Diverse mosquito-specific flaviviruses in the Bolivian Amazon basin · 2021. 6. 2. · 1 1 Title: Diverse Mosquito Specific Flaviviruses in the Bolivian Amazon basin 2 3 Authors:
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Title Diverse mosquito-specific flaviviruses in the Bolivian Amazon basin
The authors declare that they have no conflicts of interest. 336
337
Funding information 338
This study was supported by grants for Scientific Research from the Ministry of Education, Culture, 339
Sports, Science and Technology, Japan (MEXT) /Japan Society for the Promotion of Science (JSPS) 340
KAKENHI (JP16H05805, JP19H03112, and 20K21298); and grants for Scientific Research on 341
Innovative Areas and International Group from the MEXT/JSPS KAKENHI (JP16H06431, 342
JP16H06429, JP16K21723, and 19H04843). 343
344
Acknowledgements 345
We appreciate cooperation of the Japan International Cooperation Agency (JICA) members, Mariko 346
Tanaka and Madoka Matsuo for mosquito sampling in Bolivia. Computations in this work were 347
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performed in part on the NIG supercomputer at ROIS National Institute of Genetics and 348
SHIROKANE at Human Genome Center (the Univ. of Tokyo). 349
350
References 351
352 1. Saba Villarroel PM, Nurtop E, Pastorino B, Roca Y, Drexler JF et al. Zika virus 353 epidemiology in Bolivia: A seroprevalence study in volunteer blood donors. PLoS Negl Trop Dis 354 2018;12(3):e0006239. 355 2. Roca Y, Baronti C, Revollo RJ, Cook S, Loayza R et al. Molecular epidemiological analysis 356 of dengue fever in Bolivia from 1998 to 2008. Vector Borne Zoonotic Dis 2009;9(3):337-344. 357 3. Venegas EA, Aguilar PV, Cruz C, Guevara C, Kochel TJ et al. Ilheus virus infection in 358 human, Bolivia. Emerg Infect Dis 2012;18(3):516-518. 359 4. LAEMMERT HW, HUGHES TP. The virus of Ilhéus encephalitis; isolation, serological 360 specificity and transmission. J Immunol 1947;55(1):61-67. 361 5. Turell MJ, O'Guinn ML, Jones JW, Sardelis MR, Dohm DJ et al. Isolation of viruses 362 from mosquitoes (Diptera: Culicidae) collected in the Amazon Basin region of Peru. J Med Entomol 363 2005;42(5):891-898. 364 6. Vieira CJDS, Andrade CD, Kubiszeski JR, Silva DJFD, Barreto ES et al. Detection of 365 Ilheus virus in mosquitoes from southeast Amazon, Brazil. Trans R Soc Trop Med Hyg 366 2019;113(7):424-427. 367 7. Organization PAH. Geographic Spred of Chkungunya in the Americas2018. 368 8. Stollar V, Thomas VL. An agent in the Aedes aegypti cell line (Peleg) which causes fusion 369 of Aedes albopictus cells. Virology 1975;64(2):367-377. 370 9. Patterson EI, Villinger J, Muthoni JN, Dobel-Ober L, Hughes GL. Exploiting insect-371 specific viruses as a novel strategy to control vector-borne disease. Curr Opin Insect Sci 2020;39:50-372 56. 373 10. (WRBU) TWRBU. 2019. MOSQUITO IDENTIFICATION RESOURCES. 374 http://wrbu.si.edu/aors/aors_Keys.html [accessed. 375 11. Segura MdNdO. Atlas de Culicídeos na Amazônia brasileira: 376 características específicas de insetos hematófagos da família Culicidae: Instituto Evandro Chagas; 377 2007. 378 12. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of 379 mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol 380 Biotechnol 1994;3(5):294-299. 381 13. Patel P, Landt O, Kaiser M, Faye O, Koppe T et al. Development of one-step quantitative 382 reverse transcription PCR for the rapid detection of flaviviruses. Virol J 2013;10:58. 383 14. Orba Y, Hang'ombe BM, Mweene AS, Wada Y, Anindita PD et al. First isolation of West 384
