bioRxiv preprint doi: https://doi.org/10.1101/2020.05.16.100149; this version posted May 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Metagenomic identification of diverse animal hepaciviruses and 2 pegiviruses 3 4 5 Ashleigh F. Porter1, John H.-O. Pettersson1,2, Wei-Shan Chang1, Erin Harvey1, Karrie Rose3, 6 Mang Shi5, John-Sebastian Eden1,4, Jan Buchmann1, Craig Moritz6, Edward C. Holmes1 7 8 9 1. Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life and 10 Environmental Sciences and School of Medical Sciences, The University of Sydney, 11 Sydney, Australia. 12 2. Zoonosis Science Center, Department of Medical Biochemistry and Microbiology, 13 Uppsala University, Uppsala, Sweden. 14 3. Australian Registry of Wildlife Health, Taronga Conservation Society Australia, 15 Mosman, Australia. 16 4. Centre for Virus Research, Westmead Institute for Medical Research, Westmead, 17 Australia. 18 5. School of Medicine, Sun Yat-sen University, Guangzhou, China. 19 6. Research School of Biology, and Centre for Biodiversity Analysis, Australian National 20 University, Acton, ACT, Australia. 21 22 23 Address correspondence to: 24 Edward C. Holmes, 25 Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life and 26 Environmental Sciences and School of Medical Sciences, The University of Sydney, 27 Sydney, Australia 28 [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.16.100149; this version posted May 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 29 Abstract 30 31 The RNA virus family Flaviviridae harbours several important pathogens of humans and 32 other animals, including Zika virus, dengue virus and hepatitis C virus. The Flaviviridae are 33 currently divided into four genera - Hepacivirus, Pegivirus, Pestivirus and Flavivirus – each 34 of which have a diverse host range. Members of the genus Hepacivirus are associated with 35 a diverse array of animal species, including humans and non-human primates, other 36 mammalian species, as well as birds and fish, while the closely related pegiviruses have 37 been identified in a variety of mammalian taxa including humans. Using a combination of 38 meta-transcriptomic and whole genome sequencing we identified four novel hepaciviruses 39 and one novel variant of a known virus, in five species of native Australian wildlife, 40 expanding our knowledge of the diversity in this important group of RNA viruses. The 41 infected hosts comprised native Australian marsupials and birds, as well as a native gecko 42 (Gehyra lauta). The addition of these novel viruses led to the identification of a distinct 43 marsupial clade within the hepacivirus phylogeny that also included an engorged Ixodes 44 holocyclus tick collected while feeding on Australian long-nosed bandicoots (Perameles 45 nasuta). Gecko and avian associated hepacivirus lineages were also identified. In addition, 46 by mining the short-read archive (SRA) database we identified another five novel members 47 of Flaviviridae, comprising three new hepaciviruses from avian and primate hosts, as well as 48 two primate-associated pegiviruses. The large-scale phylogenetic analysis of these novel 49 hepacivirus and pegivirus genomes provides additional support for virus-host co- 50 divergence over evolutionary time-scales. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.16.100149; this version posted May 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 51 Introduction 52 As the vast majority of emerging infectious disease in humans are caused by viral zoonoses 53 [1], the identification and characterization of animal viruses is critical for identifying potential 54 disease reservoirs and providing models for the study of human viruses [2,3]. Two related 55 groups of viruses that have recently received considerable attention are the genera 56 Hepacivirus and Pegivirus from the family Flaviviridae of single-strand positive-sense RNA 57 viruses. Hepaciviruses infect a broad range of vertebrate hosts, including humans, non- 58 human primates [4,5], and a variety of other mammals including rodents [6-10], horses [11], 59 bats [12], and cows [13,14]. Hepaciviruses have also been detected in birds [15, 16], fish 60 and a variety of other vertebrates [17, 18]. Additionally, two hepacivirus-like sequences 61 have been identified in arthropods (a mosquito and tick), although their true host is 62 uncertain [19, 20]. Despite such a wide diversity of animal hosts, hepaciviruses remain 63 synonymous with liver infection, with the most notable example being human hepatitis C 64 virus (HCV). While some non-primate hepaciviruses have been well characterized, 65 particularly equine hepacivirus (also known as Hepacivirus AK, or non-primate hepacivirus, 66 NPHV) and canine hepacivirus (CHV), it seems likely that HCV, along with NPHV and CHV, 67 arose from a currently unknown zoonotic source, with rodents and bats suggested as 68 possible reservoirs [21-24]. 