerging and Infectious Diseases 20:1370.

4. Azaouzi Z, Jemli MH, Messadi L, Zaouia I, Ben Hadj Kacem MA (2018) Molecular detection of

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Tunisian dromedary camels

(Camelus dromedaries). Rev. Mar. Sci. Agron. Vet. 6:260.

5. Ben Chehida F, Ayari Fakhfakh E, Caufour P, Amdouni J, Nasr J, Mesaoudi L, HajAmmar H,

Sghaier S, Bernard C, Ghram A and Cetre Soossah C (2017) Sheeppox in Tunisia: Current status and

perspectives. Transboundary and Emerging Diseases 65:50.

Projects

- European Project “Establishment of a platform and a Tunisian-Italian network for the surveillance of

emerging diseases transmitted by ticks and Culicidae” (Projet n°2AS1.3/023 2014-2016)

- National Project “Epidemiology of enzootic infections of domestic herbivores” funded by the

Ministry of Higher Education and Scientific Research (LR02AGR03 2014-2018)

- National project on antimicrobial resistance of bacteria isolated from animals (2016-2018) funded by

the Ministry of Agriculture

- National Project Study of the main bacterial and viral agents responsible for abortions in ewes

(research agreement CNVZ-ENMV, 2017-2018)

- Research project “Optimization of vaccine efficacy of the Capripoxvirus vector by a strategy of

subtraction of genes” in the framework of the PHC-Utic program managed by the CMCU (13G 0906)

Infrastructures

The microbiology laboratory of the ENMV has several rooms for diagnostic and research, some of

which are equipped with microbiological safety station and an ELISA reader. It also includes a

molecular biology unit equipped with thermocyclers, electrophoresis equipment, amplicons

visualization system, centrifuges, tissue grinder, etc.



Page 58 of 61


ENSV

ENSV (National High Veterinary School of Algiers) has three research laboratories: i) SPA, Animal

health and productions laboratory (composed of 6 teams), ii) HASAQ, Food hygiene and quality

assurance system laboratory (composed of 4 teams) and iii) GRAL, Local animal resource

management (composed of 4 teams). Our team is affiliated to the SPA laboratory (head: Pr Hacina

Ain Baziz) and is in charge of research and scientific/technical studies on rabbit and poultry health,

nutrition and production with the objective of controling infectious diseases, the development of tools

for diagnosis, prevention and treatment, and the improvement of national production.

Curriculum vitae

Name: Hacina

Surname: Ain Baziz

Gender: female


Nationality: Algerian

Position: Senior researcher (SPA research laboratory-ENSV) and professor in poultry and rabbit

productions at ENSV.

Education:

1980: Graduate (Engineer), National Agronomic Institute of Algiers, Algeria

1990: Magistere in Agronomic Sciences. Specialities: Animal productions, National Agronomic

Institute of Algiers, Algeria

1996: PhD in Life Science, University of Tours, France


1981-2000: Head of the monogastric department at Institut Technique de l’Elevage (ITELV), Algiers,

Algeria

1993-1996: PhD student at INRA – Tours, France. Involved in research on the effect of high ambient

temperature on lipid metabolism in growing chicken.

2001-until now: Professor on rabbit and poultry productions.

2006-2012: Head of SPA laboratory- ENSV, Algiers, Algeria

2006-until now: Head of research team (rabbit and poultry productions), SPA research laboratory,

ENSV Algiers.

Since 2011: Member of the Algerian national association of agricultural engineers

Since 2012: Member of the Algerian branch of World Poultry Science Association (WPSA)

Publications

1. Saidj D, Aliouat S, Arabi F, Kirouani S, Merzem K, Merzoud S, Merzoud I, Ain Baziz H (2013) Farming rabbits in Algeria: a not negligible source of meat for rural families. Livestock Research for

Rural Development 25(8).

2. Belabbas R, Ilès I, Ain Baziz H, Theau-Clément M, Berbar A, Boumahdi Z, Boulbina I, Benali N,

Temim S (2013) Characterization of Local Algerian Population of Rabbit: Factors Influencing Fetal

and Placental Development. Journal of Agricultural Science 5:76.

