DNA metabarcoding is widely used to study prokaryotic and eukaryotic microbial diversity. Technological constraints limit most studies to marker lengths below 600 base pairs (bp). Longer sequencing reads of several thousand bp are now possible with third-generation sequencing. Increased marker lengths provide greater taxonomic resolution and allow for phylogenetic methods of classification, but longer reads may be subject to higher rates of sequencing error and chimera formation. In addition, most bioinformatics tools for DNA metabarcoding were designed for short reads and are therefore unsuitable. Here, we used Pacific Biosciences circular consensus sequencing (CCS) to DNA-metabarcode environmental samples using a ca. 4,500 bp marker that included most of the eukaryote SSU and LSU rRNA genes and the complete ITS region. We developed an analysis pipeline that reduced error rates to levels comparable to short-read platforms. Validation using a mock community indicated that our pipeline detected 98% of chimeras de novo. We recovered 947 OTUs from water and sediment samples from a natural lake, 848 of which could be classified to phylum, 397 to genus and 330 to species. By allowing for the simultaneous use of three databases (Unite, SILVA and RDP LSU), long-read DNA metabarcoding provided better taxonomic resolution than any single marker. We foresee the use of long reads enabling the cross-validation of reference sequences and the synthesis of ribosomal rRNA gene databases. The universal nature of the rRNA operon and our recovery of >100 nonfungal OTUs indicate that long-read DNA metabarcoding holds promise for studies of eukaryotic diversity more broadly.

Knowledge of Kobuvirus (Family Picornaviridae) infection in carnivores is limited and has not been described in domestic or wild carnivores in Africa. To fill this gap in knowledge we used RT-PCR to screen fresh feces from several African carnivores. We detected kobuvirus RNA in samples from domestic dog, golden jackal, side-striped jackal and spotted hyena. Using next generation sequencing we obtained one complete Kobuvirus genome sequence from each of these species. Our phylogenetic analyses revealed canine kobuvirus (CaKV) infection in all four species and placed CaKVs from Africa together and separately from CaKVs from elsewhere. Wild carnivore strains were more closely related to each other than to those from domestic dogs. We found that the secondary structure model of the IRES was similar to the Aichivirus-like IRES subclass and was conserved among African strains. We describe the first CaKVs from Africa and extend the known host range of CaKV.

The Desmodus rotundus endogenous betaretrovirus (DrERV) is fixed in the vampire bat D. rotundus population and in other phyllostomid bats but is not present in all species from this family. DrERV is not phylogenetically related to Old World bat betaretroviruses but to betaretroviruses from rodents and New World primates, suggesting recent cross-species transmission. A recent integration age estimation of the provirus in some taxa indicates that an exogenous counterpart might have been in recent circulation. The common vampire bat (Desmodus rotundus) is a phyllostomid bat species with a broad geographical distribution and lives in close proximity with humans and domestic animals (1). Recently described retroviruses in chiropterans are diverse, some potentially representing the oldest viral lineages in mammalian taxa (2-5). However, retroviral characterization has been restricted to bat species distributed in Eurasia, Africa, and Australia (2, 4, 5). Little is known about retroviruses in bats from the neotropics, and nothing is known about those in vampire bats. Endogenous retroviruses (ERVs) are present in the genomes of all vertebrates examined (2-13). As different species may share ERV sequences, it is assumed that, in many cases, an exogenous retrovirus infected the common ancestor of multiple species and became fixed in the genome prior to species divergence (14-16). Most ERVs are inactive "genetic fossils," whereas some may still retain the ability to transcribe active elements or can become reactivated, having potential health implications for the host (7,(17)(18)(19)(20). As retroviruses are transmitted primarily via blood-toblood contact, we postulate that vampire bat retroviruses are particularly prone to jumping from one species to another. In this study, we characterized an endogenous betaretrovirus present in D. rotundus and searched for evidence indicating cross-species transmission events within its evolutionary history.DrERV is a type D endogenous betaretrovirus. Genomic Illumina MiSeq shotgun sequencing from a population of free-ranging and captive D. rotundus bats from Mexico and the Berlin Zoological Garden (see Table S1 in the supplemental material) revealed the presence of a novel retrovirus, designated here Desmodus rotundus endogenous retrovirus (DrERV). Read assignment analysis showed that DrERV is homologous to squirrel monkey retrovirus (SMRV), a type D retrovirus found in New World squirrel monkeys (Saimiri genus) (21); they share a global percentage nucleotide similarity of 72% (E value, 0.0) as determined by BLAST analysis. To retrieve the complete DrERV genome and integration sites, reads were assembled against the SMRV genome through a combined mapping and de novo assembly approach using Bowtie version 2 0.2.2, Burrows-Wheeler Aligner (BWA) version 0.7.9, and Velvet 1.2.10 against the SMRV genome to build a consensus sequence (SMRV-H; GenBank accession number M23385) (22-24), and sequence gaps were covered by PCR (see Table S2 in the supplemental material). The full DrERV genome was ...