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Towards rational design of vaccines against African swine fever in East- and Southern Africa: Correlation of viral genome differences with their impact virulence and by analysis of viral and target cell transcription and protein expression. (AVSOA)
Project
Project code: 2815DOKP21, FLI-IMVZ+IVD-08-Ri-0492
Contract period: 01.06.2016
- 31.10.2019
Budget: 94,628 Euro
Purpose of research: Applied research
In many African countries the importance of pig breeding for nutrition and economy has increased substantially during the last decades. However, this development is inhibited by various animal diseases, among which African swine fever (ASF) is particularly problematic. While the infection is usually fatal in domestic pigs, the soft tick-borne African swine fever virus (ASFV) causes only mild symptoms in wart hogs and bush pigs, and is therefore widely distributed in these species, and transmitted to their domestic cognates. Although laboratory experiments suggest that vaccination against ASF should be possible in principle, development of a feasible vaccine was not successful up to now. Since ASFV isolates frequently exhibit different virulence, the complete genomes of several viruses of different genotypes, which are currently relevant in Africa, have to be compared. Representative isolates will be characterized in pigs, and the corresponding virus and host cell transcriptomes and proteomes will be analyzed using next generation sequencing and mass spectrometry. This might reveal a correlation between genetic markers (mutations leading to deletion or modified expression of viral and cellular genes) and virulence. To verify the biological relevance of the genome differences, they will be introduced by genetic engineering into closely related cell culture adapted ASFV strains. Therefore, reporter genes for fluorescent proteins (e.g. GFP) or other selectable markers will be transiently inserted into the ASFV genome, and state-of-the-art molecular biology techniques like genome modification by CRISPR/Cas (clustered regularly interspaced short palindromic repeats/Cas9 nuclease) will be applied. As soon as isogenic groups of virulent parental ASF viruses and recombinants with potential virulence gene mutations are available, their replication in vitro and their pathogenicity in vivo will be comparatively investigated. As far as possible, protective efficacy will be evaluated by subsequent challenge infections with virulent ASFV. The studies will include quantification of clinical symptoms and mortalities, pathological, virological and serological parameters (viral load in blood and organs, antibody production, leucocyte differentiation, cytokine and chemokine induction).
The project focused on a virulent ASFV isolate from Kenya (genotype IX), which could be easily adapted to efficient growth in a wild boar lung cell line. Next generation sequence analyses of the parental virus, and different cell culture passages revealed no significant genome alterations. Based on virulence studies and proteome analyses of other ASFV strains, it was attempted to delete known pathogenicity factors, and abundantly expressed virus genes from ASFV Kenya using improved reverse genetics methods. Whereas deletion of the gene encoding viral ribonucleotide reductase, membrane protein p12 and pB119L (9GL) did not result in replication competent virus mutants, the viral thymidine kinase- and dUTPase genes, as well as the abundantly expressed K145R und A104R genes could be removed successfully. Animal experiments will show, whether these mutations can contribute to development of attenuated live virus vaccines. In an alternative approach WSL cell lines stably expressing the CRISPR/Cas9 system with specific guide RNAs against ASFV Kenya. Since virus replication was severly inhibited in these cells, it will be evaluated whether transgenic, ASFV resistant swine can be generated in the same way.
Section overview
Subjects
- Animal Husbandry
- Animal health
- Wildlife Biology
- Biotechnology
Framework programme
Funding programme
Excutive institution
FLI - Institute of Molecular Virology and Cell Biology (FLI - IMVZ)
