Publikationsdatenbank

Cowpox virus virulence determinants (2017)

Art

Hochschulschrift

Autor

Tamošiūnaitė, Aistė (WE 5)

Quelle

Berlin: Mensch und Buch Verlag, 2017 — 91 Seiten

ISBN: 978-3-86387-832-0

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/25131

Kontakt

Institut für Virologie

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51833
virologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

New cowpox virus (CPXV) infections occur naturally in Germany in increasing numbers. It is striking that an infection of the pet rats with these newly occurring CPXV strains often leads to a lethal outcome. Likewise, a CPXV isolate from a pet rat that as well caused human infection in Southern Germany, RatPox09, causes severe disease in Wistar rats, whereas infection of rats with the well-characterized strain Brighton Red (BR) results only in mild symptoms. Nevertheless, these two strains are highly similar on genomic level: the calculated sequence identity between RatPox09 and BR is 92%. However, with the help of proteome similarity plots, six products with major differences between RatPox09 and BR were identified. Though, minor sequence modifications of unknown impact were found dispersed all over the genome too. The thesis at hand, concentrates on genomic differences between the two CPXV strains, mentioned above. First of all, we analyzed the sequence of the genes of major differences and we generated gain of function mutants with single or multiple genes exchanged. Secondly, for identification of additional virulence factors, we used a novel approach to generate BR-RatPox09 chimeric viruses with approximately 20-40 kbp genome segments exchanged. This was achieved by combining markerless en passant Red recombination and a classical method, homologous recombination in infected vertebrate cells. Since, these CPXV strains of different pathogenic potential in vivo; shows similar growth characteristics in vitro, animal model seems to be the only suitable method to test the virulence of the recombinant viruses. Nevertheless, in vivo models, are not favorable experimental models due to many reasons, including: aspects of animal protection, their long duration and high requirement of labor. In search of a way to reduce the need for animal experiments for testing new recombinant viruses, we evaluated a three-dimensional (3D) skin model as a possible surrogate for animal experiments. We monitored CPXV lesion formation, viral gene expression and cell cycle patterns after infection of 3D skin cultures with RatPox09 and BR. Infected 3D skin cultures exhibited histological alterations that were similar to those of mammal skin infections, but there were no differences in gene expression patterns and tissue damage between the two CPXV strains in the model system. To be brief, 3D skin cultures reflect the development of pox lesions in the human skin, but seem not to allow differentiation between more or less virulent virus strains, a distinction that is made possible by experimental infection in suitable animal models. Therefore, in vivo infection model was applied. CPXV RatPox09 and NMDAr and CrmE mutants, but not segment F chimera, infected rats attained significally higher clinical scores than BAC derived BR or wt BR infected rats (p ≤ 0.05, Anova). However, 6 out of the 8 RatPox09 infected Wistar rats had to be euthanized as their health deteriorated dramatically. Although only 2 out of 10 with BR_RatPox09 NMDAr and 1 out of 10 BR_RatPox09 CrmE infected rats succumbed to the disease. But none of CPXV BR and BR_RatPox09 sF infected rats did. Summing up the results, it can be concluded that, insertion of sequence of putative RatPox09 genes, NMDAr and CrmE, but not the genomic segment F, into strain BR, results in increase of pathogenicity. However, the insertion of these individual ORFs is not sufficient to recreate the pathological effect of RatPox09.