Final Report Summary - ASFRISK (Evaluating and controlling the risk of African swine fever in the EU)
Executive Summary:
African swine fever (ASF) in EU member states is currently confined to Italy (Sardinia). It was recently introduced to Caucasian countries and Russia and it is highly prevalent in sub-Saharan African countries. Surveys during outbreaks occurred previously in Eastern and Southern African regions have shown that although ASFV prevalence in the domestic pigs is high, in many cases serum conversion was not detected. These events pose a great challenge for the prevention and control of ASF, not only locally, but also worldwide. The complex epidemiological scenarios of ASF, the increasing mobility of people, animals and goods across the globe, emphasize the serious menace the disease presents to the growing pig farming sector in Africa and to currently ASF-free EU member states permanently threatened by the occurrence of the disease in different geographical regions.
In order to improve tools and strategies for the prevention and control of ASF, the work developed under the ASFRISK project aimed at fulfilling four objectives contained in the Major Tasks of the work-plan:
i) to improve and further develop the epidemiological, risk assessment and control measures of African swine fever (ASF), ii) to develop and provide new and robust tests for laboratory and front-line diagnosis, through the achievement, standardization and validation of more rapid, sensitive and inexpensive serological and molecular procedures iii) to establish new strategies for vaccine development against ASF, iv) to transfer knowledge on relevant aspects of ASF and transfer of diagnostic and epidemiologic tools through training courses and traineeships to provide an harmonized international training, in order to prevent and combat this highly devastating, OIE-listed transboundary animal disease.
The four main tasks of the project were outlined and harmonized in the work-plan providing a logical process for the improvement of knowledge on critical aspects of ASF and development of tools towards the achievement of the objectives through the contribution of highly qualified partners from EU, Africa and Asia, with expertise in all the areas concerned. Major outcomes of the project emphasize its successful contribution for “Evaluating and controlling the risk of ASF in the EU”:
- Development of epidemiological methods and decision support tools provide information to policy makers on ASF epidemiological situation and will assist in the prioritization of strategies to better prevent and control ASF in a potential incursion into the EU and in all currently affected territories.
- Development of a range of valuable serological and molecular diagnostic tools suitable for use in well-equipped international and national reference laboratories, in basic regional and local laboratories, or even for rapid first line on-site application, allow and complement available tests thus promoting more efficient and earlier diagnosis of the disease.
- Characterization of several ASFV-host interactions at cellular and molecular levels and the production of different attenuated recombinant virus strains that may be potentially used as future candidates for a vaccine, open new insights on the development of control strategies against ASF.
- Transfer of knowledge on relevant aspects of ASF and transfer diagnostic and epidemiologic tools through training courses and traineeships to a large number of professionals from Animal Health Institutions in EU, Africa, Asia, Russia and Caucasian countries. These harmonized training actions will contribute for the strengthening of knowledge and technological efficacy to face problems caused by ASF in endemic countries and to build up preparedness capacity against the threat of the disease in free countries.
The outcomes of the project will undoubtedly contribute to the sustained development of EU agri-food business by improving prevention, early detection and control of ASF and therefore reducing the socio-economic consequences of potential incursions of the disease in EU from infected regions.
Project Context and Objectives:
African swine fever (ASF) in EU member states is currently confined to Italy (Sardinia), it was recently introduced to Caucasian countries and Russia and it is highly prevalent in sub-Saharan African countries. Surveys during outbreaks occurred previously in Eastern and Southern African regions have shown that although the average prevalence of viral infection in the domestic pigs is high, in many cases serum conversion was not detected. These events pose a great challenge for the prevention and control of ASF, not only locally, but also worldwide. The complex epidemiological scenarios of ASF and the increasing mobility of people, animals and goods across the globe, emphasize the serious threat the disease presents to the growing pig farming sector in Africa and to currently disease-free EU member states.
In order to improve tools and strategies for the prevention and control of ASF, the work developed under the ASFRISK project aimed at fulfilling four objectives contained in the Major Tasks of the work-plan:
Objective 1 (Task 1)
To evaluate the epidemiological situation of ASF in Sardinia and Africa, in particular to characterize, from the molecular and biological point of view, currently circulating field viruses of epidemiological interest and relate the possible occurrence of genome changes with new occurring forms of the disease and diagnosis problems described during recent outbreaks namely in Africa. Furthermore this project aimed at establishing a risk assessment framework that can be applied for any European country to assess the risk of introducing ASF virus into the EU, producing an ASF risk map for Africa and to develop and evaluate control strategies appropriate for the situation of ASF in Africa. Work developed under this Task was focused on three major approaches:
• Molecular and biological characterization of currently circulating ASFV strains of epidemiological interest.
• Development and validation of a generic risk assessment framework for the introduction of ASF into EU countries
• Development of control strategies
Objective 2 (Task 2)
To develop, validate and apply improved, robust and/or simple antibody and nucleic acid detection methods for ASFV for the rapid detection and differential diagnosis of suspected cases of ASF, taking into account the newly emerging viruses circulating in Africa and Sardinia. Particular emphasis will be placed on inexpensive simple-to-use assays suitable for first-line and/or pen-side diagnosis and the deployment of these tests to regional laboratories in the EU and African states. Work developed under this Task was focused on the following approaches:
• Development and validation of:
▪ New rapid, sensitive and specific assays based on ELISA and Immunoblotting tests to detect early and late appearing ASFV antibodies, raised in the course of infection with the new circulating strains of ASFV;
▪ New powerful assays based on robust highly sensitive and specific real-time PCR techniques, for rapid detection of ASFV nucleic-acid;
▪ New rapid diagnostic procedures based on simple highly sensitive and specific isothermal amplification assays;
• Adaptation and evaluation of:
▪ Antibody detection assays based on dot-blot techniques to dipstick format, suitable for pen-side and first-line on-site diagnosis;
▪ Current and newly developed real-time PCR and isothermal amplification assays to portable platforms, suitable for front-line diagnosis of ASF;
• To standardise and validate selected developed assays to OIE standards in order to harmonise ASFV diagnosis in EU member states.
• To use the phage display procedure to search for monoclonal antibodies that could be used in the development of ELISAs to detect antibodies to ASFV and to further develop a strip test using gold nano particles;
• To transfer technologies to diagnostic facilities in Africa for validation of the tests in various regions of the continent.
Objective 3 (Task 3)
The main objective in this Task is the implementation of new strategies for the development of a vaccine against African swine fever (ASF) using and further characterizing a protection model that includes the use of two naturally occurring ASFV isolates of different virulence in which the low virulent virus (ASFV/NH68) induces protective immunity against the highly virulent isolate (ASFV/L60) but with a high residual level of virulence to be used as a vaccine.
Work developed under this Task was focused on the following approaches:
• The characterization of viral-host interactions
• The in vitro and in vivo characterization of pig immune mechanisms relevant for animal survival against ASFV.
• The construction of recombinant viruses by conventional and/or new high throughput methods
• The optimization of OprI presentation model of ASFV antigens as a new system for the production of virus antigens fused with lipoproteins
• The development of complementary methods (antivirals) for the control of the ASF.
Objective 4 (Task 4)
The main objective of the task is to transfer knowledge on relevant aspects of ASF and improved tools for ASF diagnosis, including pen-side, conventional and reference diagnostic tests, and epidemiological modelling and risk assessment strategies aiming at the establishment of control measures, both in Europe if the disease occurs and in Africa where it is endemic. The developed tools within this project in the mentioned fields will be transferred to Asian and African partners within the project and to other Animal Health institutions in EU, Africa, Russia and in the Caucasian region.
Work developed under this Task was focused on the following approaches:
• Organization of training courses and workshops on ASF epidemiology, risk assessment and laboratory diagnosis in African and Asian partners of the project and also targeting Caucasian countries and Russia where the disease was recently introduced.
• Organization of individual short-term training at expert institutions on ASF epidemiology, risk assessment and laboratory diagnostics, for beneficiaries in the project and to other institutions interested namely from Africa, Caucasian countries and Russia were the disease was recently introduced
• Individual long-term training at expert institutions carried out under PhD preparation.
Project Results:
In order to improve tools and strategies for the prevention and control of ASF, the work developed under the ASFRISK project aimed at fulfilling four objectives contained in the Major Tasks of the work-plan:
TASK I
EPIDEMIOLOGY, RISK ASSESSMENT AND CONTROL STRATEGIES
The work developed under Task 1 aimed at fulfilling the following objectives:
To evaluate the epidemiological situation of ASF in Sardinia and Africa, in particular to characterize, from the molecular and biological point of view, currently circulating field viruses of epidemiological interest and relate the possible occurrence of genome changes with currently circulating field viruses of epidemiological interest and relate the possible occurrence of genome changes with new occurring forms of the disease and diagnosis problems described during recent outbreaks namely in Africa. Furthermore this project aimed at establishing a risk assessment framework that can be applied for any European country to assess the risk of introducing ASF virus into the EU, producing an ASF risk map for Africa and to develop and evaluate control strategies appropriate for the situation of ASF in Africa.
The work was developed under seven work-packages as described ahead:
WP1 - Epidemiological and molecular characterization of currently circulating field viruses in African Countries and in Sardinia
Objectives
The main target of this WP has been focused on to increase the knowledge about the epidemiological and molecular features of currently circulating ASFV isolates related to the occurrence of atypical forms of disease and diagnosis problems recently reported in Africa and in Sardinia. The molecular characterization has been based on standardized and new developed protocols for characterizing the genomes of ASFV isolates by targeting independent ASFV genomic regions.
Results
In East Africa the role of warthog was studied by the analysis of samples obtained from warthogs, ticks (Ornithodorus porcinus) and domestic pigs from different parts of Kenya and Tanzania. These results suggest that ticks play the key role in maintenance of the virus, whilst adult warthogs represent a terminal host. The role of bushpigs in ASFV transmission to domestic pigs was studied in connection to the detection of a relatively high presence of ASFV in domestic pigs in which no clinical signs or antibodies were found. These data, together with the genetic data described below, suggest a possible role of bush pigs in the sylvatic cycle, that may harbor a low virulent or avirulent isolate of the virus, or that local pigs exhibit a degree of innate or acquired immunity to the specific virus that is currently circulating. The presence of the domestic cycle in East Africa was widely demonstrated following the ASF outbreaks occurred in Kenya and Uganda since 2007.
These epidemiological findings were supported by molecular characterization of 63 East Africa ASFV isolates from warthogs, bushpigs, ticks and domestic pigs that confirms the concurrent existence of three different transmission cycles of ASF in East Africa; i) Domestic pig-pig without ticks in Western Kenya and in Uganda; ii) domestic pig-warthog or domestic pig-bushpig and iii) domestic pig-tick/warthog sylvatic transmission. The observation of segregation of different genotypes between ticks and adult warthogs is surprising and suggests that further studies in warthogs and ticks are required to better understand the nature of the sylvatic transmission cycle of ASFV.
In West Africa sampling activities supports the fact that the recovered ASF pigs are involved in stock trade and breeding activities and play and essential role in the spreading of the disease in West Africa, which is mainly linked to the movement of infected pigs or pig products. To confirm this hypothesis, 53 West African domestic pigs ASFV isolates were selected for genotyping analysis using standardized procedures and by the analysis of new regions by the use of new set of primers CV1 and CV2. West Africa ASFV isolates were separated in 10 different CVR groups comprising eighteen different variants, revealing a cross-virus circulation between neighbouring countries Togo, Benin and Nigeria in Maritime areas and the co-circulation of multiple tetramer types in one country such as Nigeria or Burkina Faso.
The genetic analysis of a wide range of ASFV from Sardinia obtained from 1978 to 2009 revealed the presence of a unique genotype (genotype I). However, two clusters were identified, accordingly to their temporal distribution, namely sub-group III and sub-group X, by the analysis of B602L gene revealing a minor difference in the number of tetrameric repeats, placing this latter showing a deletion of 12 tetramer repeats located in the centre of the array.
In order to facilitate genotyping and characterization of ASFV, a microarray was designed, developed and tested in this project. The area targeted for the array was the C-terminal end of the p72 protein, the primary region used to place ASFV into one of the twenty-two established genotypes. The technology chosen to develop the array was the Luminex 200 system which provides an established, practical technology for multiplex assays of proteins and nucleic acids. The microarray developed within this project provides complete and correct identification for all described genotypes with a comparable sensitivity to real-time PCR, with the detection sensitivity possibly somewhat more sensitive than the typing.
WP 2 - Biological characterization of currently circulating field viruses in African countries and in Sardinia
Objectives
This WP has been focused on the characterization of biological aspects of ASFV related with the virulence of different isolates present in Africa and in Sardinia, the lesions induced and the immunological features triggered upon infection with those isolates. The main objective of this WP has been to evaluate clinically and pathologically the forms of disease currently observed in the endemic areas and compare them with the previous findings on acute and subacute forms of the disease.
Summary of results on the WP
Four different ASFV strains were selected and several experiments were developed by the inoculation of these isolates in domestic pigs (European and African breeds) for the study of clinical, pathological and immunological (cytokines expression) findings in these animals. The experiment developed with the supposed attenuated Sardinian isolate suggests that this isolate is highly virulent capable of inducing a subacute to acute form disease with even severe clinical and pathological reactions. ASFV could be detected from the blood of one animal during the incubation period, but this Sardinian isolate is clearly characterized by clinical and pathological signs, which may facilitate the diagnosis of the disease in field conditions and therefore the implementation of control measures including stamping out practices.
The study of African isolates found to be responsible for the appearance of non evident ASF clinical signs in combination to lack of humoral response co-existing with a high viral load (as described in WP1) was done by three different experiments. The inoculation of these isolates in European breeds suggested that the ASF Kenya/05 virus isolate is of low-moderate virulence capable to induce a subacute picture of disease not clearly characterized by typical clinical and pathological signs but with a conventional antibody response. However, the inoculation of the same isolate in Kenyan “indigenous pigs shown a significant delay of onset of with an unclear, unspecific, non-patognomonic picture of the disease that easily goes unrecognized. These animals present a detectable viraemia at 14 dpi with intermittent peaks and a very different humoral response, with delayed or absence seroconversion. These findings, the presence of unrecognized diseased animals with viraemia but no antibodies, have serious implications on control and success of ASF eradication in East Africa.
WP 3 - Risk Assessment of the introduction of ASFV into the EU
Objectives
This WP aims at developing a framework that allows assessing the risk of introduction of ASFV into any European Union country, taking into account the current situation of ASF in endemic areas. The framework estimates the overall risk of ASFV in each of the EU countries and specifically details the risk by each of the analyzed pathways of introduction, providing and interesting tool for supporting risk manager decisions.
Summary of results on the WP
The potential ASFV introduction routes into EU countries were selected and evaluated independently based on the availability and quality of data available. Several expert opinion elicitation sessions were developed during the project in order to assess the relative importance of some parameters, or validate the models and the results obtained by them. Finally, all these pathways were conveniently adapted and standardized to be included in the final framework to obtain the overall risk for ASFV introduction into EU countries.
For legal imports assay, two quantitative models were developed revealing a low annual risk for ASFV introduction by these routes. This risk is mainly concentrated in some countries such as Poland and Lithuania in the case of legal pigs; and Romania and Bulgaria for pig products. In the case of illegal import pathway, was performed as a semi- quantitative model obtained by linear weighted combination of the risk level for each indicator. Result reveals that no country poses the highest level of risk (very high) of this route, but several countries were in the high risk category: France, Germany, Italy and United Kingdom. For the transport fomite pathway, three different means of transport were included in the model: returning trucks from affected areas and waste disposal from boats and planes, using a similar approach than illegal import pathway. Results shown that returning trucks is the transport pathway that nowadays poses the highest risk in the EU and Lithuania and Poland (high) pose a considerable relative risk by this pathway. For the wild boar movement analysis, a previous suitability map of the potential wild boar distribution in EU was built. Then, a semiquantitative model was performed, revealing that Romania (high), followed by Finland, Latvia and Poland (medium) are the EU countries at highest risk for ASFV introduction borders by wild boar natural movement. A common exposure assessment for the illegal, transport and wild boar pathway was developed in order to be combined with pathways in the final framework. The consequence assessment was a stochastic spatio-temporal model for the spread of ASF within and between EU Member States. The model aimed at identified the MS that most likely spread ASF, “super-spreaders”, Denmark and The Netherlands; or MS that receive cases from other MS “super-receivers”, Germany and Poland during the high risk period (HRP).
All this information was conveniently adapted and incorporated in a single Excel file for the final framework for ASFV introduction into EU. Detailed Summary of results on the WP of each of the analyzed pathways are published or in process to be published and the final framework will be provided to European Commission and EU countries to be used as an interesting tool for risk management.
WP 4 - Risk mapping in ASFV endemic areas
Objectives
This WP was aimed at compiling existing knowledge and epidemiological data to produce a risk map for Africa and to spatially explore the epidemiology of ASF in Sardinia. The outcome of this WP has increased the understanding of the pig production chain, identifying cultural, sociological and economical factors that contribute to the ASF occurrence in Africa and Sardinia and providing a better description of the ASF dynamics in rural and urban areas.
Summary of results on the WP
The main conclusions obtained within this WP for Africa can be summarized in the following points: 1) North and south-western Africa are likely to be unsuitable for occurrence of ASF, 2) there is a strong evidence for suitability for outbreaks in large parts of eastern, south-eastern and western Africa but with substantial within-country heterogeneity, 3) there are many areas with high levels of uncertainty, particularly in central Africa and several countries in West Africa, 4) comparison of suitability estimates with the limited official outbreak data available revealed a good degree of agreement.
In Sardinia, the Bayesian model indicate that the probability of ASFV occurrence in Sardinia was mainly associated to a high number of confined (closed) farms, high density of roads, high mean altitude, high number of open fattening farms and high total number of pigs per commune. The high risk of confined farms may be the effect of higher notification of outbreaks or a high use of waste containing pork products to feed pigs in confined farms, particularly in small ones. It was also suggested that the system of farmer´s compensation after an outbreak in Italy may be excessive for Sardinian pigs, bred using non-professional type of farming, and mainly when referring to fattening pigs which may promote the intentional introduction of disease. The association between the total number of pigs, the density of roads and the ASFV occurrence per commune suggest that illegal trade of pigs may be a source of infection in some areas. Moreover, Summary of results on the WP reveal that Communes with most of their farms censed (i.e. controlled by the veterinary authority) are less likely to have ASF outbreaks than not censed (i.e. unsupervised) farms. This result highlights the importance that relative simple measures, such as a compulsory registration of the pig population, may have in the ASF control in Sardinia. Wild boars seem not to be important way of ASFV transmission in Sardinia. Finally, farms for self-consumption as well as areas with high pig density and with large herd sizes and intense trade/movement of animals were found to have less risk of ASFV occurrence than the background risk of the region, which may be associated with less high risk contacts or higher biosecurity measures, respectively.
WP5 - Risk Assessment for African countries
Objectives
To perform risk assessments for endemically and currently uninfected African countries, taking into account the particular epidemiology of a region, data collection about ASF epidemiology and pig production systems in selected areas is needed.
Summary of results on the WP
West African countries (Côte d´Ivoire, Ghana, Benin, Togo and Burkina Faso) were selected as target countries for the analysis. Lot of information was collected from these countries by official data sources, surveys sent to the veterinary services and expert opinion developed in Côte d´Ivoire during a workshop with the expert veterinary epidemiologist from the neighbouring countries. The study of this information suggests that a traditional connection of trade and cultural relations is present in West African countries that hardly influence the presence and spread of ASF in the area. The pig population in these countries is not very high, being Burkina Faso the country with highest number of pigs. Most of the swine population is rare in a free range production system, followed by semi confined familiar systems.
The use of the information obtained by the expert opinion session and their incorporation into a simple model structure, allow us to develop a semiquantitative model that analyze the different pathways of ASF introduction and spread in the area. According to this risk assessment, the pathway responsible for most of the ASF outbreaks in the area is the movement of infected animals. Wild animals and ticks seem not to have an important role in the ASF transmission in the area. Coast regions represent a very important source of infection due to the contaminated leftovers present around the ports. These Summary of results on the WP also point out that Togo is a key country in the area that easily connects all countries around it, being a spreader of the disease.
WP6 - Development of control and preventive strategies for African Countries
Objectives
Control strategies currently carried out in Africa and other parts of the world would be assessed. Several recommendations will be developed considering the outcomes of the risk assessments performed under WP5.
Summary of results on the WP
Information about ASF control activities and veterinary capacities in many African countries was collected. This information suggests that from West African countries, Ghana is the country with veterinary services at highest level, with higher number of veterinarians, laboratories and resources to fight against animal diseases. Second place is for Burkina Faso or Côte d´ Ivoire, depending on the analyzed parameter. These results could be considered as an indicator of the development and quality level of veterinary services in these countries as should be considered in future development and control strategies for this African countries.
Some of the critical points considered for ASF control in West Africa were the traditional type of pig production system by free range system, without control of feeding practices, the lack of knowledge by the farmers, dealers and pig drivers; and very important the high connection between ethnic groups of different countries (Benin-Togo, Togo-Ghana). These facts facilitate disease persistence in the area and potential spread within countries. These points were considered for the development of the suggestions and advices for ASF control in the area.
WP7 - DNA sequencing and sequence data bank
Objectives
Further developments for genome characterization will be attempted by applying high throughput very rapid sequencing for complete genome sequences of ASFV. These investigations will lead to a series of new insights into viral genome sequence conservation and variability, genetic diversity in nature and phylogenetic classification of isolates.
Summary of results on the WP
Sardinia represents a unique model for studies on ASFV evolution over time in a rather confined geographical region where a single virus introduction is supposed to have occurred. Therefore, a group of 9 isolates and 13 total samples representing the time-line since introduction to Sardinia was selected. One isolate (Sardinia2008) was selected for sequencing using 454 technology (performed during the first reporting period). For the remaining 13 samples, the illumina platform was chosen. The selection of technology was based on the properties that technologies possess and the price. An assessment of the technologies concluded that illumina could provide the data needed at the best price.
In terms of methodology, in the 454 run, random amplification was employed, while for the illumina sequencing a specific long PCR (10-20 kb) protocol was employed. For most segments the PCRs were successful. Segment 1 did not produce product for several samples, therefore based on the 454 sequence of Sardinia2008 new primers were designed. The newly designed primers allowed this segment to be amplified for all the samples. To date only the 454 sequence of Sardinia2008 is available. The other 12 sequences have had their genomes amplified by 15 specific PCRS, gel purified and sent to the sequencing platform. The platform has prepared libraries and now sequencing is being developed. To determine the genetic relationships among the AFSV genomes we used the concatenated core conserved set of genes. This analysis of the full genome sequences reveals that the major differences among AFSV genomes were reported in the copy number of five Multi Gene Families (MGF). It has been proposed that non-pathogenic isolates present a deletion of 8 to 10 Kbp at the left end of the sequence. This sequence is present in pathogenic strains and contains six copies of the MGF 360.
TASK 2
DEVELOPMENT AND VALIDATION OF SENSITIVE DIAGNOSTIC TECHNIQUES, INCLUDING PEN-SIDE TESTS, WITH THE EXISTING FIELD VIRUSES
This Task focused on the development, validation and application of improved, robust and/or simple antibody and nucleic acid detection methods for ASFV for the rapid detection and differential diagnosis of suspected cases of ASF, taking into account the currently circulating isolates in Sardinia and newly emerging ones circulating in Africa. Particular emphasis has been dedicated to inexpensive simple-to-use assays suitable for first-line and/or pen-side diagnosis and the deployment of these tests to regional laboratories in the EU and African states.
The work was developed under six work-packages as described ahead:
WP8 - Antibody detection techniques based on the existing circulating virus strains
and
WP 9 - Development of new ELISA tests based on the use of new antigens and monoclonal antibodies obtained from emerging “new” isolates of ASFV in Eastern, Southern Africa and Sardinia
Objectives:
Assessment and further validation, new rapid, sensitive and specific assays based on ELISA format, using different antigens and new ELISAs based on the determination of specific IgM antibodies with selected antigens. Development and assessment of new ELISAs based on the use of new recombinant proteins, semi-purified antigens and monoclonal antibodies, in order to establish robust ASF serum-diagnostics in the endemic regions of Europe and Africa following different strategies.
