Genomes from living organisms contain the remnants of the ongoing battle between pathogens and hosts, both as integrated viruses and as selective sweeps in immune-related genes. To look for an easy-accessible source of the most ancient pathogens, all we have to do to study those carefully propagated leftovers of the past is to sequence and analyze the corresponding DNA stretches in modern chromosomes. For the older viruses, those invading host genomes over 50–100 million years ago (mya), most of their genomes are likely to be useful only in retracing evolution by descent, rather than informing us of putative transmissions or epidemics caused by those parasites. However, when sufficient endogenous viral elements (EVEs) are present in a species, or in related species to reconstruct a detailed evolutionary history, their past transmission patterns may become visible, especially since more and more complete genomes are being sequenced and becoming accessible from public databases. Comparison of simian endogenous retrovirus (SERV) genomes in Old World monkey species, for instance, showed that this virus was spreading among African and Asian monkeys less than 8 million years ago (mya) [3], while tracking down single-stranded RNA virus-related integrations suggested that around 40 mya, ancestral mammals suffered from a heavy load of borna- and filovirus infections [25,26]. Of course, virulence and pathology cannot be estimated from endogenous viral genomes alone, although similarity to modern pathogens could suggest this. In theory, transmission of ancient viruses could have resulted in little or no morbidity and mortality, which would then disqualify them from being the cause of bona fide epidemics. However, witnessing recent, successful novel introductions of microbes into human populations or domestic animal populations provides ample evidence of those microorganisms being real pathogens, with at least part of a population experiencing disease. It is not likely that this would have been different in the past.

Modern genomes can likewise show evidence of ancient battles by rapid local evolution, or by selection, positive or negative, of existing alleles from genes involved in either immunity or in the replication cycle of ancient parasites. Examples are, for instance, the acquisition and fast evolution of the APOBEC3G gene in primates (reviewed in [27]), and the dominance of a dysfunctional Toll-like receptor 1 variant associated with protection against leprosy in people of European descent [28]. A 32-base pair deletion in the CCR5 gene, already found in prehistoric Europeans, has likewise been attributed to such a selective sweep, although the causative pathogen remains a mystery, after bubonic plague was ruled out [29,30,31]. However, selection of certain immunity-related alleles, such as those encoding Ficolin-2, NLRP14, and HLA-DRB1*13, involved in the response to pathogenic bacteria, was obvious from a genetic comparison of thirty-six 16th century plague victims buried in Ellwangen, Germany, with modern inhabitants of that city [32]. It is therefore likely that the plague pandemics did influence the genetic make-up of surviving populations. High carrier frequencies for pyrin—an inflammasome protein—mutations seen in Mediterranean populations have indeed been associated with resistance to Y. pestis [33]. Other allelic selections associated with epidemic pathogens are, for instance, the highly prevalent FUT2 (an α(1,2) fucosyltransferase involved in H type 1 blood group antigen synthesis) non-secretor alleles with resistance to noro- and rotavirus infections [34,35,36], IFITM3 alleles (encoding an interferon-inducible transmembrane protein) with influenza morbidity and mortality [37], DARC gene (encoding a chemokine receptor) variation with malaria resistance [38], a tyrosine kinase 2 (TYK2) polymorphism with clinical TB [39], and IFNL4 (encoding an antiviral interferon) pseudogenization with hepatitis C virus clearance [40]. In a Bangladeshi population, multiple genomic regions were found to be selected in association with cholera susceptibility [41]. These stretches, for instance, encode potassium channel genes, nuclear factor κB (NF-κB) signaling components, and proteins of the NF-κB/inflammasome-dependent pathway [41]. Glucose-6-phosphate dehydrogenase (G6PD) deficiency, and beta thalassemia- and sickle-cell disease inducing mutations, prevalent in Africa and the Mediterranean, are all linked to malaria resistance (reviewed in [42]). A more complete overview of pathogen-induced selection on human genetics can be found in [43,44].

Due to improved technology and decreased costs, the number of complete genomes is rapidly increasing, which will greatly enhance our knowledge of ancient host-microbe battles.