Nile virus in Zambia from mosquitoes. Transbound Emerg Dis 2018;65(4):933-938. 385 15. Torii S, Orba Y, Hang'ombe BM, Mweene AS, Wada Y et al. Discovery of Mwinilunga 386 alphavirus: A novel alphavirus in Culex mosquitoes in Zambia. Virus Res 2018;250:31-36. 387 16. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M et al. SPAdes: a new genome 388 assembly algorithm and its applications to single-cell sequencing. J Comput Biol 2012;19(5):455-477. 389 17. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA et al. Full-length 390 transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 391 2011;29(7):644-652. 392 18. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: 393 improvements in performance and usability. Mol Biol Evol 2013;30(4):772-780. 394 19. Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: a tool for automated 395 alignment trimming in large-scale phylogenetic analyses. Bioinformatics 2009;25(15):1972-1973. 396 20. Le SQ, Gascuel O. An improved general amino acid replacement matrix. Mol Biol Evol 397 2008;25(7):1307-1320. 398 21. Darriba D, Taboada GL, Doallo R, Posada D. ProtTest 3: fast selection of best-fit models 399 of protein evolution. Bioinformatics 2011;27(8):1164-1165. 400 22. Kozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A. RAxML-NG: a fast, scalable 401 and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics 402 2019;35(21):4453-4455. 403 23. Larkin M, Blackshields G, Brown N, Chenna R, McGettigan P et al. Clustal W and Clustal 404 X version 2.0. Bioinformatics 2007;23(21):2947-2948. 405 24. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis 406 Version 7.0 for Bigger Datasets. Mol Biol Evol 2016;33(7):1870-1874. 407 25. Almagro Armenteros JJ, Tsirigos KD, Sønderby CK, Petersen TN, Winther O et al. 408 SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat Biotechnol 409 2019;37(4):420-423. 410 26. Duckert P, Brunak S, Blom N. Prediction of proprotein convertase cleavage sites. Protein 411 Eng Des Sel 2004;17(1):107-112. 412 27. Möller S, Croning MD, Apweiler R. Evaluation of methods for the prediction of membrane 413 spanning regions. Bioinformatics 2001;17(7):646-653. 414 28. Hirokawa T, Boon-Chieng S, Mitaku S. SOSUI: classification and secondary structure 415 prediction system for membrane proteins. Bioinformatics 1998;14(4):378-379. 416 29. Buchan DWA, Jones DT. The PSIPRED Protein Analysis Workbench: 20 years on. Nucleic 417 Acids Res 2019;47(W1):W402-W407. 418 30. Jones DT. Protein secondary structure prediction based on position-specific scoring matrices. 419 J Mol Biol 1999;292(2):195-202. 420 31. Jones DT, Cozzetto D. DISOPRED3: precise disordered region predictions with annotated 421 protein-binding activity. Bioinformatics 2015;31(6):857-863. 422
15
32. Clark DC, Lobigs M, Lee E, Howard MJ, Clark K et al. In situ reactions of monoclonal 423 antibodies with a viable mutant of Murray Valley encephalitis virus reveal an absence of dimeric NS1 424 protein. J Gen Virol 2007;88(Pt 4):1175-1183. 425 33. Takhampunya R, Kim HC, Tippayachai B, Lee DK, Lee WJ et al. Distribution and 426 mosquito hosts of Chaoyang virus, a newly reported flavivirus from the Republic of Korea, 2008-2011. 427 J Med Entomol 2014;51(2):464-474. 428 34. Huhtamo E, Putkuri N, Kurkela S, Manni T, Vaheri A et al. Characterization of a novel 429 flavivirus from mosquitoes in northern europe that is related to mosquito-borne flaviviruses of the 430 tropics. J Virol 2009;83(18):9532-9540. 431 35. Vázquez A, Sánchez-Seco MP, Palacios G, Molero F, Reyes N et al. Novel flaviviruses 432 detected in different species of mosquitoes in Spain. Vector Borne Zoonotic Dis 2012;12(3):223-229. 433 36. Huhtamo E, Cook S, Moureau G, Uzcátegui NY, Sironen T et al. Novel flaviviruses from 434 mosquitoes: mosquito-specific evolutionary lineages within the phylogenetic group of mosquito-borne 435 flaviviruses. Virology 2014;464-465:320-329. 436 37. Korkusol A, Takhampunya R, Hang J, Jarman RG, Tippayachai B et al. A novel 437 flavivirus detected in two Aedes spp. collected near the demilitarized zone of the Republic of Korea. J 438 Gen Virol 2017;98(5):1122-1131. 439 38. Harrison JJ, Hobson-Peters J, Colmant AMG, Koh J, Newton ND et al. Antigenic 440 Characterization of New Lineage II Insect-Specific Flaviviruses in Australian Mosquitoes and 441 Identification of Host Restriction Factors. mSphere 2020;5(3). 