Summary of results on the WP:
Validation of ELISAs using pp62, the co-expressed p15 and p32 proteins in baculovarus systems and the fused p15-p32 protein:
Four standardized recombinant-ELISAs (rec-ELISAs) developed in previous projects and based on the baculovirus expressed proteins pp62, the co-expressed p15 and p30, the fused p15-p30 (HT) and the p54 protein were selected for further validation purposes. To this end a comparative study was initially performed by the analysis of i) 252 field samples collected in 2008-2009 in Sardinia and East Africa, and ii) 89 experimental samples obtained from in vivo experimental infections using current Kenyan and Sardinian circulating ASFV strains. From the sensitivity and specificity results obtained the rec-ELISA based on the fused p15-p30 (HT) recombinant protein was selected as the most promising protein to be used in ASF serological tests. A complete validation of the selected rec-HT-ELISA was subsequently performed through the analyses of a total of 1,873 field sera collected since 2004 until 2010 from endemic areas in Sub-Saharan Africa, including East, Central and West African countries (Kenya, Uganda, Tanzania, Cote de Voire, Togo, Ghana, Burkina Faso, Benin, Rwanda, Congo-Brazzaville and Nigeria) and Europe (Sardinia, Italy). The OIE prescribed assays (ELISA and IB) were used as gold standards comparative methods. From the analysis of 329 positive and 1,544 negative field sera, the sensitivity and specificity of the rec-HT-ELISA was established in 95% and 99% respectively. Also, the competence of the rec-HT- ELISA to detect specific antibodies during acute, sub-acute and chronic infections was assessed by the analyses of 446 sera collected at different stages from seven independent experimental infections using ASFV circulating isolates. In addition it was a perfect agreement between the specific antibody response detectable using the OIE prescribed assays and that detectable using the rec-HT-ELISA.
Further validation of ELISAs using p54, histone-like protein (gene A104R), and the uncharacterized virus proteins encoded by genes B602L and K205R expressed in E coli.: An initial validation showed that there was almost perfect agreement between the results of the “gold standard” tests and the results obtained by the p54-ELISA, the pK205R-ELISA or the pB602L-ELISA, but not with those obtained using the pA104R-ELISA. Complete validation of the selected rec-ELISAs was performed using a collection of 669 field sera collected from Sardinia and Africa since 2004 up to 2010. The pB602L protein did not allow full validation due to its insoluble properties. After the analysis of 235 positive East African sera the sensitivity of the p54-ELISA was less than 75%, not recommended for the detection of specific antibodies in East Africa. Interestingly, the pK205R protein was identified as a potentially useful serological diagnostic antigen for the detection of IgM responses, as it was able to detect all infected animals as early as 7 days post infection. In addition , a new ELISA based on combination of rec-ASFV proteins HT, p30 and pp62 was evaluated using a selection, short collection, of positive (20) and negative field sera (40) from East Africa. These results obtained did not improve those obtained using HT recombinant protein.
Standardisation of commercial prototype ELISA for IgG ASFV detection based on the fused p15-p30 (HT) protein: The p15-p30 fused (HT) protein expressed in the baculovirus expression system was transferred for the development, standardization and further validation of a new commercial ELISA. The results were compared with the commercially available competition ELISA from Ingenasa, based on VP72 protein. The results with the HT antigen indicated a similar analytical sensitivity than those obtained with the OIE ELISA and the commercial INGEZIM PPA COMPAC ELISA The specificity of this assay using serum samples from ASFV-free areas was > 99%. From the results obtained it was concluded that the HT protein is useful for detection of IgG antibodies specific for ASF virus when used in parallel with a negative antigen to reduce the background. This HT commercial ELISA could be and additional, complementary useful tool for ASF diagnosis.
Standardization of a commercial prototype ELISA for IgM ASFV detection: Three different antigens were used for the development of ELISAs for IgM ASFV detection; i) the HT recombinant protein, ii) the K205R recombinant protein produced in E.coli as protein fusion with GST and iii) the Vp72 ASFV purified protein (from Vero cells cultures). Finally for ASFV IgM specific detection the most promising results were obtained using the vp72 major antigenic protein. However, further validation is required to obtain a final IgM ELISA prototype to be used in Animal Health Laboratories.
Target Antigenic proteins from different ASFV isolates as new candidates for ELISA: The analysis of the specific sequences of the target antigenic ASFV proteins -p72, pp62, p54, p30, pB602L- from recent ASFV African and Sardinia isolates showed the East Africa Kenya 1950 isolate belonging to p72 genotype X as the most variable one. This isolate presented identity scores which dropped to 89% and 85% when the full length p30 and p54-gene sequences were compared with the Spanish ASFV isolates from which conventional serological antigens are obtained. Additionally, the analysis of p30 and p54 sequences showed the regions with the highest sequence variability also presented high antigenic indexes and theoretical low structural order. These findings were supported in a further comparative study of the 149 VP54 and 84 VP30 sequences obtained from current circulating isolates in Europe and Africa and representatives of the 22 P72 genotypes. Nevertheless, due to results from other workpackages there was not a need for new ELISAs using new antigens (see below point f). Instead, the efforts were directed to study the origin/cause for the documented low incidence of detectable serological response to ASFV infection in ASF virus positive pigs in East and southeast Africa using OIE-prescribed ASF serological methods and towards to the development, standardization and validation of a new confirmatory antibody detection test, the indirect. Immunoperox. technique (IPT) (see below, and results of task 1 as well).
New ELISA Tests based on Soluble cytoplasmatic Antigen (Cp-Ag) from different ASFV isolates of Africa and Caucasus region: Since the OIE-prescribed methods are based on the use of total semipurified antigen obtained from the Spanish ASFV E70 isolate belonging to genotype I, a possible explanation for the unexpectedly low seropositive response with east African sera may reside in the polymorphisms in immunodominant viral antigens. So, it was not clear at the beginning of the project whether current OIE sero-diagnostic techniques will be sufficiently sensitive and specific to detect specific antibody response against the different ASFV circulating variants in Africa and in the Caucasus areas. To sort out this question, the work was focused on determining the capability and competence of the OIE formal serological diagnostic techniques employed under different epidemiological scenarios currently occurring in Europe and Africa. To this end six ASFV isolates selected following the genome variability criteria and date collection were used for the preparation of six new soluble cytoplasmic antigens (Cp-Ags). The ASFV isolates selected included, i) the most variable p72 genotypes X, IX, VIII and V as so far described circulating in East African countries (Ken08/Tk2.1; Ken06.Bus Moz64 and MwLil20/1), ii) the historical circulating European isolate belonging to p72 genotype I (E70), ,and a new objective not included at the beginning of the project: iii) the use of current European p72 genotype II (Arm07). COS-1 cells (donated by CBM partner) were employed for antigen production by infection and later semipurification using each of the six selected ASFV strains according the procedure described in the OIE Manual of diagnosis (OIE 2008). The new Cp-Ags were used as antigens in six different ELISA assays based on selected viruses. Subsequently, a validation study was carried out through a comparative analysis of 1,062 serum field samples collected from 2003 up to 2010 from both domestic and wild pigs in endemic areas of Sub-Saharan African regions (Kenya, Uganda, Tanzania, Cote de Voire, Togo, Ghana, Burkina Faso, Benin, Rwanda, Congo-Brazzaville and Nigeria) and Europe (Sardinia (Italy), as well as 214 experimental sera collected at various stages from six independent experimental infections. There was a perfect agreement of the new developed Cp-Ag-ELISAs and the OIE serological techniques in the analysis of both experimental and field samples collected from current affected areas in Africa and Europe. This clearly demonstrates the capability of the OIE formal diagnostic techniques to perform a serological diagnosis with high sensitivity, specificity and confidence independently of the genetic variants circulating. In addition, all results were confirmed by a new confirmatory serological technique based on Indirect Immunoperoxidase Test (IPT) that was simultaneously developed and validated for each genotype under study.
Other approaches performed during the project included the development of phage display library to produce monoclonal single chain variable fragments (scFv) that could be used in the development of ELISAs to detect antibodies to ASFV in a near future.
Adaptation and evaluation antibody detection assays based on dot-blot techniques to dipstick format, suitable for pen-side and first-line on-site diagnosis.
The epidemiological situation of ASF worldwide has highlighted the need for front-line/penside tests as good tools for disease diagnosis allowing earlier implementation of sanitary control measures. To this end, a rapid, one-step immunochromatographic strip (pen-side test) capable of specifically detecting anti-ASF antibodies in serum specimens has been developed by INGENASA in collaboration with CISA-INIA. The qualitative assay is based on a direct immunochromatographic assay in which the detector reagent consisted of red carboxyl-modified latex microparticles covalently coated with VP72 purified protein. The capture reagent was the same protein adsorbed on the nitrocellulose membrane strip to form a test line. In addition, blue latex microparticles coated with a control protein and a second line created above the test line, by the immobilization of anti-control protein antibodies, was used as a control of test. The limit detection of the new assay was established in 9 ng/ml (1.35 ng/test). Once standardized, a complete validation of the new developed assay was performed by the analysis of a wide collection of field and experimental samples previously classified as positive or negative using the OIE-prescribed assays as “gold standards”. The diagnostic sensitivity was established in 99% by testing a total of 208 ASF antibody positive field sera collecting in East and West endemic African countries. The comparative Summary of results on the WP obtained using the new pen-side test and the conventional OIE and INGENASA tests demonstrated a diagnostic specificity of 99,9% by testing a total of 1,210 ASF antibody negative field sera collecting in East and West endemic African countries and free areas of Europe. As a conclusion a first pen side test for ASF antibody detection have been developed and validated for rapid on site ASF detection. The test, registered and commercially available by INGENASA [INGEZIM PPA CROM R.11.PPA.K41] allows sensitivity and specific ASF antibody detection in 10 minutes and can be employed an easy single step performing method where laboratory support and skilled personnel are limited.
WP 10 – Nucleic acid-based assays for detection of currently circulating ASFV isolates
Objectives:
Development and validation of new powerful assays based on robust highly sensitive and specific real-time PCR techniques, for rapid detection of ASFV nucleic-acid, and rapid diagnostic procedures based on simple highly sensitive and specific isothermal amplification assays;
Summary of results on the WP:
Further development of the TaqMan real-time PCR technology for the improved detection of ASFV
A new real-time PCR technology based on the use of universal commercial probes, Universal Probe Library (UPL), commercialized by Roche Applied Science, main advantages being reasonably low cost, short time of delivery, and ready-to-use presentation. Optimization and analytical sensitivity and specificity assays of the developed UPL PCR (using UPL#162) showed a a sensitivity quite similar to that provided by designed TaqMan PCR, while it is 10-fold more sensitive than the established reference real-time method. The detection limit of the UPL PCR showed to be between 4 and 18 DNA copies. A comprehensive study to assure the detection range of the developed UPL PCR was carried out during this time with the analysis of a collection of 26 ASFV isolates, belonging to 17 of the 22 reported p72 genotypes. Afterwards, validation experiments using an extensive collection of field porcine and tick samples (n=256), coming from Eastern and Western African regions affected by ASF, demonstrated that the UPL PCR technique was able to detect over a 10% more positive samples compared to the OIE TaqMan PCR, confirming the superior diagnostic sensitivity of the new molecular assay. Clinical material collected during experimental infections with different ASFV p72 genotypes, specifically from ASFV genotypes I, II, IX, and X, was useful to assure both the capacity of the UPL PCR for an early viral DNA detection and the competence of the technique to be applied in any ASF diagnostic target sample. An internal control PCR assay based on the detection of a β actin gene DNA fragment using a specific primer set and the same UPL probe (UPL#162) than that selected for ASFV detection was developed. The complete study offers a new validated real-time PCR technique, together with a simple internal control test, ready for its application in the routine diagnosis of ASF. The final standard operation procedure (SOP) of the developed real-time PCR method using UPL#162 probe was transferred to all consortium partners participating in the ring trial conducted under WP 13, and to several ASF National Reference Laboratories of the EU. In addition, INGENASA is working now to adapt this robust PCR technique in a commercial format.
Development of a LATE PCR system for detection of ASFV
The development and optimisation of a LATE (Linear-After-The-Exponential) PCR assay for ASFV detection has been carried out. The technology developed at Brandeis University (MA, USA) and the developed ASF assay was aimed at adaptation to their portable PCR platform BioSeeq. This work was carried out by SVA in collaboration with Brandeis University and Smiths Detection. Optimization and analytical sensitivity and specificity assays of the developed LATE PCR was carried out and the assay was further validated and also tested in a ring trial organized within EPIZONE with good results, further demonstrating the specificity and sensitivity of the new assay. The assay was ready for adaptation to portable platform but this step was dependent on the commercial collaborator.
Rapid diagnostic procedures based on simple highly sensitive and specific Loop-Mediated Isothermal Amplification (LAMP) assays
LAMP is based on amplification of nucleic acids without the need of PCR equipment. The comparative simplicity of the technology makes LAMP adaptable to front-line testing in regional laboratories, simple diagnostic situations and even to pen-side testing. This work was mainly carried out by QUBand SVA. Seven accelerated LAMP assays were designed against the 4CL, 9GL and vp72 genes using on line software (PrimerExplorer version 3&4) provided by Eiken Ltd. The assays were designed using alignments based on 23 (4CL), 32 (9GL) or 50 (vp72) sequences. DNA from the fourteen ASFV strains described above was used for the evaluation of the assays.Upon further optimization one assay was singled out and consequently used in all experiments (ASFV vp72 F3-354). Ten-fold dilution series of ASFV Moz64, Ang72, MwLil 20/1, CV97, Ug03H, Ken06 and Ken07 were further tested to determine the detection limit, indicating detection limits around 40-50 copies of viral DNA. Protocol for this assay was created and adapted to two different LAMP reaction assays using two different portable platforms (see below). The protocol was also distributed to other partners and used within the ring trial conducted under WP13. A panel of LAMP assays for cost-efficient and rapid detection of ASFV has been developed, suitable for use in basic regional and local laboratories.
WP 11 – Pen-side test and front line diagnostic tests for ASF
Objectives
A general disadvantage of all laboratory-based methods for diagnosis of ASF (including current nucleic-acid-based detection systems) is the inevitable delay caused by the time needed for samples to reach the central reference laboratory. To overcome this problem, this WP has been focused on the development of, or adaptation to robust, rapid and portable assays for front-line diagnosis of ASF, to be used near (at the pen-side of) the suspect cases. Simplified nucleic-acid and antibody purification techniques suitable for field-testing has been evaluated and used.
Summary of results on the WP
Development of a simple and robust dot-blot assay for detection of specific ASFV antibodies
A rapid, one-step immunochromatographic strip (pen-side test) capable of specifically detecting anti-ASF antibodies in serum specimens has been developed by INGENASA in collaboration with CISA-INIA (P4). The qualitative assay is based on a direct immunochromatographic assay in which the detector reagent consisted of red carboxyl-modified latex microparticles covalently coated with VP72 purified protein. The capture reagent was the same protein adsorbed on the nitrocellulose membrane strip to form a test line. In addition, blue latex microparticles coated with a control protein and a second line created above the test line, by the immobilization of anti-control protein antibodies, was used as a control of test
The diagnostic specificity was established in 99,9% by testing a total of 1,210 ASF antibody negative field sera collecting in East and West endemic African countries and free areas of Europe.
The analytical sensitivity was determined by the analyses of 160 serum samples obtained at different days post infection from experimentally infected animals using ASFV isolates belonging to p72 genotype I and X under WP2
As a conclusion a first pen side test for ASF antibody detection have been developed and validated for rapid on site ASF detection. The test, registered and commercially available by INGENASA [INGEZIM PPA CROM R.11.PPA.K41] allows sensitivity and specific ASF antibody detection in 10 minutes and can be employed an easy single step performing method where laboratory support and skilled personnel are limited.
Adaptation and evaluation current and newly developed real-time PCR and isothermal amplification assays to portable platforms, suitable for front-line diagnosis:
a) UPL system was tested and adapted to other available portable instruments. Several alternative systems were investigated and finally two systems were tested using the UPL 162 ASFV PCR developed. These platforms have been also investigated or tested for using UPL ASFV real-time PCR. The SmartCycler is perhaps the most established real-time PCR instrument that can qualify as a portable instrument, It does not have a battery, so there is a need to connect a high voltage power source, It has 16 individual thermocyclers, so 16 assays can be run independently. Also, the specialized tubes are expensive, at 1 euro each. Several enzymes kits were tested including TaqGold, Superscript III (Platinum Taq), TaqMan fast advanced, AgPath. All kits worked fine with UPL. Because of tubes used, it is not compatible with FTA/3MM paper-in procedures. The TCOR-4 instrument from Tetracore is new on the market and as part of this project, access was provided to prototypes. The instrument itself weighs 2.5 kg and has an 8 hour battery life. It has four independent wells, runs RT-PCR and Isothermal assays and has two colours to run duplex assays. As with the SmartCycler, the issue of extraction is not addressed. The system worked with UPL and different PCR kits and the sensitivity matches other real-time instruments, such as Stratagene Mx3005. In our testing the complete platform involved the SmartCycler or TCOR-4 combined with extracting ASFV DNA from FTA cards. Given that all-in-one platforms are not yet foreseen in the near future as a practical option, platforms need to be assembled with sample preparation and detection that are compatible.
b) LAMP system: Optigene Ltd, Horsham, UK is a new company specialising in LAMP technology which recently released a new LAMP reaction kit. The new kit contains a new improved polymerase in a convenient buffer which includes everything, including an intercalating dye, making it suitable for real time detection. All one has to add are the oligos, water and target. The ASFV vp72 F3-1739 (previously F3-354) assay was developed using the LAMP kit from Eiken. Running both kits side by side using two different targets, ASFV strains Ba71v (13400 copies) and MwLil 20/1 (1000 copies) showed that the new kit from Optigene is a lot faster than the Eiken kit. The kit was further evaluated on the 14 ASFV strains provided by CISA-INIA to be used in all optimisation work within the consortium. Other results included that the linear response seems to be genotype dependent; the detection point is verified to be around 50 copies ; the F3-354/optigene system developed is faster than the TopoII/NEB system (developed at Pirbright) and slightly more sensitive.
A LAMP assay is made LFD compatible by attaching Biotin and Digoxigenin labels to the loop primers. In this way the product would be captured in the LFD device and in turn capturing the beads giving a positive signal. It is a good cheap alternative to detect LAMP products in a low cost setup. The F3-1739 LFD LAMP assay was successfully tested on a selection of ASFV strains. The LFD LAMP assay is as fast and works well on the LFD device. the LFD assay worked well on the clinical material tested and even on low copy numbers; We can summarize the ASFV vp72 F3-354 assay has been successfully transferred to a portable LAMP platform.
Evaluation of nucleic acid and antibody purification techniques and application to on site diagnosis of ASF.
The use of filter papers (FTA and 3MM) for collection and storage of virus, nucleic acid and antibodies, from the current circulating strains, were also evaluated. Sensitivity using FTA cards is enough to detect viraemic animals, mainly by real-time PCR. Interestingly, the new UPL real-time PCR developed under this project seems to be more sensitive than reference methods for ASFV detection in FTA cards. This could be a Practical system to monitor wild boars
WP 12 - Establishment of virus strain and sample bank, and selection of samples on the basis of their epidemiological and geographical relevance (to be started on Month 6)
Objectives
A collection of serum and tissue samples from domestic pigs, European wild boars, bushpigs and warthogs wildlife reservoirs has ben established in collaboration with the veterinary services of Italy and several African countries, through the connections existing through OVI-South Africa, LANADA-LCPA-Côte d’Ivoire and Kenya. This collection represents numerous variants of the new circulating virus, including serum samples.
Summary of results on the WP
A sample bank database (http://www.izsum.it/IZSUM/EN/pages/wfLoginDB.aspx(si apre in una nuova finestra)) has been built at IZS-UM to collect and organize a collection of ASFV positive and negative samples. This sample bank is a web application created to archive and to manage information on the availability and the characteristics of samples. This web application has been developed with the ASP dot net technology and has been implemented using DMS Oracle. The authentication database has been realised in Microsoft SQL Server. Basically, the database is a repository of samples stored by the users after their authentication. Each user is allowed to search and obtain data on sample details and particularly it is possible to (i) view a summary list of the samples of the whole bank (ii) access to an advanced search functionality and (iii) get all the details of a single sample.
WP 13 International standardisation and validadation of the developed diagnostic assays
Objectives
Novel robust and simple antibody detection methods and nucleic acid based assays developed under WP 8, 9, 10 and 11 have been further evaluated for their suitability for a rapid detection and differential diagnosis of suspected cases of ASF, taking into account the newly emerging viruses circulating in Africa and Sardinia.
Summary of results on the WP
An initial validation of the PCR tests developed within WP10 was carried out in the context of the ring trial on ASFV real-time PCR within the EPIZONE network.
Further a ring trial on ASFV real-time PCR (UPL-PCR and LAMP assay, both developed under the project) was organized within partners of the ASFRISK.
• UPL-PCR method was selected on the basis of its high sensitivity and specificity; its possibility for analysis also in degraded DNA samples; the possibility to use of standard protocol in all Real Time machines and its features, easy and fast to be performed.
• LAMP assay (Reaction of amplification by a strand displacement Taq in isothermal conditions, was selected on the basis of the possibility to use wet and dry assays, its high amplification efficiency and high specificity, no need different steps or temperature profiles, no need sophisticated equipments and possible use on site and in not well equipped laboratories.
TASK 3
HOST-PATHOGEN INTERACTIONS AND VIRAL IMMUNE RESPONSE IN VIEW OF THE DEVELOPMENT OF A VACCINE
The main objective in this Task is the implementation of new strategies for the development of a vaccine against African swine fever (ASF) using and further characterizing a protection model that includes the use of two naturally occurring ASFV isolates of different virulence in which the low virulent virus (ASFV/NH68) induces protective immunity against the highly virulent isolate (ASFV/L60) but with a high residual level of virulence to be used as a vaccine. Work developed under this Task aimed at:
- The characterization of viral host interactions
- The in vitro and in vivo characterization of pig immune mechanisms relevant for animal survival against ASFV.
- The construction of recombinant viruses by conventional and/or new high throughput methods
- The optimization of OprI presentation model of ASFV antigens as a new system for the production of virus antigens fused with lipoproteins
- The development of complementary methods (antivirals) for the control of the ASF.
The work was developed under five work-packages as described ahead:
WP 14 - Role of virus and host genes in infection: virus genes and cellular factors involved in the control of host defences, immune evasion and virus productivity
Objectives
Viruses have developed different strategies for their maintenance in the host. One of these strategies, used by ASFV, is to produce proteins able to evade the host immune response and thus contribute to the pathogenicity of the virus. The identification and definition of the mechanisms of virus encoded “evasion” genes which interfere with host defences and immune responses and/or affect virus productivity and virulence, will provide a rational basis for the construction and testing of attenuated, non-pathogenic, gene deletion vaccines. We propose to study the role of ASFV genes involved in the control of host defenses and immune evasion, and the characterization of host cell factors important for virus productivity. The associated understanding of virus host cell manipulation of host defense mechanisms may provide new approaches for the manipulation of cell immune responses in health and disease.
Summary of results on the WP:
ASFV genes involved in immune response
The role of the A238L viral gene in the modulation of COX-2, TNF-alpha, iNOS and IL-6 expression has been shown to be mediated through the control of their promoter activities. The mechanism involves the modulation of NF-kB, NF-AT and c-Jun transactivation by the control of CREB binding protein/p300 co-activators function. Taken together, these results have established a novel mechanism in the control of gene expression by the viral protein A238L which could represent an efficient strategy used by ASFV to evade the innate immune response. The role of another ASFV gene involved in immune response, the EP153R C-type lectin homolog, has been also studied with the use of virus deletion mutants generated in this project (SWP 18.2) demonstrating an interference of the virus gene with the expression of MHC-I in the plasma membrane, as it is shown below in the summary of work developed in SWP 16.4.
ASFV genes involved in the control of apoptosis
In this Project it has been reported the effect of several ASFV genes (A179L, which encodes a Bcl-2 homologue, A224L, which encodes a protein homologous to IAP family members, and the viral lectin encoded by EP153R gene) involved in the control of apoptosis. It has been also reported that ASFV induces apoptosis in the infected cell in a post binding step, during or after virus uncoating, through the activation of caspases. Hence, we showed that p53 expression is enhanced in Vero cells, phosphorylated and located in the nucleus from early times after ASFV infection. By the use of the virus recombinants generated in SWP 18.2 we have also been able to further confirm that the deletion of genes like A224L and EP153R resulted in an increase of the apoptotic process induced by the virus infection in porcine IPAM and WSL cells, supporting the role of these ASFV genes in the control of the programmed cell death developed into the extensive antiviral response presented by the cell after virus infection.
The expression of the IAP homologue gene was characterized along parallel in vitro infections of porcine macrophages by L60 and NHV ASFV isolates. L60 inhibited caspase-3 activation by 18 hpi and induced less DNA intranucleosomic fragmentation at 8 hpi than NHV. The transcription of IAP homologue initiates very early after infection with both isolates. However, the protein was produced later, from 4 to 8 hpi, and was more abundant in L60 than in NHV-infected cells. Overall these results suggest the existence of a post-transcriptional step in the regulation of the viral gene expression.
