Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses

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1 ACCEPTED MANUSCRIPT Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses Ci-Xiu Li, Mang Shi, Jun-Hua Tian, Xian-Dan Lin, Yan-Jun Kang, Liang-Jun Chen, Xin-Cheng Qin, Jianguo Xu, Edward C Holmes, Yong-Zhen Zhang DOI: Cite as: elife 2015; /eLife Received: 29 October 2014 Accepted: 27 January 2015 Published: 29 January 2015 This PDF is the version of the article that was accepted for publication after peer review. Fully formatted HTML, PDF, and XML versions will be made available after technical processing, editing, and proofing. Stay current on the latest in life science and biomedical research from elife. Sign up for alerts at elife.elifesciences.org

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4 MononegaviralesArenaviridaeBunyaviridae OphioviridaeOrthomyxoviridae

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6 ArenaviridaeBunyaviridae OrthomyxoviridaeMononegaviralesTenuivirus EmaravirusVaricosavirus ArenaviridaeBunyaviridae Nairovirus Tenuivirus Emaravirus OrthobunyavirusTospovirus

7 Heterodera glycinesprocotyla fluviatilis

8 BunyaviridaeOrthomyxoviridae Mononegavirales

9 Mononegavirales Phlebovirus

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16 de novo

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22 Journal of virology Bioinformatics PloS one Virology Annual review of phytopathology Molecular marine biology and biotechnology Nature biotechnology Systematic biology Current opinion in microbiology Molecular biology and evolution PLoS genetics

23 Nature methods Bioinformatics PloS one Journal of virology Journal of virology Proceedings of the National Academy of Sciences of the United States of America Nucleic acids research Bioinformatics Science Virology journal Journal of virology

24 Journal of virology Zhonghua liu xing bing xue za zhi = Zhonghua liuxingbingxue zazhi

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31 Aedes sp, Armigeres subalbatus, Anopheles sinensis, Culex quinquefasciatus, Culex tritaeniorhynchus Aedes albopictus, Armigeres subalbatus, Anopheles paraliae, Anopheles sinensis, Culex pipiens, Culex sp, Culex tritaeniorhynchus, Atherigona orientalis, Chrysomya megacephala, Lucilia sericata, Musca domestica, Sarcophaga dux, S. peregrina, S. sp Tabanidae Blattella germanica Gerridae Abraxas tenuisuffusa, Hermetia illucens, Chrysopidae, Coleoptera, Psychoda alternata, Diptera, Stratiomyidae HippoboscidaeCimex hemipterus Pseudothemis zonatanepidae Camponotus japonicus, Diplonychus sp, Asellus sp Psychoda alternata,velarifictorus micado, Crocothemis servilia, Phoridae, LampyridaeAphelinus sp Hyalopterus pruni, Aulacorthum magnolia, Dermacentor marginatus, Dermacentor sp, Haemaphysalis doenitzi, H. longicornis, H. sp, H. formosensis, Hyalomma asiaticum, Rhipicephalus microplus, Argas miniatus Hyalomma asiaticum Neoscona nautica, Parasteatoda tepidariorum, Plexippus setipes, Pirata sp, Araneae Exopalaemon carinicauda, Metapenaeus sp, Solenocera crassicornis, Penaeus monodon, Litopenaeus vannamei Capitulum mitella, Charybdis hellerii, C. japonica, Uca arcuata Polydesmidae

32 Hyalomma asiaticum Dermacentor sp Dermacentor sp Parasteatoda tepidariorum Atherigona orientalis Diptera Diptera, Abraxas tenuisuffusa, Chrysopidae Chrysopidae Argas miniatus Dermacentor marginatus Charybdis japonica, Charybdis lucifera, Charybdis hellerii Charybdis japonica Blattella germanica unidentified Hippoboscidae unidentified Hippoboscidae Culex tritaeniorhynchus, C. quinquefasciatus, Anopheles sinensis, Armigeres subalbatus Rhipicephalus microplus Culex tritaeniorhynchus, C. quinquefasciatus, Anopheles sinensis, Armigeres subalbatus, Aedes sp Hyalopterus pruni OR Aphelinus sp Hyalopterus pruni OR Aphelinus sp Hyalopterus pruni OR Aphelinus sp unidentified Hippoboscidae Haemaphysalis hystricis Musca domestica, Chrysomya megacephala Musca domestica, Sarcophaga sp Musca domestica Hippoboscidae Hippoboscidae Hippoboscidae Hyalomma asiaticum Haemaphysalis doenitzi Dermacentor marginatus Haemaphysalis hystricis Rhipicephalus microplus Hyalopterus pruni OR Aphelinus sp unidentified Araneae unidentified Gerridae Argas miniatus Psychoda alternata Anopheles sinensis Capitulum mitella, Charybdis japonica, Charybdis lucifera Argas miniatus Sarcophaga sp Gerridae Chrysomya megacephala, Atherigona orientalis Cimex hemipterus Diptera, Abraxas tenuisuffusa Camponotus japonicus Musca domestica, Sarcophaga sp Musca domestica Culex tritaeniorhynchus, C. quinquefasciatus, Aedes sp Hippoboscidae

