https://cpw.cvlcollections.org/files/original/3886bb954c6a3549bfa69318df440174.pdf 94fb867ed7a556912ff0abcc06741c19 PDF Text Text The research in this publication was partially or fully funded by Colorado Parks and Wildlife. Dan Prenzlow, Director, Colorado Parks and Wildlife • Parks and Wildlife Commission: Marvin McDaniel, Chair • Carrie Besnette Hauser, Vice-Chair Marie Haskett, Secretary • Taishya Adams • Betsy Blecha • Charles Garcia • Dallas May • Duke Phillips, IV • Luke B. Schafer • James Jay Tutchton • Eden Vardy �Virology 562 (2021) 176–189 Contents lists available at ScienceDirect Virology journal homepage: www.elsevier.com/locate/virology Complex evolutionary history of felid anelloviruses Simona Kraberger a, *, Laurel EK. Serieys b, c, Cécile Richet a, Nicholas M. Fountain-Jones d, Guy Baele e, Jacqueline M. Bishop c, Mary Nehring f, Jacob S. Ivan g, Eric S. Newkirk h, John R. Squires i, Michael C. Lund a, Seth PD. Riley j, Christopher C. Wilmers b, Paul D. van Helden k, Koenraad Van Doorslaer l, Melanie Culver m, n, Sue VandeWoude e, Darren P. Martin o, Arvind Varsani a, p, ** a The Biodesign Center of Fundamental and Applied Microbiomics, School of Life Sciences, Center for Evolution and Medicine, Arizona State University, Tempe, AZ, 85287, USA b Environmental Studies, University of California, Santa Cruz, CA, 95064, USA c Institute for Communities and Wildlife in Africa, Department of Biological Sciences, University of Cape Town, Private Bag X3, Rondebosch, Cape Town, 7701, South Africa d School of Natural Sciences, University of Tasmania, Hobart, 7001, Australia e Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium f Department of Microbiology, Immunology & Pathology, Colorado State University, Fort Collins, CO, 80523, USA g Colorado Parks and Wildlife, 317 W. Prospect Rd., Fort Collins, CO, 80526, USA h Speedgoat Wildlife Solutions, Missoula, MT, 59801, USA i US Department of Agriculture, Rocky Mountain Research Station, 800 E. Beckwith Ave., Missoula, MT, 59801, USA j Santa Monica Mountains National Recreation Area, National Park Service, Thousand Oaks, CA, 91360, USA k DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research/SAMRC Centre for TB Research/Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, 7505, South Africa l School of Animal and Comparative Biomedical Sciences, The BIO5 Institute, Department of Immunobiology, Cancer Biology Graduate Interdisciplinary Program, UA Cancer Center, University of Arizona, Tucson, AZ, 85724, USA m U.S. Geological Survey, Arizona Cooperative Fish and Wildlife Research Unit, University of Arizona, Tucson, AZ, 85721, USA n School of Natural Resources and the Environment, University of Arizona, Tucson, AZ, 85721, USA o Computational Biology Group, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, 7925, South Africa p Structural Biology Research Unit, Department of Integrative Biomedical Sciences, University of Cape Town, 7925, Cape Town, South Africa A R T I C L E I N F O A B S T R A C T Keywords: Bobcat Puma Caracal Canada lynx Domestic cat Torque teno virus Anelloviridae Anellovirus infections are highly prevalent in mammals, however, prior to this study only a handful of anello­ virus genomes had been identified in members of the Felidae family. Here we characterise anelloviruses in pumas (Puma concolor), bobcats (Lynx rufus), Canada lynx (Lynx canadensis), caracals (Caracal caracal) and domestic cats (Felis catus). The complete anellovirus genomes (n = 220) recovered from 149 individuals were diverse. ORF1 protein sequence similarity network analysis coupled with phylogenetic analysis, revealed two distinct clusters that are populated by felid-derived anellovirus sequences, a pattern mirroring that observed for the porcine anelloviruses. Of the two-felid dominant anellovirus groups, one includes sequences from bobcats, pumas, do­ mestic cats and an ocelot, and the other includes sequences from caracals, Canada lynx, domestic cats and pumas. Coinfections of diverse anelloviruses appear to be common among the felids. Evidence of recombination, both within and between felid-specific anellovirus groups, supports a long coevolution history between host and virus. * Corresponding author. ** Corresponding author. The Biodesign Center of Fundamental and Applied Microbiomics, School of Life Sciences, Center for Evolution and Medicine, Arizona State University, Tempe, AZ, 85287, USA. E-mail addresses: simona.kraberger@asu.edu (S. Kraberger), arvind.varsani@asu.edu (A. Varsani). https://doi.org/10.1016/j.virol.2021.07.013 Received 8 July 2021; Received in revised form 22 July 2021; Accepted 23 July 2021 Available online 29 July 2021 0042-6822/© 2021 Elsevier Inc. All rights reserved. �S. Kraberger et al. Virology 562 (2021) 176–189 1. Introduction 2. Materials and methods Anelloviruses (also referred to as torque teno viruses) are small nonenveloped circular single-stranded negative sense DNA viruses in the Anelloviridae family (Biagini, 2009; Biagini et al., 2011; Lefkowitz et al., 2018). This family is currently comprised of ~30 genera, all of which have constituent species that have been sampled exclusively in mam­ mals, with the exception of gyroviruses which are primarily associated with birds (Biagini, 2009; Biagini et al., 2011; Kraberger et al., 2021; Lefkowitz et al., 2018; Varsani et al., 2021). Many anelloviruses have yet to be taxonomically classified. Anellovirus genomes range in size from ~2.0 to 3.9 kb and typically encode three genes referred to as ORF1, ORF2 and ORF3, the latter two of which produce several different viral proteins through alternative splicing (Kaczorowska and van der Hoek, 2020). Although anelloviruses have genomes that are among the smallest and simplest of known animal-infecting viruses, little is known about the functions of these genes. Based on the arginine-rich region found in the ORF1, which is a feature also found in the capsid proteins of distantly related ssDNA viruses in the family Circoviridae, it is thought this protein may be involved in replication and packaging of the viral DNA (Kaczorowska and van der Hoek, 2020). First discovered in a human patient from Japan in 1997 (Nishizawa et al., 1997), anelloviruses have subsequently been identified in non-human primates (Catroxo et al., 2008; Hrazdilova et al., 2016; Spandole et al., 2015), pinnipeds (Crane et al., 2018; Fahsbender et al., 2017), birds (Rijsewijk et al., 2011; Sauvage et al., 2011), pigs (Ara­ mouni et al., 2013; Bigarre et al., 2005), pandas (Zhang et al., 2017), rodents (de Souza et al., 2018; Khalifeh et al., 2021; Nishiyama et al., 2014), and many more hosts. Prevalence studies have revealed that anelloviruses are ubiquitous in many mammalian host populations, and present across a range of tissue types. For example, estimates of the prevalence of anellovirus infections in humans range from 5 % to 90 % (Kaczorowska and van der Hoek, 2020), with anelloviral DNA being detectable in blood, brain, gut tissues and faeces (Arze et al., 2021; Kraberger et al., 2020b; Ng et al., 2017; Pollicino et al., 2003; Tisza et al., 2020). Although not conclusively shown to cause disease, several studies have found potential associations with; hepatitis (Al-Qahtani et al., 2016), cancer (Pan et al., 2018), a range of infections with other viruses (Biagini et al., 2003; McElvania TeKippe et al., 2012; Smits et al., 2012; Yu et al., 2020), and several other disease states. A hypothesis that is