Archeological and historical burial sites can be good sources to look for patterns of epidemics and their causative pathogens in the distant past. For instance, a sudden increase in the number of individuals interred, or a high frequency of adolescents and young adults among the deceased; abnormal burials, as demonstrated by multiple or mass graves; bodies stocked in layers; the use of lime; un-coffined burials; and/or other sloppy burial practices can all point to a major disaster, possibly in the form of an epidemic, especially when there is no evidence of physical trauma [45,46]. Such locations have been termed “mortality crisis” graves [47]. Grave artifacts, such as coins and clothing, ¹⁴C dating, and written evidence in the form of tombstones, or parish records, can be used to date the burial grounds. For instance, three mass burials discovered in the center of Ellwangen, Germany, concerning a total of 101 skeletal remains buried close together, with other evidence, such as the majority being children, a characteristic of later plague epidemics, suggested that the site harbored 16th–17th century plague casualties [48]. Indeed, the presence of Yersinia pestis DNA was confirmed in dental pulp extracts [48]. The remains were also used to investigate the genetic variation of the victims [32]. In a plethora of other studies, teeth and bone samples from putative 14th–18th plague “pits” in many locations in Eurasia have been confirmed to contain Y. pestis DNA [7,47,48,49,50,51]. From the 17th century onwards, direct written evidence of plague epidemics and dedicated graveyards is common [7,47,51]. Retrieval of Y. pestis DNA from victims of earlier epidemics, such as the 6th century “Justinian plague”, has also relied on evidence for local mortality crises, such as when a “striking number” of double and multiple burials was seen in a grave-yard in Bavaria, Germany; when collective burials were found in Valencia, Spain; and when an existing ditch was used as an emergency cemetery near Bourges in France [52,53]. In contrast, burial patterns in Neolithic and Bronze Age graves have not been found to be useful in this regard, likely due to a lower population density, so that samples from those periods are usually tested at random for pathogens [13,54,55,56]. Similarly, in Africa, where mass burial sites, which could represent plague pits, have not yet been found, it has been speculated that two- to fourfold simultaneous interments may already be indicative of epidemic disaster, as could be a population reduction resulting in abandonment of settlements, infrastructure decay, or fluctuations in industrial activities [57,58]. Of course, such developments could easily be attributed to other causes, such as famine or climatic disaster, but epidemics should certainly be considered when no war or other type of major violence is present.

In contrast to plague emergency burials, for other common medieval diseases, such as leprosy, dedicated leprosaria had been erected from medieval times onwards to house and isolate the large numbers of patients. Leprosy, or Hansen’s disease, is caused by Mycobacterium leprae. It runs a chronic, slow course, leaving sufficient time to bury the dead peacefully in cemeteries within the institution. Ancient DNA analysis of remains from medieval European leprosaria, resulted in the retrieval of partial and complete genomes of M. leprae, with a surprisingly high genetic diversity [59,60,61]. Complete M. leprae genomes were likewise retrieved from bones collected from a 10th-12th century British leprosarium [62], confirming that dedicated hospital sites are fertile hunting grounds for research on pathogen evolution. Evidence from ancient remains of another chronic disease, namely tuberculosis (TB), which is caused by another Mycobacterial species complex, Mycobacterium tuberculosis, also shows the substantial worldwide genetic diversity of this ancient pathogen in the past, compared with modern isolates [63,64,65,66]. Detection of M. tuberculosis DNA and mycolic acids from a double burial of a ~25-year old female and a ~12-month old infant dating to around 9000 years BCE in what is now Israel, demonstrated that infection with the bacterium could be rapidly fatal in the past [67]. The child’s skeleton showed extensive bone pathology indicative of disseminated neonatal TB, to which young children are highly susceptible when infected by someone with contagious pulmonary TB [67].

