African Swine Fever is a notifiable devastating hemorrhagic fever with high mortality rates in pigs. It affects all members of the Suidae family and is one of the most important pig diseases due to its severe socio-economic consequences for affected countries, the difficulty of preventing spread across country boundaries, and the lack of vaccine and therapeutic control measures. We will use genome-wide DNA technologies to understand the Sus scrofa genomic response to the infection. Specifically, we will compare data on both healthy and infected individuals to (1) identify the possible presence of regions under selection. We will also assess (2) the hybridization rate and (3) the interaction strategies between the domestic pig and the wild boar to (4) identify the possible transmission routes in pig diseases.
African Swine Fever, the Sus scrofa evolutionary genomic response
Summary - African Swine Fever is a notifiable devastating hemorrhagic fever with high mortality rates in pigs. It affects all members of the Suidae family and is one of the most important pig diseases due to its severe socio-economic consequences for affected countries, the difficulty of preventing spread across country boundaries, and the lack of vaccine and therapeutic control measures. We will use genome-wide DNA technologies to understand the Sus scrofa genomic response to the infection. Specifically, we will compare data on both healthy and infected individuals to (1) identify the possible presence of regions under selection. We will also assess (2) the hybridization rate and (3) the interaction strategies between the domestic pig and the wild boar to (4) identify the possible transmission routes in pig diseases.
Purpose of the project - This project aims at identifying the presence of regions under selection for resistance to African Swine Fever (ASF) in S. scrofa genome and the possible increase in risk of infection related to wild/domestic swine interactions. Using Genome Wide (GW) analysis we will:
Objective (O) 1a: identify the possible presence of regions under selection (resistance/sensitivity) for ASF through the comparison of infected individuals with healthy ones (exposed to the virus) and with species (e.g. Phacochoerus africanus, Potamochoerus larvatus) resistant to the virus.
O1b: investigate polymorphisms and heterozygosity levels in genes involved in the innate and adaptive immune response of the host. This sort of information would be of paramount importance for the development of new treatments and future selective processes in domestic pig (DP). Considering there is currently no vaccine against the virus causing this infection and the alarming scenarios that a diffusion in Europe could cause (Khomenko et al 2013; Nigsch et al 2013), it is of paramount importance to better understand the possible presence of resistant lines in pigs (wild or domestic) or, the other way round, the possible cause of sensitivity.
O2: analyse the genetic composition of both the domestic and wild population in Europe and use this information to evaluate the level of hybridization between the two stocks in order to assess the risks connected to the interactions between them. We think that a better evaluation of the degree of hybridization and interaction between wild boar (WB) and DP, with the possible presence of hybrid zones, would improve the understanding of the two forms population genetics and the possible pathways of infection of ASF. The understanding of the effect of the WB as a reservoir and the risk associated to the interactions with the DP would provide important information for the development of prevention and contingency plans. The evaluation of the hybridization levels and the possibility of contact between the WB and the DP population would constitute the base for the understanding of transmission patterns not only of AFS but it could be integrated in the analysis of several pig diseases, including zoonoses.
The evaluation of the degree of hybridization between the wild and the domestic form is of paramount importance for the development of proper management strategies aimed at preserving the biodiversity of both stocks. It is our opinion that the integration of all the available information in the management plans can improve their success, with effects not only on the species but on the human health and economy as well. While the understanding of the influence of the host genome on the degree of virulence of ASF virus would be provided to the organizations currently monitoring and preventing the diffusion of the virus and its effects on the animal production. This essential work is currently based only on the virus and its diffusion patterns, our analysis will integrate these fundamental information with insights on the host sensitivity. We expect our findings to produce long term benefits for genomic assessment of hybridization patterns and the comparative study of both domestic and wild species, arising the interest applying the tools developed for domestic or model species in closely related wild species that will represent a promising instrument for research education and for coping with public interest topics.
Research methods, data and approach - Two sampling schemes will applied. The first one (S1), aimed at addressing O1 will be based exclusively on DNA samples provided by the Institutes (e.g. Państwowy Instytut Weterynaryjny-Państwowy Instytut Badawczy, Poland) in charge of monitoring the presence of the virus within their territory and will include similar numbers of healthy and infected individuals coming from the same farm (for DP) or hunting area (for WB). This should ensure an high probability of exposure of controls and the epidemiological comparability of the two groups, increasing the accuracy of phenotype definition. Additionally, the handling of DNA material will avoid the risk of accidental infection and avoid the unnecessary killing of additional individuals. S2 will involve several (minimum 5) European countries with at least 20 DP and 20 WB coming from the same area, will be based on an already established network of collaborations, will include animals killed during the hunting season (WB), blood from veterinary controls or tissues collected at abattoirs (DP).
