To better contribute to identify and solve main relevant issues regarding the prevention of ASF entry in the EU, the ASFORCE work plan is organized in five major Themes (Theme 1; Theme 2; Theme3; Theme 4 and Theme 5, corresponding to the objectives of the project), each divided into Work Packages (WP) containing several Tasks.
The participation of different partners in the majority of WP and the fact that several are considered horizontal activities, will assure a strong interaction between the collaborators. A multidisciplinary approach of this proposal further provides a strong scientific and technological basis towards the achievement of the project's objectives.
The coordination and management of scientific, administrative and financial aspects of the project will be conducted by the coordinator, in close collaboration with the Executive Board and Project Management Team (PMT), assisted by the project manager, in accordance with the grant agreement and decisions taken by the consortium
The specific goals of the work within this theme will be reached through the development of the work-package (WP) 1 (Coordination and Management).
Theme 2 will aim to provide essential information to design more cost-effective surveillance and control strategies for ASF into the different risk scenarios, providing valuable tools for policy makers, administrations and pig producers. The ultimate goal would be to better prevent and control ASF and minimize the economical loses in endemic regions or in countries at risk;
The specific goals of the research within this theme will be reached through the development of eight work-packages:
WP 2 - Characterization of the pig industry in the EU Member States and ASF affected areas in Europe.
Pig production within the European Union and beyond is a divers and multifaceted industry. Huge, highly engineered farms with exceptionally high biosecurity standards exist along with backyard holdings and small family owned enterprises. Moreover, the density of pig farming, biosecurity standards as well as the possibility of contact to wild species such as European wild boar is most variable. It is obvious that all risk assessments as well as prevention and control measures with chances of success must take into account these factors. For this reason, WP 2 will aim at the description, evaluation and mapping of existing production systems within the EU Member States and neighbouring countries, especially in the East and South-East. To this end, data on pig density, structure of industry, herd sizes, management, biosecurity measures and standards, productivity, and price differentials will be collected and analysed. This WP has strong links to all other WP within this Theme, providing the basis for the rational design of effective prevention and control strategies.
WP 3 - Characterization of contact patterns between pig populations through social network analysis (with special attention to small pig producers).
This work package intends to evaluate the spatial and temporal patterns of pig and pork movements within and between member states and other participant countries. This movement patterns will be integrated in spread models to be developed, to more realistically simulate the potential spread of ASF within and between countries.
WP 4 - Update of the epidemiological disease status in Europe, the genetic properties of ASFV (and geographical and molecular spatial-temporal patterns).
The work package will focus on updating the epidemiological disease status in Europe, the genetic properties of ASFV and geographical and molecular spatial-temporal patterns. This WP will intend to increase the knowledge about the epidemiological and molecular features of currently circulating ASFV isolates in Europe with special regards to those present in Trans-Caucasian countries (TCC) and Russia Federation. The combination of epidemiological findings, epidemiologic surveillance and molecular typing will allow to determine factors such as the prevalence of the disease at the domestic pig and wild boar population levels as well as to trace the dynamic of the infections. The work schedule will be based on molecular epidemiological analysis on selected ASFV isolates taking into consideration the results obtained from the basic surveillance studies and the epidemiological findings.
WP 5 - Evaluation and cost benefit analysis of existing surveillance, contingency plans and control strategies (in EU Member states and eastern European countries).
This WP will review and evaluate the historical and current surveillance and control programs in EU Member States and in Eastern European countries. Advantages and drawbacks of those preventive and control strategies will be elucidated in order to find an optimized strategy for each epidemiological scenario.
WP 6 - Dynamic disease modeling to identify key spread mechanisms and quantify potential local spread (and evaluate the impact of different control methods).
The work planned under this WP aims at developing a dynamic disease model of the spread of ASF in the EU, to evaluate effectiveness of different possible control strategies and to assess the potential of development of ASFV infection reservoirs in wildlife and/or domestic pigs and its impact on local spread. A key parameter for this model will be the study of survival of infectious virus stored at different temperatures, levels of humidity and pH over varying time periods. The relevant data will be generated though laboratory studies.