3. Saidj D, Ainbaziz H, Salhi O, Hornick JL, Moula N (2016) Effect of Dietary Energy on Productive

and Reproductive Performance of Algerian Local Rabbit Does and Their Litters. Animal Nutrition

and Feed Technology 16:107.

4. Belabbas R, García ML, Ain Baziz H, Berbar A, Zitouni G, Lafri M, Bouzouan M, Merrouche R,

Ismail D, Boumahdi Z, Benali N, Argente MJ (2016) Ovulation rate and early embryonic survival rate

in female rabbits of a synthetic line and a local Algerian population. World Rabbit Science 24:275.

5. Benali N, Ain Baziz H, Dahmani Y, Djellout B, Belabbas R, Tennah S, Zenia S, Cherrane M,

Temim S (2018) Effect of dietary energy content on performances and some biological parameters in

growing rabbits. Livestock Research for Rural Development 30 (3).

Projects

2011-2013: CNEPRU project



Page 59 of 61

Study of factors influencing the growth, reproduction, and health status of local rabbit populations

(Oryctogalus cuniculus).

2013: PNR project with the collaboration of ITELV (Institut technique de l’élevage).

Impact of ambient heat on the production and health status of the local rabbit population: development

of control methods.

Infrastructures

SPA laboratory is composed of a BSL1 building fully equipped for the handing of pathogenic animal

(viruses, bacteria and parasites) and for serological, virological (ultracentrifuge) and molecular

biology analyses (thermocyclers, real-time thermocyclers) and BSL1 animal facilities equipped for

experimental on rabbits.

4.2. Third parties involved in the project (including use of third party resources)

Please complete, for each participant, the following table (or simply state "No third parties

involved", if applicable):

Does the participant plan to subcontract certain tasks (please note

that core tasks of the project should not be sub-contracted)

YES NO

x

If yes, please describe and justify the tasks to be subcontracted

Does the participant envisage that part of its work is performed by

linked third parties5

YES NO

x

If yes, please describe the third party, the link of the participant to the third party, and

describe and justify the foreseen tasks to be performed by the third party

Does the participant envisage the use of contributions in kind

provided by third parties (Articles 11 and 12 of the PRIMA Model

Grant Agreement)

YES NO

x

If yes, please describe the third party and their contributions

Does the participant envisage that part of the work is performed by

International Partners6

(Article 14a of the PRIMA Model Grant

Agreement)?

YES NO

x

If yes, please describe the International Partner(s) and their contributions Participant number 6/Partner 5/INRA-ENVT: Analyses of myxoma virus – adjuvants in vitro

interactions (manufacturing and quality control of adjuvant formulations): Vaccine Formulation

Laboratory, University of Lausanne, Department of Biochemistry Chemin des Boveresses 155 1066

Epalinges, Switzerland (Nicolas Collin).

Other participants: no third parties involved.

5 A third party that is an affiliated entity or has a legal link to a participant implying a collaboration not limited

to the action. (Article 14 of the PRIMA Model Grant Agreement). 6 ‘International Partner’ is any legal entity established in a non-associated third country which is not eligible for

funding under Article 10 of the Rules for Participation Regulation No 1290/2013.



Page 60 of 61

5. Ethics and Security

5.1 Ethics

The project will involve the use of animals for collecting lagoviruses (rabbits and hares, WP1) and in

vivo viral infections (rabbits, WP3). To isolate pathogenic lagoviruses, sampling will be done on

carcasses of animals that died from RHD. Therefore, no animal will be killed during this project for

lagoviruses collection. Collection of blood samples in live animals will be performed or supervised by

veterinarians or scientists officially authorized to perform such procedures. Nonetheless, coordinator

and partners 1-9 hold the category B or C of handling animals, in accordance to the requirements of the European Council (Convention ETS 123, Article 26) and the European Union (Council Directive

86/609/EEC, Article 14).

