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Human cowpox virus (CPXV) infections are rare, but can result in severe and sometimes fatal outcomes. The majority of recent cases were traced back to contacts with infected domestic cats or pet rats. The aim of the present study was to evaluate a three-dimensional (3D) skin model as a possible replacement for animal experiments. We monitored CPXV lesion formation, viral gene expression and cell cycle patterns after infection of 3D skin cultures with two CPXV strains of different pathogenic potential: a recent pet rat isolate (RatPox09) and the reference Brighton red strain. 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. In conclusion, 3D skin cultures reflect the development of pox lesions in the skin very well, 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.

The incidence of human cowpox virus (CPXV) infections has increased significantly in recent years. Serological surveys have suggested wild rodents as the main CPXV reservoir. We characterized a CPXV isolated during a large-scale screening from a feral common vole. A comparison of the full-length DNA sequence of this CPXV strain with a highly virulent pet rat CPXV isolate showed a sequence identity of 96%, including a large additional open reading frame (ORF) of about 6,000 nucleotides which is absent in the reference CPXV strain Brighton Red. Electron microscopy analysis demonstrated that the vole isolate, in contrast to the rat strain, forms A-type inclusion (ATI) bodies with incorporated virions, consistent with the presence of complete ati and p4c genes. Experimental infections showed that the vole CPXV strain caused only mild clinical symptoms in its natural host, while all rats developed severe respiratory symptoms followed by a systemic rash. In contrast, common voles infected with a high dose of the rat CPXV showed severe signs of respiratory disease but no skin lesions, whereas infection with a low dose led to virus excretion with only mild clinical signs. We concluded that the common vole is susceptible to infection with different CPXV strains. The spectrum ranges from well-adapted viruses causing limited clinical symptoms to highly virulent strains causing severe respiratory symptoms. In addition, the low pathogenicity of the vole isolate in its eponymous host suggests a role of common voles as a major CPXV reservoir, and future research will focus on the correlation between viral genotype and phenotype/pathotype in accidental and reservoir species. IMPORTANCE We report on the first detection and isolation of CPXV from a putative reservoir host, which enables comparative analyses to understand the infection cycle of these zoonotic orthopox viruses and the relevant genes involved. In vitro studies, including whole-genome sequencing as well as in vivo experiments using the Wistar rat model and the vole reservoir host allowed us to establish links between genomic sequences and the in vivo properties (virulence) of the novel vole isolate in comparison to those of a recent zoonotic CPXV isolated from pet rats in 2009. Furthermore, the role of genes present only in a reservoir isolate can now be further analyzed. These studies therefore allow unique insights and conclusions about the role of the rodent reservoir in CPXV epidemiology and transmission and about the zoonotic threat that these viruses represent.