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Title Diverse mosquito-specific flaviviruses in the Bolivian Amazon basin

Author(s)Orba, Yasuko; Matsuno, Keita; Nakao, Ryo; Kryukov, Kirill; Saito, Yumi; Kawamori, Fumihiko; Vega, Ariel Loza;Watanabe, Tokiko; Maemura, Tadashi; Sasaki, Michihito; Hall, William W.; Hall, Roy A.; Pereira, Juan Antonio;Nakagawa, So; Sawa, Hirofumi

Citation Journal of general virology, 102(3), 001518https://doi.org/10.1099/jgv.0.001518

Issue Date 2021-01-08

Doc URL http://hdl.handle.net/2115/81625

Rights

© Orba, Yasuko; Matsuno, Keita; Nakao, Ryo; Kryukov, Kirill; Saito, Yumi; Kawamori, Fumihiko; Vega, Ariel Loza; Watanabe, Tokiko; Maemura, Tadashi; Sasaki, Michihito; Hall, William W.; Hall, Roy A.; Pereira, Juan Antonio; Nakagawa, So; Sawa, Hirofumi, 2021. The definitive peer reviewed, edited version of this article is published in Journal of generalvirology, 102 (3), 2021, http://doi.org/10.1099/jgv.0.001518.

Type article (author version)

File Information JGV for huscap orba BVFlavi 2100601.pdf

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

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Title: Diverse Mosquito Specific Flaviviruses in the Bolivian Amazon basin 1

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Authors: Yasuko Orba 1,2, Keita Matsuno 2,3, Ryo Nakao 4, Kirill Kryukov 5, Yumi Saito 1, 3

Fumihiko Kawamori 6, Ariel Loza Vega 6, Tokiko Watanabe 7, Tadashi Maemura 8, Michihito Sasaki 4

1, William W. Hall 2,9,12, Roy A. Hall 10, Juan Antonio Pereira 6, So Nakagawa 11, Hirofumi Sawa 1,2,12 5

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Author affiliations: 7

1 Division of Molecular Pathobiology, Research Center for Zoonosis Control, Hokkaido University, 8

Sapporo, Japan 9

2 International Collaboration Unit, Research Center for Zoonosis Control, Hokkaido University 10

3 Unit of Risk Analysis and Management, Research Center for Zoonosis Control, Hokkaido 11

University 12

4 Laboratory of Parasitology, Faculty of Veterinary Medicine, Hokkaido University 13

5 Department of Genomics and Evolutionary Biology, National Institute of Genetics, Shizuoka, Japan 14

6 Faculty of Veterinary Sciences, Gabriel Rene Moreno Autonomous University, Santa Cruz, Bolivia 15

7 Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 16

Osaka, Japan 17

8 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, 18

Madison, Wisconsin, USA 19

9 Centre for Research in Infectious Diseases, University College Dublin, Dublin, Ireland 20

10 Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, 21

University of Queensland, St. Lucia, Queensland, Australia 22

11 Department of Molecular Life Science, Tokai University School of Medicine, Kanagawa, Japan 23

12 Global Virus Network, Baltimore, Maryland, USA 24

Address for correspondence: 25

Yasuko Orba, Division of Molecular Pathobiology, Research Center for Zoonosis Control, Hokkaido 26

University, N20, W10, Kita-ku, Sapporo 001-0020, Japan; E-mail: orbay@czc.hokudai.ac.jp 27

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Keywords: Flavivirus, Insect-specific flavivirus, Mosquito, Bolivia, Amazon 29

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Repositories: LC567151, LC567152, LC567153 30

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Abstract 32

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The genus Flavivirus includes a range of mosquito-specific viruses in addition to well known 34

medically-important arboviruses. Isolation and comprehensive genomic analyses of viruses in 35

mosquitoes collected in Bolivia resulted in the identification of three novel flavivirus species. 36

Psorophora flavivirus (PSFV) was isolated from Psorophora albigenu. The coding sequence of the 37

PSFV polyprotein shares 60% identity with that of the Aedes-associated lineage II insect-specific 38

flavivirus (ISF), Marisma virus. Isolated PSFV replicates in both Aedes albopictus- and Aedes aegypti-39

derived cells but not in mammalian Vero or BHK-21 cell lines. Two other flaviviruses, Ochlerotatus 40

scapularis flavivirus (OSFV) and Mansonia flavivirus (MAFV) which were identified from 41

Ochlerotatus scapularis and Mansonia titillans, respectively, group with the classical lineage I ISFs. 42

The protein coding sequences of these viruses share only 60% and 40% identity with the most closely-43

related of known lineage I ISFs, including Xishuangbanna aedes flavivirus and Sabethes flavivirus, 44

respectively. Phylogenetic analysis suggests that MAFV is clearly distinct from the groups of the 45

current known Culicinae-associated lineage I ISFs. Interestingly, the predicted amino acid sequence of 46

the MAFV capsid protein is approximately two-times longer than that of any of the other known 47

flaviviruses. Our results indicate that flaviviruses with distinct features can be found at the edge of the 48

Bolivian Amazon basin at sites that are also home to dense populations of human-biting mosquitoes. 49

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Introduction 51

52

Major Aedes-borne flaviviruses, including Dengue virus 1–4 (DENV) and Zika virus (ZIKV), 53

circulate in the Bolivian lowland areas but not in the highlands. In lowland tropical regions, these 54

viruses typically circulate during the rainy season from November to April [1, 2]. A human case of 55