WP 7 - Economic evaluation of different alternative disease spread and control scenarios.
Development of a decision support tool for EU member states to identify appropriate and cost-effective ASF prevention and control strategies, trough the development of an economic model for the evaluation of different disease spread and control scenarios. This Task will be closely linked to the work conducted under WP 6.
WP 8 - Guidelines for a cost-effective control and prevention including communication.
Aims at producing a set of guidelines for cost-effective prevention and control of ASF by incorporating the results obtained in the previous Work Packages (WP 2-7). Guidelines will detail in a clear and concise language the most recommended actions to be taken in each considered risk scenario. These guidelines will be produced in different languages to facilitate the comprehension in different European territories.
This Theme will seek to provide essential data to identify key points for designing new control strategies including wildlife considerations. The understanding of the epidemiology of ASF in wild boar in its natural habitat and on the role of argasids, constitutes an important aspect for the international control of ASF.
The various aspects of the data collected and the analysis of the information on the transmission dynamics between species will provide a basis for the development of more reliable control measures and biosecurity practices, with the goal of abrogating ASF threat to EU;
The specific goals of the research within this theme will be reached through the development of four work- packages:
WP 9 - Ornithodorosticks’ distribution, competence and interactions with wild boar and domestic pigs (in EU Member States and neighboring countries).
Aims at developing a predictive map of the tick’s distribution in some EU and neighboring countries (Russian Federation and Trans-Caucasian Countries) based in the identification of suitable habitats, tick capture and the use of anti-ticks serology determination in domestic and wild boar of the area. Interaction between domestic pigs, wild boar and ticks will be also evaluated by an ELISA test development in this project. Finally the vector competence will be quantitative characterized.
WP 10 - Ecology of contacts between domestic and wild pigs in different epidemiological settings.
This WP will assess and quantify the contacts between wild and domestic suids that could potentially favor the transmission of ASFV by comparing different methods:
- Test and assess the use of questionnaires among farmers and hunters in order to identify contacts between domestic and wild pigs in different interface areas.
- Assess wildlife interaction through the use of GPS radio-tracking of domestic and wild pigs in different interface areas.
- Test and assess the use of biomarkers (fecal E. coli) to identify contacts between domestic and wild pigs. Study areas where domestic and wild pigs can interact will be chosen in the different regions of the Russian Federation, Bulgaria and some Mediterranean countries (France, Spain and Italy).
- Assessment of potential interactions between domestic pigs and wild boar populations based on their modeled, spatial distributions.
WP 11 - Modeling of ASF transmission in wildlife and between wild boars and domestic pigs.
Risk assessments are a useful tool for early detection of disease and better management of
resources, as evidenced by legislation recently adopted by the European Commission (EC, 2010) related to Avian Influenza, which emphasizes the surveillance strategy should have a risk-based approach. In this WP, the modeling of ASF transmission in wildlife and between wild boars and domestic pigs will be conducted by special analysis using mainly data obtained in WPs 9, and 10 in countries at risk in Europe and neighboring countries (such as Russian Federation, Georgia, Armenia, Azerbaijan or Iran) to improve strategy of both prevention and
control of the disease. Previously, transmission rate, wild boar distribution and risk factors will be determined to get better the understanding of the dynamic of ASF.
WP 12 - Biological characterization of the Caucasian ASFV isolates in wild boar.
Despite the fact that European wild boar are involved in several outbreak scenarios of ASF, little is known so far about biological characteristics of ASFV infections in this species. To develop effective control strategies, both epidemiological issues and disease dynamics are important to understand. Especially the knowledge about disease courses and modes of virus shedding and transmission is of paramount importance.