Other locations where emergency burials might be expected are battlefields and sites of temporary army settlements. There, death from infectious diseases is equally likely as dying from violence, as military camps are, and have been, sites of mass crowding, stress, poor hygiene, poor nutrition, as well as the gathering of soldiers from different backgrounds. In the absence of battle injuries, mass graves near army quarters may be an indication of an epidemic of some sort. Local archives, for instance, suggested that mass graves excavated near Vilnius, Lithuania, contained the remains of soldiers from Napoleon’s Great Army, retreating from Moscow in December 1812 [68]. The deceased had been quickly interred, usually before rigor mortis had set in, and were buried close to each other. DNA from the louse-borne pathogens Bartonella quintana, the causative agent of trench fever, and of typhus-inducing Rickettsia prowazekii, was found in up to one-third of the soldiers, with the first also being found in three lice samples, indicating that these diseases had had a great impact on those surviving the combat of Napoleon’s Russian campaign [68]. DNA of the above mentioned louse-borne pathogens was also found in soldiers buried in a mass grave near the besieged city of Douai in northern France, dating to 1710–1712 CE [69], while B. quintana and also Y. pestis DNA were retrieved from two French multiple-burial sites in Bondy, dating to the 11th–15th centuries CE, and putatively containing plague victims buried closely together without coffins [70]. Finding ancient DNA from louse-borne diseases in mass graves together with Y. pestis DNA has led to the hypothesis that in medieval times, the spread of human louse- and not rat flea-borne Y. pestis was the source of the plague epidemics [71]. DNA from another louse-borne pathogen, Borrelia recurrentis, the bacterial cause of relapsing fever, was detected in dental pulp from an individual buried in a 15th century CE double grave in Oslo, Norway [72], while paleo-serology was able to identify seven Borrelia infections (most likely B. recurrentis, as Borrelia spp. inducing Lyme disease do not cause epidemics) and one B. quintana seropositive tooth from a 16th–17th century military garrison cemetery in Auxi-le-Château, France [73], again indicating that louse-borne pathogens were common in historical Europe.

The emergence of ancient DNA research several decades ago, saw molecular biologists begging for pieces of valuable or, at least in the beginning, not so valuable, archeological and historical specimens, to serve as a possible source of ancient DNA. Pathogen DNA retrieved from such unique objects cannot on its own cover epidemics, but it can significantly contribute to our knowledge of the earliest features and evolution of microbes known to be able to cause epidemics. (Electron) microscopy, targeted PCR amplification, employing microarrays, and microbial reads being present as a byproduct of deep sequencing have all been used to detect ancient microbes or their DNA in (pre)historic samples. Identification of a pathogen can already give valuable information on its appearance in history, but to learn something about the virulence and genetic variation, more information is needed. Fortunately, with the advance of ancient DNA techniques, generating complete or near-complete ancient pathogen genomes is increasingly feasible (reviewed in [13]). The results from such studies are already changing our view on ancient pathogens and epidemics. For instance, using multiple specimens collected from variable periods and geography, it was shown for Y. pestis, M. leprae, M. tuberculosis, VARV, and HBV that the genetic variation of these pathogens was much larger in the past. Another interesting observation was the presence of multiple pathogens in individuals, highlighting the infectious disease burden that plagued our ancestors. Examples here are the presence of both Y. pestis and Haemophilus influenzae genomes in the dental pulp of a 6th century English child, where the observed extensive scarring of the bones could be explained by the invasive H. influenzae infection causing septic arthritis; and the finding of both Y. pestis and T. pallidum pertenue DNA in a 15-16th century CE Lithuanian plague victim [81,82].

Variant strains of Treponema pallidum, the bacterial cause of syphilis, yaws, and bejel (endemic syphilis), depending on the subspecies, were shown to be widespread in early modern times in both Europe and Colonial Mexico [83,84]. As a result, the mystery as to what was the cause of a late 15th century epidemic in Europe, described in 16th century medical literature as an acute, severe venereal disease, has not yet been solved [82,85,86]. Was it due to the introduction of T. pallidum pallidum from the New World or elsewhere, or due to the introduction of a novel strain of that bacterium, or was the disease not syphilis at all, as until the 19th century, the various venereal diseases were often confused with each other and with leprosy, while the symptoms described in those early manuscripts are very different from the chronic, relatively mild disease that syphilis soon became [84,86,87,88]. As T. pallidum sequences have been successfully obtained from both bone and teeth, it is likely that in the near future more samples will be located, so that further bacterial genomes can be added to help solve the puzzle. In particular, analyzing a well-dated sample from the early years of the epidemic, 1495–1545 CE, would help significantly.