All the samples (S1+S2) will be genotyped with the Infinium PorcineSNP60 v2 BeadChip. Whole genome next generation sequencing (NGS) will be performed on a subset of meaningful individuals, results will be compared with data collected on other Suids species, within the Sus genome sequencing project leaded by the PigEvoDiv group.
The Infinium PorcineSNP60 v2 BeadChip allows the genotyping of 64,232 uniformly distributed SNPs (average spacing 43.4 Kb). This genome coverage will allow us to estimate within population diversity and genetic structure of both domestic and wild samples, as well as an empirical estimation of linkage disequilibrium (LD). The statistical analysis (allele frequency, bayesian analysis, etc) of differences between WB and DP genotypes will consent to identify hybrid individuals and to evaluate their occurrence in the two populations (O2). All the analysis will be performed with specific software (e.g. Plink, Purcell et al 2007; Admixture, Alexander et al 2009) or R packages (e.g. Adegenet, Jombat 2008; Genetics, Warnes 2013). Data on breeding strategies, present and past, and occurrence of interactions between the two forms will be specifically collected for this project through open online surveys and direct enquiries with local experts. These information will be integrated with spatial data to identify the possible presence of practices and variables that could increase the chance of contact between the WB and the DP. Hybridization is reported to be limited in EU (Scandura et al 2011), but it has been described in several EU countries (Frantz et al 2013, Goedbloed et al 2013, Canu et al 2014) and there is currently no data related to practices that may influence the degree of admixture. Additionally, spatial and human related information will be integrated in the analysis for the first time. We expect this novel approach will bring new knowledge to the admixture process between farmed and wild specimens.
At the same time, GW association analysis will be performed on the dataset to identify the presence of loci under selection (O1a) for the disease, using specific software (e.g. Bayescan, Narum & Hess 2011) and R packages (e.g. rehh, Gautier & Vitalis 2012). Genomic regions where significant results will be obtained will be further explored in the attempt of identifying candidate genes underlying the loci. Polymorphisms and heterozygosity levels in the identified regions, together with those of genes involved in the innate and adaptive immune response will be investigated (O1b) with specific software (e.g. Plink, Purcell et al 2007) or R packages (e.g. Adegenet, Jombat 2008; Genetics, Warnes 2013). This would be the first attempt to address ASF by the host perspective. Furthermore, the comparison of SNP and NGS data on different Suids species, including some resistant to the virus, will provide additional evidence on the biological and evolutive meaning of the identified regions. This last part of the research will be conducted in collaboration with the PigEvoDiv network, that has an ongoing project on the Sus genome sequencing.
Hosting institution and relevance to area of research - The work will be based at the Section of Biology and Environmental Science (SBES) of Aalborg University (AAU) under supervision of Prof. Cino Pertoldi, who is my PhD supervisor. AAU has all the facilities, tools and staff for genomic research required by the project. AAU is currently expanding its research areas ranging from basic research and applied research and is also increasing its multidisciplinary research activities and its collaboration across different research and knowledge areas. SBES staff have experience in Conservation and Evolutionary biology but also in Disease Ecology which will be of great support to the implementation of the project. Additionally the section of Biotechnology have expertise in molecular biotechnology and will provide guidance for the statistical analysis of genomic data. The collaboration with several members of AAU staff will provide the multidisciplinary supervision necessary for the success of the project in all its aspects.
Research plan and practical implementation of the project – The project falls within an ongoing research at AAU. The previously established international network of collaborations for both sampling and scientific discussion, the availability of a genotyping service (GenoSkan) and the collaboration with several members of AAU staff for data analysis and interpretation make the project feasible in one year.
Publication, research education and dissemination of results - I anticipate spreading results of this study through peer-reviewed scientific papers. In addition, I plan to spread the results and the work to scientific audience by publication and presentations in conferences and international meetings. Divulgation will be expected to involve multiple audiences, such as internet blogs, printed articles (e.g. newspaper), and oral presentations for specialized and popular audiences.
Ethical aspects - No animal will be killed specifically for this project, infected samples and controls will be collected, according to local legislation, by Institutions in charge of monitoring the virus presence. To minimise any chance of possible accidental diffusion of the virus, we will ask those Institutions to provide us with DNA samples, thus avoiding any risk for both animals and human operators. Samples for the hybridization study will be collected during regular slaughter activities and hunting season, according to local legislation. For this reason we don't believe the research will arise ethical issues.