key: cord-0802433-4ipimv1a authors: Kraberger, Simona; Serieys, Laurel; Fountain-Jones, Nicolas; Packer, Craig; Riley, Seth; Varsani, Arvind title: Novel smacoviruses identified in the faeces of two wild felids: North American bobcat and African lion date: 2019-06-22 journal: Arch Virol DOI: 10.1007/s00705-019-04329-3 sha: a98278e14dd8ec57deb815bc5f5acb1bdd967f4f doc_id: 802433 cord_uid: 4ipimv1a Smacoviruses are small circular single-stranded DNA viruses that appear to be prevalent in faeces of a range of animals and have also been found in a few insect species. In this study, we report the first viral genomes from faeces of free-roaming wild felids on two continents. Two smacoviruses were recovered from the faeces of two North American bobcats (Lynx rufus), and one was recovered from an African lion (Panthera leo). All three genomes are genetically different, sharing 59-69% genome-wide sequence identity to other smacoviruses. These are the first full smacovirus genome sequences associated with a large top-end feline predator, and their presence in these samples suggests that feline faeces are a natural niche for the organisms that these viruses infect. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00705-019-04329-3) contains supplementary material, which is available to authorized users. The Smacoviridae are a family of circular single-stranded DNA viruses that have been identified in the faecal excrement of many mammals and birds, and a few arthropods [19] . The genomes of smacoviruses range from 2.3 to 3 kb in size and contain at least two bidirectionally transcribed open reading frames (ORFs), which encode a replicationassociated protein (Rep) and a capsid protein (CP). These ORFs are separated by two intergenic regions, one of which contains an origin of replication. At present, more than 170 smacoviruses have been documented, with six established genera; Bovismacovirus, Cosmacovirus, Dragsmacovirus, Drosmacovirus, Huchismacovirus and Porprismacovirus. Recently, it was shown that DNA matching a smacovirus was found in the archaeon "Candidatus Methanomassiliicoccus intestinalis", suggesting that smacoviruses may infect faeces-dwelling archaea [7] . Despite the broad range of animals with which smacoviruses have been associated, none have been documented in a member of the family Felidae. Felids, both domestic and wild, are known to harbour a multitude of viruses, including; feline immunodeficiency virus [13, 20] , feline foamy virus [4, 11] , feline leukaemia virus [5, 16] , feline anellovirus [9, 21] , feline gammaherpesvirus [2, 18] , feline coronavirus [15] , and several more. In two separate studies, faecal samples were collected from bobcats (Lynx rufus) in California, USA, and African lions (Panthera leo) in the Serengeti National Park in Tanzania. Faecal samples described in this study were collected from three wild cats. One bobcat sample was collected during necropsy, the other from a live trap on the day of capture, and the African lion sample was collected shortly after defecation was observed. Viral DNA was extracted, and circular molecules were enriched from the faecal samples according to a previously described protocol [12] . Enriched DNA was sequenced on an Illumina 2500 platform, and contigs were generated through de novo assembly of paired-end reads using metaSPAdes v. 3.12.0 [1] . Three contigs with similarities to smacoviruses were identified in BLASTx searches and amplified from the three individual samples using abutting primers (Table 1 ) by PCR. The amplicons were then cloned and sequenced using the Sanger method [12] . We have tentatively named the three novel smacoviruses reported in this study "Lynx rufus associated smacovirus 1" (LruSmV 1, MK796234), "Lynx rufus associated smacovirus 2" (LruSmV 2, MK796235) and "Panthera leo associated smacovirus" (PlSmV, MK796236). Their genomes are 2,435 [14] . Genome-wide pairwise comparison showed that the three genomes share 54-57% nucleotide sequence identity. With all other smacoviruses, the closest relative for each is as follows: LruSmV 1 shares 59% sequence identity with chimpanzee associated porprismacovirus 2 (GQ351273) [3] , LruSmV 2 shares 61% sequence identity with Chlorocebus cynosuros associated smacovirus (LC386199), and PlSmV shares 67% sequence identity with sheep faeces associated smacovirus 3 (KT862219) [17] (Supplementary Data 1). Rep and CP amino acid comparison with all smacoviruses showed they share 39-63% sequence identity in Rep and 35-60% sequence identity in CP ( Fig. 1 and Supplementary data 1). Smacovirus Rep amino acid sequences were aligned using MAFFT [10] , and the resulting alignment was used to construct a maximum-likelihood phylogenetic tree using PhyML 3.0 [8] with the best-fit amino acid model rtREV+G+I+F selected using ProtTest [6] . Branches with less than 0.8 aLRT