Our relationship to the pathogens whose epidemics has been shaping human society has changed through time. Professor Tom Gilbert and his research colleagues are studying the role of human genetic variation in the process, using the second plague pandemic as their model. By Tom Gilbert, professor, DPhil, Section for Evolutionary Genomics, University of Copenhagen For most of our past humans lived as small, isolated huntergatherer groups, never staying in any one place for long. However about 10,000 years ago, things began to change. Humans underwent a technical revolution, and transitioned to sedentary farming societies where the first permanent settlements were born. This transition, the so-called Neolithic Revolution, had enormous impact on humankind. For example, it allowed both our population sizes and densities to increase by many orders of magnitude. This changed how we lived, structured, and managed our societies, and also how we performed the tasks required to live in this way, whether technological developments for farming, building, or conducting warfare. But, this transition was not without its cost. For example direct consequences included slavery, economic inequality, conflict, and genocide, while one indirect consequence of settling down into large, interconnected populations was the changes in the disease challenges that our ancestors faced. And of particular relevance to this Semper Ardens project “The population genomic consequences of the second plague pandemic”, the rise of infectious disease epidemics and pandemics. Pathogen epidemics An epidemic is the simultaneous widespread occurrence of an infectious disease in a community, a pandemic arises when its geographic range expands further still. Population size is a critical pre-requisite of both, as survivors of the infection often develop immunity against future infection. Thus there must be sufficient uninfected people in the population, to allow the transmission chain to continue. Should this not be the case, the pathogen simply dies out. Many are aware of these terms today as a result of the COVID-19 crisis, but there is nothing unusual about epidemics and pandemics. Every year the Influenza-A virus cycles across the globe, incapacitating hundreds of millions, and killing tens, if not hundreds, of thousands. Fortunately there are effective vaccines against Influenza A, thus reducing the pool of people who can be infected (slowing its transmission rate), and allowing society to function throughout the epidemic. Naturally prior to the advent of modern medical treatment the number of mortalities associated with pandemics was extremely severe. One of the most notable pandemics in recorded history is our particular focus, the second plague pandemic caused by the bacteria Yersinia pestis, that spread from East Asia to Europe in 1348, and repeatedly devastated its populations for over 300 years, with mortalities estimated to possibly >50% of the population in some cities. However in shear terms of numbers, there can be few, if any groups of pathogens, that lead to more infections a year than the common cold viruses. It has been estimated that the average kindergarten attending child gets a common cold >6 times a year. Thankfully, although there are no effective vaccines against cold viruses, unlike plague or Influenza, they are rarely lethal. Which raises a fundamental question, as to why? Many European cities have enormously rich human skeletal records – often stored in large warehouses such as this example at NTNU University Museum, Trondheim. Photo: Åshild Vågene Human-Pathogen coevolution Evolutionary theory predicts there could be several answers to this question. On the one hand, to continue to exist, a pathogen must successfully transmit. For example, any pathogen that kills its host too quickly, reduces its chances of transmission. Thus there may be selection on the pathogen to reduce its lethality, and indeed there is clear evidence from many pathogens that this is the case. However the pathogen is not the only actor in this play - the host may also be under strong selection to fight the pathogen. Quite simply, any host that is genetically predisposed to more efficiently clear the pathogen has an immediate evolutionary advantage, not least if the difference is between whether the host survives or not. Testing this attractive hypothesis is at our project’s heart, but obtaining direct evidence for it is hard to come by in humans, as selection typically has to happen over many generations in order to leave a clear footprint in the population. But how can we study the impact of a pathogen over these timescales? A genetic basis for survival? The solution is to turn from the present, and indeed look directly into the past. And this is the power of the so-called discipline of ‘palaeogenomics’ within which our project is based. Specifically, we are drawing on Scandinavia’s remarkable legacy of human skeletal remains, and in particular sampling human teeth from collections that contain material that fulfills several key criteria – there must be large numbers of well-preserved samples, from single geographic locations (for example the cities of Copenhagen, Lund and Trondheim). And within each location, there need to be both samples that are confidently dated to before, and after, the pandemic hit. Every year the Influenza-A virus cycles across the globe, incapacitating hundreds of millions, and killing tens, if not hundreds, of thousands Once samples are obtained, we then extract the fragmented DNA that is preserved within the teeth in our dedicated clean laboratories at the GLOBE Institute, and use Illumina sequencing techniques to sequence this DNA. The sequence reads are then used in two principal analyses that meet our overall aim of describing how our genomes changed as a result of the plague pandemic, exploring for evidence that there was a genetic basis to survival, and identifying what genes in the genome conferred this trait. Within each storage box lie meticulously documented human remains, representing a treasure trove for the study of ancient genomes. Photo: Anna Fotakis To do this we first computationally align the sequences to the human reference genome sequence, to identify specific nucleotide positions that vary in each ancient human. Secondly, we use a similar approach to identify epigenetic modifications (such as methylated cytosines) present within the samples – these actually tell us not just which genes are present, but which genes might have been active in the human prior to death. Both of these datasets then form the basis of our selection analyses. In essence we look for specific variants (conventional or epigenetic) that are at low frequency in the skeletal cohorts prior to, and at high frequency, following, the pandemic (or vice-versa). However, while changes in frequency may be a sign that there was selection on these variants due to the plague, other factors (such as rapid population crashes and recoveries) can also cause similar signals. Fortunately computational simulations can be used to tease these apart, and we are able to draw on the power of the Danish supercomputer Computerome 2.0 in this regard. Ultimately the output of our analyses is a list of candidate genetic modifications that clearly changed in frequency during the pandemic period, which we can both compare between geographic locations to look for convergent signals, and use reference databases to identify their putative functional role. For example, in this regard we anticipate that mutations linked to the immune system might be particularly common, and if so, providing strong evidence that some people were simply more resistant to the infection, and thus left more descendants into the present day.