33 Haemaphysalis doenitzi Dermacentor marginatus Haemaphysalis hystricis Neoscona nautica, Parasteatoda tepidariorum, Plexippus setipes Neoscona nautica, Parasteatoda tepidariorum Gerridae Hippoboscoidea Chrysopidae, Psychoda alternata Blattella germanica Ostrinia furnacalis Abraxas tenuisuffusa, unidentified diptera Culex tritaeniorhynchus, Anopheles sinensis, Culex quinquefasciatus Culex tritaeniorhynchus, Anopheles sinensis, Culex quinquefasciatus, Aedes sp Haemaphysalis sp Hyalomma asiaticum Dermacentor sp Haemaphysalis longicornis Rhipicephalus microplus Dermacentor marginatus Haemaphysalis hystricis Pararge aegeria Procotyla fluviatilis Ostrinia furnacalis unidentified Chrysopidae, unidentified Diptera unidentified Hippoboscoidea Asellus sp, unidentified Nepidae, Camponotus japonicus Phoridae Aulacorthum magnoliae unidentified Polydesmidae Culex quinquefasciatus Culex quinquefasciatus Anopheles sinensis Anopheles sinensis, Armigeres subalbatus Penaeus monodon Neoscona nautica, Parasteatoda tepidariorum, Plexippus setipes Atherigona orientalis, Chrysomya megacephala, Sarcophaga sp, Musca domestica Tabanidae Culex tritaeniorhynchus Cimex hemipterus Culex tritaeniorhynchus Armigeres subalbatus Penaeus monodon, Exopalaemon carinicauda Neoscona nautica, Pirata sp, Parasteatoda tepidariorum, Araneae Hyalopterus pruni OR Aphelinus sp Gerridae Polydesmidae Asellus sp

34 Atherigona orientalis, Chrysomya megacephala, Sarcophaga sp, Musca domestica Tabanidae Gerridae Neoscona nautica Diptera, Stratiomyidae Hippoboscoidea Hippoboscoidea Culex tritaeniorhynchus, Culex quinquefasciatus, Armigeres subalbatus Culex tritaeniorhynchus, Culex quinquefasciatus, Armigeres subalbatus Culex tritaeniorhynchus, Culex quinquefasciatus, Armigeres subalbatus Culex quinquefasciatus Anopheles sinensis, Culex quinquefasciatus Psychoda alternata Blattella germanica

35 Ixodes scapularis Tetranychus urticae Daphnia pulex Eurytemora affinis Hyalella azteca Lepeophtheirus salmonis Dendroctonus ponderosae Tribolium castaneum Aedes aegypti Anopheles spp. Culex quinquefasciatus Drosophila spp. Zootermopsis nevadensis Acyrthosiphon pisum Rhodnius prolixus Atta cephalotes Acromyrmex echinatior Camponotus floridanus Harpegnathos saltator Linepithema humile Nasonia spp. Pogonomyrmex barbatus Solenopsis invicta Bombyx mori Melitaea cinxia Plutella xylostella Spodoptera frugiperda Strigamia maritima