Factors involved in the control of cell cycle
The relevance of the modulation of Cdk2 and cyclin E expression in the ASFV DNA replication and protein synthesis has been confirmed. We have also identified two new targets for the viral evasion strategy: one is a cellular factor (eIF4E) whose depletion with siRNA blocked the ASFV infection, and the other is a viral gene (g5R) with a possible activity on decapping RNAs during virus infection. Besides, the involvement of AKT phosphorylation cascades and specific cell kinases (like PI3K) in the productive ASFV infection, has been also reported, revealing new viral genes and host factors candidates for antiviral approaches
WP 15 - Genome sequencing of viral isolates used in protective models and deletion of ASFV virulence genes
Objectives
The genome sequences of the virus model strains NHV and L60 in conjunction with the sequence analyses of a number of field ASFV isolates (Task 1), will provide insight into ASFV evolution and will identify further proteins or sequence elements highly conserved (or variable), providing important information on the possible relevance of gene functions for ASFV biology. In addition, the determination of the complete sequence of strains NHV/L60 is needed for the design of virus mutants to be generated and analyzed in other WPs (14, 16 and 17). Comparative genomics will also give valuable information on the genetic divergence of ASFV, important for the development of sensitive diagnostic techniques with high specificity (Tasks 1 and 2). Another objective of this task is the deletion of factors that determine the virulence of ASFV in the model system L60 (highly virulent)/NHV (attenuated) strains. We will generate mutant viruses with deletions on genes putatively involved in virulence, previously described in other systems or analyzed in this Project for their ability to modulate the cell and host response to ASFV infection. Selected mutations will be used as attenuated models for ASFV vaccination in WP17
Summary of results on the WP
ASFV genome sequencing
In the first periodic report it was reported the determination of the sequence of a number of ASFV genes needed to develop the virus recombinants proposed in SWP 18.2 and SWP 15.2. Results regarding several ASFV genes (A179L, A224L, A238L, A276R, E248R, G5R, I329L, I268L, K205R, and L356L) showed no significant differences within the NHV and L60 encoded proteins. A new strategy for sequencing was proposed by optimizing the 454 genome sequencing technology, significantly less laborious than traditional methods. At the end of the second period, we have obtained the full genome sequences (with exception of the terminal repeats) for both NHV and L60. This information has been used to compare the sequences of several ASFV vaccine strains currently in use (NHV, OURT88, E75,...). The analyses showed so far that the 172.050 kbp NHV genome is to 99 % identical to the OURT88/3 isolate whereas the 181185 kbp L60 genome is to nearly 100 % identical to the ASFV strain E75.
Deletion of ASFV virulence genes
The objective of this SWP was the deletion of ASFV genes involved in virulence in the model system L60 / NHV strains. During the development of the Project we have reported the completion of the construction of EP153R-deleted ASFV mutants from L60, NHV and E70 isolates, and also the generation of A238L and A224L-deleted virus mutants from both L60 and NHV strains. Several of them have been used in the analysis of the role of these genes in the ASFV infection (WPs 14, 16 and 17). Preparations of the NHV/L60 mutants were available around month 24, and the evaluation of virulence was analyzed in the in vivo protective experiment with the deleted ASFV recombinants (attenuated model viruses) at the end of the project (SWP 17.1).
WP 16 - Host in vitro responses to infection
Objectives
It was proposed in this WP to analyse the infection of sensitive cells with deletion mutants and their corresponding parental strains NHV and L60, with the evaluation of cytokines, chemokines, inflammatory molecules and SLA-I expression, which help in the definition of in vitro indicators of pathogenesis and protection. A particular interest is proposed in A238L or EP153R virus genes, candidates for ASFV models in protective experiments.
The impact of ASFV “unassigned” novel genes on host gene transcription was also proposed to be investigated in IPAM macrophages infected with lentivirus vectors. These investigations will help to define whether these ASFV-encoded proteins have completely or partially overlapping functions, thus prioritizing candidates for construction of deletion mutant attenuated virus vaccines
Summary of results on the WP
Modulation of specific cytokines, chemokines and inflammatory molecules induced during virus infection and regulation of IFNα expression and production
Preliminary studies (by qRT-PCR and Elisa) suggested an increased expression at mRNA levels of IFNβ and of the factors involved in the IFN pathway namely Jak1, Tyk2 and STAT1, in NHV infection when compared to L60 at early stages of infection. Further analysis of Real Time PCR (qRT-PCR) of mRNA quantification of relevant cytokines (IFNα, IFNβ, TNFα, IL12p40, TGFβ), chemokines (RANTES, MCP1, MIP1α), IFN relating factors (IRF3, IRF7, IRF9), signal transducers (STAT1, STAT2) and kinases (Tyk2 and Jak1) was pursued in macrophages infected with the highly virulent ASFV/L60. Results obtained as the ratio between levels of mRNA expression between L60 infected and control (non-infected) macrophages revealed an increase of the mRNA expression levels of IFNβ (2 hours after infection), TNFα, chemokines and Tyk2 (from 4 to 6 hours after infection), IFNα and signal transducers (STAT1 and STAT2) (6 hours after infection). In opposition, mRNA expression levels of IL12p40, potentially involved in the induction of cellular immune responses seem to be reduced in L60 in vitro infection.
In order to get insights on the regulation of interferon and inflammatory cytokines by the above mentioned ASFV isolates (NHV and L60) porcine macrophages were infected in vitro and the kinetics of the expression of 9 unassigned ASFV genes implicated in the regulation of the interferon response (I329L, A276R, K205R, DP146L, A528R, DP148R, J268L, E248R and P1192R) as well as of the A238L gene (IkappaB homolog) was evaluated. In addition, the expression of IFN-alpha and IFN-beta was also determined in the same cultures. In general, a similar pattern of expression was observed for the unassigned viral genes, with increased expression from 12h onwards, in almost all cases with higher levels for NHV than for L60.
Study of the role of A238L gene on the regulation of different pro-inflammatory molecules
Above mentioned results regarding the role of the A238L viral gene show that the modulation of COX-2, TNF-alpha, iNOS and IL-6 expression is mediated through the control of their promoter activities. The mechanism of inhibition has been further analyzed and shown to involve the modulation of the transactivation of several cell transcription factors (like NF-kB or NF-AT) by the control of CREB binding protein/p300 coactivators function.
The effect of the deletion of this viral gene from E70 isolate in the in vivo modulation of cytokine m-RNA expression was also studied during this Project, and confirmed in the virus model NHV during the second period of the project by CSIC
Study of the role of EP153R gene on the modulation of SLA expression and modulation of NK cell cytotoxicity
The modelization of the 3D structure of the viral EP153R C-type lectin homologue protein was presented in the first period of this Project, together with the modulation of the expression of MHC-I molecules in the plasma membrane after transfection of the ASFV EP153R gene into different model systems. These studies were extended to the mechanism of interference of EP153R with the exocytosis of the class I antigens to the plasma membrane, and to the determination of the expected inhibitory effect of the viral gene during ASFV infection, by the use of the virus recombinants with specific genes deleted from NHV and L60 isolates generated as described above. It was also demonstrated the importance of the integrity of the lectin-C domain and the R133 residue to establish the inhibition of MHC-I expression. This new role reported for the ASFV lectin EP153R provides a new mechanism for ASFV evasion of host antiviral responses.
The EP153R gene did not modulate the activity of NK cells when transfected in in vitro assays.
Impact of ASFV “unassigned” novel genes on host gene transcription
The analysis of the possible role of a number of unassigned ASFV genes with inhibitory activities on IFN responses, presented in the first period, has been extended and updated in the second period. A total of 7 genes were found to inhibit IFN responses in the luciferase assays, with four of these genes (A276R, K205R, DP148R and I329L) promoted as as best candidates for construction of attenuated ASFV vaccines by specific gene deletion. As a general conclusion the non-assigned, non-homologous virus genes so far analyzed seemed to be a repository of host evasion viral genes, to be used into the multiple strategies evolved by the ASFV to manipulate the IFN responses.
WP 17- Host in vivo responses to infection
Objectives
The main goal proposed is to evaluate how the deletions of specific genes from the genome of NHV isolate will affect the pathogenesis of the disease in pigs. The information available was completed with the results obtained in WPs 14, 15 and 16, and used to select the best ASFV genes candidates to be manipulated in the NHV isolate (WP 18), in order to induce a protective response in swine. As optimization of antigen presentation by pig macrophages could be relevant to improve the response in ASFV immunization, we will also study the OPRi based expression system to assay different viral proteins expressed as fusion lipoproteins.
Summary of results on the WP
Attenuated models for ASFV vaccination
As indicated in the initial proposal, and considering the information obtained throughout the three years of the project regarding the role of different ASFV genes in virus infection and pathogenicity, we have finally selected 3 virus genes whose specific deletion should result in a possibly more attenuated infection, to use them in experiments of ASFV vaccination. The ASFV genes selected (A238L, A224L and EP153R) were deleted from the NHV strain of ASFV by conventional methods based on transfection-infection and homologous recombination. After the selection, purification and amplification of the virus clones, stocks were then grown and used in an in vivo protective experiment in swine, which confirmed the ability of the new attenuated NHV virus models to induce a protective response against virulent ASFV L60 isolate. A limited extension of the experiment, performed with a second challenge with a heterologous virulent Arm07 strain (genotype II), indicated that the NHV isolate was also able to further protect against the second virulent infection, and that not all of the selected ASFV genes must be manipulated by deletion (but instead by overexpression, as in the case of A238L) to obtain an attenuated strain.
Optimization of OPRi presentation of ASFV antigens
The objective in this SWP is the development and characterization of a new system for the production of antigens in fusion with the OprI lipoprotein, a TLR ligand. This new system allows higher stability of recombinant plasmids and enables easy purification of fusion proteins by affinity chromatography. Results presented on the expression of a heterologous fused antigen, the ASFV ORF A104R, and the extension of these studies to obtain the cloned antigens in 3 different immunogenic formulations, showed that all of them have been characterized using the model antigen ovalbumin through in vivo and in vitro studies in a mouse model and in vitro studies in the swine model, and demonstrated adjuvant capacity. A number of ASFV antigens (including A104R, K205R, A528R, E248R, DP148R, J268L, A276R, DP146L and I329L), have been cloned in this system, opening the possibility of their future use to study the immune response against the fused antigens and the recognition of ASFV proteins by leukocytes from pigs surviving infection with attenuated isolates.
WP 18 - Improving conventional and developing new high throughput methods to facilitate genome manipulation and vaccine development
Objectives
Two approaches were proposed to facilitate the ASFV genome manipulation needed for the construction of virus recombinants described in this Project. The “conventional” approach requires the use of cell-adapted virus strains, although many ASFV field isolates are expected to infect directly either COS-1 or IPAM macrophage-derived cell lines. The manipulation is performed by transfection of engineered plasmids with specific inactivated virus genes into cells infected by the parental virus, and selection of recombinant viruses (generated by homologous recombination) by expression of reporter genes, in successive rounds of plaque purification.
A novel approach, which involves the construction of an infectious ASFV clone in E. coli, was also proposed in this Project. Successful cloning of large DNA virus genomes as bacterial artificial chromosomes (BACs) was achieved in the last years, and it was expected that the availability of an ASFV-BAC would significantly improve efficacy of ASFV genome manipulation for functional analysis of genes involved in immune evasion and pathogenicity.
Summary of results on the WP
Growth and plaque assay of ASFV isolates in established lines and conventional deletion of specific ASFV genes
In this Project we have performed the studies regarding the optimization of growth and plaque assays of ASFV isolates in established cell lines and the conventional deletion of specific ASFV genes. All of them have been confirmed regarding the inactivation of the corresponding genes (by PCR and RT-PCR in different cell lines) and their effective purification. These tools supported the works performed during the second period regarding the production of virus models to WPs 15 and 18, the purification of new virus antigens for ASFV diagnosis (WP9) and the analysis of the role of these genes in the evasion of antiviral responses, both in vitro and in vivo (WPs 14, 16 and 17). As a resume, the following ASFV recombinants were constructed for the Consortium in this project: NHV deleted in A238L, A224L and EP153R, L60 deleted in A238L, A224L and EP153R, and E70 deleted in A238L and EP153R.
Cloning of ASFV genome into a BAC (E. coli) and engineering of virus genes
To facilitate genetic engineering of ASFV, it was envisaged to clone the viral DNA genome as a BAC in E. coli and to generate infectious virus. After all the preliminary steps, only one colony contained a BAC in which the entire ASFV genome with exception of the terminal repeats (TRs) was present. Purified BAC-DNA was transfected into WSL cells which were then super-infected with wt virus. Unfortunately, the rescued virus finally proved to be a recombinant of sequences contained in the BAC with the wt virus genome. Since the TR sequences of the ASFV genome were missing in all the other clones, it appears reasonable to assume that they are not stabile in E. coli. Consequently, generation of ASFV mutants using BAC technology failed. Nevertheless, work in this SWP resulted in development of a novel methodology to create ASFV recombinants with deletions in the viral TK-locus which will be used to generate virus mutants that express heterologous genes and thus will be beneficial for future vaccine developments.
WP 19 - Development of antiviral molecules
Objectives
The objective of this WP was the evaluation of antiviral molecules applicable for the preventive protection of domestic pigs, including:
1) In vitro evaluation of toxicity and antiviral properties of molecules.
2) In vitro evaluation of the antiviral interaction with the viral replication cycle.
3) In vitro evaluation of the mechanism of action and detection of resistance to the antiviral.
4) In vivo evaluation of the toxicity of the antiviral candidate.
5) In vivo evaluation of the antiviral activity of the candidate.
Summary of results on the WP
Screening of potential molecules based on their antiviral activity/toxicity ratio and in vitro evaluation of the interference of antiviral molecules with the viral replication cycle, the mechanism of antiviral actions and the possible appearance of resistance in response to antiviral treatment
The efficiency of three types of antiviral molecules (chemicals compounds, siRNA and gallate esters) to inhibit the production of ASFV has been evaluated during the first period.
For antivirals of type 1 (chemical compounds), selected classes of nucleoside and nucleotide analogues, with inhibitory activity against DNA and retroviruses, were evaluated. The most selective compounds were HPMPO-DAPy, HPMPA and most of HPMPA derivates, with active concentration below 0.8μg/ml. Antivirals were active in the first six first hpi, in concordance with the previously described inhibitory activity of certain nucleotide analogues.
Antivirals type 2 are siRNAs specific against several ASFV genes (A104R, A118R, A204L, A151R and B646L (vp72)). After screening of about 25 siRNAs on the BA71V virus model in Vero cells, only 4 of them were active in virus inhibition, 3 directed against A151R gene and 1 specific for vp72. The inhibition obtained ranged from 3 to 4 Log TCID50. The 4 active siRNAs were synthesised as shRNAs and cloned in the pENT/U6 expression vector.
Results on antivirals type 3 (gallate esters) have demonstrated the efficiency of the selected drug (LG, lauryl gallate) to inhibit the production of several DNA and RNA viruses. Up to 7 ASFV isolates (E70, Brazil81, Uganda vir., Malawi82, Mozam86, NHV, Ba71V) have been proved in 3 different virus-sensitive cell lines to be inhibited by concentrations of LG that were far from producing cytotoxic effects on the host cell. No extracellular infective virus was produced in the presence of the drug. The inhibitory effect of LG in the cellular and viral DNA synthesis, and in the late ASFV-specific protein synthesis, has been also confirmed.
A remarkable success was obtained in all of the three approaches proposed. This is more interesting considering that their inhibitory actions might be complementary, and the reduced cytotoxicity observed in all the cases.
In vivo evaluation of the toxicity and antiviral activity of the candidate molecules
The toxicity of the antiviral molecules was evaluated in vivo in mice and/or domestic pigs. One of the chemical antivirals, the HPMPDAP, has been further evaluated in vivo for toxicity and pharmacokinetics in domestic pigs. Toxicity has been observed at i.m. injection of 20, 5 and 2,5 mg/kg/day, but no more observed with a daily i.m. dose of 0,5 mg/Kg/day during 15 days. Regarding toxicity of LG, no toxicity was found in mice associated to the treatment with LG at dose up to 10 mM (1000X the therapeutical dose of in vitro experiments). In domestic pigs, no toxicity was observed in case of administration of 2 mg LG orally and gastrically (1 mg) 3 times a week during 3 weeks. Similarly, there was no evidence of toxicity in mice when siRNA were administrated intramucosally at 40 µg/mice during 3 days or in goats after intraveinous administration of 12 mg of liposome encapsulated siRNA/goat during 3 days.
The effectiveness of the antiviral molecules LG and HPMPDAP was evaluated in the course of infection with ASFV and with respect to physiological parameters, clinical signs and body weight gain. As a resume, LG and HPMPDAP were well tolerated and did not show any negative effect on appetite, sensorium, gait and enteric function. However, regarding the antiviral activity of both the molecules, no significant differences were observed between the animal groups.
TASK 4
TRAINING AND TECHNOLOGY TRANSFER
The work was developed under two work-packages as described ahead:
WP 20 Local training/workshops and technology transfer
Objectives
The objectives of this WP were to transfer knowledge on relevant aspects of ASF and tools developed within the project in the field of epidemiology, risk assessment and laboratory diagnostics (namely to the African and Asian partners) and to set up new disease control strategies locally through trainings and workshops. Trainings and workshops were organized in African countries, based on institutions involved in the project, namely at LANADA in Ivory Coast, targeting the participation of Western African countries, at OVI in South Africa targeting the participation of Southern African countries and at Entebe, Uganda (initially planned to be organized at ILRI, Kenya) targeting Eastern African countries. As also further anticipated by the consortium, a training action-workshop was developed in China (LVRI-Lanzhou) targeting students and collaborators of LVRI and representatives from Chinese Veterinary Laboratories and in Madrid for East-European epidemiologist and laboratory diagnosticians. At last, several short-term individual trainings were organized for Chinese and Vietnamese fellows.
TRAINING COURSES
The first training course on “Training Course on African swine fever (ASF) diagnostic techniques”, was initially planned to be held in Kenya, was organized in Entebe, Uganda at the Ministry of Agriculture Animal Industry and Fisheries (MAIF) from 15 to 24th of September 2008. The main purpose of the course was to transfer the ASF diagnostic tools available at CISA as EU reference laboratory for ASF to 25 participants from Department of Veterinary Services (DVS) and National systems staff from Kenya, Uganda and Tanzania with an opportunity to improve and reinforce their diagnostic technology and control measures for ASF. This course also aimed to create a good coordination and cooperation for ASF disease control measures in Eastern African countries. In addition, all participants were expected to convey the information provided in the course to other technical staff in their laboratories and to use the course information to improve ASF diagnostic testing in their home laboratories as needed. The course was organized in cooperation between Centro de Investigación en Sanidad Animal (CISA-INIA) in Madrid in collaboration with ILRI in Kenya..
The second training course on “African swine fever (ASF) Epidemiology and Diagnosis” and was held in Lanzhou Veterinary Research Institute (LVRI), Chinese Academy of Agricultural Science, at Lanzhou, Gansu in the People's Republic of China (20-24 September 2010). Thirty participants attended the course, including young scientists from Lanzhou Veterinary Research Institute, CAAS and several representatives from Veterinary Laboratories from different regions in China namely from boarding areas with the Russian Federation.
The course was organized in cooperation between LVRI, Centro de Investigación en Sanidad Animal (CISA-INIA) in Madrid, Spain and Faculdade de Medicina Veterinária (FMV-UTL) in Lisbon, Portugal.
The third and fourth training courses were held at the Laboratoire Central de Pathologie Animale /LANADA, located in Bingerville, Côte d´Ivoire. It was a combination of laboratory and epidemiology-based training. The ASF laboratory diagnosis-based training course was delivered in LANADA, at Bingerville, Ivory Coast on days 18-22nd of October 2010 with the contribution of VAR and CIRAD (Logistics). The main purpose of the laboratory-based course was to transfer the ASF diagnosis tools available at VAR and LANADA as Reference laboratories for ASF to 10 participants from Department of Veterinary Services and National systems staff from Ivory Coast, Burkina Faso, Togo, Benin and Ghana with an opportunity to improve and reinforce their diagnostic technology and control measures for ASF. This course was aimed to create a good coordination and cooperation for ASF disease control measure in West African countries. The ASF epidemiology course and expert opinion workshop were delivered at the same place from November 1 - 5, 2010 and it was organized by UCM. The main objective of the course was to provide basic information related with the epidemiology of the disease, focusing on the study of risk factors, spread and control of the disease. Some important epidemiology knowledge and tools were also introduced to the participants, like the use of surveys to collect epidemiological data from the outbreaks, the use and creation of databases, sampling methods, introduction to Geographic Information Systems (GIS), cluster analysis and risk assessment. For the epidemiology course, 10 veterinarians from the neighboring countries of West Africa (Benin, Burkina Faso, Côte d´Ivoire, Ghana and Togo) attended to the course. All of them work in animal health departments of their countries and have a daily contact with the disease.
The fifth “Training course on African swine fever (ASF) Diagnostic techniques” was held at the Agriculture Research Council in Onderstepoort (ARC-OVI), Republic of South Africa, from May 30th to June 3rd, 2011. The main purpose of this training course was to cover relevant aspects related to the disease, its epidemiology, prevention and control and eradication measures. Particular attention was dedicated to serological and virological laboratory diagnosis of ASF through theoretical and hands-on diagnostic training sessions using ASF suspicious samples. Seventeen participants, representative from 13 of the Southern African Development Community (SADC) countries, namely from Angola, Botswana , Democratic Republic of Congo, Lesotho, Malawi, Mauritius, Mozambique, Namibia, Seychelles , South Africa, Swaziland, United Republic of Tanzania, Zambia, Zimbabwe, and a young researcher from the National Institute for Veterinary Research Vietnam in Hanoi, partner of the ASFRISK project, attended the course. The course was organized in cooperation between ARC – Onderstepoort Veterinary Institute (OVI), Centro de Investigación en Sanidad Animal (CISA-INIA) in Madrid, Spain and Faculdade de Medicina Veterinária (FMV-UTL) in Lisbon, Portugal.
The sixth training course on “African swine fever (ASF) epidemiology and laboratory diagnosis (ASF EPI-LAB)” and was held in Madrid from June 27 to July 1, 2011. The course was organized in theoretical sessions and practical hands-on exercises covering relevant aspects related to the disease, its epidemiology, prevention, control and eradication and laboratory diagnosis. Twenty eight participants attended the course: thirteen specialists on epidemiology and control of diseases (from Russia, Bulgaria, Sweden, Great Britain, Portugal and Spain, participated on the ASF-Epidemiology component of the course and fifteen specialists on laboratory diagnosis of animal infectious diseases (From Russia, Belarus, Great Britain, Poland, Germany, Vietnam). During the first day of the course, all participants attended theoretical sessions and participated on discussions on general aspects of ASF (African swine fever: A State of Art and Future Trends; African Swine Fever Diagnosis: A general overview, and recent developments; Experience of ASF control in several countries). During the three following days, participants attended specific training on ASF epidemiology and ASF diagnosis. The training on Epidemiology covered hands-on practices using EPI-tools and theoretical lectures and discussions held at the Veterinary Faculty (UCM) in Madrid. The training on ASF diagnosis covered theoretical lectures, discussions and hands-on practices on serological and virological laboratory diagnosis of ASF using ASFV infected and suspicious biological samples at the Centre for Research on Animal Health (CISA-INIA), Valdeolmos, in Madrid. All participants gathered at the Vererinary Faculty (UCM) during the last day of the course. Final topics on “Control strategies of African Swine Fever; Models for Eradication” and on “Operating Duties and Main Activities of Reference Laboratories” were introduced and discussed. Final interactive and enthusiastic discussions and conclusions of the work developed on both areas of the course among trainers and trainees confirmed the success of this training action under the ASFRISK project. All the participants received a CD containing relevant information on the course.
Multimedia Course on ASF Diagnosis
A course on African swine fever diagnosis was developed by UCM and CISA-INIA in collaboration with OIE on CD format and it is available at http://sanidadanimal.info/cursos/asf/(si apre in una nuova finestra). This comprehensive course includes theoretical lessons and practical videos on clinical and laboratory diagnosis of ASF. This CD (so far edited in English, Russian and Spanish) was spread within the ASFRISK beneficiaries and also to representatives of different countries attending the different courses organized by the project, and to others through UCM, CIRAD and CISA-INIA networks of collaboration.
LABORATORY-BASED TRAINING ACTIONS
The main goal of traineeships was to transfer the ASF diagnostic knowledge and tools available at the hosting EU partners in the project, to Lanzhou Veterinary Research Institute (LVRI) in China and National Institute of Veterinary Research (NIVR) in Vietnam, towards the implementation of ASF serological, virological and molecular diagnostic techniques in both Institutions. For this purpose training was provided to collaborators of both institutions.
Seven short-term laboratory-based trainings were organized:
- One Chinese fellow (Aihong Liu) at SVA in Uppsala from 20th September – 16th October 2009
- Two Chinese fellows at CISA-INIA (Madrid) from 21st September – 28th October 2009 (Jifey Yang) and from 17 February 2011 – 13 May 2011 (Yuefeng Wang)
- One Vietnamese fellow (Dr. Bui Ngoc Anh) at CISA-INIA in Madrid on 01-28 March 2010
- One Vietnamese fellow (Dr. Dao Duy Tung) at the National Veterinary Institute in Uppsala (SVA) from 6 – 28 March 2010.
