BACKGROUND: Retroviruses infect a wide range of vertebrates. However, little is known about the diversity of retroviruses in basal vertebrates. Endogenous retrovirus (ERV) provides a valuable resource to study the ecology and evolution of retrovirus. FINDINGS: I performed a genome-scale screening for ERVs in the elephant shark (Callorhinchus milii) and identified three complete or nearly complete ERVs and many short ERV fragments. I designate these retroviral elements "C. milli ERVs" (CmiERVs). Phylogenetic analysis shows that the CmiERVs form three distinct lineages. The genome invasions by these retroviruses are estimated to take place more than 50 million years ago. CONCLUSIONS: My results reveal the extensive retroviral diversity in the elephant shark. Diverse retroviruses appear to have been associated with cartilaginous fishes for millions of years. These findings have important implications in understanding the diversity and evolution of retroviruses.

Recent discoveries indicate that the foamy virus (FV) (Spumavirus) ancestor may have been among the first retroviruses to appear during the evolution of vertebrates, demonstrated by foamy endogenous retroviruses present within deeply divergent hosts including mammals, coelacanth, and ray-finned fish. If they indeed existed in ancient marine environments hundreds of millions of years ago, significant undiscovered diversity of foamy-like endogenous retroviruses might be present in fish genomes. By screening published genomes and by applying PCR-based assays of preserved tissues, we discovered 23 novel foamy-like elements in teleost hosts. These viruses form a robust, reciprocally monophyletic sister clade with sarcopterygian host FV, with class III mammal endogenous retroviruses being the sister group to both clades. Some of these foamy-like retroviruses have larger genomes than any known retrovirus, exogenous or endogenous, due to unusually long gag-like genes and numerous accessory genes. The presence of genetic features conserved between mammalian FV and these novel retroviruses attests to a foamy-like replication biology conserved for hundreds of millions of years. We estimate that some of these viruses integrated recently into host genomes; exogenous forms of these viruses may still circulate.

Retroviruses infect a wide range of vertebrates. The understanding of the deep history and host distribution of retroviruses remains far from complete. Retroviruses can be integrated into their host genomes and occasionally become vertically inherited genomic loci. These integrated retroviruses, known as endogenous retroviruses (ERVs), provide "molecular fossils" for past retroviral infections and are useful for studying the deep history and ecology of retroviruses. ERVs are highly abundant in vertebrate genomes. However, endogenous foamy viruses and lentiviruses appear to be extremely rare. The primary focus of the research presented here is to discover and analyze novel endogenous foamy viruses and lentiviruses in animal genomes. Foamy virus has been thought to exclusively infect three placental mammal superorders (Laurasiatheria, Euarchontoglires, and Xenarthra). The discovery of endogenous foamy viral elements (CoeEFV) in the genome of the coelacanth (Latimeria chalumnae) extends the host range of foamy viruses to fish lineages (Appendix A). I demonstrate that foamy viruses have likely codiverged with their vertebrate hosts for more than 407 million years. The discovery of CoeEFV provides evidence for an ancient marine origin of retroviruses. Endogenous foamy virus-like elements (PSFVaye) were also identified within the genome of a Malagasy lemur, the aye-aye (Daubentonia madagascariensis) (Appendix B). Phylogenetic analysis shows that PSFVaye is divergent from all currently known simian foamy viruses, suggesting a potentially ancient association between foamy viruses and primate species. Another novel endogenous foamy virus (CaEFV) was identified in the genome of the Cape golden mole (Chrysochloris asiatica). The discovery of CaEFV reveals foamy virus infection in the placental mammal superorder Afrotheria and the long-term cospeciation between foamy viruses and placental mammals (Appendix C). Lentivirus has been thought to have a relatively recent origin. Endogenous lentivirus insertions (MELV) were discovered within the genomes of some species of the Weasel family (Mustelidae) (Appendix D). I verified the presence of MELV insertions in the genomes of several species of the Lutrinae and Mustelinae subfamilies but not the Martinae subfamily, which suggests that the lentiviral invasion likely occurred between 8.8 and 11.8 million years ago. Phylogenetic analysis suggests MELV might represent a novel lentiviral group. The discovery of MELV extends the host range of lentiviruses to the Caniformia. Endogenous lentiviruses (GvaELV) were also identified in the genome of the Sunda flying lemur (Galeopterus variegatus) (Appendix E). Phylogenetic analysis shows that GvaELV is a sister group of all known lentiviruses. The discovery of GvaELV might give a clue to the early evolution of lentiviral genome architecture. In summary, the discoveries and analyses of these novel ERVs provide important insights into the deep history and ecology of foamy viruses and lentiviruses as well as the retroviruses as a whole.

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Endogenous viral elements, or viral genomic fossils, have proven extremely valuable in the study of the macroevolution of viruses, providing important, and otherwise unobtainable, insights into the ancient origin of viruses, and how their ancestors might have co-evolved with their hosts in the distant past. This type of investigation falls within the realm of paleovirology—the study of ancient viruses. Investigations of extant viruses and paleovirological analyses, however, often give conflicting results, especially those concerning viral evolutionary rates and timescales. Reconciling these two types of analyses is a necessary step towards a better understanding of the overall long-term evolutionary dynamics of viruses. The main study system of this thesis is foamy viruses (FVs). FVs are characterised by their stable co-speciation history with their hosts, allowing their evolutionary dynamics to be modelled and investigated over various timescales. This unique evolutionary feature makes FVs one of the best subjects for connecting recent and ancient viral evolution. The work here reports the discovery of several endogenous mammalian FVs, and examines how mammalian FVs co-evolve with their hosts. Analyses reveal a co-diversifying history of the two that could be dated back to the basal radiation of eutherians more than 100 million years ago. However, a small number of ancient FV cross-species transmissions could still be found, mostly involving New World monkey FVs. Based on this extended FV-mammal co-speciation pattern, this thesis investigates the long-term evolutionary rate dynamics of FVs, and shows that the rate estimates of FV evolution appear to decrease continuously as the rate measurement timescale increases, following a power-law decay function. The work presented here also shows that this so-called 'time-dependent rate phenomenon' is in fact a pervasive evolutionary feature of all viruses, and surprisingly, the rate estimates of evolution of all viruses seem to decay at the same speed, decreasing by approximately half for every 3-fold increase in the measurement timescale. Based on this power-law rate-decay pattern, we could infer evolutionary timescales of modern-day lentiviruses that are consistent with paleovirological analyses for the first time. Finally, this thesis reports the discovery of basal FV-like endogenous retroviruses (FLERVs) in amphibian and fish genomes. Phylogenetic analyses reveal that the progenitors of ray-finned fish FLERVs co-diversify broadly with their fish hosts, but also suggest that there might have been several ancient viral cross-class transmissions, involving lobe-finned fish, shark, and frog FLERVs. Again, by using the power-law rate-decay model, analyses in this thesis suggest that this major retroviral clade has an ancient Ordovician marine origin, originating together with their jawed vertebrate hosts more than 450 million years ago. This finding implies that the origin of retroviruses as a whole must be in the early Paleozoic Era, if not earlier. The results presented here bridge ancient and recent viral evolution.