36 MosHB (mosquitos) Culex quinquefasciatus C. tritaeniorhynchus MosZJ (mosquitos) C. tritaeniorhynchus C. quinquefasciatus C. sp Horseflies (unidentified Tabanidae) sp1 Common flies Musca domestica Water striders (unidentified Gerridae) 97 sp Armigeres subalbatus Aedes sp. Anopheles sinensis Insect mix 1 (lacewings, moths, beetles, moth flies, soldier flies) Coleoptera sp. Abraxas tenuisuffusa unknown Chrysopidae unknown Diptera Psychoda alternata unknown Stratiomyidae unknown Stratiomyidae Insect mix 2 (louseflies and bed bugs) 0.1 unknown Hippoboscidae Cimex hemipterus C. sp 99 Armigeres Aedes sp. Anopheles sinensis Insect mix 3 (dragonflies, ants, beettles, waterscorpion, etc) Insect mix 4 (moth flies, crickets aphids, dragonflies, etc) unknown Nepidae Diplonychus sp Camponotus japonicus unknown Phalangiidae Pseudothemis zonata Asellus sp Velarifictorus micado Velarifictorus ornatus Crocothemis servilia Unknown Phoridae 96 Psychoda alternata Unknown Lampyridae Aulacorthum magnoliae Hyalopterus pruni Aphelinus sp Ticks Shrimps sp3 sp5 sp2 sp4 49 Penaeus monodon (X12) Litopenaeus vannamei (X12) Metapenaeus sp (X6) Solenocera crassicornis (X6) Chrysomya megacephala Hyalomma asiaticum 92 Dermacentor sp 97 D. marginatus 96 Rhipicephalus microplus Haemaphysalis formosensis Haemaphysalis sp H. hystricis H. longicornis H. doenitzi Argas miniatus Exopalaemon carinicauda (X12) Sarcophaga sp Lucilia sericata Atherigona orientalis Spiders Neoscona nautica sp2 cockroaches (single species: Blattella germanica) Parasteatoda tepidariorum Plexippus setipes Millipedes (two species: unknown Polydesmidae) Pirata sp unknown Araneae Crabs and barnacles Charybdis japonica (X9) Charybdis lucifera (X1) Charybdis hellerii (X2) Uca arcuata (X11) Capitulum mitella (X12)

37 Frequency ~ ~ ~ 0 > 0 Host mtdna COI gene Abundance (TPM) Japanese encephalitis virus (TPM = 4.19) Gill-associated virus (TPM = 20.05)

38 Bunya-arena-like Mononega-like rthomyxolike Ophioviridae A B 0.5 Phlebovirus Tenuivirus Arenavirus Nairovirus Hantavirus Tospovirus Emaravirus Orthobunyavirus 0.5 Sigmavirus Vesiculovirus, Ephemerovirus, etc Lyssavirus Paramyxovirus and Filovirus Novirhabdovirus Nucleorhabdovirus Cytorhabdovirus Phasmavirus Varicosavirus Bornaviridae Nyamiviridae C D Gouleako virus / 98/ 98/ 91/ 96/ / 92/ 88/99 96/ 97/ 98/ 99/99 97/ / 99/ Chuviridae Chuviridae 75/85 85/ Influenza A-D ISAV Thogotovirus 98/ 89/99 Quaranjavirus

39 Arenavirus (N=2) Phlebovirus (N=3) Tenuivirus (N>4) Phlebo-like viruses (N=3) Emaravirus (N>4) Tospovirus (N=3) Nairovirus (N=3) Orthobunyavirus (N=3) Segmented Phasmavirus (N=3) Hantavirus (N=3) Ophiovirus (N>3) Orthomyxovirus (N=6-8) Unsegmented Varicosavirus (N=2) Chuviridae Orchid fleck virus (N=2) Various Genome Organizations Type I Type II Tacheng Tick Virus 5 10,017bp Wuchang Cockroach Virus 3 L seg 7152bp L seg 6808bp Bole Tick Virus 3 11,183kb S seg 4788bp Wuhan Louse Fly Virus 7 S seg 2343bp Type III Wenzhou Crab Virus 3 12,860bp

40 (Arenavirus) (Phlebovirus) millipede (Nairovirus) tick spider water strider aphis,moths mosquito cockroach water strider (Ophiovirus) (Paramyxovirus and filovirus) tick (Tenuivirus) fly lousefly spider aphid moth mosquito aterscorpion butterfly millipede Asellus sp shrimp humbleback- (Emaravirus) fly tick, crab fly, spier, lousefly unknown diptera mosquito cockroach (Pneumovirus) mosquito (Tospovirus) midges (orthobunyvirus) mosquito bed bug spider (Hantavirus) shrimp cockroach (Influenza virus) spider water strider tick, lousefly coleoptera mosquito spider moth fly horsefly dragonfly water strider tick tick (Vesiculovirus, Ephemerovirus, etc) (Nucleorhabdovirus) (Cytorhabdovirus), aphid (Lyssavirus) moth, crab mosquito fly, lousefly, bed bug, ant, mosquito, water strider fly, lousefly mosquito tick