- One Vietnamese fellow (Dr. Dao Than Van) attended the course “African swine fever (ASF) epidemiology and laboratory diagnosis (ASF EPI-LAB)” held in Madrid from June 27 to July 1, 2011
- One Vietnamese fellow (Dr. Nguyen The Vinh), attended “Training course on African swine fever (ASF) Diagnostic techniques” held at the Agriculture Research Council in Onderstepoort (ARC-OVI), Republic of South Africa, from May 30th to June 3rd, 2011
WP 21 Individual long-term training
Long and medium term laboratory-based training developed under the project have contributed to the development of three PhD and four MSc degrees:
PhD thesis
- Basto, A.P. “A new cloning system based on the OprI lipoprotein for the production of bacterial cell wall-derived immunogenic formulations. PhD Thesis. Approved. Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa. October 2011
- Quembo, C. “A study of the epidemiology of African swine fever in Mozambique”. PhD Thesis, ongoing, to be submitted to the University of Pretoria. 2008-2012.
- Kouakou Kouame Valere. “ASF Epidemiological characterization on West African countries”.
PhD Thesis. Ongoing, to be submitted to UFR Biosciences de l'Université d'Abidjan-Cocody, Côte d'Ivoire.
Msc dissertations
- Annette Nigsch. “Introduction of African Swine Fever into the European Union: development of a generic model to assess disease spread within and between Member States”. As part fullfillment of the MSc Veterinary Epidemiology of the Royal Veterinary College, August 2011
- Anja Bauer. “Analyse früher Stadien während der Infektion von Zellkulturen mit dem Virus der afrikanischen Schweinepest (Analysis of early stages of the infection of cultured cells with African Swine fever virus). MSc thesis approved, 2010-09-16. University of Rostock, Faculty of Medicine/ Medical Biotechnology.
- Lina Mur Gil. Evaluación cuantitativa del riesgo de introducción del virus de la Peste Porcina Africana en la Unión Europea por importación legal de porcinos vivos y sus productos cárnicos derivados. MSc Thesis, approved Universidad Complutense de Madrid June 2010.
- Ricardo Gomez-Soute. “Pathogenicity of an African swine fever virus isolated from domestic pigs in Mozambique”. MSc dissertation, ongoing. 2009-2012
Potential Impact:
African Swine Fever (ASF) is the most complex and economically devastating viral disease of domestic pigs producing great socio-economic impact in affected countries. It is caused by a complex virus, the only member of the Asfarviridae family, with different virulence properties causing hyperacute, subacute, chronic and unapparent forms of disease. It is maintained in nature under different and complex epidemiological scenarios involving domestic and wild swine and arthropod vectors (soft ticks Ornithodoros sp). Due to the fact that no vaccine has been obtained so far, prevention control and eradication of the disease are solely based on the implementation of strict sanitary measures.
The disease in EU member states is currently confined to Italy (Sardinia). It was recently introduced to Caucasian countries and Russia and it is highly prevalent in sub-Saharan African countries.
Serological surveys during outbreaks occurred previously in Eastern and Southern African regions have shown that although ASFV prevalence in the domestic pigs is high, in many cases serum conversion was not detected. These events pose a great challenge for the prevention and control of ASF, not only locally, but also worldwide. The complex epidemiological scenarios of ASF, the increasing mobility of people, animals and goods across the globe, emphasize the serious menace the disease presents to the growing pig farming sector in Africa and to currently ASF-free EU member states permanently threatened by the occurrence of the disease in different geographical regions.
In order to improve tools and strategies for the prevention and control of ASF, the work developed under the ASFRISK project aimed at fulfilling four objectives contained in the Major Tasks of the work-plan:
i) To improve and further develop the epidemiological, risk assessment and control measures of African swine fever (ASF),
ii) To develop and provide new and robust tests for laboratory and front-line diagnosis, through the achievement, standardization and validation of more rapid, sensitive and inexpensive serological and molecular procedures,
iii) To establish new strategies for vaccine development against ASF and,
iv) To transfer knowledge on relevant aspects of ASF and improved tools for ASF diagnosis and epidemiological modeling and risk assessment strategies by providing an harmonized international training, in order to prevent and combat this highly devastating, OIE-listed transboundary animal disease.
The four main tasks of the project were outlined and harmonized in the work-plan providing a logical process for the improvement of knowledge on critical aspects of ASF and development of tools towards the achievement of the objectives through the contribution of highly qualified partners from EU, Africa and Asia, with expertise in all the areas concerned.
Several outcomes of the project emphasize its successful contribution for “Evaluating and controlling the risk of ASF in the EU”:
- Development of epidemiological methods and decision support tools provide information to policy makers on ASF epidemiological situation and will assist in the prioritization of strategies to better prevent and control ASF in a potential incursion into the EU and in all currently affected territories.
- Development of a range of valuable serological and molecular diagnostic tools suitable for use in well-equipped international and national reference laboratories, in basic regional and local laboratories, or even for rapid first line on-site application. New tests and reinforcement of currently available ones will promote more efficient and earlier diagnosis of the disease.
- Characterization of several ASFV-host interactions at cellular and molecular levels and the production of different attenuated recombinant virus strains that may be used as future candidates for a vaccine, open new insights on the development of control strategies against ASF.
- Transfer of knowledge on relevant aspects of ASF and transfer of diagnostic and epidemiologic tools through training courses and traineeships to a large number of professionals from Animal Health Institutions in EU, Africa, Asia, Russia and Caucasian countries, enhance preparedness capacity against the threat of the disease in different scenarios.
The project provided employment, education and training for young post-doctoral and post-graduate researchers. The employed researchers and assistants gained intra and inter-disciplinary experience and training by working within an international, multidisciplinary team and, importantly, experience of working with industry. In addition, young researchers were trained in the presentation of results and the compilation of manuscripts for publication. This experience and training contribute to both consolidating the EU scientific infrastructure of young scientists and reducing the drain of young scientists elsewhere.
The proposed objectives and deliverables of this project provide essential information for prevention and control of ASF, a devastating disease causing acute welfare problems in Africa and in Sardinia and more recently in Caucasian countries and Russia.
By doing so, the outcomes of the project will benefit EU consumer driven concerns with regard to animal welfare, food safety and food quality and will undoubtedly contribute to the sustained development of EU agri-food business by improving prevention, early detection and control of ASF and therefore reducing the socio-economic consequences of potential incursions of the disease from infected regions. In addition, assistance in the preservation of small to medium sized family farms, as viable economic units within a rural environment will contribute to maintaining rural infrastructure of EU regions and member states
Main Dissemination activities and exploitation of results
Dissemination and exploitation of the results considered by the consortium to be a vital component of the proposed work program was fulfilled through internal and external dissemination activities:
1. Internal dissemination
The consortium was composed by a multidisciplinary scientific team based on thirteen European partners from eight countries: FMV- UTL and FCG-IGC from Portugal, UCM, CISA-INIA, CSIC and INGENASA (a SME) from Spain; CIRAD from France; IZS-UM from Italy; VAR from Belgium; FLI from Germany; QUB and RVC from United Kingdom; SVA from Sweden and four partners from ICPC countries: OVI from South Africa; LANADA-LCPCA from Ivory Coast; LVRI from Popular Republic of China and NIVR from Vietnam. The ability of partners to successfully complete their program within the general context of the project was supported by their relevant scientific expertise in different fields including epidemiology and risk assessment, serological and nucleic acid based diagnostics, molecular biology, genetics, immunology, virology and infectious pathology.
Internal dissemination aiming at scientific and technological exchange between partners was achieved through the organization of project meetings, laboratory-based training actions (traineeships) and short exchange visits of collaborators from different partners.
Collaborations developed among partners at different levels during the lifelong of the project consolidated and enhanced knowledge on basic and applied science of the international scientific community working on ASF.
2. External dissemination:
External dissemination of research results was achieved through a series of activities:
- Publications in scientific peer reviewed journals
- Presentations in conferences, workshops, scientific meetings
- Phd thesis and MSc dissertations
- Transfer of knowledge and technology trough the organization of training courses and traineeships
- Co-organization of the “VIII Simpósio da Sociedade Científica de Suinicultura “Peste Suína Africana”
- Organization of the “ASFRISK Symposium”
- Exploitation of results
- Website (www.asfrisk.eu)
2.1 Publications in peer-reviewed scientific journals
So far the following scientific papers have been published:
1. Basto AP, Piedade J, Ramalho R, Alves S, Soares H, Cornelis P, Martins C, Leitão A. A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations. J Biotechnol. 2011, 157 (1):50-63.
2. Carrascosa, AL, Bustos MJ, de Leon, P. Methods for growing and titrating African swine fever virus field and laboratory virus samples. Current Protocols in Cell Biology. 2011. Chapter21.Unit 26.14.
3. Fasina FO, Shamaki D, Makinde AA, Lombin LH, Lazarus DD, Rufai SA, Adamu SS, Agom D, Pelayo V, Soler A, Simón A, Adedeji AJ, Yakubu MB, Mantip S, Benshak AJ, Okeke I, Anagor P, Mandeng DC, Akanbi BO, Ajibade AA, Faramade I, Kazeem MM, Enurah LU, Bishop R, Anchuelo R, Martin JH, Gallardo C. Surveillance for African swine fever in Nigeria, 2006-2009. Transbound Emerg Dis. 2010 Aug 1;57(4):244-53.
4. Fernández-Pinero, J, Gallardo, C, Elizalde, M, Robles, A, Gómez, C, Bishop, R, Heath, L, Couacy-Hymann, E. Fasina, FO, Pelayo, V, Soler, A, Arias, M. Molecular diagnosis of African swine fever (ASF) by a new real-time PCR using Universal Probe Library (UPL). Transbound Emerg Dis . 2012. In press
5. Gallardo C, Ademun AR, Nieto R, Nantima N, Arias M, Martín E, Pelayo V and Bishop RP. “Genotyping of African swine fever virus (ASFV) isolates associated with disease outbreaks in Uganda in 2007”. African Journal of Biotechnology (AJB). April 2011; 10 (17): pp. 3488-3497
6. Gallardo C, Anchuelo R, Pelayo V, Poudevigne F, Leon T, Nzoussi J, Bishop R, Pérez C, Soler A, Nieto R, Martín H, Arias M. African swine fever virus p72 genotype IX in domestic pigs, Congo, 2009. Emerg Infect Dis. 2011. Aug; 17(8):1556-8.
7. Gallardo C, Okoth E, Pelayo V, Anchuelo R, Martín E, Simón A, Llorente A, Nieto R, Soler A, Martín R, Arias M, Bishop RP. African swine fever viruses with two different genotypes, both of which occur in domestic pigs, are associated with ticks and adult warthogs, respectively, at a single geographical site. J Gen Virol. 2011 Feb; 92(Pt 2):432-44.
8. Gallardo C, Reis AL, Kalema-Zikusoka G, Malta J, Soler A, Blanco E, Parkhouse RM, Leitão A. Recombinant antigen targets for serodiagnosis of African swine fever. Clin Vaccine Immunol. 2009 Jul; 16(7):1012-20.
9. Giammarioli M, Gallardo C, Oggiano A, Iscaro C, Nieto R, Pellegrini C, Dei Giudici S, Arias M, De Mia GM. Genetic characterisation of African swine fever viruses from recent and historical outbreaks in Sardinia (1978-2009). Virus Genes. 2011 Jun; 42(3):377-87.
10. Hurtado C, Bustos MJ, Carrascosa AL. The use of COS-1 cells for studies of field and laboratory African swine fever virus samples. J Virol Methods. 2010 Mar; 164(1-2):131-4.
11. Hurtado C, Bustos MJ, Granja AG, de León P, Sabina P, López-Viñas E, Gómez-Puertas P, Revilla Y, Carrascosa AL. The African swine fever virus lectin EP153R modulates the surface membrane expression of MHC class I antigens. Arch Virol. 2011 Feb; 156(2):219-34.
12. Keita D, Heath L, Albina E. Control of African swine fever virus replication by small interfering RNA targeting the A151R and VP72 genes. Antivir Ther. 2010; 15(5):727-36.
13. Martínez, M., Martínez-López, B., Muñoz, M.J. de la Torre, A., Mur, L., Sánchez-Vizcaíno, JM., 2009. Expansión de peste porcina africana en el mundo: Riesgo de entrada en España. Gaceta Sanitaria. Vol.23 Especial Congreso 3, 28.
14. McKillen J, McMenamy M, Hjertner B, McNeilly F, Uttenthal A, Gallardo C, Adair B, Allan G. Sensitive detection of African swine fever virus using real-time PCR with a 5' conjugated minor groove binder probe. J Virol Methods. 2010 Sep; 168(1-2):141-6.
15. Mur L, Martínez-López B, Martínez-Avilés M, Costard S, Wieland B, Pfeiffer DU, Sánchez-Vizcaíno JM. Quantitative Risk Assessment for the Introduction of African Swine Fever Virus into the European Union by Legal Import of Live Pigs. Transbound Emerg Dis. 2011 Aug 10.
doi: 10.1111/j.1865-1682.2011.01253.x.
16. Mur L., Martínez-López B., Gallardo C., Gortazar C. and Sánchez-Vizcaíno JM. Monitoring of African Swine Fever in the Wild Boar Population of the Most Recent Endemic Area of Spain. Transboundary and Emerging Diseases. In Press. doi: 10.1111/j.1865-1682.2012.01308.
17. Portugal R, Leitão A, Martins C. Apoptosis in porcine macrophages infected in vitro with African swine fever virus (ASFV) strains with different virulence. Arch Virol. 2009;154 (9):1441-50.
18. Portugal R, Leitão A, Martins C. Characterization of African swine fever virus IAP homologue expression in porcine macrophages infected with different virulence isolates. Vet Microbiol. 2009 Oct 20; 139(1-2):140-6.
19. Ronish B, Hakhverdyan M, Ståhl K, Gallardo C, Fernandez-Pinero J, Belák S, LeBlanc N, Wangh L. Linear-After-The-Exponential PCR (LATE-PCR) assay for the detection of African swine fever virus”. J Virol Methods. 2011 Mar; 172(1-2):8-15.
20. Tignon M, Gallardo C, Iscaro C, Hutet E, Van der Stede Y, Kolbasov D, De Mia GM, Le Potier MF, Bishop RP, Arias M, Koenen F. Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus. J Virol Methods. Sep 17. 2011 Dec;178 (1-2):161-70
21. Sánchez-Vizcaíno JM., Mur L., and Martínez-López B. African Swine Fever: An Epidemiological Update. Transboundary and Emerging Diseases. 2012 Jan 9. doi: 10.1111/j.1865-1682.2011.01293.x. [Epub ahead of print]
2.2 Presentations at scientific meetings, conferences, workshops
Around one hundred presentations were introduced at different scientific meetings, conferences, workshops and others and they are shown in the website www.asfrisk.eu (Dissemination)
2.3 PhD thesis and MSc dissertations under the project
Long and medium term laboratory-based training developed under the project have contributed to the development of three PhD and four MSc degrees:
PhD thesis
- Basto, A.P. “A new cloning system based on the OprI lipoprotein for the production of bacterial cell wall-derived immunogenic formulations. PhD Thesis. Approved. Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa. October 2011
- Quembo, C. “A study of the epidemiology of African swine fever in Mozambique”. PhD Thesis, ongoing, to be submitted to the University of Pretoria. 2008-2012.
- Kouakou Kouame Valere. “ASF Epidemiological characterization on West African countries”.
PhD Thesis. Ongoing, to be submitted to UFR Biosciences de l'Université d'Abidjan-Cocody, Côte d'Ivoire.
Msc dissertations
- Annette Nigsch. “Introduction of African Swine Fever into the European Union: development of a generic model to assess disease spread within and between Member States”. As part fullfillment of the MSc Veterinary Epidemiology of the Royal Veterinary College, August 2011
- Anja Bauer. “Analyse früher Stadien während der Infektion von Zellkulturen mit dem Virus der afrikanischen Schweinepest (Analysis of early stages of the infection of cultured cells with African Swine fever virus). MSc thesis approved, 2010-09-16. University of Rostock, Faculty of Medicine/ Medical Biotechnology.
- Lina Mur Gil. Evaluación cuantitativa del riesgo de introducción del virus de la Peste Porcina Africana en la Unión Europea por importación legal de porcinos vivos y sus productos cárnicos derivados. MSc Thesis, approved Universidad Complutense de Madrid June 2010.
- Ricardo Gomez-Soute. “Pathogenicity of an African swine fever virus isolated from domestic pigs in Mozambique”. MSc dissertation, ongoing. 2009-2012
2.4 Training courses
Dissemination activities aiming at transferring knowledge on relevant aspects of ASF and transfer of tools developed within the project in the field of epidemiology, risk assessment and laboratory diagnostics was achieved through the organization of training courses and traineeships. Based on institutions involved in the project, four Training Courses were organized in Africa, one course in China and one in Spain. One Course on ASF diagnosis was launched on a CD version, available on Internet
- Multimedia Course on ASF Diagnosis
A course on African swine fever diagnosis was developed by UCM and CISA-INIA in collaboration with OIE on CD format and it is available at http://sanidadanimal.info/cursos/asf/(si apre in una nuova finestra). This comprehensive course includes theoretical lessons and practical videos on clinical and laboratory diagnosis of ASF. This CD (so far edited in English, Russian and Spanish) was spread within the ASFRISK beneficiaries and also to representatives of different countries attending the different courses organized by the project, and to others through UCM, CIRAD and CISA-INIA networks of collaboration.
- “Training Course on African swine fever (ASF) diagnostic techniques”, held in Entebe, Uganda from 15 to 24th of September 2008. Twenty five participants from Department of Veterinary Services (DVS) and National systems staff from Kenya, Uganda and Tanzania attended the course which was organized in cooperation between Centro de Investigación en Sanidad Animal (CISA-INIA) in Madrid and ILRI in Kenya at the Ministry of Agriculture Animal Industry and Fisheries (MAIF) in Uganda.
- “Course on African swine fever (ASF) Epidemiology and Diagnosis”, held at the Veterinary Research Institute (LVRI), Chinese Academy of Agricultural Science, at Lanzhou, Gansu in the People's Republic of China (20-24 September 2010). Thirty participants attended the course, including young scientists from Lanzhou Veterinary Research Institute, CAAS and several representatives from Veterinary Laboratories of different regions in China namely from boarding areas with the Russian Federation. The course was organized in cooperation between LVRI, Centro de Investigación en Sanidad Animal (CISA-INIA) in Madrid, Spain and Faculdade de Medicina Veterinária (FMV-UTL) in Lisbon, Portugal.
- “The ASF laboratory diagnosis-based training course and the The ASF epidemiology course and expert opinion workshop”, held at Laboratoire Central de Pathologie Animale /LANADA , Bingerville, Ivory Coast respectively from 18-22nd of October 2010 and November 1st - 5th , 2010). Twenty participants from the Departments of Veterinary Services, Animal Health Departments from Ivory Coast, Burkina Faso, Togo, Benin and Ghana attended these courses organized by VAR and UCM in collaboration with LANADA –LCPA.
- “Training course on African swine fever (ASF) Diagnostic techniques”, held at the Agriculture Research Council in Onderstepoort (ARC-OVI), Republic of South Africa, from May 30th to June 3rd, 2011. Seventeen participants, representative from 13 of the Southern African Development Community (SADC) countries, namely from Angola, Botswana , Democratic Republic of Congo, Lesotho, Malawi, Mauritius, Mozambique, Namibia, Seychelles , South Africa, Swaziland, United Republic of Tanzania, Zambia, Zimbabwe, and a young researcher from the National Institute for Veterinary Research Vietnam in Hanoi, partner of the ASFRISK project, attended the course. The course was organized in cooperation between ARC – Onderstepoort Veterinary Institute (OVI), Centro de Investigación en Sanidad Animal (CISA-INIA) in Madrid, Spain and Faculdade de Medicina Veterinária (FMV-UTL) in Lisbon, Portugal.
- Taining course on “African swine fever (ASF) epidemiology and laboratory diagnosis (ASF EPI-LAB)”, held in Madrid from June 27 to July 1, 2011. Twenty eight participants attended the course: thirteen specialists on epidemiology and control of diseases (from Russia, Bulgaria, Sweden, Great Britain, Portugal and Spain, participated on the ASF-Epidemiology component of the course and fifteen specialists on laboratory diagnosis of animal infectious diseases (From Russia, Belarus, Great Britain, Poland, Germany, Vietnam). The course was organized by UCM in collaboration with CISA-INIA.
- For each training course, participants received documents in informatics support (CD) and others, containing the programs followed, lectures, laboratory procedures, reference manuals and relevant bibliography.
2.5 Laboratory-based traineeships
The main goal of these trainings was to transfer the ASF diagnostic knowledge and tools available at the hosting EU partners in the project to Lanzhou Veterinary Research Institute (LVRI) in China and National Institute of Veterinary Research (NIVR) in Vietnam, providing training to collaborators of both institutions towards the implementation of ASF serological and virological diagnostic techniques in their laboratories. Five short-term laboratory-based trainings were organized to three Chinese fellows from LVI, Lanzhou and to two Vietnamese fellows At SVA (Sweeden) and CISA-INIA (Spain)
2.6 VIII Simpósio, Sociedade Científica de Suinicultura, “Peste Suína Africana”, held at the Faculdade de Medicina Veterinária, Lisbon, November 11th , 2010. Organization: Sociedade Científica de Suinicultura (SCS) in collaboration with the ASFRISK Project. This meeting targeted the associated members of SCS (pig farm veterinarians and professionals, pig farmers and associates to pig industry organizations (Federação Portuguesa de Associações de Suinicultores - FPAS ), representatives of the Portuguese Ministry of Agriculture and students. Around forty participants attended this Symposium. Major lectures were introduced by different authors covering “General aspects of the disease” (Carlos Martins, Portugal), “Epidemiology, risk assessment, control and eradication” (Jose Manuel Sanchez –Vizcaino, Madrid), “Laboratory diagnosis” (Margarida Duarte, Portugal), “ASF situation in Russia and Caucasian countries” (Denis Kolbasov, Russia), “The official veterinary services and ASF” (Pina Fonseca, Portuguese Ministry of Agriculture).
2.7 The ASFRISK Symposium
The ASFRISK Symposium, organized by the Coordinator Partner (Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa (FMV-UTL) was held in Lisbon at the Cento Cultural de Belém, at the end of the project on September 23rd, 2011.
The main objective of this meeting aimed at the dissemination of the ASFRISK achievements to representatives of international organizations (EC, FAO and OIE); Chief Veterinary Officers (Animal Health) of Member States of the European Union and Candidate and Associated Countries; representatives of Animal Health Institutions from Russia and Caucasian Countries; representatives of professional and policy maker organizations related to pig industry.
A total of 80 participants attended the meeting: 35 guests among CVOs or their representatives, EC, FAO, OIE representatives, and others from different organizations related to pig industry, Faculty members and 45 delegates of the ASFRISK project. Major lectures on general aspects relating to research on Animal Health and on critical aspects of ASF were introduced by the coordinator and guest speakers (Anne Sophie Lequarré, EC, DG RTD; Mary Louise Penrith, University of Pretoria, South Africa; Serghey Dudnikoff, Institute of Animal Health, Russia; Francisco Riviriego, EC, DG SANCO). Major achievements of the ASFRISK project were introduced by the Task Coordinators and complemented by presentations of different delegates involved in the work developed, promoting an opened discussion on different matters with the audience.
2.8 Exploitation of results
A penside immunochromatography test was developed, standardized and validated. The test, which is now commercially available through INGENASA (INGEZIM PPA-CROM), detects anti-ASFV antibodies in serum in less than 10 minutes, with 99% diagnostic sensitivity and 99.8% diagnostic specificity compared to OIE methods. Currently, this assay is being adapted for blood sample use.
A set of nucleic acid-based assays for laboratory and front-line/penside application were also developed, and included three systems based on commercial Universal Probe Library (UPL-PCR, Roche®) technology, Linear-After-The-Exponential PCR (LATE-PCR) technology, and Loop-mediated isothermal amplification (LAMP) technology, respectively, targeting highly conserved regions of the ASFV genome. A large collection of isolates was used for the evaluation, and the performance of the assays was then tested in two different European ring trials, with excellent results. The UPL- PCR and the LAMP were adapted to commercially available portable platforms, and tested in the European partner laboratories as well as in regions with endemic ASF. The assays were also transferred to ready-to-use diagnostic kit formats, and prototypes are now under evaluation for their potential upcoming launching in the market.
The use of filter papers (FTA and 3MM) for collection, storage and shipping of samples for detection of virus, nucleic acid and antibodies was evaluated and standardized. This may be a practical system to monitor wild boars, but also for sampling, storage and shipping of samples in regions with endemic ASF and poor veterinary surveillance infrastructure.
2.9 Development of the Project website
The website for the ASFRISK was released to the public on May 7th, 2009.
The address of the web page is: www.asfrisk.eu
Information is contained in Public and Internal Sections
- The Public Section is opened to general public and it contains information on the organization of the project (Summary; Objectives; Impact; Map; Management; Consortium), consortium meetings, Training activities; Dissemination (containing internal and external dissemination activities); Summary of activities; News and Links. It is updated accordingly to the progress of the Project.
- The Internal section exclusive to the use of the participants in the project contains the following Topics: Project Handbook; Official Documents; Management; Participants; Meetings; Laboratory Procedures; Publications; Links and a Bulletin Board.