branch support were collapsed. The phylogenetic tree was rooted with the Rep sequences of the nanoviruses. Based on the taxonomic guidelines put forward by the Smacoviridae subcommittee for the International Committee on Taxonomy of Viruses [19] , the results of phylogenetic analysis and sequence comparisons support the assignment of each of these three smacoviruses to a distinct species. Phylogenetic analysis showed that the Rep of LruSmV 1 is divergent, forming a singleton clade that is closest to the huchismacoviruses. The Reps of LruSmV 2 and PlSmV group in a clade consisting of the members of the genus Porprismacovirus (Fig. 1) . The Rep proteins of LruSmV 2 and PlSmV share a common ancestor with porcine associated porprismacovirus 10 (KT862225) and sheep faeces associated smacovirus 3 (KT862219), respectively (Fig. 1 ) [17] . It is worth noting that these two LruSmVs were recovered from the faeces of individual bobcats living in the Los Angeles area (California, USA), but they are genetically distinct from each other. If smacoviruses do in fact infect archaea [7] , this may indicate that these two distinct viruses are hosted by different archaeal species. Here, we describe the first smacoviruses associated with felid faeces and show that they are highly diverse, representing three distinct species. Continued research in this area is needed to determine the true hosts of these viruses and to elucidate what relationship they have to other felid viruses and those infecting other animals. TTT CAG CAA GCT AGT AAG AAG GTG TTC ATC GATGG TGT ATT CAT CCT TCC ATT ACC TTC TCT ATA TCCGC Bobcat Lynx rufus smacovirus 2 2,595 CAA CTT ACT GGT GAT TCG TCC TCT AAT TAT CCG AGA TAC ATT CTC ATA CAC ACC CTG ACC ATA 39 39 39 39 40 39 40 39 40 39 41 39 39 37 38 41 39 39 34 39 38 42 41 42 43 41 42 40 42 42 40 38 39 47 47 43 42 42 42 42 42 42 42 42 42 42 43 42 42 43 42 40 36 44 46 46 45 46 45 45 46 46 45 46 45 46 46 46 46 47 47 44 42 43 46 45 44 41 41 41 40 41 43 50 49 49 46 48 50 50 50 50 50 51 53 52 50 50 51 51 52 51 51 52 51 49 49 49 49 51 51 51 52 52 52 51 52 54 54 42 42 43 43 43 43 42 48 46 47 47 39 40 39 39 39 39 39 40 40 39 39 40 40 40 39 39 36 40 39 42 39 39 44 44 44 43 44 44 44 44 42 36 39 47 47 41 39 40 40 39 40 40 41 41 42 42 42 40 40 41 43 63 44 48 48 46 46 46 46 50 50 47 45 45 47 47 47 45 46 46 44 43 43 39 38 44 41 42 42 42 42 48 49 47 47 46 41 40 40 42 41 41 44 45 45 46 44 43 44 44 44 45 45 47 46 42 42 42 43 44 47 46 45 45 45 45 46 44 44 40 42 41 42 42 42 41 46 37 39 39 P lS m V L r u S m V 2 LruSmV 2 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 Pairwise amino acid rep identity (%) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing Felis catus gammaherpesvirus 1; a widely endemic potential pathogen of domestic cats Novel circular DNA viruses in stool samples of wild-living chimpanzees Clinical and molecular features of feline foamy virus and feline leukemia virus co-infection in naturally-infected cats Multiple introductions of domestic cat feline leukemia virus in endangered florida panthers ProtTest 3: fast selection of best-fit models of protein evolution CRISPR analysis suggests that small circular single-stranded DNA smacoviruses infect Archaea instead of humans New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0 Whole genome sequencing and phylogenetic analysis of feline anelloviruses MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform Complete genome sequences of two novel puma concolor foamy viruses from california Identification of circular single-stranded DNA viruses in faecal samples of Canada lynx (Lynx canadensis), moose (Alces alces) and snowshoe hare (Lepus americanus) inhabiting the Colorado San Juan Mountains Feline immunodeficiency virus cross-species transmission: implications for emergence of new lentiviral infections SDT: a virus classification tool based on pairwise sequence alignment and identity calculation The paradox of feline coronavirus pathogenesis: a review Feline leukemia virus disease outcomes in a domestic cat breeding colony: relationship to endogenous FeLV and other chronic viral infections Circular replication-associated protein encoding DNA viruses identified in the faecal matter of various animals in New Zealand Novel gammaherpesviruses in North American domestic cats, bobcats and pumas: identification, prevalence and risk factors Smacoviridae: a new family of animal-associated single-stranded DNA viruses Epidemiology of feline foamy virus and feline immunodeficiency virus infections in domestic and feral cats: a seroepidemiological study Identification and genomic characterization of a novel species of feline anellovirus Acknowledgements Sample collection of bobcat faecal material was supported by the National Park Service and Santa Monica Mountains Fund, and Laurel Serieys was supported by a NSF graduate student fellowship.