currently supported is that anelloviruses may have a presently unde­ termined commensal role in the biology of their hosts (Kaczorowska and van der Hoek, 2020). Given the high prevalence of anelloviruses in apparently healthy hosts and difficulties with culturing these viruses, it has remained very difficult to study interactions between these viruses and their hosts. Despite having no known causal association with disease states, the ubiquity of infections and the small sizes of anellovirus genomes have meant that large numbers of anellovirus genomes have been charac­ terized for several mammalian host groups. Although known to occur in felids, they have not been extensively investigated in this host group. Prior to this study only twelve anellovirus genome sequences were available in GenBank from domestic cats (Felis catus) in Japan (Okamoto et al., 2002), China (Zhang et al., 2016; Zhu et al., 2011), France (Biagini et al., 2007), USA (unpublished) and Czech Republic (Jarosova et al., 2015). One unpublished sequence is also available from a Brazilian ocelot (Leopardus pardalis). Interestingly, these felid anellovirus se­ quences and that of their hosts present surprisingly incongruent phy­ logenies, which suggests that domestic cats, and possibly other felids too, harbour diverse anelloviruses. In order to investigate this in greater depth, we undertook a comprehensive study characterising anellovirus genomes from puma (Puma concolor), bobcats (Lynx rufus), Canada lynx (Lynx canadensis), caracals (Caracal caracal) and domestic cats. Further, we analysed these feline-derived anelloviruses to determine their di­ versity, recombination patterns and ancestral relationships. 2.1. Ethics statement Mountain lion samples were obtained as part of an ongoing collab­ orative study with Colorado Parks and Wildlife (CPW) and provided to Colorado State University (CSU) for viral screening. Domestic cat sam­ ples were collected by collaborating shelters and sent to CSU. Blood samples from these studies have been archived and used for several studies. CSU and CPW Institutional Animal Care and Use Committees reviewed and approved this work prior to commencement (CSU IACUC protocol 05–061A). This work was performed in accordance with United States Department of Agriculture Animal Welfare Act and The Guide for the Care and Use of Laboratory Animals. CSU Public Health Assurance number is D16-00345. CSU is accredited by AAALAC International. Bobcats from California were captured, handled, collared, and samples collected under approval of the Institutional Animal Care and Use Committee (IACUC) of the University of California, Santa Cruz (Seril1701). Scientific collecting permits were authorized by the Cali­ fornia Department of Fish and Wildlife (Aromas, SCP-11968; Coyote Valley, SCP-13565). Further those from the Los Angeles area were approved by the University of California, Los Angeles Office of Animal Research Oversight of UCLA (Protocol ARC#2007-167-12). Scientific collecting permits were authorized by the California Department of Fish and Wildlife (SCP-9791). Caracal handling was approved by the University of Cape Town Animal Ethics Committee (2014/V20/LS), Cape Nature (AAA007-01470056), and South Africa National Parks (SANParks; 2014/CRC/ 2014–017, 2015/CRC/2014–017, 2016/CRC/2014–017, 2017/CRC/ 2014–017). 2.2. Nucleic acid extraction and high-throughput sequencing Faecal and/or blood samples were collected from domestic cats, Canada lynx, bobcats and mountain lions from North America, and blood collected from caracals from South Africa between the years of 1999–2018 (see Table 1 for details). Faecal samples were processed according to a protocol described in Steel et al. (2016). 200 μl of faecal sample resuspensions or blood samples were individually processed using the High Pure Viral Nucleic Acid Kit (Roche Diagnostics, USA) to extract viral DNA according to the manufacturer’s specifications. In order to target the amplification of anelloviruses, TempliPhi™ (GE Healthcare, USA) was used to preferentially amplify circular DNA through rolling-circle amplification (RCA). Circular amplified DNA was then pooled according to sample type, host and location, and used to prepare Illumina sequencing libraries with a TruSeq Nano DNA kit (Illumina, USA) and sequenced on an Illumina HiSeq 4000 at Psomagen Inc. USA. Raw reads were de novo assembled using metaSPAdes v3.12.0 (Bankevich et al., 2012) and contigs >1000 nts analysed using BLASTx (Altschul et al., 1990) against a RefSeq viral protein database NCBI GenBank website to identify anellovirus-like contigs. Based on the identified anellovirus-like de novo assembled contigs, back-to-back primers were designed and used with Kapa HiFi Hotstart DNA polymerase (Kapa Biosystems, USA) in a polymerase chain reaction (PCR) to recover full anellovirus genomes from individual samples. Primers used to amplify the anellovirus genomes are provided in Sup­ plementary Data 1 and cycling conditions were applied as per manu­ facturer’s instructions and primer annealing temperatures. PCR products were resolved on 0.7% agarose gels, ~2–2.7 kb amplicons were gel excised, purified, ligated into pJET 1.2 vector (Thermo Fisher Sci­ entific, USA) and transformed into XL blue Escherichia coli competent cells. Recombinant plasmids with viral sequences were purified and Sanger sequenced at Macrogen Inc. Korea. 177 �S. Kraberger et al. Virology 562 (2021) 176–189 Table 1 Summary of sample information for all anelloviruses recovered in this study including source/host, feline demographic information, sampling location, year, type, anellovirus species grouping and accession number. Sample ID Host Sex Sampling location Sample date Sample type Virus Accession # 55 85 105 LSF3 Bobcat Bobcat Bobcat Bobcat unknown unknown unknown F Colorado, USA Colorado, USA Colorado, USA California, USA unknown unknown unknown 2003 Blood Blood Blood Faeces LSF4 Bobcat M California, USA 2004 Faeces LSF9 Bobcat unknown California, USA unknown Faeces LSF12 Bobcat M California, USA 2008 Faeces LSF14 Bobcat M California, USA 2008 Faeces LSF19 LSF30 Bobcat Bobcat M M California, USA California, USA 2010 2010 Faeces Faeces LSF32 LSF33 Bobcat Bobcat F F California, USA California, USA 2010 2010 Faeces Faeces LSF44 LSF48 LSF55 LSF56 LSF58 LSF59 LSF61 LSF62 LSF123 LSF125 Bobcat Bobcat Bobcat Bobcat Bobcat Bobcat Bobcat Bobcat Puma Puma M F unknown unknown F M M M M M California, USA California, USA California, USA California, USA California, USA California, USA California, USA California, USA California, USA California, USA 2011 2014 2014 2014 2011 2011 2011 2011 2008 2010 Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces LSF126 LSF128 Puma Puma F unknown California, USA California, USA 2015 2014 Faeces Faeces LSF129 Puma unknown California, USA 2011 Faeces LSF132 Puma unknown California, USA unknown Faeces LSF134 LSF140 LSF141 Puma Bobcat Bobcat unknown unknown M California, USA California, USA California, USA 2012 2012 2017 Faeces Faeces Faeces LSF147 Bobcat M California, USA 2017 Faeces LSF48 LSF68 LSF69 LSF70 LSF72 LSF73 LSF77 LSF89 Bobcat Bobcat Bobcat Bobcat Bobcat Bobcat Bobcat Bobcat F M unknown unknown unknown unknown unknown unknown California, USA California, USA California, USA California, USA California, USA California, USA California, USA California, USA 2014 2012 unknown 2012 2012 2012 2010 unknown Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces LSF91 LSF101 