To this means, previous pilot studies on the biological characterization of the Caucasian ASFV isolates in European wild boar of different age classes, have shown that those isolates are highly virulent for domestic pigs and wild boar of all investigated age classes. So far, no obvious differences were seen in the disease course in wild boar or domestic pigs. Contact controls got infected with moderate efficiency (mainly related to contact with blood), and the viral load in secretions and excretions was rather low. Under field conditions, it can be assumed that the infection dose might be low in several cases (feeding on food waste/swill, direct contact to infected sounder mates without blood involvement, carcasses). As a dose response effect was seen with some moderately virulent isolates, especially in older animals, it cannot be ruled out that such an effect also applies for highly virulent isolates in wild boar. This could lead to the development of carriers and/or chronically infected animals, and this could be of crucial impact for the evolution of an epidemic.
In order to supplement and complete existing data on the behavior of the Caucasian ASFV isolates in wild boar that could help to understand the epidemiology and transmission modes in affected areas, studies will be carried out in collaboration to assess whether a dose response effect can be seen after experimental infections with the Armenian ASFV isolate (identical with Georgia 2007). Data will be generated on clinical course, viraemia, virus shedding and transmission to domestic contact controls. In addition, immunological tests will be carried out.
Theme 4 will aim to advance work leading to vaccine development through two routes: 1) Rational deletion of genes to produce attenuated and non-replicating candidate ASFV vaccine strains; 2) Identification of protective antigens and their incorporation into vectored virus vaccines.
Further to these studies, assessment of pig-carrier state induced in experimentally “vaccinated animals” will be pursued and also applied in field conditions by using improved diagnostic tests for viral and antibody detection to be developed under this Theme.
The specific goals of this research collaborative work will be reached through four work packages:
WP 13 - Development of deletion mutants as attenuated and DISC vaccine strains.
The objective of this WP is to construct candidate ASFV vaccine strains by targeted gene deletion from the virus genome. In one approach, non-essential virus genes involved in virulence and immune evasion will be deleted to produce replicating attenuated virus strains. In another approach defective infectious single cycle (DISC) ASFV strains will be produced.
Previous work has established that immunization of pigs with attenuated replication competent ASFV isolates can induce good levels of protection against lethal challenge with virulent strains. Low levels of replication of the attenuated and challenge virus are observed. However in some pigs adverse clinical signs occur following immunization, demonstrating that currently available strains are not sufficiently attenuated for release in the field. One objective of this WP will be to delete additional genes from current attenuated strains of genotype I, NHV/P68, and OURT88/3, in order to abrogate adverse post-vaccination reactions but maintain high efficacy of protection. The additional genes to be deleted will be selected from current knowledge of genes known to be involved in immune evasion and virulence and new knowledge of the role of additional genes. Single or multiple genes will be deleted. A second objective will be to construct candidate attenuated replication competent strains by targeted sequential gene deletions from ASFV strains circulating in the Caucasus and Russian Federation. This is necessary since these strains are genotype II and the genotype I isolates do not provide full cross-protection. This will be achieved by sequential gene deletions based on those deleted from the NHV/P68 and OURT88/3 isolates. A third objective will be to construct DISC ASFV strains for evaluation as vaccine candidates. These strains will be constructed by making a helper cell line expressing an essential ASFV gene. This would then be used to construct and replicate an ASFV deletion mutant in lacking the same essential gene. This approach would provide replication defective ASFV candidate vaccine strains lacking potential for spread in the environment. The effect of these gene deletions on virus replication and modulation of host responses will be studied in WP 14. Selected ASFV mutants will be tested in immunization and challenge experiments as described in WP 15. Two of them, selected from phase I, will be also tested for assessment of carrier state in WP15.
WP 14 - ASFV - cell interactions aimed at the identification of genes for construction of attenuated (and DISC) ASFV strains.