Pathology collections, containing the actual body parts, tissue, or blood samples of those who lived less than a few centuries ago, the oldest of which can be found in museums, and the more recent in biobanks, medical archives, or hospital freezers, are a valuable source when looking for historical pathogens in “wet” samples. As such collections are likely to contain only the most interesting specimens, it is improbable that epidemics could be reconstructed by analyzing their samples, yet information regarding the genetic make-up of an example microbe from a documented outbreak can be very valuable. For instance, analysis of an intestinal sample collected from a 1849 victim of the second cholera pandemic showed that Vibrio cholerae in the specimen indeed belonged to, as assumed, the classical O1 biotype, which was replaced in the 20th century by the El Tor O1 biotype [112]. Both a plasma sample and a lymph node biopsy from 1959 and 1960, respectively, stored in what is now the Democratic Republic of the Congo, have been used to amplify human immunodeficiency virus type 1 (HIV-1) genomic fragments [113,114]. The results indicated that the virus was not only definitely present by then, but had also diversified into the genetic subtypes seen at present, implying that its origin in humans dates back to at least the early 20th century. Formalin-fixed, paraffin-embedded lung tissue from victims of the 1918 influenza virus pandemic, as well as lung tissue biopsies taken from those buried in permafrost, have likewise suggested the probable origin (novel re-assortment of avian and human flu viruses), characteristics (hemagglutinin (HA) and polymerase genes contributed to its virulence), and genetic variation (evidence for early adaptation to humans in HA) from the causative agent of one of the deadliest pandemics in human history [12,115,116,117,118]. Influenza virus sequences obtained from a formalin-fixed, ethanol-preserved North American wild goose collected in 1917, however, suggested that the 1918 HA segment was not directly acquired from birds, but had likely already circulated for some time in humans or other mammals [119]. Poliovirus (PV) RNA fragments were retrieved from fifty-year old formalin-fixed, paraffin-embedded spinal cord samples of Norwegian poliomyelitis fatalities dating to 1951–1952 [120]. Subsequent sequencing suggested that the outbreak could most likely be attributed to wild type PV1. A further RNA virus sequenced from fixed autopsy material is measles morbillivirus (MeV), one of the most infectious viruses known. As MeV infection induces life-long sterilizing immunity, the virus needs large groups of naïve hosts to sustain itself. Although an effective vaccine is available, measles continues to break out, as it did in the past. After retrieval of an almost complete MeV genome from a 1912 formalin-fixed lung specimen, Düx et al. calculated the entry of MeV, likely from a bovine precursor, into the human population as early as the 6th century BCE, a time period that saw the rise of big cities, supplying the virus with its required number of targets [121].

From the texts and inscriptions of early human civilizations up to contemporary scientific publications, ever since the invention of writing, people have documented evidence of disease and disaster. For instance, the ancient Greek tragedian Sophocles and historian and general Thucydides described the Plagues of Thebes (430–420 BCE) and Athens (430–426 BCE), respectively, while accounts of the Justinian Plague (541–543 CE) destroying the Roman empire, appeared in Arabic, Greek, Latin, and Syriac works [158]. Several ancient Egyptian “medical” papyri, such as the Ebers, Smith, and Hearst papyri, describe disease symptoms and epidemics that have been interpreted to refer to current pathogens [159]. (Critical) reviews of ancient epidemics in literature can be found in [159,160,161].

