MICROBIAL GENOMICS REVIEWS Ancient pathogen genomics as an emerging tool for infectious disease research Maria A. Spyrou *, Kirsten I. Bos, Alexander Herbig and Johannes Krause * Abstract | Over the past decade, a genomics revolution, made possible through the development of high- throughput sequencing, has triggered considerable progress in the study of ancient DNA , enabling complete genomes of past organisms to be reconstructed. A newly established branch of this field, ancient pathogen genomics, affords an in- depth view of microbial evolution by providing a molecular fossil record for a number of human-associated pathogens. Recent accomplishments include the confident identification of causative agents from past pandemics, the discovery of microbial lineages that are now extinct, the extrapolation of past emergence events on a chronological scale and the characterization of long-term evolutionary history of microorganisms that remain relevant to public health today. In this Review , we discuss methodological advancements, persistent challenges and novel revelations gained through the study of ancient pathogen genomes. Ancient pathogen genomics The long shared history between humans and infectious techniques to this study, providing a diachronic genetic A scientific field focused on disease places ancient pathogen genomics within the inter­ perspective to infectious disease research. Initial attempts the study of whole pathogen est of several fields such as microbiology, evolutionary relied on PCR technology5–9, which restricted the study genomes retrieved from biology, history and anthropology. Research on this of ancient microbial DNA to targeted, short genomic ancient human, animal or plant remains. topic aims to better understand the interactions between fragments that were amplified from ancient human pathogens and their hosts on an evolutionary timescale, remains. This method made infectious disease detection Neolithic revolution to uncover the origins of pathogens and to disentangle possible but gave limited information on the evolution­ The cultural transition the genetic processes involved in their epidemic emer­ ary history of the patho gen. In addition, complications associated with the adoption gence among human populations. Over the past 10,000 associated with the study of aDNA, which is typically of farming, animal husbandry and domestication as well as years, major transitions in human subsistence strategies, present at low quantities, is heavily fragmented and har­ 1 10–12 the practice of a sedentary such as those that accompanied the Neolithic revolution , bours chemical modifications , hampered efforts to lifestyle among human likely exposed our species to a novel range of infectious reproduce and authenticate early findings13–15. populations. agents2. Closer contact with domesticated animals would Over the past decade, major advancements in geno­ zoonotic transmission Zoonotic transmission have increased the frequency of mics, in particular, the development of high­throughput The infectious disease events, and higher human population densities would sequencing, also called next­ generation sequencing transmission from animals have enhanced the potential of pathogens to propagate (NGS)16, radically increased the amount of data that to humans. within and between groups. Throughout human his­ can be retrieved from ancient remains. This techno­ tory, a number of epidemics and pandemics have been logy has assisted the development of quantitative meth­ recorded or are hypothesized to have occurred (Fig. 1). ods for aDNA authentication11,12,17–19 and has enabled Although most of their causative agents still remain the retrieval of whole ancient pathogen genomes from speculative, robust molecular methods coupled with archaeological specimens. The first such genome, pub­ archaeological and historical data can confidently lished in 2011 (REF.20), was that of the notorious bacterial demonstrate the involvement of certain pathogens in pathogen Yersinia pestis, the causative agent of plague. Department of Archaeogenetics, these episodes. Since then, the field has expanded its directions to the Max Planck Institute for the The investigation of past infectious diseases has tra­ in­depth study of infectious disease evolution, providing Science of Human History, palaeopathological Jena, Germany. ditionally been conducted through a unique resource for understanding human history. assessment 3,4 *e- mail: [email protected]; of ancient skeletal assemblages , although Here, we review the latest methodological innovations [email protected] this approach is limited by the fact that most acute infec­ that have aided the whole­ genome retrieval and evolu­ https://doi.org/10.1038/ tions do not leave visible traces on