LSF107 LSF112 LSF117 LSF140 LSF145 LSF150 LSF153 LSF154 LSF156 LSF165 LSF170 Bobcat Bobcat Bobcat Bobcat Bobcat Bobcat Bobcat Bobcat Bobcat Bobcat Bobcat Bobcat Bobcat unknown unknown unknown unknown unknown unknown M F F M M M M California, USA California, USA California, USA California, USA California, USA California, USA California, USA California, USA California, USA California, USA California, USA California, USA California, USA 2009 2010 2010 2010 2011 2012 2017 2017 unknown 2017 2009 2011 2010 Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces TTFV5 TTFV5 TTFV5 TTFV20 TTFV18 TTFV5 TTFV19 TTFV5 TTFV5 TTFV23 TTFV5 TTFV5 TTFV19 TTFV25 TTFV5 TTFV5 TTFV24 TTFV5 TTFV5 TTFV18 TTFV5 TTFV26 TTFV24 TTFV24 TTFV24 TTFV5 TTFV24 TTFV24 TTFV18 TTFV5 TTFV18 TTFV18 TTFV19 TTFV19 TTFV19 TTFV21 TTFV19 TTFV20 TTFV19 TTFV25 TTFV27 TTFV19 TTFV19 TTFV3 TTFV19 TTFV19 TTFV19 TTFV25 TTFV5 TTFV25 TTFV20 TTFV24 TTFV20 TTFV20 TTFV18 TTFV20 TTFV18 TTFV5 TTFV24 TTFV20 TTFV18 TTFV5 TTFV5 TTFV24 TTFV23 TTFV5 TTFV20 TTFV19 TTFV5 TTFV24 TTFV5 TTFV5 TTFV18 MT537965 MT537966 MT537967 MT538039 MT538040 MT538041 MT538042 MT538043 MT538044 MT538045 MT538046 MT538049 MT538050 MT538051 MT538052 MT538053 MT538054 MT538055 MT538056 MT538060 MT538061 MT538062 MT538064 MT538065 MT538066 MT538073 MT538074 MT538075 MT538076 MT538077 MT538078 MT538079 MT538080 MT538081 MT538083 MT538084 MT538085 MT538086 MT538087 MT538088 MT538089 MT538090 MT538091 MT538092 MT538093 MT538094 MT538095 MT538096 MT538097 MT538098 MT538099 MT538100 MT538101 MT538102 MT538103 MT538104 MT538105 MT538106 MT538107 MT538108 MT538109 MT538110 MT538111 MT538112 MT538113 MT538114 MT538115 MT538116 MT538117 MT538118 MT538119 MT538122 MT538124 (continued on next page) 178 �S. Kraberger et al. Virology 562 (2021) 176–189 Table 1 (continued ) Sample ID Host Sex Sampling location Sample date Sample type Virus Accession # LSF176 UoA1 UoA2 UoA3 UoA4 Bobcat Puma Puma Puma Puma unknown unknown unknown unknown unknown California, USA Sonora, Mexico Sonora, Mexico Sonora, Mexico Sonora, Mexico 2011 2013 2014 2014 2014 Faeces Faeces Faeces Faeces Faeces UoA9 Puma unknown Sonora, Mexico 2014 Faeces x183 x262 x269 x271 x272 x906 Bobcat Bobcat Bobcat Bobcat Bobcat Bobcat M M M M M F California, USA Colorado, USA Colorado, USA Colorado, USA Colorado, USA California, USA 2000 2008 2008 2008 2008 2007 Blood Blood Blood Blood Blood Blood TTFV20 TTFV22 TTFV23 TTFV19 TTFV23 TTFV21 TTFV3 TTFV3 TTFV5 TTFV19 TTFV5 TTFV19 TTFV5 TTFV19 TTFV19 TTFV20 TTFV5 TTFV5 TTFV5 TTFV20 TTFV26 TTFV20 TTFV24 TTFV24 TTFV24 TTFV20 TTFV25 TTFV5 TTFV5 TTFV5 TTFV5 TTFV19 TTFV3 TTFV20 TTFV18 TTFV18 TTFV5 TTFV25 TTFV20 TTFV19 TTFV20 TTFV19 TTFV5 TTFV24 TTFV5 TTFV5 TTFV19 TTFV23 TTFV19 TTFV20 TTFV5 TTFV5 TTFV20 TTFV5 TTFV19 TTFV5 TTFV5 TTFV5 TTFV23 TTFV19 TTFV19 TTFV24 TTFV19 TTFV19 TTFV19 TTFV8 TTFV9 TTFV9 TTFV9 TTFV9 TTFV10 TTFV9 TTFV8 TTFV9 TTFV9 TTFV8 MT538125 MT538131 MT538132 MT538134 MT538135 MT538136 MT538137 MT538138 MT538140 MT538141 MT538142 MT538143 MT538144 MT538145 MT538146 MT538147 MT538047 MT538048 MT538148 MT538149 MT538152 MT538153 MT538120 MT538121 MT538154 MT538155 MT538156 MT538157 MT538158 MT538159 MT538057 MT538160 MT538161 MT538163 MT538164 MT538058 MT538059 MT538165 MT538166 MT538167 MT538123 MT538168 MT538063 MT538169 MT538170 MT538171 MT538172 MT538173 MT538174 MT538175 MT538176 MT538177 MT538067 MT538068 MT538178 MT538069 MT538070 MT538071 MT538179 MT538072 MT538180 MT538181 MT538182 MT538183 MT538184 MT537968 MT537969 MT537970 MT537971 MT537972 MT537973 MT537974 MT537975 MT537976 MT537977 MT537978 Faeces x913 x1172 x1294 X1296 Bobcat Bobcat Bobcat Bobcat M F M F California, USA California, USA California, USA California, USA 2007 2010 2009 2009 Blood Blood Blood Blood Faeces x1299 Bobcat M California, USA 2010 Blood x1301 X1303 X1307 Bobcat Bobcat Bobcat M F M California, USA California, USA California, USA 2010 2010 2010 Blood Blood Blood X1350 Puma F Nevada, USA 2010 Faeces Blood x1508 x1509 Bobcat Bobcat F F California, USA California, USA 2011 2010 Blood Blood Faeces x1511 Bobcat M California, USA 2010 Blood x1513 Bobcat F California, USA 2010 x1514 M F California, USA California, USA California, USA 2010 x1516 Bobcat Bobcat Bobcat 2010 Blood Faeces Blood Faeces Blood x1520 x1522 Bobcat Bobcat M F California, USA California, USA 2010 2010 Blood Blood x1523 Bobcat M California, USA 2010 Blood x1525 x1529 Bobcat Bobcat F M California, USA California, USA 2010 2010 Blood Blood Faeces x1532 Bobcat F California, USA 2010 Blood Faeces x1535 Bobcat M California, USA 2011 x1542 x1543 x1574 Bobcat Bobcat Bobcat F M California, USA California, USA California, USA 2011 2011 2009 Blood Faeces Blood Blood Blood x1576 CCB9 CCB10 CCB16 CCB21 CCB22 CCB23 Bobcat Caracal Caracal Caracal Caracal Caracal Caracal M M M F M F F California, USA Western Cape, South Western Cape, South Western Cape, South Western Cape, South Western Cape, South Western Cape, South Africa Africa Africa Africa Africa Africa 2011 2014 2016 2016 2017 unknown 2015 Blood Blood Blood Blood Blood Blood Blood CCB25 CCB27 CCB28 CCB29 Caracal Caracal Caracal Caracal M M M M Western Cape, Western Cape, Western Cape, Western Cape, Africa Africa Africa Africa 2015 2015 2015 2015 Blood Blood Blood Blood South South South South (continued on next page) 179 �S. Kraberger et al. Virology 562 (2021) 176–189 Table 1 (continued ) Sample ID Host Sex Sampling location Sample date Sample type Virus Accession # CCB31 CCB36 CCB37 CCB38 CCB39 CCB40 CCB41 CCB42 CCB43 CCB44 CCB46 CCB48 CCB49 CCB52 Caracal Caracal Caracal Caracal Caracal Caracal Caracal Caracal Caracal Caracal Caracal Caracal Caracal Caracal F M F M M M M F M M F F F M Western Cape, Western Cape, Western Cape, Western Cape, Western Cape, Western Cape, Western Cape, Western Cape, Western Cape, Western Cape, Western Cape, Western Cape, Western Cape, Western Cape, Africa Africa Africa Africa Africa Africa Africa Africa Africa Africa Africa Africa Africa Africa 2015 2015 2015 2015 2016 2015 2015 2016 2016 2016 2016 2016 2016 2016 Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood CCB55 CCB56 Caracal Caracal F M Western Cape, South Africa Western Cape, South Africa CCB59 CCB60 Caracal Caracal F M Western Cape, South Africa Western Cape, South Africa CCB62 CCB63 CLB2 Caracal Caracal Canada lynx M F Western Cape, South Africa Western Cape, South Africa Quebec, Canada 2016 2016 2016 2018 2017 2017 2018 2018 2003 Blood Blood Blood Blood Blood Blood Blood Blood Blood CLB3 CLB4 Canada lynx Canada lynx F F Quebec, Canada Quebec, Canada 2004 2004 Blood Blood CLB5 CLB7 CLB8 CLB9 CLB10 Canada lynx Canada lynx Canada lynx Canada lynx Canada lynx F F F F F Quebec, Canada Quebec, Canada Quebec, Canada Colorado, USA Quebec, Canada 2004 2002 2002 2011 2002 Blood Blood Blood Blood Blood CLB11 Canada lynx M Alaska, USA 1999 Blood CLB12 CLB13 CLB14 Canada lynx Canada lynx Canada lynx F M M Colorado, USA Alaska, USA Yukon, Canada 2011 1999 1999 Blood Blood Blood CLB16 Canada lynx F Colorado, USA 2006 Blood CLB17 Canada lynx F British Columbia, Canada 2005 Blood CLB18 Canada lynx M British Columbia, Canada 2005 Blood CLB20 Canada lynx M British Columbia, Canada 2005 Blood CLB21 Canada lynx M Yukon, Canada 2006 Blood CLB22 Canada lynx M Yukon, Canada 2006 Blood CLB24 LSF125 MAF4 MAF5 MAF11 MAF12 Canada lynx Puma Canada lynx Canada lynx Canada lynx Canada lynx M M M M F M Yukon, Canada California, USA Montana, USA Montana, USA Montana, USA Montana, USA 2010 2010 