Outnumbering the ancient writings mentioning epidemic diseases are present papers speculating on the causative microorganisms from these accounts. However, not only are disease symptoms hardly diagnostic, translating or interpreting historical descriptions can be equally challenging. For instance, the Septuagint (LXX) and the Masoretic Text (MT), used in Old Testament translations, differ significantly as to the account of the plague of Ashdod (1190, or 1141 BCE), as described in Samuel 5:6–12. The coincident arrival of mice (or rats?) is not described in MT, although later on in both texts, five golden rats (or mice) must be provided as a sacrifice to free the town’s inhabitants, both young and old, from the plague [162]. The disease symptoms of that plague are nowadays translated as tumors, or boils, of the groin, which together with the rats would make an excellent case for bubonic plague as a putative cause (discussed in [163]). However, if those rats were mice, the case would be much weaker. Regarding pathology, the original texts mention “boils” or “sores” on the anus [162], which is quite unlike Y. pestis infection as we know it.

Descriptions of the Plague of Athens and the Justinian Plague inspired researchers to explore their actual causes. After analysis of three teeth samples from a mass burial pit at Kerameikos, Greece, the likely cause of the first was determined to be typhoid fever due to Salmonella enterica infection [11]. That claim was refuted by others, based on subsequent phylogenetic analysis of the initial results [164]. Teeth samples from individuals buried in Bavaria, Germany at the time of the Justinian pandemic, which affected large parts of Europe, were confirmed by two ancient DNA laboratories to contain DNA from an early, independent, lineage of Yersinia pestis, making it the first plague pandemic in history [52,144]. Ethiopian sources from the 13th–15th centuries also contain references to epidemics, possibly of plague, one of which wiped out so many people, that “no one was left to bury the death” [57,165]. Arabic, Egyptian, Syriac, and Sudanese texts likewise refer to epidemic diseases, which may or may not have been plague [57]. The 16th century Mexican “catastrophy” burials, which tested positive for S. enterica serovar Paratyphi C DNA, were backed up by evidence from Aztec painted manuscripts, the Codex Telleriano-Remensis and the Codex en Cruz, in which drawings of stacks of corpses and of figurines displaying symptoms of nose bleeds (epistaxis) and a body rash, respectively, are believed to represent an epidemic starting around 1544 in New Spain [75]. Indeed, epistaxis, and a so-called “rose spot” rash are clinical symptoms of enteric fever, although they are not the most prominent ones. Age, geography, and the specific causative organism are known to widely influence the signs and complications of enteric fever [166,167].

From around the 16th century onwards, parish registers in Europe kept track of baptisms, marriages, and deaths, and can be used as sources of unexpected or increased mortality in a neighborhood. Often, the churchyards related to the registers are known, and could be a source of ancient DNA, although many of them, and the skeletal remains, have been destroyed over time. Sometimes, records provide the cause of death, for instance, death registers from Bergen op Zoom, the Netherlands, provide this information from 1779 onwards. From that source, for instance, we learn that there was a particular severe outbreak of smallpox in the summer of 1790, with more than half of fatalities in July and August of that year being due to young children dying from it [168]. In the winter of 1797–1798, smallpox raged again, with an even higher mortality this time. In the autumn of 1807, there was a deadly epidemic of something called “nervous fever” (Febris nervosa epidemica), most likely enteric fever, to which about 45% of adult deaths were attributed. Interestingly, no casualties were seen in children. Likewise, from the 16th century onwards, medical doctors in Europe and the USA started to prepare lists and publications on health and disease from their own observations. Many 19th century publications were, for instance, devoted to cholera, then a new disease that was spreading around the world and causing deadly pandemics [169]. From medical publications listing the annual diseases prevalent in town and villages, it is obvious that in these times of poverty, crowding, malnutrition, and lack of vaccines, infectious diseases were common. A problem with the historical descriptions, however, is that the names of the maladies, or the symptoms listed, do not always ring a bell in our present times. Rubeola, scarlatina, and febres intermittentes likely indicate measles, scarlet fever, and malaria, respectively, but the medical conditions that would nowadays best describe morbi inflammatorii pectoris and erysipelas intestinorum, as given in 19th century publications, are difficult to ascertain [170]. In addition, biological samples connected to the reports are likely to be rare. An exception is the analysis of a wet intestinal sample derived from a 1849 victim of the second cholera pandemic in the USA, from which a complete genome of Vibrio cholerae has been reconstructed [112].