41 DG3-2 DG3-4 JJ3-9 JJ3-6 DG3-9 DG3-10 DG3-1 JJ3-1 DG2-5 JJ2-1 JJ2-5 JJ2-6 JJ2-8 DG2-6 DG2-4 DG2-2 DG2-3 DG DG3-4 DG2-3 DG3-1 DG2-4 DG1-1 DG2-5 DG3-10 DG3-2 DG3-9 DG2-6 JJ2-5 DG2-2 JJ2-6 JJ2-1 JJ3-6 JJ3-9 JJ3-1 JJ Wuhan Horsefly Virus horseflies (host) SYY4-2 SYY2-2 SYY4-8 SYY2-4 SYY4-1 SYY4-5 SYY3-1 SYY2-6 SYY SYY2-1 SYY2-2 SYY2-4 SYY2-6 SYY4-5 SYY4-2 SYY4-8 SYY3-1 SYY Wuhan Fly Virus 1 flies (host) WT3-14 WT3-15 WT3-13 QN3-6 QN3-5 JX1-14 JX1-12 JX1-13 QN2-6 XC2-6 XZ3-11 XC1-9 XC1-8 XC1-7 XC1-6 XC1-5 XZ XZ1-6 QN3-5 XC1-9 XC2-6 JX1-12 QN3-6 XC1-5 XC1-8 XC1-7 WT3-13 XC1-6 WT3-15 JX1-14 JX1-13 WT3-14 QN2-6 XZ Wuhan Mosquito Virus 1 mosquitos (host) XC1-5 XC1-6 XC1-9 QN2-7 JX1-13 WT3-14 WT3-13 JX1-14 WT3-15 XZ3-11 QN3-5 QN3-6 JX WT3-14 WT3-15 WT3-13 QN3-6 QN3-5 JX1-14 JX1-12 JX1-13 QN2-7 XZ3-11 XC1-5 XC1-6 XC Wuhan Mosquito Virus 2 mosquitos (host) (A) (B) (D) (C)

42 A RdRP Putative Glycoprotein Putative Nucleoprotein Bole Tick Virus 3 Changping Tick Virus 2 Tacheng Tick Virus 5 Changping Tick Virus 3 Wuhan Tick Virus 2 Wuhan Louse Fly Virus 7 Wuhan Louse Fly Virus 6 Wenzhou Crab Virus 2 Wuchang Cockraoch Virus 3 Lishi Spider Virus 1 Wuhan Mosquito Virus 8 Tacheng Tick Virus 4 Shayang Fly Virus 1 Wenzhou Crab Virus Bole Tick Virus 3 Changping Tick Virus 2 Wuhan Tick Virus 2 Wuchang Cockraoch Virus 3 Lishi Spider Virus 1 Wuhan Mosquito Virus 8 Wenzhou Crab Virus 2 Tacheng Tick Virus Bole Tick Virus 3 Changping Tick Virus 2 Tacheng Tick Virus 5 Changping Tick Virus 3 Wuhan Tick Virus 2 Wuhan Louse Fly Virus 6 Wuhan Louse Fly Virus 7 Wuhan Mosquito Virus 8 Wuchang Cockraoch Virus 3 Lishi Spider Virus 1 Wenzhou Crab Virus 2 Tacheng Tick Virus 4 B Changping Tick Virus 3 Tacheng Tick Virus 5 Wuhan Tick Virus 2 L 252.7kDa L 252.5kDa nts 9527 nts N? 47.6kDa 17 nts N? 47.2kDa VP3 12.5kDa Wuhan Mosquito Virus nts L 247.3kDa G 73.4kDa Wenzhou Crab Virus nts L 251.3kDa Lishi Spider Virus nts G 72.4kDa N? VP4 70.6kDa 11.2kDa 4698 nts N? VP4 67.9kDa 17.6kDa 4426 nts L 250.6kDa G 75.7kDa N? VP4 45.0kDa 13.8kDa Changping Tick Virus nts L 252.1kDa Wuchang Cockroach Virus nts G 75.1kDa N? VP4 46.3kDa 14.5kDa 4788 nts L 244.0kDa G 73.8kDa N? 51.6kDa Bole Tick Virus nts L 254.2kDa Tacheng Tick Virus 4 G 75.7kDa N? VP4 47.0kDa 20.2kDa nts Wuhan Louse Fly Virus 6 L 244.7kDa G 72.3kDa 6777 nts N? VP4 51.3kDa 12.2kDa 2291 nts G 72.9kDa N? 54.7kDa Wenzhou Crab Virus 3 L 247.2kDa nts L 249.1kDa Wuhan Louse Fly Virus nts N? VP3 G1 96.8kDa N? G2 61.3kDa L 238.1kDa VP5 47.0kDa 15.1kDa 19.6kDa Shayang Fly Virus nts nts L 247.7kDa N? 47.5kDa G 121.6kDa VP2 N? 69.5kDa 11.7kDa L 281.4kDa C 0-899X 14X TTTTATTT------GAATACAAGGG TTTTATTCAAA---GAATACAAGGG TTTTATTCGG----GAATACAAGGG TTTTATTCAATTTTGAATACAAGGG TTTTATTTTT----GAATACAAGGG TTTTATTCAATT--GAATACAAGGG TTTTATTCTTT---GAATACAAGGG L G N? L G N?