The ASFV genome has between 150 and 165 genes, including those which are essential for virus replication and genes that are not essential but have roles in virus-host interactions that are important for virus replication in its hosts. The objective of this WP is to identify additional genes that are targets for deletion from the genome to construct candidate attenuated vaccine or DISC vaccine strains. The innate immune system is very important as a first line of defense both for elimination of pathogens and to orchestrate the pathogen specific adaptive immune response. Some ASFV genes involved in evading the innate immune response have already been identified but we believe that many more remain to be identified and their role in protection analysed. New genes involved in immune evasion will be identified and their function better understood. Other studies will focus on the characterization of ASFV genes involved in virus genome replication and transcription. Some of these genes, such as enzymes involved in DNA repair or nucleotide metabolism may be not essential for replication but their deletion can reduce virus replication and virulence. These may be good targets for constructing attenuated vaccines strains. Essential genes involved in replication and transcription of the ASFV genome could be targets for construction of DISC ASFV strains as it will be assessed under the work plan of the WP. The process of virus entry is a critical step for virus replication and should involve interaction of virus proteins with host cells and the activation of host cell signaling pathways. These pathways may activate innate host defense systems such as toll-like receptor signaling of apoptosis or they may activate processes essential for the virus to replicate. Further characterization of the virus proteins involved in the binding and entry process will identify new virus targets for vaccine development and will identify virus genes which may be deleted to construct attenuated ASFV strains. The need for qualitatively specific protective immune responses implies the need for tailor-made adjuvants. TLR-based adjuvants will be tested in those cultures in combination with mutants for induction of the appropriate immune responses.
The information from this WP will be used for selection of genes to delete from the virus genome. The deletion mutants generated will be tested in cell cultures and the effect of the gene deletions on virus replication, activation of host innate defense responses will be determined including IFN induced antiviral responses, cytokine and chemokine responses and activation of host signaling pathways including PI3K-Akt pathway. This information will be used to select which virus mutants are included in the in vivo vaccination and protection experiments in pigs described in WP 15.
WP 15 - In vivo testing of selected mutants ASFV and assessment of the carrier state (and development of penside/frontline diagnostic tests).
This WP focuses on testing selected ASFV deletion mutants constructed in WP 13 and tested in WP 14 for their ability to induce protection in pigs against a lethal dose of virulent ASFV challenge virus.
Pigs immunized with the deletion mutants constructed in genotype I strains NHV/P68 and OURT88/3, will be challenged with homologous and/or heterologous virulent isolates belonging to genotype I (L60 or OURT88/1) and to genotype II from the Caucasus regions. For the deletion mutants constructed, based on the Georgia 2007/1 isolate, the parental strain will be used for challenge.
In order to generate comparable results the immunization, challenge, clinical scoring and collection of samples will be standardized. A total of six deletion mutants will be tested starting after month 9. In addition, a carrier state study will be performed in one of the experiments. In a second phase of the “in vivo” testing, the effectiveness of administration of adjuvants in boosting the immune response to ASFV deletion mutants and directing the response to a T helper 1 or 2 type response will be measured in comparison to not administering an adjuvant in selected mutant. In the third year of the project, the most promising deletion mutants will be used for oral vaccination of a small number of European wild boar.
Depending on the outcome of the vaccination phase, the animals will be challenged with the highly virulent Caucasian ASFV isolate. Samples collected from immunized pigs before and after challenge will be monitored for levels of viraemia, ASFV specific antibody response and T cell responses.
For the eventual licensing of vaccines regulatory requirements have to be fulfilled. At an early stage in the project these requirements will be reviewed so that meeting these can be considered as much as possible in the design of in vivo experiments for the third year.
A crucial point for the control and eradication of ASFV is the rapid and reliable diagnosis in the early stages of infection. For that reason, it is very important to provide to veterinarians in the field and to small non-specialized laboratories around world, with user-friendly, inexpensive and reliable diagnostic systems. New test for virus and antibodies detection that fulfill those characteristics will be developed.
WP 16 - Identification of immunogenic ASFV antigens and testing in vaccination challenge in pigs.