2018 2018 2018 2018 Blood Faeces Faeces Faeces Faeces Faeces UoA2 X1259 X1498 X1272 UoA20 Puma Domestic Cat Domestic Cat Domestic Cat Bobcat unknown M M F unknown Sonora, Mexico Colorado, USA Colorado, USA Colorado, USA Sonora, Mexico 2014 2010 2011 2010 2014 Faeces Blood Blood Blood Faeces TTFV8 TTFV9 TTFV9 TTFV9 TTFV9 TTFV9 TTFV9 TTFV9 TTFV9 TTFV9 TTFV9 TTFV8 TTFV9 TTFV10 TTFV7 TTFV9 TTFV10 TTFV11 TTFV8 TTFV9 TTFV9 TTFV10 TTFV9 TTFV8 TTFV10 TTFV10 TTFV10 TTFV15 TTFV15 TTFV10 TTFV10 TTFV15 TTFV8 TTFV12 TTFV10 TTFV16 TTFV8 TTFV7 TTFV10 TTFV14 TTFV8 TTFV15 TTFV10 TTFV16 TTFV14 TTFV10 TTFV11 TTFV8 TTFV10 TTFV16 TTFV8 TTFV8 TTFV10 TTFV15 TTFV8 TTFV13 TTFV15 TTFV10 TTFV14 TTFV10 TTFV17 TTFV12 TTFV15 TTFV16 TTFV12 TTFV15 TTFV17 TTFV7 TTFV7 TTFV7 TTRodFV 1 MT537979 MT537980 MT537981 MT537982 MT537983 MT537984 MT537985 MT537986 MT537987 MT537988 MT537989 MT537990 MT537991 MT537992 MT537993 MT537994 MT537995 MT537996 MT537997 MT537998 MT537999 MT538000 MT538001 MT538002 MT538038 MT538003 MT538004 MT538005 MT538006 MT538007 MT538008 MT538009 MT538010 MT538011 MT538012 MT538013 MT538014 MT538015 MT538016 MT538017 MT538018 MT538019 MT538020 MT538021 MT538022 MT538023 MT538024 MT538025 MT538026 MT538027 MT538028 MT538029 MT538030 MT538031 MT538032 MT538033 MT538034 MT538035 MT538036 MT538037 MT538082 MT538126 MT538127 MT538128 MT538129 MT538130 MT538133 MT538150 MT538162 MT538151 MT538139 South South South South South South South South South South South South South South 180 �S. Kraberger et al. Virology 562 (2021) 176–189 2.3. Sequence assembly, annotation and network analyses 3. Results and discussion Contigs were assembled, annotated and datasets compiled in Gene­ ious v11.0.3 (Biomatters Ltd., New Zealand). Datasets of the ORF1 protein sequences of all the anellovirus genomes recovered in this study together with those from GenBank (downloaded 1 December 2020) were compiled and a sequence similarity network (SSN) was generated using EST-EFI (Gerlt et al., 2015) using a threshold of 75. Cytoscape (V3.8.1) (Shannon et al., 2003) was used to visualize ORF1 protein SSN. Three network clusters containing feline-derived sequences resulted from these analyses and these will hereafter be referred to as feline network cluster 1 (sequences originating from puma, bobcats and domestic cats), 2 (sequences originating from caracals, Canada lynx, puma and do­ mestic cats) and felid rodent network cluster 1 (sequences originating from rodent species and a bobcat faecal sample). 3.1. Characterisation of anelloviruses from five feline species In this study, a total of 220 complete anellovirus genomes were determined from blood or faecal samples from five felid species. These were recovered from bobcats (n = 117), Canada lynx (n = 42), caracals (n = 34), domestic cats (n = 3), and pumas (n = 24). One or more anellovirus genomes were recovered and characterized from 149 indi­ vidual animals: bobcats (n = 78) - from Mexico (n = 1) and USA (n = 77); Canada lynx (n = 23) - Canada (n = 14), USA (n = 8); caracals (n = 30) - all South Africa; domestic cats (n = 3) - all USA; pumas (n = 15) Mexico (n = 6), USA (n = 9). All of these samples were collected be­ tween 1999 and 2018, see Table 1 for full details. In all the anellovirus genomes the putative ORF1 and ORF2 open reading frames (ORFs) were identified and annotated. The genomes range in size from 1829–2653 nts, varying dramatically between felid species (Fig. 1A). Bobcats had the smallest anellovirus genomes on average ranging from 1829– 2156 nts (excluding the anellovirus which is most similar to rodent anelloviruses which is 2352 nts, MT538139, referred to as torque teno rodfelid virus 1). The largest average genome sizes are those from caracals 2397–2586 and Canada lynx 2429–2622 nts, and the two groups with the most variable genome sizes are those from domestic cats 2012–2653 nts and pumas 1974–2560 nts. With the exception of one isolate previously recovered from a domestic cat in China (KX262893) (Zhang et al., 2016) which has a genome of 2409 nts and three domestic cat isolates from the USA recovered in this study (MT538162, MT538151, MT538150) which have genomes of ~2600 nts, all other domestic cat genomes are ~2000 nt. Although the moun­ tain lion isolates exhibited a broad range of sizes, only two (MT538133 and MT538082) are ~2500 nts, while the remainder are ~2000 nts. 2.4. Recombination analyses Sequence UoA20_55_BC (MT538139) was excluded from these ana­ lyses because it was recovered from a bobcat faecal sample and is most closely related to anelloviruses found in rodents and might have there­ fore been prey-animal-associated. A full genome dataset of the felinederived anelloviruses, with the exception of UoA20_55_BC (MT538139) was aligned using MUSCLE (Edgar, 2004) and recombi­ nation analyses performed using RDP v5.5 (Martin et al., 2021). Se­ quences were set as circular with similar sequences auto-masked. Events were deemed as credible if they were detected by three or more of the seven recombination detection methods implemented in RDP5.5 with an associated p-value <0.05 and were supported by phylogenetic evidence. 2.5. Phylogenetic and pairwise analyses The compiled ORF1 protein sequence dataset was aligned using MAFFT (Katoh et al., 2002) and an approximate maximum-likelihood phylogenetic tree was constructed using FastTree (Price et al., 2010) with the default JTT + CAT substitution model (Jones et al., 1992; Si Quang et al., 2008), branches having less than 0.6 SH-like branch sup­ port were collapsed using TreeGraph2 v2.14 (Stover and Muller, 2010). Full genome sequence datasets were compiled for each of the three groups of isolates identified in the network analyses shown in Fig. 2. For feline network clusters 1 and 2, referred to as feline groups 1 and 2, recombination-free datasets were generated following recombination analyses. The third dataset, comprised of felid rodent network cluster 1 (a single bobcat-derived anellovirus together with rodent anellovirus sequences), referred to as felid rodent group 1, was aligned using MUSCLE (Edgar, 2004) but was not analysed for recombination, given how small this group is and genetically distant the members are. Maximum-likelihood phylogenies were then constructed for these three datasets. For the recombination-free sequences in the feline group 1 and 2 datasets, phylogenies were constructed using RAxML implemented in RDP5 (Martin et al., 2021) which explicitly accounts for large amounts of missing data (Stamatakis, 2014). A maximum-likelihood tree for the rodent group-1 dataset was constructed in Seaview (v4) (Gouy et al., 2010) using PhyML (Guindon et al., 2010) with the GTR + G substitu­ tion model. The phylogenetic trees were all midpoint rooted and branches with less than 0.6 bootstrap support were collapsed using TreeGraph2 v2.14 (Stover and Muller, 2010). A phylogram depicting the evolutionary history of Felidae and Viverridae was constructed with TimeTree (Hedges et al., 2015). Pairwise identity analyses were undertaken for the ORF1 nucleotide and amino acid datasets of feline group 1, 2, and rodent group 1 with SDT v1.2 (Muhire et al., 2014). 