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Projekt
Aus dem Reservoir: Identifizierung und Charakterisierung viraler und wirtsspezifischer Faktoren zoonotischer Kuhpockenviren
Antragsteller
Professor Dr. Martin Beer; Professor Dr. Nikolaus Osterrieder
Fachliche Zuordnung
Virologie
Förderung
Förderung von 2013 bis 2020
Projektkennung
Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 226372197
CPXV, a member of the genus Orthopoxvirus (OPV), has a very wide host range including rodents, cats, cattle, but also several zoo animals and humans. The pathogenic potential of different CPXV strains is rather variable. A recent CPXV isolate from pet rats and human infections in Southern Germany, Bay09, causes severe disease in Wistar rats including poxvirus lesions. On the other hand results the infection of rats with the CPXV strain BR only in mild clinical symptoms. Knowledge of virulence factors of CPXV field strains is crucial to allow a risk assessment for the viruses circulating in rodents and cats. Comparison of all available CPXV sequences show that there is no obvious virulence associated pattern of viral genome sequences nor an individual ORF which directly correlates with the pathotype of the isolates. Nevertheless there is a set of genes which are encoded by the pathogenic strain Bay09, but not by the low-virulent BR nor the majority of the sequenced CPXV. Two of the gene products are predicted to have a potential impact on cell cycle progression, one is of unknown function and one, CrmE, is a known virulence factor. Our hypothesis is that all four have an impact on the pathotype of Bay09 in rats. To challenge this, we plan to test single deletion viruses of all four genes in BAC derived Bay09 mutants in 3D skin cultures and in infected Wistar rats. The impact of individual mutant viruses on cell cycle progression will be tested in non-permanent cultured cells. Also the immunohistological staining of skin lesions in organotypic rafts, in rats, and of infected chicken chorioallantoic membranes (CAMs) will be used to address this question. Beside those negative mutants we also plan to test chimeras of Bay09 segments in BR to allow gain of virulence assays. For this purpose we want to use markerless en passant mutagenesis to swap approx. 40 kbp segments of Bay09 into the BRBAC clone. This shall allow the identification of virulence factors beside the four predicted genes. The overall objective of this proposal is the identification of new OPV virulence markers in the original rodent host to allow a prospective screening of newly isolated CPXV. Chimeric viruses and negative mutants with detected modified pathotype shall be analyzed in the future with proteome analysis to allow the examination of cellular mechanisms involved in CPXV virulence. Also changes in CPXV morphology and replication of mutant viruses will be addressed by electron microscopy analysis.
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