Genome-wide characterization of endogenous retroviruses in the bat Myotis lucifugus reveals recent and diverse infections

Abstract

Bats are increasingly recognized as reservoir species for a variety of zoonotic viruses that pose severe threats to human health. While many RNA viruses have been identified in bats, little is known about bat retroviruses. Endogenous retroviruses (ERVs) represent genomic fossils of past retroviral infections and, thus, can inform us on the diversity and history of retroviruses that have infected a species lineage. Here, we took advantage of the availability of a high-quality genome assembly for the little brown bat, Myotis lucifugus, to systematically identify and analyze ERVs in this species. We mined an initial set of 362 potentially complete proviruses from the three main classes of ERVs, which were further resolved into 13 major families and 86 subfamilies by phylogenetic analysis. Consensus or representative sequences for each of the 86 subfamilies were then merged to the Repbase collection of known ERV/long terminal repeat (LTR) elements to annotate the retroviral complement of the bat genome. The results show that nearly 5% of the genome assembly is occupied by ERV-derived sequences, a quantity comparable to findings for other eutherian mammals. About one-fourth of these sequences belong to subfamilies newly identified in this study. Using two independent methods, intraelement LTR divergence and analysis of orthologous loci in two other bat species, we found that the vast majority of the potentially complete proviruses identified in M. lucifugus were integrated in the last ~25 million years. All three major ERV classes include recently integrated proviruses, suggesting that a wide diversity of retroviruses is still circulating in Myotis bats.

Fig 1

Potential complete ERV identification pipeline. We identified 25,239 LTR candidates with pairs of putative LTRs and TSD using LTRharvest, and all of them were annotated using LTRdigest. LTR candidates with canonical retroviral features were extracted as potential complete ERVs. We also used the independent pipeline MGEScan-LTR to identify potential complete ERVs. By combining the two independent pipelines, we identified 362 potential complete ERVs. They were further classified into 13 families based on RT domain phylogeny and into 86 subfamilies based on LTR sequence similarity.

Fig 2

Phylogeny of 13 MLERV families and reference retroviral sequences. Class I, class II, and class III ERVs are illustrated with blue, yellow, and green, respectively, and reference retroviral sequences are shown in red. MLERV families with neighbor joining bootstrap values higher than 95 are labeled with an asterisk at the root, and those with a bootstrap value of between 75 and 95 are labeled with a dot at the root. KoRV, koala retrovirus; GaLV, gibbon ape leukemia virus; MDEV, Mus dunni endogenous virus; PERV, porcine endogenous retrovirus; MuLV, murine leukemia virus; FELV, feline leukemia virus; BaEV, baboon endogenous virus; HERV, human endogenous retrovirus; ZFERV, zebrafish endogenous retrovirus; WDSV, walleye dermal sarcoma virus; SnRV, snakehead fish retrovirus; FeFV, feline foamy virus; HFV, human foamy virus; BLV, bovine leukemia virus; RSV, Rous sarcoma virus; GH-G18, Golden hamster intracisternal A-particle H18; RERV, rabbit endogenous retrovirus; HML, human MMTV-like; SRV, simian type D retrovirus; MMTV, mouse mammary tumor virus.

Fig 3

Comparison of ERV abundance and dynamics in different genomes. Different ERV classes and DNA transposons are labeled with different colors. (a) Comparison of percentages of genomes derived from different classes of ERVs in little brown bat and other mammals. (b) ERV and DNA transposon dynamics in little brown bat, human, and mouse genomes. Distance to consensus was corrected using the Jukes-Cantor model. Older elements are more distant from the consensus. The abundance is illustrated also, using the percentage of the genome.

Fig 4

Recent ERV invasion in M. lucifugus genome. (a) Most of the 362 complete ERVs invaded the M. lucifugus genome recently. Copy numbers of different ERV classes are shown in different colors; age was estimated by LTR pair divergence. (b) An example of an MLERV integration event (TG…CA) specific to the Myotis lineage. The MLERV is present in the M. davidii genome with target site duplication (TCTC), but a precise empty site is found in E. fuscus. (c) Orthologous loci of MLERVs in E. fuscus and M. davidii indicate that most of the ERVs invaded after divergence from E. fuscus, and ERVs were active before and after speciation of M. lucifugus from M. davidii. The speciation times between M. lucifugus and M. davidii and between M. lucifugus and E. fuscus were around 13 My and around 25 My, respectively. Numbers of ERV insertions are labeled between time points.