African swine fever (ASF) in EU member states is currently confined to Italy (Sardinia). It was recently introduced to Caucasian countries and Russia and it is highly prevalent in sub-Saharan African countries. Surveys during outbreaks occurred previously in Eastern and Southern African regions have shown that although ASFV prevalence in the domestic pigs is high, in many cases serum conversion was not detected. These events pose a great challenge for the prevention and control of ASF, not only locally, but also worldwide. The complex epidemiological scenarios of ASF, the increasing mobility of people, animals and goods across the globe, emphasize the serious menace the disease presents to the growing pig farming sector in Africa and to currently ASF-free EU member states permanently threatened by the occurrence of the disease in different geographical regions.
In order to improve tools and strategies for the prevention and control of ASF, the work developed under the ASFRISK project aimed at fulfilling four objectives contained in the Major Tasks of the work-plan:
i) to improve and further develop the epidemiological, risk assessment and control measures of African swine fever (ASF), ii) to develop and provide new and robust tests for laboratory and front-line diagnosis, through the achievement, standardization and validation of more rapid, sensitive and inexpensive serological and molecular procedures iii) to establish new strategies for vaccine development against ASF, iv) to transfer knowledge on relevant aspects of ASF and transfer of diagnostic and epidemiologic tools through training courses and traineeships to provide an harmonized international training, in order to prevent and combat this highly devastating, OIE-listed transboundary animal disease.
The four main tasks of the project were outlined and harmonized in the work-plan providing a logical process for the improvement of knowledge on critical aspects of ASF and development of tools towards the achievement of the objectives through the contribution of highly qualified partners from EU, Africa and Asia, with expertise in all the areas concerned. Major outcomes of the project emphasize its successful contribution for “Evaluating and controlling the risk of ASF in the EU”:
- Development of epidemiological methods and decision support tools provide information to policy makers on ASF epidemiological situation and will assist in the prioritization of strategies to better prevent and control ASF in a potential incursion into the EU and in all currently affected territories.
- Development of a range of valuable serological and molecular diagnostic tools suitable for use in well-equipped international and national reference laboratories, in basic regional and local laboratories, or even for rapid first line on-site application, allow and complement available tests thus promoting more efficient and earlier diagnosis of the disease.
- Characterization of several ASFV-host interactions at cellular and molecular levels and the production of different attenuated recombinant virus strains that may be potentially used as future candidates for a vaccine, open new insights on the development of control strategies against ASF.
- Transfer of knowledge on relevant aspects of ASF and transfer diagnostic and epidemiologic tools through training courses and traineeships to a large number of professionals from Animal Health Institutions in EU, Africa, Asia, Russia and Caucasian countries. These harmonized training actions will contribute for the strengthening of knowledge and technological efficacy to face problems caused by ASF in endemic countries and to build up preparedness capacity against the threat of the disease in free countries.
The outcomes of the project will undoubtedly contribute to the sustained development of EU agri-food business by improving prevention, early detection and control of ASF and therefore reducing the socio-economic consequences of potential incursions of the disease in EU from infected regions.
Project Context and Objectives:
African swine fever (ASF) in EU member states is currently confined to Italy (Sardinia), it was recently introduced to Caucasian countries and Russia and it is highly prevalent in sub-Saharan African countries. Surveys during outbreaks occurred previously in Eastern and Southern African regions have shown that although the average prevalence of viral infection in the domestic pigs is high, in many cases serum conversion was not detected. These events pose a great challenge for the prevention and control of ASF, not only locally, but also worldwide. The complex epidemiological scenarios of ASF and the increasing mobility of people, animals and goods across the globe, emphasize the serious threat the disease presents to the growing pig farming sector in Africa and to currently disease-free EU member states.
In order to improve tools and strategies for the prevention and control of ASF, the work developed under the ASFRISK project aimed at fulfilling four objectives contained in the Major Tasks of the work-plan:
Objective 1 (Task 1)
To evaluate the epidemiological situation of ASF in Sardinia and Africa, in particular to characterize, from the molecular and biological point of view, currently circulating field viruses of epidemiological interest and relate the possible occurrence of genome changes with new occurring forms of the disease and diagnosis problems described during recent outbreaks namely in Africa. Furthermore this project aimed at establishing a risk assessment framework that can be applied for any European country to assess the risk of introducing ASF virus into the EU, producing an ASF risk map for Africa and to develop and evaluate control strategies appropriate for the situation of ASF in Africa. Work developed under this Task was focused on three major approaches:
• Molecular and biological characterization of currently circulating ASFV strains of epidemiological interest.
• Development and validation of a generic risk assessment framework for the introduction of ASF into EU countries
• Development of control strategies
Objective 2 (Task 2)
To develop, validate and apply improved, robust and/or simple antibody and nucleic acid detection methods for ASFV for the rapid detection and differential diagnosis of suspected cases of ASF, taking into account the newly emerging viruses circulating in Africa and Sardinia. Particular emphasis will be placed on inexpensive simple-to-use assays suitable for first-line and/or pen-side diagnosis and the deployment of these tests to regional laboratories in the EU and African states. Work developed under this Task was focused on the following approaches:
• Development and validation of:
▪ New rapid, sensitive and specific assays based on ELISA and Immunoblotting tests to detect early and late appearing ASFV antibodies, raised in the course of infection with the new circulating strains of ASFV;
▪ New powerful assays based on robust highly sensitive and specific real-time PCR techniques, for rapid detection of ASFV nucleic-acid;
▪ New rapid diagnostic procedures based on simple highly sensitive and specific isothermal amplification assays;
• Adaptation and evaluation of:
▪ Antibody detection assays based on dot-blot techniques to dipstick format, suitable for pen-side and first-line on-site diagnosis;
▪ Current and newly developed real-time PCR and isothermal amplification assays to portable platforms, suitable for front-line diagnosis of ASF;
• To standardise and validate selected developed assays to OIE standards in order to harmonise ASFV diagnosis in EU member states.
• To use the phage display procedure to search for monoclonal antibodies that could be used in the development of ELISAs to detect antibodies to ASFV and to further develop a strip test using gold nano particles;
• To transfer technologies to diagnostic facilities in Africa for validation of the tests in various regions of the continent.
Objective 3 (Task 3)
The main objective in this Task is the implementation of new strategies for the development of a vaccine against African swine fever (ASF) using and further characterizing a protection model that includes the use of two naturally occurring ASFV isolates of different virulence in which the low virulent virus (ASFV/NH68) induces protective immunity against the highly virulent isolate (ASFV/L60) but with a high residual level of virulence to be used as a vaccine.
Work developed under this Task was focused on the following approaches:
• The characterization of viral-host interactions
• The in vitro and in vivo characterization of pig immune mechanisms relevant for animal survival against ASFV.
• The construction of recombinant viruses by conventional and/or new high throughput methods
• The optimization of OprI presentation model of ASFV antigens as a new system for the production of virus antigens fused with lipoproteins
• The development of complementary methods (antivirals) for the control of the ASF.
Objective 4 (Task 4)
The main objective of the task is to transfer knowledge on relevant aspects of ASF and improved tools for ASF diagnosis, including pen-side, conventional and reference diagnostic tests, and epidemiological modelling and risk assessment strategies aiming at the establishment of control measures, both in Europe if the disease occurs and in Africa where it is endemic. The developed tools within this project in the mentioned fields will be transferred to Asian and African partners within the project and to other Animal Health institutions in EU, Africa, Russia and in the Caucasian region.
Work developed under this Task was focused on the following approaches:
• Organization of training courses and workshops on ASF epidemiology, risk assessment and laboratory diagnosis in African and Asian partners of the project and also targeting Caucasian countries and Russia where the disease was recently introduced.
• Organization of individual short-term training at expert institutions on ASF epidemiology, risk assessment and laboratory diagnostics, for beneficiaries in the project and to other institutions interested namely from Africa, Caucasian countries and Russia were the disease was recently introduced
• Individual long-term training at expert institutions carried out under PhD preparation.
Project Results:
In order to improve tools and strategies for the prevention and control of ASF, the work developed under the ASFRISK project aimed at fulfilling four objectives contained in the Major Tasks of the work-plan:
TASK I
EPIDEMIOLOGY, RISK ASSESSMENT AND CONTROL STRATEGIES
The work developed under Task 1 aimed at fulfilling the following objectives:
To evaluate the epidemiological situation of ASF in Sardinia and Africa, in particular to characterize, from the molecular and biological point of view, currently circulating field viruses of epidemiological interest and relate the possible occurrence of genome changes with currently circulating field viruses of epidemiological interest and relate the possible occurrence of genome changes with new occurring forms of the disease and diagnosis problems described during recent outbreaks namely in Africa. Furthermore this project aimed at establishing a risk assessment framework that can be applied for any European country to assess the risk of introducing ASF virus into the EU, producing an ASF risk map for Africa and to develop and evaluate control strategies appropriate for the situation of ASF in Africa.
The work was developed under seven work-packages as described ahead:
WP1 - Epidemiological and molecular characterization of currently circulating field viruses in African Countries and in Sardinia
Objectives
The main target of this WP has been focused on to increase the knowledge about the epidemiological and molecular features of currently circulating ASFV isolates related to the occurrence of atypical forms of disease and diagnosis problems recently reported in Africa and in Sardinia. The molecular characterization has been based on standardized and new developed protocols for characterizing the genomes of ASFV isolates by targeting independent ASFV genomic regions.
Results
In East Africa the role of warthog was studied by the analysis of samples obtained from warthogs, ticks (Ornithodorus porcinus) and domestic pigs from different parts of Kenya and Tanzania. These results suggest that ticks play the key role in maintenance of the virus, whilst adult warthogs represent a terminal host. The role of bushpigs in ASFV transmission to domestic pigs was studied in connection to the detection of a relatively high presence of ASFV in domestic pigs in which no clinical signs or antibodies were found. These data, together with the genetic data described below, suggest a possible role of bush pigs in the sylvatic cycle, that may harbor a low virulent or avirulent isolate of the virus, or that local pigs exhibit a degree of innate or acquired immunity to the specific virus that is currently circulating. The presence of the domestic cycle in East Africa was widely demonstrated following the ASF outbreaks occurred in Kenya and Uganda since 2007.
These epidemiological findings were supported by molecular characterization of 63 East Africa ASFV isolates from warthogs, bushpigs, ticks and domestic pigs that confirms the concurrent existence of three different transmission cycles of ASF in East Africa; i) Domestic pig-pig without ticks in Western Kenya and in Uganda; ii) domestic pig-warthog or domestic pig-bushpig and iii) domestic pig-tick/warthog sylvatic transmission. The observation of segregation of different genotypes between ticks and adult warthogs is surprising and suggests that further studies in warthogs and ticks are required to better understand the nature of the sylvatic transmission cycle of ASFV.
In West Africa sampling activities supports the fact that the recovered ASF pigs are involved in stock trade and breeding activities and play and essential role in the spreading of the disease in West Africa, which is mainly linked to the movement of infected pigs or pig products. To confirm this hypothesis, 53 West African domestic pigs ASFV isolates were selected for genotyping analysis using standardized procedures and by the analysis of new regions by the use of new set of primers CV1 and CV2. West Africa ASFV isolates were separated in 10 different CVR groups comprising eighteen different variants, revealing a cross-virus circulation between neighbouring countries Togo, Benin and Nigeria in Maritime areas and the co-circulation of multiple tetramer types in one country such as Nigeria or Burkina Faso.
The genetic analysis of a wide range of ASFV from Sardinia obtained from 1978 to 2009 revealed the presence of a unique genotype (genotype I). However, two clusters were identified, accordingly to their temporal distribution, namely sub-group III and sub-group X, by the analysis of B602L gene revealing a minor difference in the number of tetrameric repeats, placing this latter showing a deletion of 12 tetramer repeats located in the centre of the array.
In order to facilitate genotyping and characterization of ASFV, a microarray was designed, developed and tested in this project. The area targeted for the array was the C-terminal end of the p72 protein, the primary region used to place ASFV into one of the twenty-two established genotypes. The technology chosen to develop the array was the Luminex 200 system which provides an established, practical technology for multiplex assays of proteins and nucleic acids. The microarray developed within this project provides complete and correct identification for all described genotypes with a comparable sensitivity to real-time PCR, with the detection sensitivity possibly somewhat more sensitive than the typing.
WP 2 - Biological characterization of currently circulating field viruses in African countries and in Sardinia
Objectives
This WP has been focused on the characterization of biological aspects of ASFV related with the virulence of different isolates present in Africa and in Sardinia, the lesions induced and the immunological features triggered upon infection with those isolates. The main objective of this WP has been to evaluate clinically and pathologically the forms of disease currently observed in the endemic areas and compare them with the previous findings on acute and subacute forms of the disease.
Summary of results on the WP
Four different ASFV strains were selected and several experiments were developed by the inoculation of these isolates in domestic pigs (European and African breeds) for the study of clinical, pathological and immunological (cytokines expression) findings in these animals. The experiment developed with the supposed attenuated Sardinian isolate suggests that this isolate is highly virulent capable of inducing a subacute to acute form disease with even severe clinical and pathological reactions. ASFV could be detected from the blood of one animal during the incubation period, but this Sardinian isolate is clearly characterized by clinical and pathological signs, which may facilitate the diagnosis of the disease in field conditions and therefore the implementation of control measures including stamping out practices.
The study of African isolates found to be responsible for the appearance of non evident ASF clinical signs in combination to lack of humoral response co-existing with a high viral load (as described in WP1) was done by three different experiments. The inoculation of these isolates in European breeds suggested that the ASF Kenya/05 virus isolate is of low-moderate virulence capable to induce a subacute picture of disease not clearly characterized by typical clinical and pathological signs but with a conventional antibody response. However, the inoculation of the same isolate in Kenyan “indigenous pigs shown a significant delay of onset of with an unclear, unspecific, non-patognomonic picture of the disease that easily goes unrecognized. These animals present a detectable viraemia at 14 dpi with intermittent peaks and a very different humoral response, with delayed or absence seroconversion. These findings, the presence of unrecognized diseased animals with viraemia but no antibodies, have serious implications on control and success of ASF eradication in East Africa.
WP 3 - Risk Assessment of the introduction of ASFV into the EU
Objectives
This WP aims at developing a framework that allows assessing the risk of introduction of ASFV into any European Union country, taking into account the current situation of ASF in endemic areas. The framework estimates the overall risk of ASFV in each of the EU countries and specifically details the risk by each of the analyzed pathways of introduction, providing and interesting tool for supporting risk manager decisions.
Summary of results on the WP
The potential ASFV introduction routes into EU countries were selected and evaluated independently based on the availability and quality of data available. Several expert opinion elicitation sessions were developed during the project in order to assess the relative importance of some parameters, or validate the models and the results obtained by them. Finally, all these pathways were conveniently adapted and standardized to be included in the final framework to obtain the overall risk for ASFV introduction into EU countries.
For legal imports assay, two quantitative models were developed revealing a low annual risk for ASFV introduction by these routes. This risk is mainly concentrated in some countries such as Poland and Lithuania in the case of legal pigs; and Romania and Bulgaria for pig products. In the case of illegal import pathway, was performed as a semi- quantitative model obtained by linear weighted combination of the risk level for each indicator. Result reveals that no country poses the highest level of risk (very high) of this route, but several countries were in the high risk category: France, Germany, Italy and United Kingdom. For the transport fomite pathway, three different means of transport were included in the model: returning trucks from affected areas and waste disposal from boats and planes, using a similar approach than illegal import pathway. Results shown that returning trucks is the transport pathway that nowadays poses the highest risk in the EU and Lithuania and Poland (high) pose a considerable relative risk by this pathway. For the wild boar movement analysis, a previous suitability map of the potential wild boar distribution in EU was built. Then, a semiquantitative model was performed, revealing that Romania (high), followed by Finland, Latvia and Poland (medium) are the EU countries at highest risk for ASFV introduction borders by wild boar natural movement. A common exposure assessment for the illegal, transport and wild boar pathway was developed in order to be combined with pathways in the final framework. The consequence assessment was a stochastic spatio-temporal model for the spread of ASF within and between EU Member States. The model aimed at identified the MS that most likely spread ASF, “super-spreaders”, Denmark and The Netherlands; or MS that receive cases from other MS “super-receivers”, Germany and Poland during the high risk period (HRP).
All this information was conveniently adapted and incorporated in a single Excel file for the final framework for ASFV introduction into EU. Detailed Summary of results on the WP of each of the analyzed pathways are published or in process to be published and the final framework will be provided to European Commission and EU countries to be used as an interesting tool for risk management.
WP 4 - Risk mapping in ASFV endemic areas
Objectives
This WP was aimed at compiling existing knowledge and epidemiological data to produce a risk map for Africa and to spatially explore the epidemiology of ASF in Sardinia. The outcome of this WP has increased the understanding of the pig production chain, identifying cultural, sociological and economical factors that contribute to the ASF occurrence in Africa and Sardinia and providing a better description of the ASF dynamics in rural and urban areas.
Summary of results on the WP
The main conclusions obtained within this WP for Africa can be summarized in the following points: 1) North and south-western Africa are likely to be unsuitable for occurrence of ASF, 2) there is a strong evidence for suitability for outbreaks in large parts of eastern, south-eastern and western Africa but with substantial within-country heterogeneity, 3) there are many areas with high levels of uncertainty, particularly in central Africa and several countries in West Africa, 4) comparison of suitability estimates with the limited official outbreak data available revealed a good degree of agreement.
In Sardinia, the Bayesian model indicate that the probability of ASFV occurrence in Sardinia was mainly associated to a high number of confined (closed) farms, high density of roads, high mean altitude, high number of open fattening farms and high total number of pigs per commune. The high risk of confined farms may be the effect of higher notification of outbreaks or a high use of waste containing pork products to feed pigs in confined farms, particularly in small ones. It was also suggested that the system of farmer´s compensation after an outbreak in Italy may be excessive for Sardinian pigs, bred using non-professional type of farming, and mainly when referring to fattening pigs which may promote the intentional introduction of disease. The association between the total number of pigs, the density of roads and the ASFV occurrence per commune suggest that illegal trade of pigs may be a source of infection in some areas. Moreover, Summary of results on the WP reveal that Communes with most of their farms censed (i.e. controlled by the veterinary authority) are less likely to have ASF outbreaks than not censed (i.e. unsupervised) farms. This result highlights the importance that relative simple measures, such as a compulsory registration of the pig population, may have in the ASF control in Sardinia. Wild boars seem not to be important way of ASFV transmission in Sardinia. Finally, farms for self-consumption as well as areas with high pig density and with large herd sizes and intense trade/movement of animals were found to have less risk of ASFV occurrence than the background risk of the region, which may be associated with less high risk contacts or higher biosecurity measures, respectively.
WP5 - Risk Assessment for African countries
Objectives
To perform risk assessments for endemically and currently uninfected African countries, taking into account the particular epidemiology of a region, data collection about ASF epidemiology and pig production systems in selected areas is needed.
Summary of results on the WP
West African countries (Côte d´Ivoire, Ghana, Benin, Togo and Burkina Faso) were selected as target countries for the analysis. Lot of information was collected from these countries by official data sources, surveys sent to the veterinary services and expert opinion developed in Côte d´Ivoire during a workshop with the expert veterinary epidemiologist from the neighbouring countries. The study of this information suggests that a traditional connection of trade and cultural relations is present in West African countries that hardly influence the presence and spread of ASF in the area. The pig population in these countries is not very high, being Burkina Faso the country with highest number of pigs. Most of the swine population is rare in a free range production system, followed by semi confined familiar systems.
The use of the information obtained by the expert opinion session and their incorporation into a simple model structure, allow us to develop a semiquantitative model that analyze the different pathways of ASF introduction and spread in the area. According to this risk assessment, the pathway responsible for most of the ASF outbreaks in the area is the movement of infected animals. Wild animals and ticks seem not to have an important role in the ASF transmission in the area. Coast regions represent a very important source of infection due to the contaminated leftovers present around the ports. These Summary of results on the WP also point out that Togo is a key country in the area that easily connects all countries around it, being a spreader of the disease.
WP6 - Development of control and preventive strategies for African Countries
Objectives
Control strategies currently carried out in Africa and other parts of the world would be assessed. Several recommendations will be developed considering the outcomes of the risk assessments performed under WP5.
Summary of results on the WP
Information about ASF control activities and veterinary capacities in many African countries was collected. This information suggests that from West African countries, Ghana is the country with veterinary services at highest level, with higher number of veterinarians, laboratories and resources to fight against animal diseases. Second place is for Burkina Faso or Côte d´ Ivoire, depending on the analyzed parameter. These results could be considered as an indicator of the development and quality level of veterinary services in these countries as should be considered in future development and control strategies for this African countries.
Some of the critical points considered for ASF control in West Africa were the traditional type of pig production system by free range system, without control of feeding practices, the lack of knowledge by the farmers, dealers and pig drivers; and very important the high connection between ethnic groups of different countries (Benin-Togo, Togo-Ghana). These facts facilitate disease persistence in the area and potential spread within countries. These points were considered for the development of the suggestions and advices for ASF control in the area.
WP7 - DNA sequencing and sequence data bank
Objectives
Further developments for genome characterization will be attempted by applying high throughput very rapid sequencing for complete genome sequences of ASFV. These investigations will lead to a series of new insights into viral genome sequence conservation and variability, genetic diversity in nature and phylogenetic classification of isolates.
Summary of results on the WP
Sardinia represents a unique model for studies on ASFV evolution over time in a rather confined geographical region where a single virus introduction is supposed to have occurred. Therefore, a group of 9 isolates and 13 total samples representing the time-line since introduction to Sardinia was selected. One isolate (Sardinia2008) was selected for sequencing using 454 technology (performed during the first reporting period). For the remaining 13 samples, the illumina platform was chosen. The selection of technology was based on the properties that technologies possess and the price. An assessment of the technologies concluded that illumina could provide the data needed at the best price.
In terms of methodology, in the 454 run, random amplification was employed, while for the illumina sequencing a specific long PCR (10-20 kb) protocol was employed. For most segments the PCRs were successful. Segment 1 did not produce product for several samples, therefore based on the 454 sequence of Sardinia2008 new primers were designed. The newly designed primers allowed this segment to be amplified for all the samples. To date only the 454 sequence of Sardinia2008 is available. The other 12 sequences have had their genomes amplified by 15 specific PCRS, gel purified and sent to the sequencing platform. The platform has prepared libraries and now sequencing is being developed. To determine the genetic relationships among the AFSV genomes we used the concatenated core conserved set of genes. This analysis of the full genome sequences reveals that the major differences among AFSV genomes were reported in the copy number of five Multi Gene Families (MGF). It has been proposed that non-pathogenic isolates present a deletion of 8 to 10 Kbp at the left end of the sequence. This sequence is present in pathogenic strains and contains six copies of the MGF 360.
TASK 2
DEVELOPMENT AND VALIDATION OF SENSITIVE DIAGNOSTIC TECHNIQUES, INCLUDING PEN-SIDE TESTS, WITH THE EXISTING FIELD VIRUSES
This Task focused on the development, validation and application of improved, robust and/or simple antibody and nucleic acid detection methods for ASFV for the rapid detection and differential diagnosis of suspected cases of ASF, taking into account the currently circulating isolates in Sardinia and newly emerging ones circulating in Africa. Particular emphasis has been dedicated to inexpensive simple-to-use assays suitable for first-line and/or pen-side diagnosis and the deployment of these tests to regional laboratories in the EU and African states.
The work was developed under six work-packages as described ahead:
WP8 - Antibody detection techniques based on the existing circulating virus strains
and
WP 9 - Development of new ELISA tests based on the use of new antigens and monoclonal antibodies obtained from emerging “new” isolates of ASFV in Eastern, Southern Africa and Sardinia
Objectives:
Assessment and further validation, new rapid, sensitive and specific assays based on ELISA format, using different antigens and new ELISAs based on the determination of specific IgM antibodies with selected antigens. Development and assessment of new ELISAs based on the use of new recombinant proteins, semi-purified antigens and monoclonal antibodies, in order to establish robust ASF serum-diagnostics in the endemic regions of Europe and Africa following different strategies.
Summary of results on the WP:
Validation of ELISAs using pp62, the co-expressed p15 and p32 proteins in baculovarus systems and the fused p15-p32 protein:
Four standardized recombinant-ELISAs (rec-ELISAs) developed in previous projects and based on the baculovirus expressed proteins pp62, the co-expressed p15 and p30, the fused p15-p30 (HT) and the p54 protein were selected for further validation purposes. To this end a comparative study was initially performed by the analysis of i) 252 field samples collected in 2008-2009 in Sardinia and East Africa, and ii) 89 experimental samples obtained from in vivo experimental infections using current Kenyan and Sardinian circulating ASFV strains. From the sensitivity and specificity results obtained the rec-ELISA based on the fused p15-p30 (HT) recombinant protein was selected as the most promising protein to be used in ASF serological tests. A complete validation of the selected rec-HT-ELISA was subsequently performed through the analyses of a total of 1,873 field sera collected since 2004 until 2010 from endemic areas in Sub-Saharan Africa, including East, Central and West African countries (Kenya, Uganda, Tanzania, Cote de Voire, Togo, Ghana, Burkina Faso, Benin, Rwanda, Congo-Brazzaville and Nigeria) and Europe (Sardinia, Italy). The OIE prescribed assays (ELISA and IB) were used as gold standards comparative methods. From the analysis of 329 positive and 1,544 negative field sera, the sensitivity and specificity of the rec-HT-ELISA was established in 95% and 99% respectively. Also, the competence of the rec-HT- ELISA to detect specific antibodies during acute, sub-acute and chronic infections was assessed by the analyses of 446 sera collected at different stages from seven independent experimental infections using ASFV circulating isolates. In addition it was a perfect agreement between the specific antibody response detectable using the OIE prescribed assays and that detectable using the rec-HT-ELISA.