43 Phlebovirus Huangpi Tick Virus 2 Yongjia Tick Virus 1 Dabieshan Tick Virus Tacheng Tick Virus 2 Changping Tick Virus 1 Bole Tick Virus 1 Lihan Tick Virus Phlebovirus_like Whenzhou Shrimp Virus 1 Wutai Mosquito Virus Wuhan Fly Virus 1 Wuhan Louse Fly Virus 2 Qingnian Mosquito Virus Wuhan Insect Virus 1 Wuhan Millipede Virus 1 Yichang Insect Virus Shuangao Insect Virus 3 ZheE Mosquito Virus Xinzhou Mosquito Virus Wuhan Spider Virus Huangshi Humpbacked Fly Virus Tenuivirus_like Wuhan horsefly Virus L segment 6441 nts L 243.0kDa 6527 nts L 243.6kDa 6549 nts L 245.6kDa 6660 nts L 247.1kDa 6637 nts L 250.3kDa 6483 nts L 244.9kDa 6517 nts L 247.4kDa 6284 nts L 233.3kDa 6754 nts L 251.3kDa 6779 nts L 252.6kDa 7082 nts L 267.2kDa 7046 nts L 252.7kDa 6520 nts L 240.7kDa 5662 nts L partial 6421 nts L 238.8kDa 6276 nts L 234.3kDa 7604 nts L 276.5kDa 6362 nts L 230.6kDa 6870 nts L 256.6kDa 6055 nts L 230.2kDa 9525 nts L 363.0kDa M segment 3568 nts 5 Gn 57.9 kda Gc 55.0 kda 2969 nts partial 3357 nts Gn 81.1 kda Gc 55.9 kda 4454 nts kda 3648 nts Gn 63.8 kda Gc 57.8 kda 3278 nts Gn 55.9 kda Gc 54.6 kda 3357 nts Gn 62.3 kda Gc 54.6 kda 2804 nts Gn 43.4 kda Gc 50.5 kda S segment 1709 nts N 27.4kDa NSs 30.9kDa 1830 nts N 29.8kDa NSs 11.3kDa 1795 nts N 30.2kDa NSs 15.1kDa 2185 nts N 46.9kDa 1227 nts N partial 1659 nts N 34.1kDa 1925 nts N 50.0kDa 898 nts N 27.2kDa 1454 nts N 31.8kDa 1116 nts N 30.4kDa 1285 nts N partial 1211 nts N partial 1030 nts N 28.1kDa 1480 nts N 45.9kDa 1889 nts N 51.6kDa Nairovirus_like Xinzhou Spider Virus Sanxia Water Strider virus 1 Shayang Spider Virus 1 Huangpi Tick Virus 1 Wenzhou Tick Virus Tacheng Tick Virus 1 Phasmavirus_like Wuhan Mosquito Virus 2 Wuhan Mosquito Virus 1 Wuchang Cockraoch Virus 1 Shuangao Insect Virus 2 Orthobunyavirus Wuhan Louse Fly Virus 1 Orthobunyavirus_like Shuangao Insect Virus 1 Unclassified segmented virus 1 Jiangxia Mosquito Virus 1 Unclassified segmented virus 2 Jiangxia Mosquito Virus 2 Shuangao Bedbug Virus 1 Shuangao Mosquito Virus Unclassified segmented virus 3 Whenzhou Shrimp Virus 2 Unclassified segmented virus 4 Shayang Spider Virus 2 Unclassified segmented virus 5 Wuhan Insect Virus 2 Sanxia Water Strider Virus 2 Unclassified segmented virus 6 Wuhan Millipede Virus 2 Unclassified segmented virus 7 Wuhan Insect Virus 3 L segment M segment S segment nts L 441.4kDa nts L 446.9kDa 6574 nts L 242.4kDa 6474 nts L 238.4kDa 6493 nts L 246.1kDa 4899 nts L partial 6787 nts L 262.4kDa 7325 nts L 264.8kDa 5754 nts L partial 5956 nts L 217.0kDa 6085 nts L 229.7kDa 6553 nts L partial 6873 nts L 258.0kDa 6646 nts L 249.4kDa 7243 nts L 272.2kDa 7151 nts L 271.6kDa nts L 417.0kDa 6743 nts L 249.0kDa Gn Gc 52.8 kda NSs (?) N 42.9kDa partial 2217 nts 13.1kDa 2111 nts Gn Gc 53.2 kda NSs(?) N 38.2kDa VP3(?) partial 2636 nts 14.1kDa 12.3kDa 1808 nts Gn 35.1 kda Gc 53.7 kda NSs (?) N 48.3kDa 2255 nts 13.9kDa 1959 nts Gn Gc 51.7 kda NSs (?) N 54.7kDa VP3 (?) 23.1 kda 15.4kDa 18.4kDa 1148 nts NSs (?) N partial 2939 nts partial 959 nts Gn NSm Gc partial partial15.9kda NSs N 27.1kDa 4753 nts 9.6kDa 1233 nts Gn NSm Gc kda N 30.0kDa 27.4kDa 2957 nts Gn partial Gc partial 2312 nts Gn Gc partial partial 3302 nts Gn 49.0 kda Gc 67.7 kda L 464.6kDa Gn 66.3 kda Gc partial nts 4193 nts L 450.0kDa Gn Gc 74.9 kda nts 52.2 kda 4805 nts L 506.5kDa Gn Gc.9 kda nts 60.2 kda 4675 nts L 444.7kDa Gn 66.1 kda Gc 77.9 kda nts 4428 nts Gn 72.0 kda Gc 73.7 kda 5147 nts Gn 73.2 kda Gc 76.1 kda 2073 nts Gn partial Gn partial 3322 nts 2466 nts Gc 56.3 kda 2852 nts Gc partial 2241 nts N 56.6kDa 2171 nts N 53.9kDa 1940 nts N 64.2kDa 1969 nts N 53.9kDa 1785 nts N 54.7kDa 1840 nts N 54.6kDa 2208 nts 1820 nts N 53.9kDa 1960 nts N 53.8kDa 1867 nts N 53.8kDa

