What a difference a gene makes – identification of virulence factors of Cowpox virus

Cowpox virus (CPXV) is a zoonotic Orthopoxvirus (OPV) that causes spill-over infections from their animal hosts to humans. In 2009, several human CPXV cases occurred through transmission from pet rats. An isolate from a diseased rat, RatPox09, exhibited significantly increased virulence in Wistar rats and caused high mortality when compared to the mildly virulent laboratory strain Brighton Red (BR). RatPox09 encodes four genes which are absent in the BR genome. We hypothesized that their gene products could be major factors influencing its high virulence. To address this hypothesis, we employed several BR/RatPox09 chimeric viruses using Red-mediated mutagenesis to generate BR knock-in mutants with single or multiple insertions of the respective RatPox09 genes based on BR. High-throughput sequencing was used to verify the genomic integrity of all recombinant viruses; and transcriptomic analyses confirmed the expression profile of genes adjacent to the ones modified be unaltered. While in vitro growth kinetics were comparable to those of BR and RatPox09, we discovered that a knock-in BR mutant containing the four RatPox09-specific genes was as virulent as the RatPox09 isolate causing death in over 75% of infected Wistar rats. Unexpectedly, the insertion of gCPXV0030 (7tGP) alone into the BR genome resulted in significantly higher clinical scores and lower survival rates matching that of RatPox09. The insertion of gCPXV0284 encoding the BTB domain protein D7L also increased the virulence of BR, while the other two ORFs failed to rescue virulence independently. In summary, our results confirmed our hypothesis that a relatively small set of four genes can contribute significantly to CPXV virulence in the natural rat animal model.



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