Virus Evolution: Current research and future directions Caister Academic Press 1 10 Paleovirology: The Study of Endogenous Viral Elements Amr Aswad & Aris Katzourakis * Department of Zoology, University of Oxford, The Tinbergen Building, South Parks Road, Oxford, OX1 3PS * Corresponding author: aris.katzourakis@zoo.ox.ac.uk Abstract Viruses sometimes heritably integrate into the genomes of their hosts, resulting in genomic features known as endogenous viral elements (EVEs). Using EVEs, the field of paleovirology investigates the long-term evolution of viruses and their impact on hosts. One of the fruitful outcomes of high throughput genomics is the widespread availability of whole genome data, offering the unprecedented opportunity to investigate EVEs at a large scale. In this chapter, we review the consequent surge in paleovirology research that can be traced to landmark work performed over half a century ago. We describe general principles of EVE biology and the main methodological techniques used to study them. We show how EVEs can only be understood within an evolutionary framework and we outline a generalized workflow for conducting paleovirology studies. We review exemplar paleovirological discoveries from each of the main viral groups, highlighting a range of approaches to paleovirology. We also discuss the major implications that certain discoveries and insights from paleovirology could have on our understanding of both virus and genome evolution. Finally, we consider the current limitations and potential pitfalls in paleovirology, and anticipate the possible future directions of this exciting and rapidly growing field. Background Usage of the term paleovirology to denote the study of ancient viruses has risen in the last decade, reflecting the establishment of a distinct specialty of evolutionary virology. The precise definition continues to expand since it has emerged from infancy, but predominantly involves the analysis of viral remnants in host genomes. Under the general term endogenous viral elements (EVEs), these ‘viral fossils’ are the consequence of a germline infection that results in the heritable genome integration of a virus (or part thereof) [1]. Alternatively, ancient viruses can be studied by examining the lasting effect they have had on the evolution of antiviral genes, a method described as indirect paleovirology in contrast to the direct approach of investigating EVEs [2]. The first EVEs were endogenous retroviruses identified in chickens and mice in the late 60’s and early 70’s, even before retroviruses were formally named [3]. Endogenous retroviruses (ERVs) are by far the most abundant group of EVEs due to an obligatory