bone. Since the 1990s, tionary analysis of various ancient pathogens (TAble 1), s41576-019-0119-1 the field of ancient DNA (aDNA) has brought molecular most of which are still relevant to public health today. NATURE REVIEWS | GENETICS VOLUME 20 | JUNE 2019 | 323 REVIEWS Copper Period of the Modern era Age Iron Age Roman Empire Period of the Byzantine Empire European Neolithic Age Bronze Age Classical and late antiquity Medieval period colonization of the Americas Frequent appearance of infectious Tuberculosis, cholera disease in archaeological record and Black Death and pandemics, third plague molecular detection of Y. pestis31,33,34,37, European plague pandemic, Spanish flu58, H. pylori49, B19V45 and HBV43,44 Plague of Athens Plague of Cyprian epidemics20,30,32,38 HIV57, SARS and Ebola 5000–1500 BCE 430–426 BCE 249–262 CE 1346–1772 CE 19th to 21st century CE 5000 BCE 1000 BCE 500 BCE 0 CE 500 CE 1000 CE 1500 CE Hittite plague Antonine Plague Plague of Justinian and Epidemics among Native Americans, 14th century BCE 165–180 CE first plague pandemic35,36 e.g. cocoliztli41 and smallpox 541–750 CE 15th to 19th century CE Fig. 1 | Selected cultural time periods and epidemics or pandemics of human history. This overview provides a timeline of key events in predominantly Eurasian history since the Neolithic period (upper panel, grey squares), which have overlapped temporally and geographically with major historical epidemics or pandemics (lower panel, beige squares). The respective citations are indicated, in which whole-genome or low-coverage genome-wide data from pathogens implicated in those events have been reconstructed by ancient DNA analysis. B19V, human parvovirus B19; bce, before current era; ce, current era; HBV, hepatitis B virus; H. pylori, Helicobacter pylori; SARS, severe acute respiratory syndrome; Y. pestis, Yersinia pestis. In the second half of this Review, we highlight the util­ Of note, the sampling methods for recovering pathogen ity of this approach by discussing evolutionary events in DNA do not generally follow a standardized procedure, the history of Y. pestis that have been uniquely revealed in part because of the great diversity in tissue tropism and through the study of ancient genomes. resulting disease progression. In addition, acute blood-​ Pandemics borne infections do not typically produce diagnostic Refers to increased, often sudden, disease occurrence Methods for isolating ancient microbial DNA bone changes as opposed to those that affect their hosts 3 within populations across more The sweet spot for ancient pathogen DNA. The chronically . Therefore, if infections have caused mortal­ than one region or continent, retrieval of DNA from ancient human, animal or plant ity in the acute phase, as is the case for individuals from whereas epidemics refers to remains carries with it a number of challenges, namely, epidemic contexts who do not display skeletal evidence increased disease occurrences within a confined region its limited preservation and hence low abundance, its of infection, the preferred study material has been the or country. highly fragmented and damaged state and the perva­ inner cavities of teeth. Pathogen aDNA is thought to sive modern­ DNA contamination that necessitates a be preserved within the remnants of the pulp chamber, Palaeopathological confident evaluation of its authenticity21,22. Efficient likely as part of desiccated blood8,17. Consequently, assessment aDNA recovery is best accomplished via sampling of the tooth sampling has proved successful in the retrieval The evaluation of the health status of ancient individuals or anatomical element that contains the highest quantity of whole genomes or genome­ wide data (that is, low-​ populations, usually through of DNA from the target organism. For human aDNA coverage genomes that have provided limited analytical the analysis of disease marker analysis, bone and teeth have been the preferred study resolution) from ancient bacteria such as Y. pestis 20,30–39, presence on skeletal material, given their abundance in the archaeological Borrelia recurrentis40 and Salmonella enterica41; ancient assemblages. record. Recent studies suggest that the inner­ ear por­ eukaryotic pathogens such as Plasmodium falciparum42; 23 43,44 Ancient DNA tion of the petrous bone and the cementum layer of and ancient viruses such as hepatitis B virus (HBV) and (aDNA). The DNA that has teeth24 have the greatest potential for successful human human parvovirus B19 (B19V)45. Even M. leprae, which been retrieved from historical, DNA retrieval. However, petrous bone sampling and commonly manifests in the chronic form, has been archaeological or shotgun NGS sequencing of aDNA from five Bronze retrieved from ancient teeth27,28. palaeontological remains.