Kurzbeschreibung
(Deutsch)
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Ein durch den SNF gefördertes Projekt
Die Afrikanische Schweinepest ist eine tödliche hämorrhagische Erkrankung von Wild- und Hausschweinen, die weltweit hohe Verluste verursacht. Aufgrund der komplexen Immunpathogenese der Krankheit gibt es derzeit keine wirksame Immunprophylaxe gegen die Afrikanische Schweinepest. In diesem Projekt sollen die Mechanismen untersucht werden, mit denen das Virus die Immunabwehr von Schweinen umgeht. Dies wird einen wichtigen Beitrag zur Entwicklung von Impfstoffen leisten.
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Abstract
(Englisch)
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Since 2007, pig meat producers from Eastern and Central Europe and Asia have been confronted with African swine fever (ASF), a devastating, nearly 100% lethal hemorrhagic disease of pigs and wild boar. The disease is endemic in wild boar, which renders its control and eradication particularly difficult, leading to regular outbreaks in pig farms. The socio-economic impact of an outbreak in domestic pigs is enormous, as all pigs of an affected farm must be culled. There are neither therapies nor registered vaccines available. The main reasons for that are the complex immunopathogenesis and the poorly understood immune evasion mechanisms of ASF virus (ASFV). The virus belongs to the nucleocytoplasmic large DNA viruses, whose hallmark is the large protein coding capacity. Accordingly, the ASFV DNA encodes 150 to 180 viral proteins, 68 of which are structural components of the virus particle, while approximately 100 non-structural ASFV proteins orchestrate disease immunopathogenesis and other accessory functions by interaction with host factors. Most structural and some non-structural proteins have essential functions that the virus needs to complete its cellular life cycle. Besides these, a considerable amount of non-structural and some structural proteins are non-essential. Their genes can be deleted from the genome without preventing the formation of progeny virus. Most of the non-essential proteins of ASFV have neither been identified as such, nor has their function been characterized yet. The main hypothesis of the present project is that non-essential viral gene products exert accessory functions that are required primarily for the virus to combat host antiviral defenses and replicate efficiently in pigs. We hypothesize also that most of these non-essential viral proteins contribute to the pathogenic phenotype of the virus. Therefore, we propose to construct gene-deleted ASFV for open reading frames - or combinations thereof - for which it is unknown yet if they are essential or not. For this, we have established a novel technique for CRISPR/Cas9-assisted recombination. An alternative approach will employ transformation-associated recombination (TAR) cloning of the full-length ASFV genome from field virus in a yeast artificial chromosome (YAC). We have cloned the ASFV genome in a YAC but need to establish virus rescue from synthetic DNA, since ASFV DNA per se is not infectious. The project will then characterize extensively the replication of the gene-deleted viruses in primary porcine cells and study the functions of the deleted viral proteins. Differential analyses of cytokine and transcriptomic profiles of gene-deleted versus parent viruses will inform on the cellular pathways targeted by the missing proteins. This will be supported by trans-complementation experiments with the viral proteins of interest. Finally, we will determine the phenotype of selected gene-deleted ASFV in pigs. For this, we will use specific-pathogen-free pigs bred at the IVI and take advantage of the BSL3-Ag facility of the IVI. The latter is the only laboratory and animal facility in Switzerland authorized to perform in vivo studies with risk class 4 pathogens like ASFV. In conclusion, this project will permit to discover and understand yet unknown functions of ASFV proteins that interfere with cellular pathways and factors of the antiviral immune response. This knowledge is of utmost importance to understand the complex immunopathogenesis of the disease and to support vaccine development.
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