69 70 The genus Pegivirus contains 11 defined virus species known to infect humans and variety 71 of other mammals. In addition, a novel avian pegivirus was recently identified in a Common 72 myna bird (Acridotheres tristis) [25]. Human pegivirus (HPgV), previously known as GB virus 73 C, is known to infect humans but with an unproven link with clinical illness [26], despite 74 being identified in the brain tissue of patients with encephalitis [27]. Non-human primate 75 pegiviruses have been identified in both New World monkeys [28, 29] and Old World apes, 76 including chimpanzees [30-32], the latter of which (SPgVcpz) are closely related to HPgV 77 [33]. Other pegiviruses have been found in horses [34], bats [12, 35] and rodents [7,8]. 78 79 Despite their broad host range, all hepaciviruses and pegiviruses described to date possess 80 a single-strand positive-sense RNA genome and a large open reading frame encoding a 81 single polyprotein flanked by untranslated regions. Although this structure is common 82 among the Flaviviridae [36, 37], a more diverse set of genome structures, including 83 segmented forms, have been identified in invertebrate flaviviruses [18]. The multifunctional 84 polyprotein is cleaved by proteases to create ten proteins: three structural (core, E1, E2) 85 and seven non-structural (NS1, NS2, NS3, NS4a, NS4b, NS5a, NS5b) [24]. It is important to 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.16.100149; this version posted May 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 86 note that while many members of the genus Flavivirus are transmitted via arthropod 87 vectors, and it has been hypothesised that biting arthropod vectors may transmit some 88 hepaciviruses [37], there is currently no evidence of vector-borne transmission in either the 89 Hepacivirus or Pegivirus genera. Recently, however, a novel hepacivirus, Brushtail possum 90 hepacivirus, was discovered in brush-tailed possums (Trichosurus vulpecula), a native 91 Australian marsupial [38]. Similarly, Collins beach hepacivirus was identified in a tick (Ixodes 92 holocyclus) feeding on another Australian marsupial (long-nosed bandicoot, Perameles 93 nasuta) [19]. These data suggest that Australian native wildlife harbour a diversity of 94 hepaciviruses worthy of further exploration. 95 96 As hepaciviruses have traditionally been difficult to culture, the recent expansion of animal 97 hepaciviruses is largely due to the advent of unbiased high-throughput sequencing. This 98 expansion has greatly impacted our understanding of the diversity and evolution of this 99 important genus. Herein, we used a bulk RNA-sequencing (meta-transcriptomic) approach 100 to identify additional novel hepaciviruses in Australian wildlife and determine their 101 abundance. This approach has previously proven successful in an Australian context, 102 identifying a variety of viruses in Australian wildlife, including native Australian and invasive 103 species [19, 38-43]. To supplement this analysis, we mined the sequence read archive 104 (SRA) database for hepacivirus-like sequences. Previously generated RNA sequencing 105 data, such as those found in the SRA, are a relatively untapped resource for novel virus 106 discovery [44]. Through SRA-mining we identified three novel hepaciviruses and two novel 107 pegiviruses, again highlighting the power of genomics to identify novel viral sequences. 108 109 110 Materials and Methods 111 Animal ethics 112 The magpie lark and pelican were handled under a series of NSW Office of Environment 113 and Heritage Licences to Rehabilitate Injured, Sick or Orphaned Protected Wildlife 114 (#MWL000100542). Samples were collected under the Opportunistic Sample Collection 115 Program of the Taronga Animal Ethics Committee, and scientific licence SL100104 issued 116 by the NSW Office of Environment and Heritage. Ticks were removed from the long-nosed 117 bandicoot under the approval of the NSW Office of Environment and Heritage Animal Ethics 118 Committee (#000214/05) and scientific license SL100104. 119 120 Sample collection 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.16.100149; this version posted May 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.