Experimental trials (WP3) will be conducted for characterisation of the immune response and for

vaccine formulation and testing. They will be carried out in accordance with the European

Communities Council Directives of 22 September 2010 (2010/63/UE) and with National laws and

regulations regarding the care and use of animals. Thus, all experiment will be previously submitted

to the national ethics committees for approval (for INIA: Animal Ethical Committee at CISA as well

as by the National Biosecurity Committee; for ANSES: ComEth Anses/ENVA/UPEC (agreement

number 16) in accordance with the Ministry of Higher Education and Research Minister; for INRA-

ENVT: the Veterinary College Scientific Council; for IZSLER: IZSLER Ethical Committee and

OPBA in accordance with the Ministry of Health). In these institutes, in vivo studies are performed

under negative pressure in BSL2/3 experimental facilities with filtered air according to biosafety

procedures. These animal facilities are regularly controlled and approved by the National Ministry

concerned (for INIA: codes ES281620002741 and ES280790002069; for ANSES: Prefectorial

Agreement number C-22-745-1; for INRA-ENVT: Prefectorial Agreement number C-31-555-27; for

IZSLER: Ministerial Decree (DM n.184/94)). Only qualified technicians who received the official

agreement for animal experimentations from competent national authorities are operating in the

facilities for handle, monitor and treat animals.

Protocols will be shared between the different partners to ensure that the best practices are applied

throughout the course of the project. The consortium will have a commitment to support the 3Rs

principles for live animal experimentation:

1. Replacement: the lagoviruses are non-cultivable, which makes impossible to assess the immune

responses triggered without in vivo studies. However, each study will be performed only by strict

necessity.

2. Reduction: the statistical power of each experimental design will be preliminarily calculated to

allow significant interpretation of the data (5% significance level and 90% statistical power) with a

minimal number of animals. The inventory of the experiments to perform will be discussed again at

each meeting of the consortium to avoid redundancies.

3. Refinement: two or three daily observations for morbidity will be done to check that the infected

animals do not suffer and in this case, they will be humanely euthanised. Thus, before the sacrifice,

these animals or the surviving animals at the end of the experiments will be anaesthetised or stuned by

electronarcosis.

For INIA, research will be carried out in compliance with current Spanish and European legal and

ethical requirements, including: Ley 6/2013, from 11th June of 2013 which updates the Ley 32/2007

from 7th of November, for the animal care, production, transport, experimentation and sacrifice, as

well as with the EU Directive 2010/63/EU which updates and replaces the 1986 Directive

86/609/EEC, regarding the protection of animals used for experimental and/or scientific purposes.

For ANSES, the achievement of immunsera from rabbits experimentally inoculated with VLPs of

calicivirus (Tasks 3.2 and 3.3) are covered by the agreement of the French ethics committee ComEth

Anses/ENVA/UPEC number 10/04/18-2 (10th April 2018, in French).



Page 61 of 61

English summary:

The ComEth Anses/ENVA/UPEC has proceeded to the examination of the project entitled

“Production of specific immune serum from pathogenic or non-pathogenic calicivirus viral-like

particles (rabbit haemorrhagic disease) on rabbits”. After thought, the ComEth informs you that the

project has received a favorable advice subject to:

- modify the project authorisation request by including the 3R rules in the non-technical summary

- classify the gravity level as “moderated” following the use of adjuvant.

The ComEth advices too to put two rabbits per cage only if the rabbits are socially and harmoniously

compatibles.

Please note that the current ComEth notification is informative and does not worth the project

authorisation, the definitive advice on the project will be given by the Research Ministry.

IZSLER states that the experiments performed in its facilities will comply with current (DLGS

26/2014) and future legislation and regulations in Italy. We are aware that requirements of the

national legislation or rules have to be followed. We are also concerned by the following animal and

welfare laws: the “European convention for the Protection of Vertebrate Animals used for

Experimental and other Scientific Purposes” - ETS No. 123 and the EU Council Directive

86/609/EEC and 2007/526/EG. The in vivo tests carried out at IZSLER within approved Research

Projects are at first submitted to the Ethical Committee of the Institute itself and, if approved, sent to

Ministry of Health for final approval. Currently the Ethical Committee is supported by the New

Scientific group (Committee for Animal Health) as required by Directive 2010/63/EU and referred to

in the Italian transposition Decree DL 4 March n° 26 of 2014 of National Ministry of Health as

“Organismo Preposto al Benessere Animale – OBPA”.

5.2 Security7

Please indicate if your project will involve:

No

-classified information' as background or results: No

7 See article 37 of the PRIMA Model Grant Agreement

PRIMA Full Proposal Template Table of Contents

Documents

Transcript of PRIMA Full Proposal Template Table of Contents