Tissue specimens from 283 principally spontaneously (naturally) desiccated human mummies from coastal and low valley sites in northern Chile and southern Peru were tested with a DNA probe directed at a kinetoplast DNA segment of Trypanosoma cruzi.

Hepatitis B virus (HBV) is a ubiquitous viral pathogen associated with large-scale morbidity and mortality in humans. However, there is considerable uncertainty over the time-scale of its origin and evolution. Initial shotgun data from a mid-16 th century Italian child mummy, that was previously paleopathologically identified as having been infected with Variola virus (VARV, the agent of smallpox), showed no DNA reads for VARV yet did for hepatitis B virus (HBV). Previously, electron microscopy provided evidence for the presence of VARV in this sample, although similar analyses conducted here did not reveal any VARV particles. We attempted to enrich and sequence for both VARV and HBV DNA. Although we did not recover any reads identified as VARV, we were successful in reconstructing an HBV genome at 163.8X coverage. Strikingly, both the HBV sequence and that of the associated host mitochondrial DNA displayed a nearly identical cytosine deamination pattern near the termini of DNA fragments, characteristic of an ancient origin. In contrast, phylogenetic analyses revealed a close relationship between the putative ancient virus and contemporary HBV strains (of genotype D), at first suggesting contamination. In addressing this paradox we demonstrate that HBV evolution is characterized by a marked lack of temporal structure. This confounds attempts to use molecular clock-based methods to date the origin of this virus over the time-frame sampled so far, and means that phylogenetic measures alone cannot yet be used to determine HBV sequence authenticity. If genuine, this phylogenetic pattern indicates that the genotypes of HBV diversified long before the 16 th century, and enables comparison of potential pathogenic similarities between modern and ancient HBV. These results have important implications for our understanding of the emergence and evolution of this common viral pathogen. Author summaryHepatitis B virus (HBV) exerts formidable morbidity and mortality in humans. We used ancient DNA techniques to recover the complete genome sequence of an HBV from the mummified remains of a child discovered in the 16 th century from Naples, Italy. Strikingly, our analysis of this specimen resulted in two contrasting findings: while the damage patterns lend credence to this HBV sequence being authentically 16 th century, phylogenetic analysis revealed a close relationship to recently sampled viruses as expected if the sequence were a modern contaminant. We reconcile these two observations by showing that HBV evolution over the last~450 years is characterized by a marked lack of temporal structure that hinders attempts to resolve the evolutionary time-scale of this important human pathogen.

Estimation of age of skeletal remains is one of the most complex questions for anthropologists. The most common macroscopic methods are based on dental wear and histological evaluation of bone remodeling. These methods are often qualitative, require great technical expertise, and have proved inexact in the estimation of ages over 50 years. Certain dental methods investigate the apposition of secondary dentine, in the study of tooth cross-sections, and X-rays to study width, height, and pulp area. The primary author previously proposed a method of estimating the age of a living person based on the pulp/tooth ratio (PTR) method in the upper canines. The aim of the present study is to verify whether the PTR method can also be used to estimate the age at death of skeletal remains. This paper investigates the study of historical samples of known age as a means to validate the proposed method.

The process of natural mummification is a rare and unique process from which little is known about the resulting microbial community structure. In the present study, we characterized the microbiome of paleofeces, and ascending, transverse and descending colon of an 11th century A.D. pre-Columbian Andean mummy by 16S rRNA gene high-throughput sequencing and metagenomics. Firmicutes were the most abundant bacterial group, with Clostridium spp. comprising up to 96.2% of the mummified gut, while Turicibacter spp. represented 89.2% of the bacteria identified in the paleofeces. Microbiome profile of the paleofeces was unique when compared to previously characterized coprolites that did not undergo natural mummification. We identified DNA sequences homologous to Clostridium botulinum, Trypanosoma cruzi and human papillomaviruses (HPVs). Unexpectedly, putative antibiotic-resistance genes including beta-lactamases, penicillin-binding proteins, resistance to fosfomycin, chloramphenicol, aminoglycosides, macrolides, sulfa, quinolones, tetracycline and vancomycin, and multi-drug transporters, were also identified. The presence of putative antibiotic-resistance genes suggests that resistance may not necessarily be associated with a selective pressure of antibiotics or contact with European cultures. Identification of pathogens and antibiotic-resistance genes in ancient human specimens will aid in the understanding of the evolution of pathogens as a way to treat and prevent diseases caused by bacteria, microbial eukaryotes and viruses.