442 39. Colmant AMG, Hobson-Peters J, Bielefeldt-Ohmann H, van den Hurk AF, Hall-443 Mendelin S et al. A New Clade of Insect-Specific Flaviviruses from Australian. mSphere 2017;2(4). 444 40. Zhang X, Guo X, Fan H, Zhao Q, Zuo S et al. Complete genome sequence of Menghai 445 flavivirus, a novel insect-specific flavivirus from China. Arch Virol 2017;162(5):1435-1439. 446 41. Fan H, Zhao Q, Guo X, Sun Q, Zuo S et al. Complete genome sequence of Xishuangbanna 447 flavivirus, a novel mosquito-specific flavivirus from China. Arch Virol 2016;161(6):1723-1727. 448 42. McLean BJ, Hobson-Peters J, Webb CE, Watterson D, Prow NA et al. A novel insect-449 specific flavivirus replicates only in Aedes-derived cells and persists at high prevalence in wild Aedes 450 vigilax populations in Sydney, Australia. Virology 2015;486:272-283. 451 43. Weaver SC, Reisen WK. Present and future arboviral threats. Antiviral Res 2010;85(2):328-452 345. 453 44. Ryan SJ, Carlson CJ, Mordecai EA, Johnson LR. Global expansion and redistribution of 454 Aedes-borne virus transmission risk with climate change. PLoS Negl Trop Dis 2019;13(3):e0007213. 455 45. Alencar J, Pacheco JB, Dos Santos Silva J, Silva SOF, Guimarães A. Influence of Climatic 456 Factors On Psorophora (Janthinosoma) Albigenu In Pantanal Landscape, Mato Grosso State, Brazil. J 457 Am Mosq Control Assoc 2018;34(3):177-181. 458 46. Turell MJ, Jones JW, Sardelis MR, Dohm DJ, Coleman RE et al. Vector competence of 459 Peruvian mosquitoes (Diptera: Culicidae) for epizootic and enzootic strains of Venezuelan equine 460
16
encephalomyelitis virus. J Med Entomol 2000;37(6):835-839. 461 47. GROOT H, MORALES A, VIDALES H. Virus isolations from forest mosquitoes in San 462 Vicente de Chucuri, Colombia. Am J Trop Med Hyg 1961;10:397-402. 463 48. Serra OP, Cardoso BF, Ribeiro AL, Santos FA, Slhessarenko RD. Mayaro virus and 464 dengue virus 1 and 4 natural infection in culicids from Cuiabá, state of Mato Grosso, Brazil. Mem Inst 465 Oswaldo Cruz 2016;111(1):20-29. 466 49. Mucci LF, Júnior RP, de Paula MB, Scandar SA, Pacchioni ML et al. Feeding habits of 467 mosquitoes (Diptera: Culicidae) in an area of sylvatic transmission of yellow fever in the state of São 468 Paulo, Brazil. J Venom Anim Toxins Incl Trop Dis 2015;21:6. 469 50. Öhlund P, Lundén H, Blomström AL. Insect-specific virus evolution and potential effects 470 on vector competence. Virus Genes 2019;55(2):127-137. 471 51. Hobson-Peters J, Harrison JJ, Watterson D, Hazlewood JE, Vet LJ et al. A recombinant 472 platform for flavivirus vaccines and diagnostics using chimeras of a new insect-specific virus. Sci Transl 473 Med 2019;11(522). 474 52. Tan TY, Fibriansah G, Kostyuchenko VA, Ng TS, Lim XX et al. Capsid protein structure 475 in Zika virus reveals the flavivirus assembly process. Nat Commun 2020;11(1):895. 476 53. Dokland T, Walsh M, Mackenzie JM, Khromykh AA, Ee KH et al. West Nile virus core 477 protein; tetramer structure and ribbon formation. Structure 2004;12(7):1157-1163. 478 54. Oliveira ERA, Mohana-Borges R, de Alencastro RB, Horta BAC. The flavivirus capsid 479 protein: Structure, function and perspectives towards drug design. Virus Res 2017;227:115-123. 480 55. Sotcheff S, Routh A. Understanding Flavivirus Capsid Protein Functions: The Tip of the 481 Iceberg. Pathogens 2020;9(1). 482 56. Jones CT, Ma L, Burgner JW, Groesch TD, Post CB et al. Flavivirus capsid is a dimeric 483 alpha-helical protein. J Virol 2003;77(12):7143-7149. 484 57. Ma L, Jones CT, Groesch TD, Kuhn RJ, Post CB. Solution structure of dengue virus capsid 485 protein reveals another fold. Proc Natl Acad Sci U S A 2004;101(10):3414-3419. 486
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Table 1. Mosquito species analyzed in this study 490 Season Place Species No.