44 Nishimuro virus like Yongjia Tick Virus 2 N P(?) M G L Kolente virus like Wuhan Louse Fly Virus 5 N P M G L Sigmavirus_like Wuhan Fly Virus 2 Shayang Fly Virus 2 Wuhan House Fly Virus nts Wuhan Louse Fly Virus nts Wuhan Louse Fly Virus nts N P(?) M G L Unclassified Dimarhabdovirus 2 Wuhan Insect virus 7 N P(?) M G L Unclassified Dimarhabdovirus 1 Huangpi Tick Virus nts Taishun Tick Virus Bole Tick Virus nts nts nts N P(?) X M G L nts N P(?) X M G L N P(?) X(?) M G L N P(?) M G L N P(?) M(?) G N 52.2 VP2 VP nts L nts L nts Unclassified Rhabdovirus 2 Sanxia Water Strider Virus nts N VP2 VP3 G L Shuangao Bedbug Virus nts VP1 G VP Wuhan Mosquito Virus 9 VP1 ORF1 VP2 ORF2 VP3 ORF3 G 69.5kDa G L 196.7kDa L Wuhan House Fly Virus nts VP1 VP2 VP3 VP4 G VP6 L Shayang Fly Virus nts VP1 VP2 VP3 G L Unclassified Rhabdovirus 1 Tacheng Tick Virus nts VP1 VP4 G VP2 VP Cytorhabdovirus_like Wuhan Insect virus 4 N P 4b M G Wuhan Insect virus 5 N P 4b M G Unclassified Mononegavirus 1 Tacheng Tick Virus 6 L Sanxia Water Strider Virus nts VP1 VP2 VP3 G VP Unclassified Mononegavirus 2 Xincheng Mosquito Virus nts L nts N P 4b M(?) G L Wuhan Insect virus nts VP1 VP2 VP3 VP4 VP L nts L nts L L nts N P(?) M G Wuhan Tick Virus 1 N 55.1 VP2 VP L nts L VP1 VP2 VP3 G Unclassified Mononegavirus 3 Wenzhou Crab Virus 1 N VP VP G 57.5 L nts L 202.2