To date, attempts to develop vaccination strategies against ASFV by using recombinant proteins, DNA vaccination alone or in prime-boost regimens, or synthetic peptides have not been successful. The application of viral vector candidate vaccine has not been reported so far. Thus, development of novel, conventional attenuated or recombinant ASFV-independent vaccination approaches needs to make an initial selection, based on in silico and early ASFV-infected cell protein analyses, of about 30 ASFV antigens from the total of 150 to 165 open reading frames (ORFs) to identify viral proteins that might induce a strong and protective cellular immune response. For this high-throughput screen, BacMam viruses, each containing genes for three different ASFV proteins, will be used for immunization of pigs which, since protection against ASFV infection is the most significant parameter for delivery vector construction, will then be challenged with virulent ASFV. Different delivery systems known to be compatible with food security needs will then be used to express the proteins with protective potential to generate vaccine candidates which will be tested for protection efficacy and safety. Master seed and working seed stocks will be prepared for candidate vaccine production and vaccine(s) will be licensed if applicable.
Theme 5 aims to increase the preparedness for ASF trough investigation of the attitude of pig farmers, butchers/middlemen and hunters and to increase preparedness for ASF among veterinarians, representatives of different governmental agencies and stakeholders.
Distance learning material on ASF will be made available through the project website coordinated by FMV-UTL. Workshops for veterinarians will be offered at different locations to facilitate attendance of practicing veterinarians from EU and non EU countries namely in Russia where the disease has become enzootic, in Bulgaria (focus on South Eastern European countries) and Spain (focus on West European countries) where there is significant pig production and where preparedness for ASF is needed.
Regional workshops for representatives of governmental agencies of EU Member States and countries recently affected by ASF will be organised. Locations are proposed to be Germany (Central Europe and neighbouring countries), Rome (SE Europe and neighbouring countries), and Lisboa where the ASFORCE Symposium will be organized addressed to international organizations (EC, OIE, FAO, EFSA), CVOs from EU and from candidate and associated countries, international stakeholders and others considered relevant.
The specific goals of this collaborative work will be reached through three work- packages:
WP 17 - Disease awareness in pig producers and hunters
The aim of this work package is to improve preparedness for ASF among pig producers in the EU, in particular in areas at risk, and in non-EU countries recently affected (e.g. Russia, Armenia, Georgia), trough: i) Investigation of the attitude of pig farmers, butchers/middlemen and hunters towards disease reporting in EU countries at risk and non-EU countries recently affected; ii) Development of materials for disease awareness campaigns to be distributed in countries at risk in the EU and outside. This will be achieved in collaboration with national pign organisations, veterinary associations, hunter associations and representatives of the pig industry as well as through government and FAO links.
WP 18 - Training and disease awareness of veterinarians
The aim of this work package is to increase preparedness for ASF among veterinarians, in particular in regions at risk in the EU and in recently affected countries in Caucasus and in Russia. The objectives are to organise workshops and provide training for veterinarians to increase risk awareness and capacity of incident response. The workshops will be offered at different locations to facilitate attendance of practicing veterinarians from EU and non EU countries namely in Russia where the disease has become enzootic, in Bulgaria (focus on South Eastern European countries) and Spain (focus on West European countries) where there is significant pig production and where preparedness for ASF is needed. Futhermore distance learning material on ASF will be produced as a Training course, organized in different modules to be available through the project website.
WP 19 - Regional workshops for governmental agencies
The aim of this work package is to provide an opportunity to representatives from different governmental agencies in the EU and in countries recently affected by ASF to discuss and update their prevention strategy and to adjust contingency plans based on the results of Themes 2-4 of ASFORCE. At least 3 regional workshops will be organized, namely to representatives: i) from Central Europe and neighboring countries, ii) from SE Europe and neighboring countries and iii) from international organizations (EC, OIE, FAO, EFSA), CVOs from EU and from candidate and associated countries, international stakeholders and others considered relevant.