100-My history of bornavirus infections hidden in vertebrate genomes

Abstract

Although viruses have threatened our ancestors for millions of years, prehistoric epidemics of viruses are largely unknown. Endogenous bornavirus-like elements (EBLs) are ancient bornavirus sequences derived from the viral messenger RNAs that were reverse transcribed and inserted into animal genomes, most likely by retrotransposons. These elements can be used as molecular fossil records to trace past bornaviral infections. In this study, we systematically identified EBLs in vertebrate genomes and revealed the history of bornavirus infections over nearly 100 My. We confirmed that ancient bornaviral infections have occurred in diverse vertebrate lineages, especially in primate ancestors. Phylogenetic analyses indicated that primate ancestors were infected with various bornaviral lineages during evolution. EBLs in primate genomes formed clades according to their integration ages, suggesting that bornavirus lineages infected with primate ancestors had changed chronologically. However, some bornaviral lineages may have coexisted with primate ancestors and underwent repeated endogenizations for tens of millions of years. Moreover, a bornaviral lineage that coexisted with primate ancestors also endogenized in the genomes of some ancestral bats. The habitats of these bat ancestors have been reported to overlap with the migration route of primate ancestors. These results suggest that long-term virus-host coexistence expanded the geographic distributions of the bornaviral lineage along with primate migration and may have spread their infections to these bat ancestors. Our findings provide insight into the history of bornavirus infections over geological timescales that cannot be deduced from research using extant viruses alone, thus broadening our perspective on virus-host coevolution.

Keywords: ancient viral infection; endogenous bornavirus-like element; paleovirology; vertebrate evolution; virus–host coevolutionary history.

Fig. 1.

Identification of EBLs in vertebrate genomes. (A) Schematic diagram of the procedure to identify EBLs. First, bornavirus-like sequences were detected from host genomes by tBLASTn, using extant bornaviral sequences as queries. Second, the detected bornavirus-like sequences were aligned with corresponding proteins of extant bornaviruses. Third, the bornavirus-like sequences were concatenated based on the host genomic locations and alignment positions with bornaviral proteins. When several bornavirus-like sequences were detected in the same genomic positions in a species genome, the sequence with higher reliability (low E-value score in the tBLASTn search) was used for EBL sequence reconstruction. (B) Alignment coverage plot of EBLs. The scales on the x-axis are marked at intervals of 100 amino acids. The y-axis indicates the number of EBLs identified in this study. (C) Numbers of EBLs in the host genomes. The x-axis indicates the vertebrate species, and the y-axis indicates the number of EBLs identified in the species genome. The bar color shows the bornaviral gene (Upper) or genus from which the EBL originated (Lower). (D) Phylogenetic trees of EBLs and extant bornaviruses. These trees were constructed by the maximum likelihood method using the amino acid sequences of EBLs and extant bornaviral proteins. The branch colors indicate the sequence groups: EBLs (gray), extant nyamivirus used as outgroup (black), extant orthobornaviruses (red), extant carboviruses (blue), and extant cultervirus (green). Colored arrows mark extant bornaviruses. Highlights correspond to the current bornaviral classifications: genus Orthobornavirus (light red), genus Carbovirus (light blue), and genus Cultervirus (light green). Representative supporting values (percent) are shown on branches. The scale bars indicate genetic distances (substitutions per site).

Fig. 2.

History of bornaviral integration events for ∼100 My. (A) Bornaviral integration events during vertebrate evolution. The evolutionary tree of vertebrates was obtained from the TimeTree database. The positions of pie charts on the tree indicate the lower limit ages of bornaviral integration events, and their size shows the number of events in each period. Annotations in the internal nodes on the tree indicate the common ancestors of Boreoeutheria (N1), Afrotheria (N2), Metatheria (N3), Primates (N4), Rodentia (N5), Tethytheria (N6), Passeriformes (N7), Australidelphia (N8), Simiiformes (N9), Lemuroidea (N10), Platyrrhini (N11), and Catarrhini (N12). The right panel shows the numbers of EBLs categorized into “EBLs with orthologs” or “EBLs without orthologs” in each host species. The definitions of these categories are described in the section titled Large-Scale Dating Analysis for Bornaviral Integration Ages. The bar colors show the viral genus as indicated on the left side of the tree. The representative hosts of extant bornaviruses are shown by animal silhouettes on the left side of the tree. (B) Schematic diagram of the geographical distributions of ancient bornaviruses and their hosts. The colored continents, except for yellow, indicate the continents where bornaviral endogenization may have occurred: Laurasia or Eurasia (blue), Africa (green), Antarctica (beige), Australia (dark brown), and South America (brown). The biogeography of hosts during their evolution was cited from previous reports (Dataset S2). Plate tectonic maps were downloaded from Ocean Drilling Stratigraphic Network (ODSN) Plate Tectonic Reconstruction Service (https://www.odsn.de/odsn/services/paleomap/paleomap.html).

Fig. 3.

Phylogenetic relationships of ancient bornaviruses that infected primate ancestors. (A–C) Phylogenetic analyses of ancient and modern bornaviral N genes. These trees were constructed by the maximum likelihood method using the amino acid sequences of EBLNs and extant bornaviral N proteins of genus Carbovirus (A), Orthobornavirus (B), or Cultervirus (C). Colored arrows mark extant bornaviruses. Square and triangle nodes indicate collapsed clades containing all and over half of the orthologs used in the phylogenetic analyses, respectively. Phylogenetic trees with all expanding nodes are available in SI Appendix, Fig. S3. The node colors indicate the host lineages of ancient bornaviruses or extant bornaviral genera as indicated in the lower right panel. Colored boxes highlight the bornaviral lineages endogenized during primate evolution. Number on the branches are bootstrap values (percent) based on 1,000 replications. The scale bars show genetic distances (substitutions per site). The genetic distance to distinguish extant bornaviral species is shown as the comparative standard for estimating the genetic diversity of ancient bornaviruses. (D) EBLN integration events during primate evolution. Arrowheads indicate the occurrence of ancient bornaviral integrations: orthobornaviral EBLN (red), carboviral EBLN (blue), and culterviral EBLN (green). The colors of highlighted boxes correspond to ancient bornaviral lineages shown in A–C.