Further validation of ELISAs using p54, histone-like protein (gene A104R), and the uncharacterized virus proteins encoded by genes B602L and K205R expressed in E coli.: An initial validation showed that there was almost perfect agreement between the results of the “gold standard” tests and the results obtained by the p54-ELISA, the pK205R-ELISA or the pB602L-ELISA, but not with those obtained using the pA104R-ELISA. Complete validation of the selected rec-ELISAs was performed using a collection of 669 field sera collected from Sardinia and Africa since 2004 up to 2010. The pB602L protein did not allow full validation due to its insoluble properties. After the analysis of 235 positive East African sera the sensitivity of the p54-ELISA was less than 75%, not recommended for the detection of specific antibodies in East Africa. Interestingly, the pK205R protein was identified as a potentially useful serological diagnostic antigen for the detection of IgM responses, as it was able to detect all infected animals as early as 7 days post infection. In addition , a new ELISA based on combination of rec-ASFV proteins HT, p30 and pp62 was evaluated using a selection, short collection, of positive (20) and negative field sera (40) from East Africa. These results obtained did not improve those obtained using HT recombinant protein.
Standardisation of commercial prototype ELISA for IgG ASFV detection based on the fused p15-p30 (HT) protein: The p15-p30 fused (HT) protein expressed in the baculovirus expression system was transferred for the development, standardization and further validation of a new commercial ELISA. The results were compared with the commercially available competition ELISA from Ingenasa, based on VP72 protein. The results with the HT antigen indicated a similar analytical sensitivity than those obtained with the OIE ELISA and the commercial INGEZIM PPA COMPAC ELISA The specificity of this assay using serum samples from ASFV-free areas was > 99%. From the results obtained it was concluded that the HT protein is useful for detection of IgG antibodies specific for ASF virus when used in parallel with a negative antigen to reduce the background. This HT commercial ELISA could be and additional, complementary useful tool for ASF diagnosis.
Standardization of a commercial prototype ELISA for IgM ASFV detection: Three different antigens were used for the development of ELISAs for IgM ASFV detection; i) the HT recombinant protein, ii) the K205R recombinant protein produced in E.coli as protein fusion with GST and iii) the Vp72 ASFV purified protein (from Vero cells cultures). Finally for ASFV IgM specific detection the most promising results were obtained using the vp72 major antigenic protein. However, further validation is required to obtain a final IgM ELISA prototype to be used in Animal Health Laboratories.
Target Antigenic proteins from different ASFV isolates as new candidates for ELISA: The analysis of the specific sequences of the target antigenic ASFV proteins -p72, pp62, p54, p30, pB602L- from recent ASFV African and Sardinia isolates showed the East Africa Kenya 1950 isolate belonging to p72 genotype X as the most variable one. This isolate presented identity scores which dropped to 89% and 85% when the full length p30 and p54-gene sequences were compared with the Spanish ASFV isolates from which conventional serological antigens are obtained. Additionally, the analysis of p30 and p54 sequences showed the regions with the highest sequence variability also presented high antigenic indexes and theoretical low structural order. These findings were supported in a further comparative study of the 149 VP54 and 84 VP30 sequences obtained from current circulating isolates in Europe and Africa and representatives of the 22 P72 genotypes. Nevertheless, due to results from other workpackages there was not a need for new ELISAs using new antigens (see below point f). Instead, the efforts were directed to study the origin/cause for the documented low incidence of detectable serological response to ASFV infection in ASF virus positive pigs in East and southeast Africa using OIE-prescribed ASF serological methods and towards to the development, standardization and validation of a new confirmatory antibody detection test, the indirect. Immunoperox. technique (IPT) (see below, and results of task 1 as well).
New ELISA Tests based on Soluble cytoplasmatic Antigen (Cp-Ag) from different ASFV isolates of Africa and Caucasus region: Since the OIE-prescribed methods are based on the use of total semipurified antigen obtained from the Spanish ASFV E70 isolate belonging to genotype I, a possible explanation for the unexpectedly low seropositive response with east African sera may reside in the polymorphisms in immunodominant viral antigens. So, it was not clear at the beginning of the project whether current OIE sero-diagnostic techniques will be sufficiently sensitive and specific to detect specific antibody response against the different ASFV circulating variants in Africa and in the Caucasus areas. To sort out this question, the work was focused on determining the capability and competence of the OIE formal serological diagnostic techniques employed under different epidemiological scenarios currently occurring in Europe and Africa. To this end six ASFV isolates selected following the genome variability criteria and date collection were used for the preparation of six new soluble cytoplasmic antigens (Cp-Ags). The ASFV isolates selected included, i) the most variable p72 genotypes X, IX, VIII and V as so far described circulating in East African countries (Ken08/Tk2.1; Ken06.Bus Moz64 and MwLil20/1), ii) the historical circulating European isolate belonging to p72 genotype I (E70), ,and a new objective not included at the beginning of the project: iii) the use of current European p72 genotype II (Arm07). COS-1 cells (donated by CBM partner) were employed for antigen production by infection and later semipurification using each of the six selected ASFV strains according the procedure described in the OIE Manual of diagnosis (OIE 2008). The new Cp-Ags were used as antigens in six different ELISA assays based on selected viruses. Subsequently, a validation study was carried out through a comparative analysis of 1,062 serum field samples collected from 2003 up to 2010 from both domestic and wild pigs in endemic areas of Sub-Saharan African regions (Kenya, Uganda, Tanzania, Cote de Voire, Togo, Ghana, Burkina Faso, Benin, Rwanda, Congo-Brazzaville and Nigeria) and Europe (Sardinia (Italy), as well as 214 experimental sera collected at various stages from six independent experimental infections. There was a perfect agreement of the new developed Cp-Ag-ELISAs and the OIE serological techniques in the analysis of both experimental and field samples collected from current affected areas in Africa and Europe. This clearly demonstrates the capability of the OIE formal diagnostic techniques to perform a serological diagnosis with high sensitivity, specificity and confidence independently of the genetic variants circulating. In addition, all results were confirmed by a new confirmatory serological technique based on Indirect Immunoperoxidase Test (IPT) that was simultaneously developed and validated for each genotype under study.
Other approaches performed during the project included the development of phage display library to produce monoclonal single chain variable fragments (scFv) that could be used in the development of ELISAs to detect antibodies to ASFV in a near future.
Adaptation and evaluation antibody detection assays based on dot-blot techniques to dipstick format, suitable for pen-side and first-line on-site diagnosis.
The epidemiological situation of ASF worldwide has highlighted the need for front-line/penside tests as good tools for disease diagnosis allowing earlier implementation of sanitary control measures. To this end, a rapid, one-step immunochromatographic strip (pen-side test) capable of specifically detecting anti-ASF antibodies in serum specimens has been developed by INGENASA in collaboration with CISA-INIA. The qualitative assay is based on a direct immunochromatographic assay in which the detector reagent consisted of red carboxyl-modified latex microparticles covalently coated with VP72 purified protein. The capture reagent was the same protein adsorbed on the nitrocellulose membrane strip to form a test line. In addition, blue latex microparticles coated with a control protein and a second line created above the test line, by the immobilization of anti-control protein antibodies, was used as a control of test. The limit detection of the new assay was established in 9 ng/ml (1.35 ng/test). Once standardized, a complete validation of the new developed assay was performed by the analysis of a wide collection of field and experimental samples previously classified as positive or negative using the OIE-prescribed assays as “gold standards”. The diagnostic sensitivity was established in 99% by testing a total of 208 ASF antibody positive field sera collecting in East and West endemic African countries. The comparative Summary of results on the WP obtained using the new pen-side test and the conventional OIE and INGENASA tests demonstrated a diagnostic specificity of 99,9% by testing a total of 1,210 ASF antibody negative field sera collecting in East and West endemic African countries and free areas of Europe. As a conclusion a first pen side test for ASF antibody detection have been developed and validated for rapid on site ASF detection. The test, registered and commercially available by INGENASA [INGEZIM PPA CROM R.11.PPA.K41] allows sensitivity and specific ASF antibody detection in 10 minutes and can be employed an easy single step performing method where laboratory support and skilled personnel are limited.
WP 10 – Nucleic acid-based assays for detection of currently circulating ASFV isolates
Objectives:
Development and validation of new powerful assays based on robust highly sensitive and specific real-time PCR techniques, for rapid detection of ASFV nucleic-acid, and rapid diagnostic procedures based on simple highly sensitive and specific isothermal amplification assays;
Summary of results on the WP:
Further development of the TaqMan real-time PCR technology for the improved detection of ASFV
A new real-time PCR technology based on the use of universal commercial probes, Universal Probe Library (UPL), commercialized by Roche Applied Science, main advantages being reasonably low cost, short time of delivery, and ready-to-use presentation. Optimization and analytical sensitivity and specificity assays of the developed UPL PCR (using UPL#162) showed a a sensitivity quite similar to that provided by designed TaqMan PCR, while it is 10-fold more sensitive than the established reference real-time method. The detection limit of the UPL PCR showed to be between 4 and 18 DNA copies. A comprehensive study to assure the detection range of the developed UPL PCR was carried out during this time with the analysis of a collection of 26 ASFV isolates, belonging to 17 of the 22 reported p72 genotypes. Afterwards, validation experiments using an extensive collection of field porcine and tick samples (n=256), coming from Eastern and Western African regions affected by ASF, demonstrated that the UPL PCR technique was able to detect over a 10% more positive samples compared to the OIE TaqMan PCR, confirming the superior diagnostic sensitivity of the new molecular assay. Clinical material collected during experimental infections with different ASFV p72 genotypes, specifically from ASFV genotypes I, II, IX, and X, was useful to assure both the capacity of the UPL PCR for an early viral DNA detection and the competence of the technique to be applied in any ASF diagnostic target sample. An internal control PCR assay based on the detection of a β actin gene DNA fragment using a specific primer set and the same UPL probe (UPL#162) than that selected for ASFV detection was developed. The complete study offers a new validated real-time PCR technique, together with a simple internal control test, ready for its application in the routine diagnosis of ASF. The final standard operation procedure (SOP) of the developed real-time PCR method using UPL#162 probe was transferred to all consortium partners participating in the ring trial conducted under WP 13, and to several ASF National Reference Laboratories of the EU. In addition, INGENASA is working now to adapt this robust PCR technique in a commercial format.
Development of a LATE PCR system for detection of ASFV
The development and optimisation of a LATE (Linear-After-The-Exponential) PCR assay for ASFV detection has been carried out. The technology developed at Brandeis University (MA, USA) and the developed ASF assay was aimed at adaptation to their portable PCR platform BioSeeq. This work was carried out by SVA in collaboration with Brandeis University and Smiths Detection. Optimization and analytical sensitivity and specificity assays of the developed LATE PCR was carried out and the assay was further validated and also tested in a ring trial organized within EPIZONE with good results, further demonstrating the specificity and sensitivity of the new assay. The assay was ready for adaptation to portable platform but this step was dependent on the commercial collaborator.
Rapid diagnostic procedures based on simple highly sensitive and specific Loop-Mediated Isothermal Amplification (LAMP) assays
LAMP is based on amplification of nucleic acids without the need of PCR equipment. The comparative simplicity of the technology makes LAMP adaptable to front-line testing in regional laboratories, simple diagnostic situations and even to pen-side testing. This work was mainly carried out by QUBand SVA. Seven accelerated LAMP assays were designed against the 4CL, 9GL and vp72 genes using on line software (PrimerExplorer version 3&4) provided by Eiken Ltd. The assays were designed using alignments based on 23 (4CL), 32 (9GL) or 50 (vp72) sequences. DNA from the fourteen ASFV strains described above was used for the evaluation of the assays.Upon further optimization one assay was singled out and consequently used in all experiments (ASFV vp72 F3-354). Ten-fold dilution series of ASFV Moz64, Ang72, MwLil 20/1, CV97, Ug03H, Ken06 and Ken07 were further tested to determine the detection limit, indicating detection limits around 40-50 copies of viral DNA. Protocol for this assay was created and adapted to two different LAMP reaction assays using two different portable platforms (see below). The protocol was also distributed to other partners and used within the ring trial conducted under WP13. A panel of LAMP assays for cost-efficient and rapid detection of ASFV has been developed, suitable for use in basic regional and local laboratories.
WP 11 – Pen-side test and front line diagnostic tests for ASF
Objectives
A general disadvantage of all laboratory-based methods for diagnosis of ASF (including current nucleic-acid-based detection systems) is the inevitable delay caused by the time needed for samples to reach the central reference laboratory. To overcome this problem, this WP has been focused on the development of, or adaptation to robust, rapid and portable assays for front-line diagnosis of ASF, to be used near (at the pen-side of) the suspect cases. Simplified nucleic-acid and antibody purification techniques suitable for field-testing has been evaluated and used.
Summary of results on the WP
Development of a simple and robust dot-blot assay for detection of specific ASFV antibodies
A rapid, one-step immunochromatographic strip (pen-side test) capable of specifically detecting anti-ASF antibodies in serum specimens has been developed by INGENASA in collaboration with CISA-INIA (P4). The qualitative assay is based on a direct immunochromatographic assay in which the detector reagent consisted of red carboxyl-modified latex microparticles covalently coated with VP72 purified protein. The capture reagent was the same protein adsorbed on the nitrocellulose membrane strip to form a test line. In addition, blue latex microparticles coated with a control protein and a second line created above the test line, by the immobilization of anti-control protein antibodies, was used as a control of test
The diagnostic specificity was established in 99,9% by testing a total of 1,210 ASF antibody negative field sera collecting in East and West endemic African countries and free areas of Europe.
The analytical sensitivity was determined by the analyses of 160 serum samples obtained at different days post infection from experimentally infected animals using ASFV isolates belonging to p72 genotype I and X under WP2
As a conclusion a first pen side test for ASF antibody detection have been developed and validated for rapid on site ASF detection. The test, registered and commercially available by INGENASA [INGEZIM PPA CROM R.11.PPA.K41] allows sensitivity and specific ASF antibody detection in 10 minutes and can be employed an easy single step performing method where laboratory support and skilled personnel are limited.
Adaptation and evaluation current and newly developed real-time PCR and isothermal amplification assays to portable platforms, suitable for front-line diagnosis:
a) UPL system was tested and adapted to other available portable instruments. Several alternative systems were investigated and finally two systems were tested using the UPL 162 ASFV PCR developed. These platforms have been also investigated or tested for using UPL ASFV real-time PCR. The SmartCycler is perhaps the most established real-time PCR instrument that can qualify as a portable instrument, It does not have a battery, so there is a need to connect a high voltage power source, It has 16 individual thermocyclers, so 16 assays can be run independently. Also, the specialized tubes are expensive, at 1 euro each. Several enzymes kits were tested including TaqGold, Superscript III (Platinum Taq), TaqMan fast advanced, AgPath. All kits worked fine with UPL. Because of tubes used, it is not compatible with FTA/3MM paper-in procedures. The TCOR-4 instrument from Tetracore is new on the market and as part of this project, access was provided to prototypes. The instrument itself weighs 2.5 kg and has an 8 hour battery life. It has four independent wells, runs RT-PCR and Isothermal assays and has two colours to run duplex assays. As with the SmartCycler, the issue of extraction is not addressed. The system worked with UPL and different PCR kits and the sensitivity matches other real-time instruments, such as Stratagene Mx3005. In our testing the complete platform involved the SmartCycler or TCOR-4 combined with extracting ASFV DNA from FTA cards. Given that all-in-one platforms are not yet foreseen in the near future as a practical option, platforms need to be assembled with sample preparation and detection that are compatible.
b) LAMP system: Optigene Ltd, Horsham, UK is a new company specialising in LAMP technology which recently released a new LAMP reaction kit. The new kit contains a new improved polymerase in a convenient buffer which includes everything, including an intercalating dye, making it suitable for real time detection. All one has to add are the oligos, water and target. The ASFV vp72 F3-1739 (previously F3-354) assay was developed using the LAMP kit from Eiken. Running both kits side by side using two different targets, ASFV strains Ba71v (13400 copies) and MwLil 20/1 (1000 copies) showed that the new kit from Optigene is a lot faster than the Eiken kit. The kit was further evaluated on the 14 ASFV strains provided by CISA-INIA to be used in all optimisation work within the consortium. Other results included that the linear response seems to be genotype dependent; the detection point is verified to be around 50 copies ; the F3-354/optigene system developed is faster than the TopoII/NEB system (developed at Pirbright) and slightly more sensitive.
A LAMP assay is made LFD compatible by attaching Biotin and Digoxigenin labels to the loop primers. In this way the product would be captured in the LFD device and in turn capturing the beads giving a positive signal. It is a good cheap alternative to detect LAMP products in a low cost setup. The F3-1739 LFD LAMP assay was successfully tested on a selection of ASFV strains. The LFD LAMP assay is as fast and works well on the LFD device. the LFD assay worked well on the clinical material tested and even on low copy numbers; We can summarize the ASFV vp72 F3-354 assay has been successfully transferred to a portable LAMP platform.
Evaluation of nucleic acid and antibody purification techniques and application to on site diagnosis of ASF.
The use of filter papers (FTA and 3MM) for collection and storage of virus, nucleic acid and antibodies, from the current circulating strains, were also evaluated. Sensitivity using FTA cards is enough to detect viraemic animals, mainly by real-time PCR. Interestingly, the new UPL real-time PCR developed under this project seems to be more sensitive than reference methods for ASFV detection in FTA cards. This could be a Practical system to monitor wild boars
WP 12 - Establishment of virus strain and sample bank, and selection of samples on the basis of their epidemiological and geographical relevance (to be started on Month 6)
Objectives
A collection of serum and tissue samples from domestic pigs, European wild boars, bushpigs and warthogs wildlife reservoirs has ben established in collaboration with the veterinary services of Italy and several African countries, through the connections existing through OVI-South Africa, LANADA-LCPA-Côte d’Ivoire and Kenya. This collection represents numerous variants of the new circulating virus, including serum samples.
Summary of results on the WP
A sample bank database (http://www.izsum.it/IZSUM/EN/pages/wfLoginDB.aspx(si apre in una nuova finestra)) has been built at IZS-UM to collect and organize a collection of ASFV positive and negative samples. This sample bank is a web application created to archive and to manage information on the availability and the characteristics of samples. This web application has been developed with the ASP dot net technology and has been implemented using DMS Oracle. The authentication database has been realised in Microsoft SQL Server. Basically, the database is a repository of samples stored by the users after their authentication. Each user is allowed to search and obtain data on sample details and particularly it is possible to (i) view a summary list of the samples of the whole bank (ii) access to an advanced search functionality and (iii) get all the details of a single sample.
WP 13 International standardisation and validadation of the developed diagnostic assays
Objectives
Novel robust and simple antibody detection methods and nucleic acid based assays developed under WP 8, 9, 10 and 11 have been further evaluated for their suitability for a rapid detection and differential diagnosis of suspected cases of ASF, taking into account the newly emerging viruses circulating in Africa and Sardinia.
Summary of results on the WP
An initial validation of the PCR tests developed within WP10 was carried out in the context of the ring trial on ASFV real-time PCR within the EPIZONE network.
Further a ring trial on ASFV real-time PCR (UPL-PCR and LAMP assay, both developed under the project) was organized within partners of the ASFRISK.
• UPL-PCR method was selected on the basis of its high sensitivity and specificity; its possibility for analysis also in degraded DNA samples; the possibility to use of standard protocol in all Real Time machines and its features, easy and fast to be performed.
• LAMP assay (Reaction of amplification by a strand displacement Taq in isothermal conditions, was selected on the basis of the possibility to use wet and dry assays, its high amplification efficiency and high specificity, no need different steps or temperature profiles, no need sophisticated equipments and possible use on site and in not well equipped laboratories.
TASK 3
HOST-PATHOGEN INTERACTIONS AND VIRAL IMMUNE RESPONSE IN VIEW OF THE DEVELOPMENT OF A VACCINE
The main objective in this Task is the implementation of new strategies for the development of a vaccine against African swine fever (ASF) using and further characterizing a protection model that includes the use of two naturally occurring ASFV isolates of different virulence in which the low virulent virus (ASFV/NH68) induces protective immunity against the highly virulent isolate (ASFV/L60) but with a high residual level of virulence to be used as a vaccine. Work developed under this Task aimed at:
- The characterization of viral host interactions
- The in vitro and in vivo characterization of pig immune mechanisms relevant for animal survival against ASFV.
- The construction of recombinant viruses by conventional and/or new high throughput methods
- The optimization of OprI presentation model of ASFV antigens as a new system for the production of virus antigens fused with lipoproteins
- The development of complementary methods (antivirals) for the control of the ASF.
The work was developed under five work-packages as described ahead:
WP 14 - Role of virus and host genes in infection: virus genes and cellular factors involved in the control of host defences, immune evasion and virus productivity
Objectives
Viruses have developed different strategies for their maintenance in the host. One of these strategies, used by ASFV, is to produce proteins able to evade the host immune response and thus contribute to the pathogenicity of the virus. The identification and definition of the mechanisms of virus encoded “evasion” genes which interfere with host defences and immune responses and/or affect virus productivity and virulence, will provide a rational basis for the construction and testing of attenuated, non-pathogenic, gene deletion vaccines. We propose to study the role of ASFV genes involved in the control of host defenses and immune evasion, and the characterization of host cell factors important for virus productivity. The associated understanding of virus host cell manipulation of host defense mechanisms may provide new approaches for the manipulation of cell immune responses in health and disease.
Summary of results on the WP:
ASFV genes involved in immune response
The role of the A238L viral gene in the modulation of COX-2, TNF-alpha, iNOS and IL-6 expression has been shown to be mediated through the control of their promoter activities. The mechanism involves the modulation of NF-kB, NF-AT and c-Jun transactivation by the control of CREB binding protein/p300 co-activators function. Taken together, these results have established a novel mechanism in the control of gene expression by the viral protein A238L which could represent an efficient strategy used by ASFV to evade the innate immune response. The role of another ASFV gene involved in immune response, the EP153R C-type lectin homolog, has been also studied with the use of virus deletion mutants generated in this project (SWP 18.2) demonstrating an interference of the virus gene with the expression of MHC-I in the plasma membrane, as it is shown below in the summary of work developed in SWP 16.4.
ASFV genes involved in the control of apoptosis
In this Project it has been reported the effect of several ASFV genes (A179L, which encodes a Bcl-2 homologue, A224L, which encodes a protein homologous to IAP family members, and the viral lectin encoded by EP153R gene) involved in the control of apoptosis. It has been also reported that ASFV induces apoptosis in the infected cell in a post binding step, during or after virus uncoating, through the activation of caspases. Hence, we showed that p53 expression is enhanced in Vero cells, phosphorylated and located in the nucleus from early times after ASFV infection. By the use of the virus recombinants generated in SWP 18.2 we have also been able to further confirm that the deletion of genes like A224L and EP153R resulted in an increase of the apoptotic process induced by the virus infection in porcine IPAM and WSL cells, supporting the role of these ASFV genes in the control of the programmed cell death developed into the extensive antiviral response presented by the cell after virus infection.
The expression of the IAP homologue gene was characterized along parallel in vitro infections of porcine macrophages by L60 and NHV ASFV isolates. L60 inhibited caspase-3 activation by 18 hpi and induced less DNA intranucleosomic fragmentation at 8 hpi than NHV. The transcription of IAP homologue initiates very early after infection with both isolates. However, the protein was produced later, from 4 to 8 hpi, and was more abundant in L60 than in NHV-infected cells. Overall these results suggest the existence of a post-transcriptional step in the regulation of the viral gene expression.
