SummarySmallpox holds a unique position in the history of medicine. It was the first disease for which a vaccine was developed and remains the only human disease eradicated by vaccination. Although there have been claims of smallpox in Egypt, India, and China dating back millennia [1, 2, 3, 4], the timescale of emergence of the causative agent, variola virus (VARV), and how it evolved in the context of increasingly widespread immunization, have proven controversial [4, 5, 6, 7, 8, 9]. In particular, some molecular-clock-based studies have suggested that key events in VARV evolution only occurred during the last two centuries [4, 5, 6] and hence in apparent conflict with anecdotal historical reports, although it is difficult to distinguish smallpox from other pustular rashes by description alone. To address these issues, we captured, sequenced, and reconstructed a draft genome of an ancient strain of VARV, sampled from a Lithuanian child mummy dating between 1643 and 1665 and close to the time of several documented European epidemics [1, 2, 10]. When compared to vaccinia virus, this archival strain contained the same pattern of gene degradation as 20th century VARVs, indicating that such loss of gene function had occurred before ca. 1650. Strikingly, the mummy sequence fell basal to all currently sequenced strains of VARV on phylogenetic trees. Molecular-clock analyses revealed a strong clock-like structure and that the timescale of smallpox evolution is more recent than often supposed, with the diversification of major viral lineages only occurring within the 18th and 19th centuries, concomitant with the development of modern vaccination.

The Capuchin Catacombs of Palermo contain over 1800 preserved bodies dating from the 16th to 20th centuries AD and showing evidence of biodeterioration. An extensive microbiological and molecular investigation was recently performed. Samples were taken from skin, muscle, hair, bone, stuffing materials, clothes, and surrounding walls as well as from the indoor air. In this study, we witnessed that the different degradation phenomena observed on the variety of materials located at the Capuchin Catacombs of Palermo are biological in origin. Molecular techniques showed the dominance of halophilic species of the domains Bacteria and Archaea on the walls and – as a result of salt emanating from the walls – on the mummies themselves. Nevertheless, specialized microorganisms belonging to taxa well-known for their cellulolytic and proteolytic activities were detected on clothes and stuffing material, and on skin, muscle, hair, and bone, respectively. This specialized microbiota is threatening the conservation of the mummies themselves. Additionally, sequences related to the human skin microbiome and to some pathogenic Bacteria (order Clostridiales) and fungi (genus Phialosimplex) were identified on samples derived from the mummies. Furthermore, a phosphate-reducing fungus, Penicillium radicum, was detected on bone. Finally, the high concentration of airborne fungal spores is not conducive to the conservation of the human remains and is posing a potential health risk for visitors.

The purpose of this study was to develop a checklist for standardized assessment of soft tissue preservation in human mummies based on whole-body computed tomography examinations, and to add a scoring system to facilitate quantitative comparison of mummies. Computed tomography examinations of 23 mummies from the Capuchin Catacombs of Palermo, Sicily (17 adults, 6 children; 17 anthropogenically and 6 naturally mummified) and 7 mummies from the crypt of the Dominican Church of the Holy Spirit of Vilnius, Lithuania (5 adults, 2 children; all naturally mummified) were used to develop the checklist following previously published guidelines. The scoring system was developed by assigning equal scores for checkpoints with equivalent quality. The checklist was evaluated by intra- and inter-observer reliability. The finalized checklist was applied to compare the groups of anthropogenically and naturally mummified bodies. The finalized checklist contains 97 checkpoints and was divided into two main categories, “A. Soft Tissues of Head and Musculoskeletal System” and “B. Organs and Organ Systems”, each including various subcategories. The complete checklist had an intra-observer reliability of 98% and an inter-observer reliability of 93%. Statistical comparison revealed significantly higher values in anthropogenically compared to naturally mummified bodies for the total score and for three subcategories. In conclusion, the developed checklist allows for a standardized assessment and documentation of soft tissue preservation in whole-body computed tomography examinations of human mummies. The scoring system facilitates a quantitative comparison of the soft tissue preservation status between single mummies or mummy collections.

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