Experimental intranasal infection of marmosets (Callithrix jacchus) with calpox virus results in fatal disease. Route and dose used for viral inoculation of the test animals mimics the natural transmission of smallpox, thus representing a suitable model to study pathogenesis and to evaluate new vaccines against orthopoxvirus infection. However, the pathogenic mechanisms leading to death are still unclear. Therefore, our study aimed at investigating the kinetics of pathological alterations to clarify the pathogenesis in calpox virus infection. Following intranasal inoculation with two different viral doses, common marmosets were sacrificed on days 3, 5, 7, 10 and 12 post inoculation. Collected tissue was screened using histopathology, immunohistochemistry, transmission electron microscopy, and virological assays. Our data suggest that primary replication took place in nasal and bronchial epithelia followed by secondary replication in submandibular lymph nodes and spleen. Parallel to viremia at day 7, virus was detectable in many organs, mainly located in epithelial cells and macrophages, as well as in endothelial cells. Based on the onset of clinical signs, the histological and ultrastructural lesions and the immunohistochemical distribution pattern of the virus, the incubation period was defined to last 11 days, which resembles human smallpox. In conclusion, the data indicate that the calpox model is highly suitable for studying orthopoxvirus-induced disease.

The use of pesticides is an inseparable and undeniable part of modern agriculture to ensure sufficient food supply for a still-growing population but, due to their intrinsic toxic nature, rigorous testing is needed before making them available on the market. In the European Union, in vitro metabolism studies with the active ingredients became mandatory in 2013. Unfortunately, in contrast to most other toxicological endpoints, a harmonised test guideline for their conduct is not yet available. More than 10 years after its introduction, we aimed to analyse the practical experience with that data requirement. For this purpose, 70 comparative in vitro metabolism studies were reviewed. Significant methodological differences were noticed among the studies, including species selection, test systems, tested concentrations, and incubation times. These differences and the deviations from existing but not legally binding recommendations limit the use that could be made of these studies, in particular beyond a simple comparison of human and rat metabolism. There is an urgent need for a harmonised guidance how to perform the in vitro biotransformation studies. Its implementation would not only increase the likelihood to identify unique or disproportionate human metabolites but might also support next-generation risk assessment.