Factors involved in the control of cell cycle
The relevance of the modulation of Cdk2 and cyclin E expression in the ASFV DNA replication and protein synthesis has been confirmed. We have also identified two new targets for the viral evasion strategy: one is a cellular factor (eIF4E) whose depletion with siRNA blocked the ASFV infection, and the other is a viral gene (g5R) with a possible activity on decapping RNAs during virus infection. Besides, the involvement of AKT phosphorylation cascades and specific cell kinases (like PI3K) in the productive ASFV infection, has been also reported, revealing new viral genes and host factors candidates for antiviral approaches
WP 15 - Genome sequencing of viral isolates used in protective models and deletion of ASFV virulence genes
Objectives
The genome sequences of the virus model strains NHV and L60 in conjunction with the sequence analyses of a number of field ASFV isolates (Task 1), will provide insight into ASFV evolution and will identify further proteins or sequence elements highly conserved (or variable), providing important information on the possible relevance of gene functions for ASFV biology. In addition, the determination of the complete sequence of strains NHV/L60 is needed for the design of virus mutants to be generated and analyzed in other WPs (14, 16 and 17). Comparative genomics will also give valuable information on the genetic divergence of ASFV, important for the development of sensitive diagnostic techniques with high specificity (Tasks 1 and 2). Another objective of this task is the deletion of factors that determine the virulence of ASFV in the model system L60 (highly virulent)/NHV (attenuated) strains. We will generate mutant viruses with deletions on genes putatively involved in virulence, previously described in other systems or analyzed in this Project for their ability to modulate the cell and host response to ASFV infection. Selected mutations will be used as attenuated models for ASFV vaccination in WP17
Summary of results on the WP
ASFV genome sequencing
In the first periodic report it was reported the determination of the sequence of a number of ASFV genes needed to develop the virus recombinants proposed in SWP 18.2 and SWP 15.2. Results regarding several ASFV genes (A179L, A224L, A238L, A276R, E248R, G5R, I329L, I268L, K205R, and L356L) showed no significant differences within the NHV and L60 encoded proteins. A new strategy for sequencing was proposed by optimizing the 454 genome sequencing technology, significantly less laborious than traditional methods. At the end of the second period, we have obtained the full genome sequences (with exception of the terminal repeats) for both NHV and L60. This information has been used to compare the sequences of several ASFV vaccine strains currently in use (NHV, OURT88, E75,...). The analyses showed so far that the 172.050 kbp NHV genome is to 99 % identical to the OURT88/3 isolate whereas the 181185 kbp L60 genome is to nearly 100 % identical to the ASFV strain E75.
Deletion of ASFV virulence genes
The objective of this SWP was the deletion of ASFV genes involved in virulence in the model system L60 / NHV strains. During the development of the Project we have reported the completion of the construction of EP153R-deleted ASFV mutants from L60, NHV and E70 isolates, and also the generation of A238L and A224L-deleted virus mutants from both L60 and NHV strains. Several of them have been used in the analysis of the role of these genes in the ASFV infection (WPs 14, 16 and 17). Preparations of the NHV/L60 mutants were available around month 24, and the evaluation of virulence was analyzed in the in vivo protective experiment with the deleted ASFV recombinants (attenuated model viruses) at the end of the project (SWP 17.1).
WP 16 - Host in vitro responses to infection
Objectives
It was proposed in this WP to analyse the infection of sensitive cells with deletion mutants and their corresponding parental strains NHV and L60, with the evaluation of cytokines, chemokines, inflammatory molecules and SLA-I expression, which help in the definition of in vitro indicators of pathogenesis and protection. A particular interest is proposed in A238L or EP153R virus genes, candidates for ASFV models in protective experiments.
The impact of ASFV “unassigned” novel genes on host gene transcription was also proposed to be investigated in IPAM macrophages infected with lentivirus vectors. These investigations will help to define whether these ASFV-encoded proteins have completely or partially overlapping functions, thus prioritizing candidates for construction of deletion mutant attenuated virus vaccines
Summary of results on the WP
Modulation of specific cytokines, chemokines and inflammatory molecules induced during virus infection and regulation of IFNα expression and production
Preliminary studies (by qRT-PCR and Elisa) suggested an increased expression at mRNA levels of IFNβ and of the factors involved in the IFN pathway namely Jak1, Tyk2 and STAT1, in NHV infection when compared to L60 at early stages of infection. Further analysis of Real Time PCR (qRT-PCR) of mRNA quantification of relevant cytokines (IFNα, IFNβ, TNFα, IL12p40, TGFβ), chemokines (RANTES, MCP1, MIP1α), IFN relating factors (IRF3, IRF7, IRF9), signal transducers (STAT1, STAT2) and kinases (Tyk2 and Jak1) was pursued in macrophages infected with the highly virulent ASFV/L60. Results obtained as the ratio between levels of mRNA expression between L60 infected and control (non-infected) macrophages revealed an increase of the mRNA expression levels of IFNβ (2 hours after infection), TNFα, chemokines and Tyk2 (from 4 to 6 hours after infection), IFNα and signal transducers (STAT1 and STAT2) (6 hours after infection). In opposition, mRNA expression levels of IL12p40, potentially involved in the induction of cellular immune responses seem to be reduced in L60 in vitro infection.
In order to get insights on the regulation of interferon and inflammatory cytokines by the above mentioned ASFV isolates (NHV and L60) porcine macrophages were infected in vitro and the kinetics of the expression of 9 unassigned ASFV genes implicated in the regulation of the interferon response (I329L, A276R, K205R, DP146L, A528R, DP148R, J268L, E248R and P1192R) as well as of the A238L gene (IkappaB homolog) was evaluated. In addition, the expression of IFN-alpha and IFN-beta was also determined in the same cultures. In general, a similar pattern of expression was observed for the unassigned viral genes, with increased expression from 12h onwards, in almost all cases with higher levels for NHV than for L60.
Study of the role of A238L gene on the regulation of different pro-inflammatory molecules
Above mentioned results regarding the role of the A238L viral gene show that the modulation of COX-2, TNF-alpha, iNOS and IL-6 expression is mediated through the control of their promoter activities. The mechanism of inhibition has been further analyzed and shown to involve the modulation of the transactivation of several cell transcription factors (like NF-kB or NF-AT) by the control of CREB binding protein/p300 coactivators function.
The effect of the deletion of this viral gene from E70 isolate in the in vivo modulation of cytokine m-RNA expression was also studied during this Project, and confirmed in the virus model NHV during the second period of the project by CSIC
Study of the role of EP153R gene on the modulation of SLA expression and modulation of NK cell cytotoxicity
The modelization of the 3D structure of the viral EP153R C-type lectin homologue protein was presented in the first period of this Project, together with the modulation of the expression of MHC-I molecules in the plasma membrane after transfection of the ASFV EP153R gene into different model systems. These studies were extended to the mechanism of interference of EP153R with the exocytosis of the class I antigens to the plasma membrane, and to the determination of the expected inhibitory effect of the viral gene during ASFV infection, by the use of the virus recombinants with specific genes deleted from NHV and L60 isolates generated as described above. It was also demonstrated the importance of the integrity of the lectin-C domain and the R133 residue to establish the inhibition of MHC-I expression. This new role reported for the ASFV lectin EP153R provides a new mechanism for ASFV evasion of host antiviral responses.
The EP153R gene did not modulate the activity of NK cells when transfected in in vitro assays.
Impact of ASFV “unassigned” novel genes on host gene transcription
The analysis of the possible role of a number of unassigned ASFV genes with inhibitory activities on IFN responses, presented in the first period, has been extended and updated in the second period. A total of 7 genes were found to inhibit IFN responses in the luciferase assays, with four of these genes (A276R, K205R, DP148R and I329L) promoted as as best candidates for construction of attenuated ASFV vaccines by specific gene deletion. As a general conclusion the non-assigned, non-homologous virus genes so far analyzed seemed to be a repository of host evasion viral genes, to be used into the multiple strategies evolved by the ASFV to manipulate the IFN responses.
WP 17- Host in vivo responses to infection
Objectives
The main goal proposed is to evaluate how the deletions of specific genes from the genome of NHV isolate will affect the pathogenesis of the disease in pigs. The information available was completed with the results obtained in WPs 14, 15 and 16, and used to select the best ASFV genes candidates to be manipulated in the NHV isolate (WP 18), in order to induce a protective response in swine. As optimization of antigen presentation by pig macrophages could be relevant to improve the response in ASFV immunization, we will also study the OPRi based expression system to assay different viral proteins expressed as fusion lipoproteins.
Summary of results on the WP
Attenuated models for ASFV vaccination
As indicated in the initial proposal, and considering the information obtained throughout the three years of the project regarding the role of different ASFV genes in virus infection and pathogenicity, we have finally selected 3 virus genes whose specific deletion should result in a possibly more attenuated infection, to use them in experiments of ASFV vaccination. The ASFV genes selected (A238L, A224L and EP153R) were deleted from the NHV strain of ASFV by conventional methods based on transfection-infection and homologous recombination. After the selection, purification and amplification of the virus clones, stocks were then grown and used in an in vivo protective experiment in swine, which confirmed the ability of the new attenuated NHV virus models to induce a protective response against virulent ASFV L60 isolate. A limited extension of the experiment, performed with a second challenge with a heterologous virulent Arm07 strain (genotype II), indicated that the NHV isolate was also able to further protect against the second virulent infection, and that not all of the selected ASFV genes must be manipulated by deletion (but instead by overexpression, as in the case of A238L) to obtain an attenuated strain.
Optimization of OPRi presentation of ASFV antigens
The objective in this SWP is the development and characterization of a new system for the production of antigens in fusion with the OprI lipoprotein, a TLR ligand. This new system allows higher stability of recombinant plasmids and enables easy purification of fusion proteins by affinity chromatography. Results presented on the expression of a heterologous fused antigen, the ASFV ORF A104R, and the extension of these studies to obtain the cloned antigens in 3 different immunogenic formulations, showed that all of them have been characterized using the model antigen ovalbumin through in vivo and in vitro studies in a mouse model and in vitro studies in the swine model, and demonstrated adjuvant capacity. A number of ASFV antigens (including A104R, K205R, A528R, E248R, DP148R, J268L, A276R, DP146L and I329L), have been cloned in this system, opening the possibility of their future use to study the immune response against the fused antigens and the recognition of ASFV proteins by leukocytes from pigs surviving infection with attenuated isolates.
WP 18 - Improving conventional and developing new high throughput methods to facilitate genome manipulation and vaccine development
Objectives
Two approaches were proposed to facilitate the ASFV genome manipulation needed for the construction of virus recombinants described in this Project. The “conventional” approach requires the use of cell-adapted virus strains, although many ASFV field isolates are expected to infect directly either COS-1 or IPAM macrophage-derived cell lines. The manipulation is performed by transfection of engineered plasmids with specific inactivated virus genes into cells infected by the parental virus, and selection of recombinant viruses (generated by homologous recombination) by expression of reporter genes, in successive rounds of plaque purification.
A novel approach, which involves the construction of an infectious ASFV clone in E. coli, was also proposed in this Project. Successful cloning of large DNA virus genomes as bacterial artificial chromosomes (BACs) was achieved in the last years, and it was expected that the availability of an ASFV-BAC would significantly improve efficacy of ASFV genome manipulation for functional analysis of genes involved in immune evasion and pathogenicity.
Summary of results on the WP
Growth and plaque assay of ASFV isolates in established lines and conventional deletion of specific ASFV genes
In this Project we have performed the studies regarding the optimization of growth and plaque assays of ASFV isolates in established cell lines and the conventional deletion of specific ASFV genes. All of them have been confirmed regarding the inactivation of the corresponding genes (by PCR and RT-PCR in different cell lines) and their effective purification. These tools supported the works performed during the second period regarding the production of virus models to WPs 15 and 18, the purification of new virus antigens for ASFV diagnosis (WP9) and the analysis of the role of these genes in the evasion of antiviral responses, both in vitro and in vivo (WPs 14, 16 and 17). As a resume, the following ASFV recombinants were constructed for the Consortium in this project: NHV deleted in A238L, A224L and EP153R, L60 deleted in A238L, A224L and EP153R, and E70 deleted in A238L and EP153R.
Cloning of ASFV genome into a BAC (E. coli) and engineering of virus genes
To facilitate genetic engineering of ASFV, it was envisaged to clone the viral DNA genome as a BAC in E. coli and to generate infectious virus. After all the preliminary steps, only one colony contained a BAC in which the entire ASFV genome with exception of the terminal repeats (TRs) was present. Purified BAC-DNA was transfected into WSL cells which were then super-infected with wt virus. Unfortunately, the rescued virus finally proved to be a recombinant of sequences contained in the BAC with the wt virus genome. Since the TR sequences of the ASFV genome were missing in all the other clones, it appears reasonable to assume that they are not stabile in E. coli. Consequently, generation of ASFV mutants using BAC technology failed. Nevertheless, work in this SWP resulted in development of a novel methodology to create ASFV recombinants with deletions in the viral TK-locus which will be used to generate virus mutants that express heterologous genes and thus will be beneficial for future vaccine developments.
WP 19 - Development of antiviral molecules
Objectives
The objective of this WP was the evaluation of antiviral molecules applicable for the preventive protection of domestic pigs, including:
1) In vitro evaluation of toxicity and antiviral properties of molecules.
2) In vitro evaluation of the antiviral interaction with the viral replication cycle.
3) In vitro evaluation of the mechanism of action and detection of resistance to the antiviral.
4) In vivo evaluation of the toxicity of the antiviral candidate.
5) In vivo evaluation of the antiviral activity of the candidate.
Summary of results on the WP
Screening of potential molecules based on their antiviral activity/toxicity ratio and in vitro evaluation of the interference of antiviral molecules with the viral replication cycle, the mechanism of antiviral actions and the possible appearance of resistance in response to antiviral treatment
The efficiency of three types of antiviral molecules (chemicals compounds, siRNA and gallate esters) to inhibit the production of ASFV has been evaluated during the first period.
For antivirals of type 1 (chemical compounds), selected classes of nucleoside and nucleotide analogues, with inhibitory activity against DNA and retroviruses, were evaluated. The most selective compounds were HPMPO-DAPy, HPMPA and most of HPMPA derivates, with active concentration below 0.8μg/ml. Antivirals were active in the first six first hpi, in concordance with the previously described inhibitory activity of certain nucleotide analogues.
Antivirals type 2 are siRNAs specific against several ASFV genes (A104R, A118R, A204L, A151R and B646L (vp72)). After screening of about 25 siRNAs on the BA71V virus model in Vero cells, only 4 of them were active in virus inhibition, 3 directed against A151R gene and 1 specific for vp72. The inhibition obtained ranged from 3 to 4 Log TCID50. The 4 active siRNAs were synthesised as shRNAs and cloned in the pENT/U6 expression vector.
Results on antivirals type 3 (gallate esters) have demonstrated the efficiency of the selected drug (LG, lauryl gallate) to inhibit the production of several DNA and RNA viruses. Up to 7 ASFV isolates (E70, Brazil81, Uganda vir., Malawi82, Mozam86, NHV, Ba71V) have been proved in 3 different virus-sensitive cell lines to be inhibited by concentrations of LG that were far from producing cytotoxic effects on the host cell. No extracellular infective virus was produced in the presence of the drug. The inhibitory effect of LG in the cellular and viral DNA synthesis, and in the late ASFV-specific protein synthesis, has been also confirmed.
A remarkable success was obtained in all of the three approaches proposed. This is more interesting considering that their inhibitory actions might be complementary, and the reduced cytotoxicity observed in all the cases.
In vivo evaluation of the toxicity and antiviral activity of the candidate molecules
The toxicity of the antiviral molecules was evaluated in vivo in mice and/or domestic pigs. One of the chemical antivirals, the HPMPDAP, has been further evaluated in vivo for toxicity and pharmacokinetics in domestic pigs. Toxicity has been observed at i.m. injection of 20, 5 and 2,5 mg/kg/day, but no more observed with a daily i.m. dose of 0,5 mg/Kg/day during 15 days. Regarding toxicity of LG, no toxicity was found in mice associated to the treatment with LG at dose up to 10 mM (1000X the therapeutical dose of in vitro experiments). In domestic pigs, no toxicity was observed in case of administration of 2 mg LG orally and gastrically (1 mg) 3 times a week during 3 weeks. Similarly, there was no evidence of toxicity in mice when siRNA were administrated intramucosally at 40 µg/mice during 3 days or in goats after intraveinous administration of 12 mg of liposome encapsulated siRNA/goat during 3 days.
The effectiveness of the antiviral molecules LG and HPMPDAP was evaluated in the course of infection with ASFV and with respect to physiological parameters, clinical signs and body weight gain. As a resume, LG and HPMPDAP were well tolerated and did not show any negative effect on appetite, sensorium, gait and enteric function. However, regarding the antiviral activity of both the molecules, no significant differences were observed between the animal groups.
TASK 4
TRAINING AND TECHNOLOGY TRANSFER
The work was developed under two work-packages as described ahead:
WP 20 Local training/workshops and technology transfer
Objectives
The objectives of this WP were to transfer knowledge on relevant aspects of ASF and tools developed within the project in the field of epidemiology, risk assessment and laboratory diagnostics (namely to the African and Asian partners) and to set up new disease control strategies locally through trainings and workshops. Trainings and workshops were organized in African countries, based on institutions involved in the project, namely at LANADA in Ivory Coast, targeting the participation of Western African countries, at OVI in South Africa targeting the participation of Southern African countries and at Entebe, Uganda (initially planned to be organized at ILRI, Kenya) targeting Eastern African countries. As also further anticipated by the consortium, a training action-workshop was developed in China (LVRI-Lanzhou) targeting students and collaborators of LVRI and representatives from Chinese Veterinary Laboratories and in Madrid for East-European epidemiologist and laboratory diagnosticians. At last, several short-term individual trainings were organized for Chinese and Vietnamese fellows.
TRAINING COURSES
The first training course on “Training Course on African swine fever (ASF) diagnostic techniques”, was initially planned to be held in Kenya, was organized in Entebe, Uganda at the Ministry of Agriculture Animal Industry and Fisheries (MAIF) from 15 to 24th of September 2008. The main purpose of the course was to transfer the ASF diagnostic tools available at CISA as EU reference laboratory for ASF to 25 participants from Department of Veterinary Services (DVS) and National systems staff from Kenya, Uganda and Tanzania with an opportunity to improve and reinforce their diagnostic technology and control measures for ASF. This course also aimed to create a good coordination and cooperation for ASF disease control measures in Eastern African countries. In addition, all participants were expected to convey the information provided in the course to other technical staff in their laboratories and to use the course information to improve ASF diagnostic testing in their home laboratories as needed. The course was organized in cooperation between Centro de Investigación en Sanidad Animal (CISA-INIA) in Madrid in collaboration with ILRI in Kenya..
The second training course on “African swine fever (ASF) Epidemiology and Diagnosis” and was held in Lanzhou Veterinary Research Institute (LVRI), Chinese Academy of Agricultural Science, at Lanzhou, Gansu in the People's Republic of China (20-24 September 2010). Thirty participants attended the course, including young scientists from Lanzhou Veterinary Research Institute, CAAS and several representatives from Veterinary Laboratories from different regions in China namely from boarding areas with the Russian Federation.
The course was organized in cooperation between LVRI, Centro de Investigación en Sanidad Animal (CISA-INIA) in Madrid, Spain and Faculdade de Medicina Veterinária (FMV-UTL) in Lisbon, Portugal.
The third and fourth training courses were held at the Laboratoire Central de Pathologie Animale /LANADA, located in Bingerville, Côte d´Ivoire. It was a combination of laboratory and epidemiology-based training. The ASF laboratory diagnosis-based training course was delivered in LANADA, at Bingerville, Ivory Coast on days 18-22nd of October 2010 with the contribution of VAR and CIRAD (Logistics). The main purpose of the laboratory-based course was to transfer the ASF diagnosis tools available at VAR and LANADA as Reference laboratories for ASF to 10 participants from Department of Veterinary Services and National systems staff from Ivory Coast, Burkina Faso, Togo, Benin and Ghana with an opportunity to improve and reinforce their diagnostic technology and control measures for ASF. This course was aimed to create a good coordination and cooperation for ASF disease control measure in West African countries. The ASF epidemiology course and expert opinion workshop were delivered at the same place from November 1 - 5, 2010 and it was organized by UCM. The main objective of the course was to provide basic information related with the epidemiology of the disease, focusing on the study of risk factors, spread and control of the disease. Some important epidemiology knowledge and tools were also introduced to the participants, like the use of surveys to collect epidemiological data from the outbreaks, the use and creation of databases, sampling methods, introduction to Geographic Information Systems (GIS), cluster analysis and risk assessment. For the epidemiology course, 10 veterinarians from the neighboring countries of West Africa (Benin, Burkina Faso, Côte d´Ivoire, Ghana and Togo) attended to the course. All of them work in animal health departments of their countries and have a daily contact with the disease.
The fifth “Training course on African swine fever (ASF) Diagnostic techniques” was held at the Agriculture Research Council in Onderstepoort (ARC-OVI), Republic of South Africa, from May 30th to June 3rd, 2011. The main purpose of this training course was to cover relevant aspects related to the disease, its epidemiology, prevention and control and eradication measures. Particular attention was dedicated to serological and virological laboratory diagnosis of ASF through theoretical and hands-on diagnostic training sessions using ASF suspicious samples. Seventeen participants, representative from 13 of the Southern African Development Community (SADC) countries, namely from Angola, Botswana , Democratic Republic of Congo, Lesotho, Malawi, Mauritius, Mozambique, Namibia, Seychelles , South Africa, Swaziland, United Republic of Tanzania, Zambia, Zimbabwe, and a young researcher from the National Institute for Veterinary Research Vietnam in Hanoi, partner of the ASFRISK project, attended the course. The course was organized in cooperation between ARC – Onderstepoort Veterinary Institute (OVI), Centro de Investigación en Sanidad Animal (CISA-INIA) in Madrid, Spain and Faculdade de Medicina Veterinária (FMV-UTL) in Lisbon, Portugal.
The sixth training course on “African swine fever (ASF) epidemiology and laboratory diagnosis (ASF EPI-LAB)” and was held in Madrid from June 27 to July 1, 2011. The course was organized in theoretical sessions and practical hands-on exercises covering relevant aspects related to the disease, its epidemiology, prevention, control and eradication and laboratory diagnosis. Twenty eight participants attended the course: thirteen specialists on epidemiology and control of diseases (from Russia, Bulgaria, Sweden, Great Britain, Portugal and Spain, participated on the ASF-Epidemiology component of the course and fifteen specialists on laboratory diagnosis of animal infectious diseases (From Russia, Belarus, Great Britain, Poland, Germany, Vietnam). During the first day of the course, all participants attended theoretical sessions and participated on discussions on general aspects of ASF (African swine fever: A State of Art and Future Trends; African Swine Fever Diagnosis: A general overview, and recent developments; Experience of ASF control in several countries). During the three following days, participants attended specific training on ASF epidemiology and ASF diagnosis. The training on Epidemiology covered hands-on practices using EPI-tools and theoretical lectures and discussions held at the Veterinary Faculty (UCM) in Madrid. The training on ASF diagnosis covered theoretical lectures, discussions and hands-on practices on serological and virological laboratory diagnosis of ASF using ASFV infected and suspicious biological samples at the Centre for Research on Animal Health (CISA-INIA), Valdeolmos, in Madrid. All participants gathered at the Vererinary Faculty (UCM) during the last day of the course. Final topics on “Control strategies of African Swine Fever; Models for Eradication” and on “Operating Duties and Main Activities of Reference Laboratories” were introduced and discussed. Final interactive and enthusiastic discussions and conclusions of the work developed on both areas of the course among trainers and trainees confirmed the success of this training action under the ASFRISK project. All the participants received a CD containing relevant information on the course.
Multimedia Course on ASF Diagnosis
A course on African swine fever diagnosis was developed by UCM and CISA-INIA in collaboration with OIE on CD format and it is available at http://sanidadanimal.info/cursos/asf/(si apre in una nuova finestra). This comprehensive course includes theoretical lessons and practical videos on clinical and laboratory diagnosis of ASF. This CD (so far edited in English, Russian and Spanish) was spread within the ASFRISK beneficiaries and also to representatives of different countries attending the different courses organized by the project, and to others through UCM, CIRAD and CISA-INIA networks of collaboration.
LABORATORY-BASED TRAINING ACTIONS
The main goal of traineeships was to transfer the ASF diagnostic knowledge and tools available at the hosting EU partners in the project, to Lanzhou Veterinary Research Institute (LVRI) in China and National Institute of Veterinary Research (NIVR) in Vietnam, towards the implementation of ASF serological, virological and molecular diagnostic techniques in both Institutions. For this purpose training was provided to collaborators of both institutions.
Seven short-term laboratory-based trainings were organized:
- One Chinese fellow (Aihong Liu) at SVA in Uppsala from 20th September – 16th October 2009
- Two Chinese fellows at CISA-INIA (Madrid) from 21st September – 28th October 2009 (Jifey Yang) and from 17 February 2011 – 13 May 2011 (Yuefeng Wang)
- One Vietnamese fellow (Dr. Bui Ngoc Anh) at CISA-INIA in Madrid on 01-28 March 2010
- One Vietnamese fellow (Dr. Dao Duy Tung) at the National Veterinary Institute in Uppsala (SVA) from 6 – 28 March 2010.