3.2. Distributions of pairwise genetic distances between anelloviruses within each felid species Anellovirus diversity within each felid species is high whether considering the ORF1 nucleotide sequence or the translated amino acid sequences (Fig. 1B–F). ORF1 nucleotide pairwise identities across all the felid species were 55–100 % with the distribution being slightly nar­ rower for bobcats at 59–100 %. The distribution of ORF1 amino acid pairwise distances, however, was much wider. Domestic cats and pumas harboured anelloviruses with the broadest ORF1 pairwise amino acid identity distribution, 24–99 % and 21–99 %, respectively. Interestingly they also made up ~8 % of the felid anelloviruses recovered, the fewest anellovirus genomes recovered from all feline species. The caracal and Canada lynx-derived anellovirus translated ORF1 sequences have similar pairwise distance distributions: 35–99 % and 37–99 %, respec­ tively. The translated ORF1 anellovirus sequences of bobcats, which incidentally have the most isolates recovered (~50 %), share between 44 and 100 % pairwise identity. According to the ICTV anellovirus taxonomy guidelines (Varsani et al., 2021) viruses exhibiting < 69 % ORF1 pairwise nucleotide simi­ larity can be considered distinct species. Based on this criterion, the feline anelloviruses discovered here fall into 24 tentative species groupings hereby named torque teno felid virus (TTFV) 3, 5, 7–27 (Supplementary Data 2, 3). The rodent-like anellovirus from a bobcat faecal sample (MT538139) was named torque teno rodfelid virus 1 (TTRFV-1) (Supplementary Data 4). 3.3. Anellovirus ORF1 protein phylogeny, network grouping and geographical distribution Phylogenetic analysis of ORF1 amino acid sequences of the newly determined sequences together with all those available in GenBank (downloaded 1 December 1 2020) indicated that the feline sequences fall into two major phylogenetic clades that correspond with the two 181 �S. Kraberger et al. Virology 562 (2021) 176–189 Fig. 1. Pairwise distributions of anelloviruses from each species show high within host diversity. A) Plot showing genome sizes of feline derived anelloviruses recovered from the five host species (this study and previously documented) and the different sample types. B–F) Pairwise distribution plots of anellovirus ORF1 nucleotide and amino acid sequences for the five feline species from which anelloviruses were recovered in this and previous studies. sequence clusters identified in the network analysis (Fig. 2A). These two groups of predominantly feline sequences also contain anellovirus se­ quences from Japanese palm civet (Paguma larvata) faecal samples. Given the faecal origin of these palm civet anelloviruses and the fact that palm civets are omnivores, these could have originated in a prey animal. Interestingly, palm civets are in the Viverridae family which is in the same suborder, Feliforma, as the Felidae family. The most recent com­ mon ancestor of the Viverridae and Felidae likely existed between 33 and 46 MYA (Hedges et al., 2015), which could indicate that the most recent common ancestor of the civet and feline anelloviruses might have 182 �S. Kraberger et al. Virology 562 (2021) 176–189 Fig. 2. Feline anelloviruses display a complex evolutionary history. A) The approximate maximum-likelihood phylogenetic tree on the left illustrates the evolutionary relationships of ORF1 proteins from all published anellovirus genomes with those recovered from felid species in this study. Protein similarity networks are shown next to the feline anellovirus clades, with each node in the network representing the ORF1 proteins from feline and palm civet derived anelloviruses. Species of sample origin are colour coded. The phylogram on the right shows the species and genus within the Feliforma Suborder; The Felidae and Viverridae families for which anelloviruses were recovered in this study, and other previously recovered anelloviruses in these groups are shown by a “*“. The numbers of isolates from each host is shown next to the general and Latin species names. B) Shows the regions from which feline and palm civet derived anelloviruses were sampled. 183 �S. Kraberger et al. Virology 562 (2021) 176–189 been an ancestral anellovirus that infected the common ancestor of cats and civets prior to their divergence (Fig. 2A). Feline grouping 1 is comprised of feline anellovirus sequences from bobcats and pumas from the USA and Mexico, domestic cats from Europe, Asia and the USA, and an ocelot from Brazil (Fig. 2). Feline grouping 2 is comprised of feline anellovirus sequences from Canada lynx from Canada, and Alaska and Montana, USA, caracal from South Africa, domestic cats from the USA, and pumas from Mexico and the USA. Given that only two of the 24 analysed puma anellovirus sequences (MT538133 and MT538082) cluster in feline grouping 2, and that these sequences are from faecal samples, it is also possible that they are derived from felid prey animals. Bobcat and Canada lynx anelloviruses sit in two separate groupings, which is noteworthy given these two felid species are close relatives in the same genus, thought to have diverged ~3.2–5.6 MYA (Hedges et al., 2015; O’Brien and Johnson, 2007) and which presently have overlapping geographic distributions. Fig. 3. Inter- and intra-species recombination events detected in bobcat and puma anelloviruses. Recombination-free maximum-likelihood phylogeny of the sequences in feline anellovirus group 1 derived from pumas, bobcats, domestic cats and an ocelot. Anellovirus species groupings are shown in the grey bar beside the tree. Recombination events are indicated in the linearized genome schematic. Accession numbers for each sequence are coloured based on the source/host and sampling location indicated by state and/or country codes. 184 �S. Kraberger et al. Virology 562 (2021) 176–189 Lastly, an anellovirus sequence from a bobcat faecal sample groups both phylogenetically and in a network grouping with rodent-derived anellovirus sequences in rodent grouping 1. Based on this finding, we hypothesise that this sequence was diet-derived from a rodent preyed upon by the bobcat. Domestication of cats has led to their high global prevalence; therefore, it is not surprising that the felid anelloviruses with the broadest geographic range and phylogenetic spread are those from do­ mestic cats. Interestingly, all those identified in previous studies from the USA, Europe and Asia (except for KX262893, which sits outside both groups) (Fig. 2B) fall in the same clade/network grouping, together with those from pumas and bobcats. Given the diverse nature of the domestic cat anelloviruses, their relationship with other feline anelloviruses and how underrepresented they are, more sampling is warranted to help unravel the most common ancestor. Both pumas and bobcats have overlapping geographical ranges in North America and therefore it is expected that their anelloviruses would fall in the same grouping. The Canada lynx anelloviruses are from samples collected in Canada and the USA, and despite dwelling in regions overlapping with puma and/or bobcats they cluster in feline grouping 2 with the caracal anelloviruses that were sampled in South Africa. 