45 Rice grassy stunt virus Wuhan horsefly Virus RNA1 9760bp) P1 RdRp RNA1 (9549bp) RdRp RNA2 4056bp) P2 Putative Gn-Gc RNA2 (2804bp) Putative Gn-Gc RNA3 3123bp) P3 PC3 RNA3 (1889bp) N RNA4 2915bp) P4 PC4 RNA4 (1698bp) NS4 RNA5 2704bp) P5 N RNA6 2584bp) NCP NS4

46 A Changping Tick Virus 2 Bole Tick Virus 3 Wuhan Tick Virus 2 EVE Anopheles maculatus EVE Acyrthosiphon pisum (N = 9) EVE Tribolium castaneum (N = 3) EVE Hyalella azteca Wenzhou Crab Virus 2 Lishi Spider Virus 1 Wuchang Cockroach Virus 3 EVE Culicidae Wuhan Mosquito Virus 8 EVE Hymenoptera Tacheng Tick Virus 4 B Pogonomyrmex barbatus (N=13) Solenopsis invicta Camponotus floridanus Linepithema humile Linepithema humile 0.5 Acromyrmex echinatior (N=4) Harpegnathos saltator (N=4) 93 Solenopsis invicta Acromyrmex echinatior 98 Pogonomyrmex barbatus 99 Nasonia vitripennis Nasonia vitripennis 99 Nasonia giraulti Nasonia longicornis Nasonia vitripennis Nasonia vitripennis Nasonia vitripennis Nasonia giraulti 92 Nasonia longicornis 98 Nasonia vitripennis 95 Nasonia longicornis 98 Nasonia vitripennis Nasonia vitripennis Nasonia longicornis 83 Nasonia vitripennis Nasonia vitripennis (N=5) Nasonia vitripennis 99 Nasonia vitripennis Nasonia vitripennis Nasonia giraulti 97 Nasonia longicornis Nasonia giraulti Nasonia vitripennis 93 Nasonia vitripennis 98 Nasonia vitripennis Solenopsis invicta Linepithema humile Camponotus floridanus 99 Camponotus floridanus Harpegnathos saltator Harpegnathos saltator Camponotus floridanus Linepithema humile (N=5) Acromyrmex echinatior (N=3) Solenopsis invicta Linepithema humile (N=4) Solenopsis invicta (N=7) 98 Pogonomyrmex barbatus (N=8) 98 Solenopsis invicta Solenopsis invicta Harpegnathos saltator 90 Solenopsis invicta Pogonomyrmex barbatus C Anopheles funestus Anopheles funestus Anopheles funestus Anopheles minimus 88 Anopheles stephensi Anopheles stephensi Anopheles stephensi Anopheles stephensi Anopheles minimus Anopheles dirus Anopheles dirus Anopheles arabiensis Anopheles gambiae Anopheles minimus A. quadriannulatus Anopheles albimanus Anopheles albimanus Anopheles sinensis Anopheles sinensis Anopheles epiroticus Anopheles atroparvus Anopheles gambiae Anopheles gambiae Anopheles quadriannulatus Anopheles dirus Anopheles nili Anopheles farauti Anopheles farauti Anopheles albimanus Aedes aegypti Aedes aegypti Aedes aegypti Aedes aegypti Aedes aegypti Aedes aegypti Culex quinquefasciatus Aedes aegypti Aedes aegypti Aedes aegypti Aedes aegypti Aedes aegypti Aedes aegypti Culex quinquefasciatus Culex quinquefasciatus Culex quinquefasciatus C. quinquefasciatus

47 Arthropods Phytophagous vectors Blood-feeding vectors Plants Vertebrates Vertebrates Tospovirus Cytorhabdovirus Nucleorhabdovirus Emaravirus Tenuivirus Nairovirus Phlebovirus Orthobunyavirus Ephemerovirus Vesiculovirus Filovirus Arenavirus Bornavirus Influenza virus Paramyxovirus Lyssavirus Hantavirus