- One Vietnamese fellow (Dr. Dao Than Van) attended the course “African swine fever (ASF) epidemiology and laboratory diagnosis (ASF EPI-LAB)” held in Madrid from June 27 to July 1, 2011
- One Vietnamese fellow (Dr. Nguyen The Vinh), attended “Training course on African swine fever (ASF) Diagnostic techniques” held at the Agriculture Research Council in Onderstepoort (ARC-OVI), Republic of South Africa, from May 30th to June 3rd, 2011
WP 21 Individual long-term training
Long and medium term laboratory-based training developed under the project have contributed to the development of three PhD and four MSc degrees:
PhD thesis
- Basto, A.P. “A new cloning system based on the OprI lipoprotein for the production of bacterial cell wall-derived immunogenic formulations. PhD Thesis. Approved. Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa. October 2011
- Quembo, C. “A study of the epidemiology of African swine fever in Mozambique”. PhD Thesis, ongoing, to be submitted to the University of Pretoria. 2008-2012.
- Kouakou Kouame Valere. “ASF Epidemiological characterization on West African countries”.
PhD Thesis. Ongoing, to be submitted to UFR Biosciences de l'Université d'Abidjan-Cocody, Côte d'Ivoire.
Msc dissertations
- Annette Nigsch. “Introduction of African Swine Fever into the European Union: development of a generic model to assess disease spread within and between Member States”. As part fullfillment of the MSc Veterinary Epidemiology of the Royal Veterinary College, August 2011
- Anja Bauer. “Analyse früher Stadien während der Infektion von Zellkulturen mit dem Virus der afrikanischen Schweinepest (Analysis of early stages of the infection of cultured cells with African Swine fever virus). MSc thesis approved, 2010-09-16. University of Rostock, Faculty of Medicine/ Medical Biotechnology.
- Lina Mur Gil. Evaluación cuantitativa del riesgo de introducción del virus de la Peste Porcina Africana en la Unión Europea por importación legal de porcinos vivos y sus productos cárnicos derivados. MSc Thesis, approved Universidad Complutense de Madrid June 2010.
- Ricardo Gomez-Soute. “Pathogenicity of an African swine fever virus isolated from domestic pigs in Mozambique”. MSc dissertation, ongoing. 2009-2012
Potential Impact:
African Swine Fever (ASF) is the most complex and economically devastating viral disease of domestic pigs producing great socio-economic impact in affected countries. It is caused by a complex virus, the only member of the Asfarviridae family, with different virulence properties causing hyperacute, subacute, chronic and unapparent forms of disease. It is maintained in nature under different and complex epidemiological scenarios involving domestic and wild swine and arthropod vectors (soft ticks Ornithodoros sp). Due to the fact that no vaccine has been obtained so far, prevention control and eradication of the disease are solely based on the implementation of strict sanitary measures.
The disease in EU member states is currently confined to Italy (Sardinia). It was recently introduced to Caucasian countries and Russia and it is highly prevalent in sub-Saharan African countries.
Serological surveys during outbreaks occurred previously in Eastern and Southern African regions have shown that although ASFV prevalence in the domestic pigs is high, in many cases serum conversion was not detected. These events pose a great challenge for the prevention and control of ASF, not only locally, but also worldwide. The complex epidemiological scenarios of ASF, the increasing mobility of people, animals and goods across the globe, emphasize the serious menace the disease presents to the growing pig farming sector in Africa and to currently ASF-free EU member states permanently threatened by the occurrence of the disease in different geographical regions.
In order to improve tools and strategies for the prevention and control of ASF, the work developed under the ASFRISK project aimed at fulfilling four objectives contained in the Major Tasks of the work-plan:
i) To improve and further develop the epidemiological, risk assessment and control measures of African swine fever (ASF),
ii) To develop and provide new and robust tests for laboratory and front-line diagnosis, through the achievement, standardization and validation of more rapid, sensitive and inexpensive serological and molecular procedures,
iii) To establish new strategies for vaccine development against ASF and,
iv) To transfer knowledge on relevant aspects of ASF and improved tools for ASF diagnosis and epidemiological modeling and risk assessment strategies by providing an harmonized international training, in order to prevent and combat this highly devastating, OIE-listed transboundary animal disease.
The four main tasks of the project were outlined and harmonized in the work-plan providing a logical process for the improvement of knowledge on critical aspects of ASF and development of tools towards the achievement of the objectives through the contribution of highly qualified partners from EU, Africa and Asia, with expertise in all the areas concerned.
Several outcomes of the project emphasize its successful contribution for “Evaluating and controlling the risk of ASF in the EU”:
- Development of epidemiological methods and decision support tools provide information to policy makers on ASF epidemiological situation and will assist in the prioritization of strategies to better prevent and control ASF in a potential incursion into the EU and in all currently affected territories.
- Development of a range of valuable serological and molecular diagnostic tools suitable for use in well-equipped international and national reference laboratories, in basic regional and local laboratories, or even for rapid first line on-site application. New tests and reinforcement of currently available ones will promote more efficient and earlier diagnosis of the disease.
- Characterization of several ASFV-host interactions at cellular and molecular levels and the production of different attenuated recombinant virus strains that may be used as future candidates for a vaccine, open new insights on the development of control strategies against ASF.
- Transfer of knowledge on relevant aspects of ASF and transfer of diagnostic and epidemiologic tools through training courses and traineeships to a large number of professionals from Animal Health Institutions in EU, Africa, Asia, Russia and Caucasian countries, enhance preparedness capacity against the threat of the disease in different scenarios.
The project provided employment, education and training for young post-doctoral and post-graduate researchers. The employed researchers and assistants gained intra and inter-disciplinary experience and training by working within an international, multidisciplinary team and, importantly, experience of working with industry. In addition, young researchers were trained in the presentation of results and the compilation of manuscripts for publication. This experience and training contribute to both consolidating the EU scientific infrastructure of young scientists and reducing the drain of young scientists elsewhere.
The proposed objectives and deliverables of this project provide essential information for prevention and control of ASF, a devastating disease causing acute welfare problems in Africa and in Sardinia and more recently in Caucasian countries and Russia.
By doing so, the outcomes of the project will benefit EU consumer driven concerns with regard to animal welfare, food safety and food quality and will undoubtedly contribute to the sustained development of EU agri-food business by improving prevention, early detection and control of ASF and therefore reducing the socio-economic consequences of potential incursions of the disease from infected regions. In addition, assistance in the preservation of small to medium sized family farms, as viable economic units within a rural environment will contribute to maintaining rural infrastructure of EU regions and member states
Main Dissemination activities and exploitation of results
Dissemination and exploitation of the results considered by the consortium to be a vital component of the proposed work program was fulfilled through internal and external dissemination activities:
1. Internal dissemination
The consortium was composed by a multidisciplinary scientific team based on thirteen European partners from eight countries: FMV- UTL and FCG-IGC from Portugal, UCM, CISA-INIA, CSIC and INGENASA (a SME) from Spain; CIRAD from France; IZS-UM from Italy; VAR from Belgium; FLI from Germany; QUB and RVC from United Kingdom; SVA from Sweden and four partners from ICPC countries: OVI from South Africa; LANADA-LCPCA from Ivory Coast; LVRI from Popular Republic of China and NIVR from Vietnam. The ability of partners to successfully complete their program within the general context of the project was supported by their relevant scientific expertise in different fields including epidemiology and risk assessment, serological and nucleic acid based diagnostics, molecular biology, genetics, immunology, virology and infectious pathology.
Internal dissemination aiming at scientific and technological exchange between partners was achieved through the organization of project meetings, laboratory-based training actions (traineeships) and short exchange visits of collaborators from different partners.
Collaborations developed among partners at different levels during the lifelong of the project consolidated and enhanced knowledge on basic and applied science of the international scientific community working on ASF.
2. External dissemination:
External dissemination of research results was achieved through a series of activities:
- Publications in scientific peer reviewed journals
- Presentations in conferences, workshops, scientific meetings
- Phd thesis and MSc dissertations
- Transfer of knowledge and technology trough the organization of training courses and traineeships
- Co-organization of the “VIII Simpósio da Sociedade Científica de Suinicultura “Peste Suína Africana”
- Organization of the “ASFRISK Symposium”
- Exploitation of results
- Website (www.asfrisk.eu)
2.1 Publications in peer-reviewed scientific journals
So far the following scientific papers have been published:
1. Basto AP, Piedade J, Ramalho R, Alves S, Soares H, Cornelis P, Martins C, Leitão A. A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations. J Biotechnol. 2011, 157 (1):50-63.
2. Carrascosa, AL, Bustos MJ, de Leon, P. Methods for growing and titrating African swine fever virus field and laboratory virus samples. Current Protocols in Cell Biology. 2011. Chapter21.Unit 26.14.
3. Fasina FO, Shamaki D, Makinde AA, Lombin LH, Lazarus DD, Rufai SA, Adamu SS, Agom D, Pelayo V, Soler A, Simón A, Adedeji AJ, Yakubu MB, Mantip S, Benshak AJ, Okeke I, Anagor P, Mandeng DC, Akanbi BO, Ajibade AA, Faramade I, Kazeem MM, Enurah LU, Bishop R, Anchuelo R, Martin JH, Gallardo C. Surveillance for African swine fever in Nigeria, 2006-2009. Transbound Emerg Dis. 2010 Aug 1;57(4):244-53.
4. Fernández-Pinero, J, Gallardo, C, Elizalde, M, Robles, A, Gómez, C, Bishop, R, Heath, L, Couacy-Hymann, E. Fasina, FO, Pelayo, V, Soler, A, Arias, M. Molecular diagnosis of African swine fever (ASF) by a new real-time PCR using Universal Probe Library (UPL). Transbound Emerg Dis . 2012. In press
5. Gallardo C, Ademun AR, Nieto R, Nantima N, Arias M, Martín E, Pelayo V and Bishop RP. “Genotyping of African swine fever virus (ASFV) isolates associated with disease outbreaks in Uganda in 2007”. African Journal of Biotechnology (AJB). April 2011; 10 (17): pp. 3488-3497
6. Gallardo C, Anchuelo R, Pelayo V, Poudevigne F, Leon T, Nzoussi J, Bishop R, Pérez C, Soler A, Nieto R, Martín H, Arias M. African swine fever virus p72 genotype IX in domestic pigs, Congo, 2009. Emerg Infect Dis. 2011. Aug; 17(8):1556-8.
7. Gallardo C, Okoth E, Pelayo V, Anchuelo R, Martín E, Simón A, Llorente A, Nieto R, Soler A, Martín R, Arias M, Bishop RP. African swine fever viruses with two different genotypes, both of which occur in domestic pigs, are associated with ticks and adult warthogs, respectively, at a single geographical site. J Gen Virol. 2011 Feb; 92(Pt 2):432-44.
8. Gallardo C, Reis AL, Kalema-Zikusoka G, Malta J, Soler A, Blanco E, Parkhouse RM, Leitão A. Recombinant antigen targets for serodiagnosis of African swine fever. Clin Vaccine Immunol. 2009 Jul; 16(7):1012-20.
9. Giammarioli M, Gallardo C, Oggiano A, Iscaro C, Nieto R, Pellegrini C, Dei Giudici S, Arias M, De Mia GM. Genetic characterisation of African swine fever viruses from recent and historical outbreaks in Sardinia (1978-2009). Virus Genes. 2011 Jun; 42(3):377-87.
10. Hurtado C, Bustos MJ, Carrascosa AL. The use of COS-1 cells for studies of field and laboratory African swine fever virus samples. J Virol Methods. 2010 Mar; 164(1-2):131-4.
11. Hurtado C, Bustos MJ, Granja AG, de León P, Sabina P, López-Viñas E, Gómez-Puertas P, Revilla Y, Carrascosa AL. The African swine fever virus lectin EP153R modulates the surface membrane expression of MHC class I antigens. Arch Virol. 2011 Feb; 156(2):219-34.
12. Keita D, Heath L, Albina E. Control of African swine fever virus replication by small interfering RNA targeting the A151R and VP72 genes. Antivir Ther. 2010; 15(5):727-36.
13. Martínez, M., Martínez-López, B., Muñoz, M.J. de la Torre, A., Mur, L., Sánchez-Vizcaíno, JM., 2009. Expansión de peste porcina africana en el mundo: Riesgo de entrada en España. Gaceta Sanitaria. Vol.23 Especial Congreso 3, 28.
14. McKillen J, McMenamy M, Hjertner B, McNeilly F, Uttenthal A, Gallardo C, Adair B, Allan G. Sensitive detection of African swine fever virus using real-time PCR with a 5' conjugated minor groove binder probe. J Virol Methods. 2010 Sep; 168(1-2):141-6.
15. Mur L, Martínez-López B, Martínez-Avilés M, Costard S, Wieland B, Pfeiffer DU, Sánchez-Vizcaíno JM. Quantitative Risk Assessment for the Introduction of African Swine Fever Virus into the European Union by Legal Import of Live Pigs. Transbound Emerg Dis. 2011 Aug 10.
doi: 10.1111/j.1865-1682.2011.01253.x.
16. Mur L., Martínez-López B., Gallardo C., Gortazar C. and Sánchez-Vizcaíno JM. Monitoring of African Swine Fever in the Wild Boar Population of the Most Recent Endemic Area of Spain. Transboundary and Emerging Diseases. In Press. doi: 10.1111/j.1865-1682.2012.01308.
17. Portugal R, Leitão A, Martins C. Apoptosis in porcine macrophages infected in vitro with African swine fever virus (ASFV) strains with different virulence. Arch Virol. 2009;154 (9):1441-50.
18. Portugal R, Leitão A, Martins C. Characterization of African swine fever virus IAP homologue expression in porcine macrophages infected with different virulence isolates. Vet Microbiol. 2009 Oct 20; 139(1-2):140-6.
19. Ronish B, Hakhverdyan M, Ståhl K, Gallardo C, Fernandez-Pinero J, Belák S, LeBlanc N, Wangh L. Linear-After-The-Exponential PCR (LATE-PCR) assay for the detection of African swine fever virus”. J Virol Methods. 2011 Mar; 172(1-2):8-15.
20. Tignon M, Gallardo C, Iscaro C, Hutet E, Van der Stede Y, Kolbasov D, De Mia GM, Le Potier MF, Bishop RP, Arias M, Koenen F. Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus. J Virol Methods. Sep 17. 2011 Dec;178 (1-2):161-70
21. Sánchez-Vizcaíno JM., Mur L., and Martínez-López B. African Swine Fever: An Epidemiological Update. Transboundary and Emerging Diseases. 2012 Jan 9. doi: 10.1111/j.1865-1682.2011.01293.x. [Epub ahead of print]
2.2 Presentations at scientific meetings, conferences, workshops
Around one hundred presentations were introduced at different scientific meetings, conferences, workshops and others and they are shown in the website www.asfrisk.eu (Dissemination)
2.3 PhD thesis and MSc dissertations under the project
Long and medium term laboratory-based training developed under the project have contributed to the development of three PhD and four MSc degrees:
PhD thesis
- Basto, A.P. “A new cloning system based on the OprI lipoprotein for the production of bacterial cell wall-derived immunogenic formulations. PhD Thesis. Approved. Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa. October 2011
- Quembo, C. “A study of the epidemiology of African swine fever in Mozambique”. PhD Thesis, ongoing, to be submitted to the University of Pretoria. 2008-2012.
- Kouakou Kouame Valere. “ASF Epidemiological characterization on West African countries”.
PhD Thesis. Ongoing, to be submitted to UFR Biosciences de l'Université d'Abidjan-Cocody, Côte d'Ivoire.
Msc dissertations
- Annette Nigsch. “Introduction of African Swine Fever into the European Union: development of a generic model to assess disease spread within and between Member States”. As part fullfillment of the MSc Veterinary Epidemiology of the Royal Veterinary College, August 2011
- Anja Bauer. “Analyse früher Stadien während der Infektion von Zellkulturen mit dem Virus der afrikanischen Schweinepest (Analysis of early stages of the infection of cultured cells with African Swine fever virus). MSc thesis approved, 2010-09-16. University of Rostock, Faculty of Medicine/ Medical Biotechnology.
- Lina Mur Gil. Evaluación cuantitativa del riesgo de introducción del virus de la Peste Porcina Africana en la Unión Europea por importación legal de porcinos vivos y sus productos cárnicos derivados. MSc Thesis, approved Universidad Complutense de Madrid June 2010.
- Ricardo Gomez-Soute. “Pathogenicity of an African swine fever virus isolated from domestic pigs in Mozambique”. MSc dissertation, ongoing. 2009-2012
2.4 Training courses
Dissemination activities aiming at transferring knowledge on relevant aspects of ASF and transfer of tools developed within the project in the field of epidemiology, risk assessment and laboratory diagnostics was achieved through the organization of training courses and traineeships. Based on institutions involved in the project, four Training Courses were organized in Africa, one course in China and one in Spain. One Course on ASF diagnosis was launched on a CD version, available on Internet
- Multimedia Course on ASF Diagnosis
A course on African swine fever diagnosis was developed by UCM and CISA-INIA in collaboration with OIE on CD format and it is available at http://sanidadanimal.info/cursos/asf/(si apre in una nuova finestra). This comprehensive course includes theoretical lessons and practical videos on clinical and laboratory diagnosis of ASF. This CD (so far edited in English, Russian and Spanish) was spread within the ASFRISK beneficiaries and also to representatives of different countries attending the different courses organized by the project, and to others through UCM, CIRAD and CISA-INIA networks of collaboration.
- “Training Course on African swine fever (ASF) diagnostic techniques”, held in Entebe, Uganda from 15 to 24th of September 2008. Twenty five participants from Department of Veterinary Services (DVS) and National systems staff from Kenya, Uganda and Tanzania attended the course which was organized in cooperation between Centro de Investigación en Sanidad Animal (CISA-INIA) in Madrid and ILRI in Kenya at the Ministry of Agriculture Animal Industry and Fisheries (MAIF) in Uganda.
- “Course on African swine fever (ASF) Epidemiology and Diagnosis”, held at the Veterinary Research Institute (LVRI), Chinese Academy of Agricultural Science, at Lanzhou, Gansu in the People's Republic of China (20-24 September 2010). Thirty participants attended the course, including young scientists from Lanzhou Veterinary Research Institute, CAAS and several representatives from Veterinary Laboratories of different regions in China namely from boarding areas with the Russian Federation. The course was organized in cooperation between LVRI, Centro de Investigación en Sanidad Animal (CISA-INIA) in Madrid, Spain and Faculdade de Medicina Veterinária (FMV-UTL) in Lisbon, Portugal.
- “The ASF laboratory diagnosis-based training course and the The ASF epidemiology course and expert opinion workshop”, held at Laboratoire Central de Pathologie Animale /LANADA , Bingerville, Ivory Coast respectively from 18-22nd of October 2010 and November 1st - 5th , 2010). Twenty participants from the Departments of Veterinary Services, Animal Health Departments from Ivory Coast, Burkina Faso, Togo, Benin and Ghana attended these courses organized by VAR and UCM in collaboration with LANADA –LCPA.
- “Training course on African swine fever (ASF) Diagnostic techniques”, held at the Agriculture Research Council in Onderstepoort (ARC-OVI), Republic of South Africa, from May 30th to June 3rd, 2011. Seventeen participants, representative from 13 of the Southern African Development Community (SADC) countries, namely from Angola, Botswana , Democratic Republic of Congo, Lesotho, Malawi, Mauritius, Mozambique, Namibia, Seychelles , South Africa, Swaziland, United Republic of Tanzania, Zambia, Zimbabwe, and a young researcher from the National Institute for Veterinary Research Vietnam in Hanoi, partner of the ASFRISK project, attended the course. The course was organized in cooperation between ARC – Onderstepoort Veterinary Institute (OVI), Centro de Investigación en Sanidad Animal (CISA-INIA) in Madrid, Spain and Faculdade de Medicina Veterinária (FMV-UTL) in Lisbon, Portugal.
- Taining course on “African swine fever (ASF) epidemiology and laboratory diagnosis (ASF EPI-LAB)”, held in Madrid from June 27 to July 1, 2011. Twenty eight participants attended the course: thirteen specialists on epidemiology and control of diseases (from Russia, Bulgaria, Sweden, Great Britain, Portugal and Spain, participated on the ASF-Epidemiology component of the course and fifteen specialists on laboratory diagnosis of animal infectious diseases (From Russia, Belarus, Great Britain, Poland, Germany, Vietnam). The course was organized by UCM in collaboration with CISA-INIA.
- For each training course, participants received documents in informatics support (CD) and others, containing the programs followed, lectures, laboratory procedures, reference manuals and relevant bibliography.
2.5 Laboratory-based traineeships
The main goal of these trainings was to transfer the ASF diagnostic knowledge and tools available at the hosting EU partners in the project to Lanzhou Veterinary Research Institute (LVRI) in China and National Institute of Veterinary Research (NIVR) in Vietnam, providing training to collaborators of both institutions towards the implementation of ASF serological and virological diagnostic techniques in their laboratories. Five short-term laboratory-based trainings were organized to three Chinese fellows from LVI, Lanzhou and to two Vietnamese fellows At SVA (Sweeden) and CISA-INIA (Spain)
2.6 VIII Simpósio, Sociedade Científica de Suinicultura, “Peste Suína Africana”, held at the Faculdade de Medicina Veterinária, Lisbon, November 11th , 2010. Organization: Sociedade Científica de Suinicultura (SCS) in collaboration with the ASFRISK Project. This meeting targeted the associated members of SCS (pig farm veterinarians and professionals, pig farmers and associates to pig industry organizations (Federação Portuguesa de Associações de Suinicultores - FPAS ), representatives of the Portuguese Ministry of Agriculture and students. Around forty participants attended this Symposium. Major lectures were introduced by different authors covering “General aspects of the disease” (Carlos Martins, Portugal), “Epidemiology, risk assessment, control and eradication” (Jose Manuel Sanchez –Vizcaino, Madrid), “Laboratory diagnosis” (Margarida Duarte, Portugal), “ASF situation in Russia and Caucasian countries” (Denis Kolbasov, Russia), “The official veterinary services and ASF” (Pina Fonseca, Portuguese Ministry of Agriculture).
2.7 The ASFRISK Symposium
The ASFRISK Symposium, organized by the Coordinator Partner (Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa (FMV-UTL) was held in Lisbon at the Cento Cultural de Belém, at the end of the project on September 23rd, 2011.
The main objective of this meeting aimed at the dissemination of the ASFRISK achievements to representatives of international organizations (EC, FAO and OIE); Chief Veterinary Officers (Animal Health) of Member States of the European Union and Candidate and Associated Countries; representatives of Animal Health Institutions from Russia and Caucasian Countries; representatives of professional and policy maker organizations related to pig industry.
A total of 80 participants attended the meeting: 35 guests among CVOs or their representatives, EC, FAO, OIE representatives, and others from different organizations related to pig industry, Faculty members and 45 delegates of the ASFRISK project. Major lectures on general aspects relating to research on Animal Health and on critical aspects of ASF were introduced by the coordinator and guest speakers (Anne Sophie Lequarré, EC, DG RTD; Mary Louise Penrith, University of Pretoria, South Africa; Serghey Dudnikoff, Institute of Animal Health, Russia; Francisco Riviriego, EC, DG SANCO). Major achievements of the ASFRISK project were introduced by the Task Coordinators and complemented by presentations of different delegates involved in the work developed, promoting an opened discussion on different matters with the audience.
2.8 Exploitation of results
A penside immunochromatography test was developed, standardized and validated. The test, which is now commercially available through INGENASA (INGEZIM PPA-CROM), detects anti-ASFV antibodies in serum in less than 10 minutes, with 99% diagnostic sensitivity and 99.8% diagnostic specificity compared to OIE methods. Currently, this assay is being adapted for blood sample use.
A set of nucleic acid-based assays for laboratory and front-line/penside application were also developed, and included three systems based on commercial Universal Probe Library (UPL-PCR, Roche®) technology, Linear-After-The-Exponential PCR (LATE-PCR) technology, and Loop-mediated isothermal amplification (LAMP) technology, respectively, targeting highly conserved regions of the ASFV genome. A large collection of isolates was used for the evaluation, and the performance of the assays was then tested in two different European ring trials, with excellent results. The UPL- PCR and the LAMP were adapted to commercially available portable platforms, and tested in the European partner laboratories as well as in regions with endemic ASF. The assays were also transferred to ready-to-use diagnostic kit formats, and prototypes are now under evaluation for their potential upcoming launching in the market.
The use of filter papers (FTA and 3MM) for collection, storage and shipping of samples for detection of virus, nucleic acid and antibodies was evaluated and standardized. This may be a practical system to monitor wild boars, but also for sampling, storage and shipping of samples in regions with endemic ASF and poor veterinary surveillance infrastructure.
2.9 Development of the Project website
The website for the ASFRISK was released to the public on May 7th, 2009.
The address of the web page is: www.asfrisk.eu
Information is contained in Public and Internal Sections
- The Public Section is opened to general public and it contains information on the organization of the project (Summary; Objectives; Impact; Map; Management; Consortium), consortium meetings, Training activities; Dissemination (containing internal and external dissemination activities); Summary of activities; News and Links. It is updated accordingly to the progress of the Project.
- The Internal section exclusive to the use of the participants in the project contains the following Topics: Project Handbook; Official Documents; Management; Participants; Meetings; Laboratory Procedures; Publications; Links and a Bulletin Board.