3.4. Feline anellovirus phylogenetic relationships and recombination patterns 3.4.1. Feline grouping 1 A recombination-free phylogenetic tree of feline grouping 1 anello­ viruses, including only those sequences sampled from members of the Felidae family was constructed using sequences from pumas, bobcats, domestic cats and an ocelot. These sequences fall into 12 tentative new species groupings and two that have already been established (Fig. 3) (Biagini, 2009; Varsani et al., 2021). Although anellovirus sequences from individual felid species do not form monophyletic clusters within this tree, it is nevertheless clear that anellovirus sequences sampled from particular felid species tend to cluster together. A noteworthy exception is one domestic cat sequence from the USA (JF304937), which clusters with bobcat-derived sequences in TTFV 5. Within this group it does however form a distinct branch and therefore it may be that as more domestic cat isolates from the USA are characterized we see the for­ mation of a related but separate grouping. Among the sequences from bobcats, pumas and domestic cats there were 24 recombination events detected (Fig. 3, Supplementary Data 5). Out of these 24 events, 13 involved parental sequences from different felid species (i.e., inter-species recombination events) and eleven involved parents belonging to the same species (i.e. intra-species recombination events). Recombination was only detected in one of the sequences from domestic cats (EF538877, sampled in France). Fig. 4. Three anelloviruses recovered from bobcat and puma faecal samples appear to be derived from prey animals. A) Recombination free maximumlikelihood phylogeny of the anelloviruses in feline group 2 sampled from caracals, Canada lynx and domestic cats. Anellovirus species groupings are displayed in the grey bar beside the tree. Detected recombination events within individual sequences are indicated in the linearized genome schematics. Accession numbers for each sequence are coloured based on the hosts from which they were sampled and locations are indicated with state and/or country codes. 185 �S. Kraberger et al. Virology 562 (2021) 176–189 3.4.2. Feline grouping 2 A feline grouping 2 phylogeny, with recombinant regions removed, indicated the evolutionary relationships between anellovirus sequences from caracals, lynx, bobcats, pumas and domestic cats (Fig. 4). These sequences fall into 11 tentative species (Fig. 4 and Supplementary Data 3). Similar to what was observed in feline group 1, the isolates cluster according to source host. A total of 17 recombination events were detected, all in the caracal and Canada lynx TTFV sequences. The Can­ ada lynx sequences from several locations across North America (Can­ ada, and Alaska and Montana, USA) are closely related and share evidence of common recombination events (Fig. 4; Supplementary Data 3). Of the 17 recombination events, eleven occurred between anellovi­ ruses in different species and six occurred between viruses in the same species. Eleven of the recombination events appear to have involved anelloviruses that seem to be associated with two different felid species (caracal and Canada lynx). This suggests, despite the strong associations of these viruses with the hosts from which they were isolated, the viruses infected another unsampled host or a common ancestor. It is unusual that there are two puma-derived sequences that are part of this group, given that the other puma-derived sequences fall in feline grouping 1. These puma-associated sequences were recovered from faecal samples collected in Mexico and the state of California, USA, and therefore are either a divergent outgroup of mountain lion-infecting anelloviruses or are derived from another felid species upon which the mountain lions have preyed upon. These two puma-associated se­ quences are most closely related to isolates recovered from a Canada lynx (sampled in Alaska), a caracal (sampled in South Africa) and a domestic cat (sampled in the USA) (Fig. 4). ORF1. Crucially, a very similar hot and cold spot pattern has been shown in anelloviruses from Weddell seals, suggesting that these patterns may be a general feature of anellovirus recombination (Fahsbender et al., 2017). 3.6. Feline rodent grouping 1 One anellovirus sequence from a bobcat faecal sample collected in Mexico was not related to the other feline derived TTFVs but instead is most closely related to anelloviruses identified from rodents (Fig. 6). Phylogenetically it sits just outside a rodent clade comprised of anello­ viruses from voles and mice from the UK (Nishiyama et al., 2014) and China (Du et al., 2018). This rodent-anellovirus-like sequence has an ORF1 that shares ~59–64 % pairwise nucleotide identity with those from anelloviruses associated with rodents (Supplementary Data 3) and therefore we have named it torque teno rodfelid virus 1 (TTRFV-1). Given that it was obtained from a faecal sample and is most closely related to anelloviruses from rodents, it is likely that this is a virus derived from a predated rodent (Fig. 6). 3.7. Co-infection dynamics Coinfections of multiple genetically diverse anelloviruses have been reported in in humans (Okamoto et al., 1999) and also other mammals several studies (Biagini et al., 2007; Fahsbender et al., 2017; Huang et al., 2010; Kraberger et al., 2020b; Leme et al., 2013; Nishiyama et al., 2014). This was also evident for the feline TTFVs where blood samples from 17 individuals harboured between two and four distinct anellovi­ rus species (Table 1). If viruses sampled from faecal samples are also included, an additional 21 individuals appear to harbour more than one TTFV species. Keeping in mind we cannot rule out the possibility that viruses sampled from faecal samples might have originated from prey animals. Out of the five felid species investigated, domestic cats were the only ones that did not display evidence of mixed infections involving multiple TTFV species in this study. This is however likely attributed to the low numbers of domestic cast samples analysed here (Table 1) as co-infections have been previously been shown in a domestic cat (Bia­ gini et al., 2007). For eight of the bobcats sampled in California we were able to obtain matching blood and faecal samples. For five of these animals, different TTFV species were detected in blood than were detected in the matched faecal samples (Table 1). There could be several possible explanations 3.5. Recombination hot and cold spots The recombination events detected within the feline anellovirus genomes were not randomly distributed (Figs. 3 and 4). Specifically, a statistically significant ~500 nt recombination breakpoint hotspot was evident in the non-coding region and a statistically significant cold-spot was evident throughout most of ORF1 (Fig. 5). The recombination hotspot colocalizes with the GC box (Kaczorowska and van der Hoek, 2020) that is highly conserved between anelloviruses and might there­ fore act as a homologous region that is particularly prone to template switching during replication (Martin et al., 2011). Conversely, it is possible that the high degrees of ORF1 diversity seen even within indi­ vidual TTFV species might impede homologous recombination within Fig. 5. Recombination hot- and coldspots within anellovirus genomes from feline groupings 1 and 2. The black vertical lines above the figure indicate the positions of detected recombination breakpoints and the black line in the plot indicates breakpoint numbers falling within a 200-nucleotide sliding window. The red regions indicate the breakpoint hotspot and the blue region the cold-spot. The light and dark grey areas respectively indicate 99 % the 95 % confi­ dence intervals of the expected degrees of breakpoint clustering under random recombination. 