BM M F W N N G N E V S N L P G G K N P F K K P H E A G E N A S N R SK G A Y G A G K L T N L K N S Q G G R A L A T G G G W G S G L G G S S G T S R T H A P L S P T S F R T R I N N F 90‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐
G W F G G S G S G G V N N P P K A H A T K A K L N Q P D G Y A H G WG S N S G S G A R T M S Q A T H T T W K N K A N L N S A S E Y G Q V G N S K A M I P Y L A G N Q P P M L G G H P 180‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M E K S R R F S R P GG G R V P S V K D G T K K R T G T V V G P Q V Q K Q A P G ‐ ‐ ‐ A Q K P R K V A S L P P G V A ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ 55‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M E Q N G K F S R P GG G R A R P R E G L G K K R T G S V V G K S S T R S T T K N V A A Q K P L S R A S L P A T S M Q T K A Q R N ‐ ‐‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M H R L R D L L P D KG K K N R S P A V R R Q G G I A K G V D R K I I P S S G S K E R K V G T E R K A K R V V A R M K P ‐ ‐ ‐ ‐ ‐ ‐ ‐
‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M S S K E ‐ ‐ ‐ ‐ ‐ ‐‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ A L K K K G I S L G G G G K R G F E M K K A P Q H ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M K R K ‐ ‐ ‐ D L E AR G K A P G R D ‐ ‐ ‐ ‐ ‐ S S T P F W G R E G R R K D K D K ‐ G G E S P S N R Q V T L K T P I Q S G R R A G K ‐‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M G K D ‐ ‐ ‐ D G K KK ‐ K G P G S S R W L L P S E R A G L G R K E E K K K K E K ‐ R S V R S T P Q L V S G G A Q H R R G G G T G P ‐
‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M E R K K ‐ ‐ ‐ ‐ ‐ ‐‐ ‐ ‐ G V N Y G R R E A I P P P P I K E K K D R K K K R D D V K K G L V L R G T K P I R R K F G ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M V T K T ‐ ‐ R K P AK ‐ R A V D I I K R R L P R V P S P M G T V ‐ R R V A Q K V V K G M G N F R A F L A F F V Y Q V ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ 55‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M R S M V F R A ‐ ‐ R R P VK ‐ R A V D I I K R K L P Y V P P P M R V A ‐ K M A A K K V M I G I G S L K A F L A L F L F M T ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M A N K P ‐ ‐ K K P AR ‐ R A I D I V R R A L P R V S G P K R V L ‐ A R A S K S I M Q S L A G L R A T V A Y L L Y M T ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M V T R P ‐ ‐ R K P AA K R A V N M L K R V A R R A L S P V E A A ‐ V K L V K N V F V G K G P T R A I M A V M A M L R ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐
‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M K N P K ‐ ‐ K K S GG F R I V N M L K R G V A R V N P L G G L ‐ ‐ K R L P A G L L L G H G P I R M V L A I L A F L R ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M S K K P ‐ ‐ G G P GK S R A V N M L K R G M P R V L S L I G L ‐ ‐ K R A M L S L I D G K G P I R F V L A L L A F F R ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐
‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M N N Q R ‐ ‐ K K T GR ‐ P S F N M L K R A R N R V S T V S Q L A ‐ K R F S K G L L S G Q G P M K L V M A F I A F L R ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐
D E R Q G R K K G Q L K T R K L G Q A Q V K D R M K Q F F N L S I TE A L E A C M L V M V Q L F S L L T L M I Q R L I W Q A T R E R K T R S V G F Y F P L I V W M A F M A T C C A ‐ 269‐ E R N K K R E G ‐ ‐ F G Q H L K W E K F S F G W V D L L R A D L MQ A L V M L M A V L T L T F R D Y N R K L A N L F M R V R R L E G Q R G H Y S V V W A F V A L A I F A Q M Y G C 141‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ I G K L S K V L G T R Y G W K D F L Q A D L IQ A L W M I F T V I T Q S I K D L Y A K V G R L N K R V V K L E K K R S G K A S L L T L I F M T M A I L L V T S
‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ N W L S F G T G N Q R S M W R Q I F S V D L ME G L L L F I A L M S N L Y E R V Q R D I A D L K R R V T R L E K E R S H P R K V P I M L L C G L I V V T G L S‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ N N S N R Q L R R S L F R M D V GR A L E L M L A S I V N A I R G L I Q R V S R L E T R V G L L E R K K T R S I Y Y S L P M F T L L S L A C C ‐ ‐
‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ R Q R V G L L G R L G V G W G S F L Q E D I VQ A L I H M A L V L H A L F A S I D R R I R S L S R R V T A L E S R R T T G N P M T L A F I L G F L T V L C G C‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ R A R ‐ G L L G R L G I G W G S I L Q E D I VQ A L M H L V L V L H S V F I A I D R R L R S L T R R V T A L E A K R S A K N A V R I T L I L T G L M M V L G A
‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ L G T K T E W E N W I G L L R S D V TT G V V Y L A M V L L M M L K Q L R D R V W G L S R R V T R L E Q R R G A Q R G L Q G I G L L T L I M V T F A ‐‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ F M G K K V S R A D H Q R F R G S D K G A T LK V L N S F R K I I G N L V K T L Q G K R S ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ R N G R R S G T Q V S V L F I M L V I G C M G F R L 126‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ F T G R K I S N A Q H K R F R S I D K T Q A MK V L A T F K K I L G N L M K T L Q M R K K ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ K G N R R S G H G V G L V T L A F I G T V M A A T S
‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ F L G N K V S N A T K Q K F R N A K K S D V IK I L S G F K R T V T N L L S S V Q ‐ K R K ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ K N G K R S K T E I S L V V L M L F G A A M A A S M‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ F L A M R P S A S L K Q R W H K V D R K E G SK V L G K F R N V I G D M L K D L N S R K R ‐ ‐ ‐ ‐ ‐ ‐ K S K T S K R G L Q Q S F V V S C L W T M A A C A T L G
‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ F T A I K P S L G L I N R W G T V G K K E A ME I I K K F K K D L A A M L R I I N A R K ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ E R K R R G A D T S I G I V G L L L T T A M A A E I‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ F T A I A P T R A V L D R W R G V N K Q T A MK H L L S F K K E L G T L T S A I N R R S S ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ K Q K K R G G K T G I A V M I G L I A S V G A V T L
‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ F L A I P P T A G I L A R W G S F K K N G A IK V L R G F K K E I S N M L N I M N R R ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ K R S V T ‐ ‐ ‐ M L L M L L P T A L A F H L T
Supplemental Table 1. Estimates of evolutionary divergence between PSFV and other flavivirus aa sequences
Supplemental Table 2. Table. Estimates of evolutionary divergence between MAFV, OSFV and other ISFs aa sequences
The number of amino acid differences per site from between sequences are shown. The p-distance values of structural proteins are shown in lower left, and that of non-structural proteins are shown in upper right cells. All standard error estimate(s) obtained by a bootstrap procedure (500 replicates) are less than 0.02. All ambiguous positions were removed for each sequence pair. Evolutionary analyses were conducted in MEGA X.