186 �S. Kraberger et al. Virology 562 (2021) 176–189 Fig. 6. Maximum-likelihood phylogeny of anelloviruses in rodent grouping 1 with one bobcat-derived anellovirus from a faecal sample. Anellovirus species are shown in grey bars. Accession numbers for the sequences are coloured based on the source/host and sampling locations are indicated with state/country codes. caracal infecting anelloviruses: it is extremely implausible given the geographic separation of these species that there are any transmissions of anelloviruses between them. The actual parents of these recombinants are much more likely to be other Canada lynx or caracal infecting anelloviruses that presently remain unsampled (Figs. 3 and 4 and sup­ plementary data 5). Genome size varied greatly between anelloviruses from each felid species (Fig. 1A). Although domestic cats harboured anelloviruses with a large range of genome sizes, if one disregards viruses from faecal sam­ ples that might represent prey-animal derived viruses, anelloviruses from each group of wild felid species fell within a narrower size range. It is likely that with more sampling there may be some additional corre­ lations between genome size and host/geographical location. With more sampling of anelloviruses in other felid species, it is likely that a clearer evolutionary picture will come to light. The high degree of anellovirus sequence heterogeneity seen within the felids is similar to that noted for anelloviruses from primates (Kac­ zorowska and van der Hoek, 2020; Spandole et al., 2015), pinnipeds (Crane et al., 2018; Fahsbender et al., 2017) and swine (Blois et al., 2014; Ghosh et al., 2020; Huang et al., 2010). The feline anelloviruses fall into 24 species-level groupings (Figs. 3, 4 and 6; Supplementary Data 2, 3 and 4), one of which is from a bobcat faecal sample and sits within a predominantly rodent-derived anellovirus group (Fig. 6). The diversity of ORF1 nucleotide sequences found within individual felid species was >54 % similarity, showing high diversity (Fig. 1). This, together with fact that the recombination analysis shows that the entire ORF1 region is a recombination cold spot, is consistent with the hy­ pothesis that there is an “arms race” between the host immune response and one or more of the proteins encoded by this ORF (such as the capsid protein): a dynamic that may have driven the diversification of ORF1 (Spandole et al., 2015). Coinfections of more than one anellovirus species add to the complexity of virus-host dynamics in the felids. When considering only virus sequences recovered from blood, 17 out of the 149 animals sampled were detectably coinfected with different anellovirus species (Table 1). Anellovirus co-infection should be considered in future studies to understand in greater depth the role these play in generating new recombinants. As more anellovirus genomes are recovered from felids the evolu­ tionary relationship between host and virus will be further elucidated, and this may also provide critical insight into whether these viruses are the friends or the foes of the species that they infect. for this, including the different anellovirus species having different cell tropisms, or low viral titres in one or the other of the sample types precluding their detection in both. For three bobcats the same anello­ virus species were detected in both blood and faeces suggesting that one might expect to find similar viruses in blood and faecal samples from the same animals. This expectation is reasonable given that anelloviruses are thought to be transmitted via the faecal-oral route (Kaczorowska and van der Hoek, 2020). 4. Concluding remarks Anelloviruses are abundant among mammals, display high degrees of genomic diversity and appear to have complex evolutionary histories characterized by frequent recombination and potential codivergence with their host species. Specifically, anelloviruses from different groups of host species such as the primates, pinnipeds or porcine cluster together phylogenetically potentially signifying long coevolutionary histories with their host lineages (Hrazdilova et al., 2016; Spandole et al., 2015). In the case of porcine anelloviruses, two distinct clusters are evident. In this study, we determine the diversity and evolutionary relationships of anelloviruses associated with members of the Felidae family by undertaking comprehensive analyses of 220 anellovirus ge­ nomes from mountain lions, bobcats, Canada lynx, caracals and do­ mestic cats. We determine that, as with the porcine anelloviruses, the felid anelloviruses fall into two distinct phylogenetic clades (Fig. 2) If indeed the anelloviruses are codiverging with their hosts this would imply that at least two the anellovirus lineages that infected the most recent com­ mon ancestor of the felids has today yielded the feline grouping 1 and 2 lineages. Other factors most likely play a role in anellovirus evolution including the geographic distribution of the felid species (both present day and historical), and their trophic interactions. Studies involving feline foamy virus, feline immunodeficiency virus and feline leukaemia virus have indicated that predation of felids on other felids can result in cross-species virus transmissions (Chiu et al., 2019; Franklin et al., 2007; Kraberger et al., 2020a). Within both felid anellovirus groups various recombination events were detected where identified parental se­ quences are found infecting different felid species. While superficially this might appear to represent evidence of felid anelloviruses infecting multiple different felid species, this is not necessarily the case. Specif­ ically, the parental sequences identified in our recombination analysis are not actual parents but rather the sequences in our sample that are most similar to the actual parents. This dynamic is best illustrated with the discovery of apparent recombinants between Canada lynx and 187 �S. Kraberger et al. Virology 562 (2021) 176–189 Disclaimer Appendix A. Supplementary data Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Supplementary data to this article can be found online at https://doi. org/10.1016/j.virol.2021.07.013. Accession numbers References Al-Qahtani, A.A., Alabsi, E.S., AbuOdeh, R., Thalib, L., El Zowalaty, M.E., Nasrallah, G. K., 2016. 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CRediT authorship contribution statement Simona Kraberger: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data curation, Writing – original draft, Writing – review & editing, Visualization. Laurel EK. Serieys: Methodology, Investigation, Writing – review & editing, Funding acquisition. Cécile Richet: Methodology, Investigation, Writing – review & editing. Nicholas M. Fountain-Jones: Methodol­ ogy, Investigation, Writing – review & editing. Guy Baele: Methodol­ ogy, Investigation, Writing – review & editing. Jacqueline M. Bishop: Investigation, Writing – review & editing. Mary Nehring: Methodology, Investigation, Writing – review & editing. Jacob S. Ivan: Methodology, Investigation, Writing – review & editing. Eric S. Newkirk: Methodol­ ogy, Investigation, Writing – review & editing. John R. Squires: Methodology, Investigation, Writing – review & editing. Michael C. Lund: Methodology, Investigation, Writing – review & editing. Seth PD. Riley: Methodology, Investigation, Writing – review & editing. Chris­ topher C. Wilmers: Methodology, Investigation, Writing – review & editing, Funding acquisition. Paul D. van Helden: Methodology, Investigation, Writing – review & editing, Funding acquisition. Koen­ raad Van Doorslaer: Methodology, Investigation, Writing – review & editing, Funding acquisition. Melanie Culver: Methodology, Investi­ gation, Writing – review & editing, Funding acquisition. Sue Vande­ Woude: Methodology, Investigation, Writing – review & editing, Funding acquisition. Darren P. Martin: Methodology, Formal analysis, Investigation, Writing – review & editing. Arvind Varsani: Conceptu­ alization, Methodology, Validation, Formal analysis, Investigation, Re­ sources, Data curation, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition. Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal re­ lationships which may be considered as potential competing interests: Acknowledgements We thank Robert Fitak for his independent review and input for improving this manuscript. Sample collection of bobcat faecal material was supported by the National Park Service and Santa Monica Moun­ tains Fund and Laurel Serieys was supported by an NSF graduate student fellowship. The caracal sampling was supported by Cape Leopard Trust and the Claude Leon Foundation. The puma and bobcat faecal material sampling from Mexico was supported by Primero Conservation (www. primeroconservation.org), Ron Thompson and Ivonne Cassaigne. G.B. acknowledges support from the Internal Fondsen KU Leuven/Internal Funds KU Leuven (Grant No. C14/18/094) and from the Research Foundation - Flanders (“Fonds voor Wetenschappelijk Onderzoek Vlaanderen”, G0E1420 N, G098321 N). The molecular work was sup­ ported by philanthropic donations from Arvind Varsani and the Klein family. 188 �S. Kraberger et al. Virology 562 (2021) 176–189 Okamoto, H., Takahashi, M., Nishizawa, T., Ukita, M., Fukuda, M., Tsuda, F., Miyakawa, Y., Mayumi, M., 1999. Marked genomic heterogeneity and frequent mixed infection of TT virus demonstrated by PCR with primers from coding and noncoding regions. Virology 259, 428–436. Pan, S., Yu, T., Wang, Y., Lu, R., Wang, H., Xie, Y., Feng, X., 2018. Identification of a torque teno mini virus (TTMV) in Hodgkin’s lymphoma patients. Front. Microbiol. 9, 1680. Pollicino, T., Raffa, G., Squadrito, G., Costantino, L., Cacciola, I., Brancatelli, S., Alafaci, C., Florio, M., Raimondo, G., 2003. 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For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Journal Articles Description An account of the resource CPW peer-reviewed journal publications Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Complex evolutionary history of felid anelloviruses Description An account of the resource <span><a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/anellovirus" title="Learn more about Anellovirus from ScienceDirect's AI-generated Topic Pages" class="topic-link" target="_blank" rel="noreferrer noopener">Anellovirus</a> infections are highly prevalent in mammals, however, prior to this study only a handful of anellovirus genomes had been identified in members of the <a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/felidae" title="Learn more about Felidae from ScienceDirect's AI-generated Topic Pages" class="topic-link" target="_blank" rel="noreferrer noopener">Felidae</a> family. Here we characterise anelloviruses in pumas (</span><span><em><a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/puma-concolor" title="Learn more about Puma concolor from ScienceDirect's AI-generated Topic Pages" class="topic-link" target="_blank" rel="noreferrer noopener">Puma concolor</a></em></span><span>), <a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/lynx-rufus" title="Learn more about bobcats from ScienceDirect's AI-generated Topic Pages" class="topic-link" target="_blank" rel="noreferrer noopener">bobcats</a> (</span><em>Lynx rufus</em><span>), <a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/lynx-canadensis" title="Learn more about Canada lynx from ScienceDirect's AI-generated Topic Pages" class="topic-link" target="_blank" rel="noreferrer noopener">Canada lynx</a> (</span><em>Lynx canadensis</em><span>), caracals (</span><em>Caracal caracal</em><span>) and domestic cats (</span><em>Felis catus</em><span>). The complete anellovirus genomes (n = 220) recovered from 149 individuals were diverse. ORF1 <a href="https://www.sciencedirect.com/topics/medicine-and-dentistry/peptide-sequence" title="Learn more about protein sequence from ScienceDirect's AI-generated Topic Pages" class="topic-link" target="_blank" rel="noreferrer noopener">protein sequence</a> similarity network  coupled with <a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/phylogeny" title="Learn more about phylogenetic analysis from ScienceDirect's AI-generated Topic Pages" class="topic-link" target="_blank" rel="noreferrer noopener">phylogenetic analysis</a>, revealed two distinct clusters that are populated by felid-derived anellovirus sequences, a pattern mirroring that observed for the porcine anelloviruses. Of the two-felid dominant anellovirus groups, one includes sequences from bobcats, pumas, domestic cats and an ocelot, and the other includes sequences from caracals, Canada lynx, domestic cats and pumas. <a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/coinfection" title="Learn more about Coinfections from ScienceDirect's AI-generated Topic Pages" class="topic-link" target="_blank" rel="noreferrer noopener">Coinfections</a> of diverse anelloviruses appear to be common among the felids. Evidence of recombination, both within and between felid-specific anellovirus groups, supports a long <a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/coevolution" title="Learn more about coevolution from ScienceDirect's AI-generated Topic Pages" class="topic-link" target="_blank" rel="noreferrer noopener">coevolution</a> history between host and virus.</span> Bibliographic Citation A bibliographic reference for the resource. Recommended practice is to include sufficient bibliographic detail to identify the resource as unambiguously as possible. Kraberger, S., L. E. K. Serieys, C. Richet, N. M. Fountain-Jones, G. Baele, J. M. Bishop, M. Nehring, J. S. Ivan, E. S. Newkirk, J. R. Squires, M. C. Lund, S. P. D. Riley, C. C. Wilmers, P. D. van Helden, K. Van Doorslaer, M. Culver, S. VandeWoude, D. P. Martin, and A. Varsani. 2021. Complex evolutionary history of felid anelloviruses. Virology 562:176–189. <a href="https://doi.org/10.1016/j.virol.2021.07.013" target="_blank" rel="noreferrer noopener">https://doi.org/10.1016/j.virol.2021.07.013</a> Creator An entity primarily responsible for making the resource Kraberger, Simona Serieys, Laurel E.K. Richet, Cécile Fountain-Jones, Nicholas M. Baele, Guy Bishop, Jacqueline M. Nehring, Mary Ivan, Jacob S. Newkirk, Eric S. Squires, John R. Lund, Michael C. Riley, Seth P.D. Wilmers, Christopher C. van Helden, Paul D. Van Doorslaer, Koenraad Culver, Melanie VandeWoude, Sue Martin, Darren P. Varsani, Arvind Subject The topic of the resource Bobcat Puma Caracal Canada lynx Domestic cat Torque teno virus <em>Anelloviridae</em> Extent The size or duration of the resource. 14 pages Date Created Date of creation of the resource. 2021-07-29 Rights Information about rights held in and over the resource <a href="http://rightsstatements.org/vocab/InC-NC/1.0/" target="_blank" rel="noreferrer noopener">In Copyright - Non-Commercial Use Permitted</a> Format The file format, physical medium, or dimensions of the resource application/pdf Language A language of the resource English Is Part Of A related resource in which the described resource is physically or logically included. Virology Type The nature or genre of the resource Article