Ilheus virus (ILHV) infection was also identified in Magdalena, in the Beni Department in Northern 56

Bolivia in 2005 [3]. ILHV is also a mosquito-borne flavivirus which has been detected in and isolated 57

from Aedes, Psorophora, Ochlerotatus, and Culex species of mosquitoes collected in the Amazon 58

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basin regions of both Peru and Brazil [4-6]. Alphaviruses, including Chikungunya virus (CHIKV), 59

Mayaro virus (MAYV), and equine encephalitis viruses (EEVs) have also spread throughout Latin 60

America. CHIKV specifically has been associated with large outbreaks of disease in the tropical areas 61

of Bolivia in 2016 [7]. 62

Cell fusing agent virus was the first identified insect-specific flavivirus (ISF) and this was originally 63

identified in cultures of Aedes aegypti cells [8]. Subsequently, a large number of ISFs have been 64

identified as a result of advances in the metagenomic analysis of field-collected mosquitos. ISFs are 65

believed to replicate exclusively in insect cells and not in vertebrate cells. The lineage I ISFs are 66

phylogenetically distinct from the mosquito- and tick-borne flavivirus pathogens that infect vertebrate 67

hosts; the lineage II ISFs (dual-host affiliated ISFs) constitute clades within the group of pathogenic 68

mosquito-borne flaviviruses (MBFVs) [9]. Replication of lineage II ISFs is also restricted to mosquito 69

cell and these viruses have never been identified in vertebrates. However, it is possible that there may 70

exist as yet uncharacterized natural life cycles in which some lineage II ISFs are transmitted from 71

mosquitoes to vertebrate animals with no associated pathogenicity. 72

In this study, mosquitoes collected in Bolivian lowland areas, including a forested area of the 73

Amazon basin, were investigated in order to identify potential novel flaviviruses and/or alphaviruses. 74

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Materials and Methods 76

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Mosquito collection 78

Mosquito collections were carried out in a forested area of Trinidad, the capital of Beni Department 79

(14°43'10"S 64°56'45"W) in October 2018 and August 2019, and in Buena Vista, a town in the Santa 80

Cruz Department at the north side of the Amboro National Park (17°22'26"S 63°39'40"W) in October 81

2018. Mosquitoes were trapped beginning in the afternoon hours until the following morning using 82

Centers for Disease Control (CDC) light traps (John W. Hock Co., Gainesville, FL, USA) with CO2 83

produced by yeast fermentation, and BG-sentinel traps (Biogents AG, Regensburg, Germany). Hand-84

nets were used in the morning and early evening collections. Sampling was carried out for one or two 85

nights in each location. Collected mosquitoes were killed by freezing. After species identification 86

based on morphology with reference to identification keys for mosquitoes, one to as many as 40 87

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female mosquitoes from each species were pooled [10, 11]. Molecular identification of each species 88

was confirmed by polymerase chain reaction (PCR) amplification and sequencing of the cytochrome 89

oxidase I (COI) gene [12]. 90

91

Detection of flavivirus and alphavirus genes by reverse transcription-PCR (RT-PCR) 92

Mosquitoes were immersed in minimum essential medium containing 2% fetal bovine serum 93

supplemented with an antibiotic antimycotic solution (penicillin, streptomycin, and amphotericin B) 94

and homogenized using a BioMasher (Nippi, Tokyo, Japan). Aliquots of supernatants from the 95

mosquito homogenates (100 µl) were used for RNA extraction using the Direct-Zol kit (Zymo 96

research, CA, USA) according to the manufacturer’s instructions. The remaining portions of the 97

supernatants were filtered and inoculated onto cells for virus propagation. Pan-flavivirus and pan-98

alphavirus RT-PCR assays were performed with a PrimeScript One-step RT-PCR kit Ver.2 (Takara, 99

Shiga, Japan) and degenerate primer sets, including Flavi all S and DEN4 and Flavi all AS2 [13, 14] 100

for identification of flaviviruses, and nsP4-6692F (5'-CAYACRYTRTTYGAYATGTCDGC-3') and 101

nsP4-7152R (5'-GCRTCDATKATYTTBACYTCCAT-3') for alphaviruses [15]. The cycling protocol 102

included 30 min of incubation at 50 °C for cDNA synthesis, followed by 2 min of incubation at 94 °C, 103

and 43 cycles each of 94 °C for 30 sec, 53 °C (for flaviviruses) or 52 °C (for alphaviruses) for 30 sec 104

and 72 °C for 30 sec, followed by 72 °C for 5 min. The amplification products were sequenced using 105

a BigDye Terminator v3.0 Cycle Sequencing kit on an ABI PRISM 3130 Genetic Analyzer (Applied 106

Biosystems, Foster City, CA, USA). 107

108

Virus isolation 109

Viruses were isolated from mosquito homogenates by inoculating C6/36 (Aedes albopictus) cells, 110

Vero (African green monkey kidney) cells and BHK-21 (baby hamster kidney) cells in minimum 111

essential medium supplemented with 2% FBS, penicillin, streptomycin, gentamycin and 2 mM L-112

glutamine. Cultures were maintained in a 5% CO2 atmosphere at 28 °C for C6/36 cells or at 37 °C for 113

Vero and BHK-21 cells. All virus isolation studies were performed in Biosafety Level-3 facilities at 114

the Research Center for Zoonosis Control in Sapporo, Japan. Isolation of PSFV by a limiting dilution-115

culture method was confirmed by RT-PCR and RNA sequencing using the supernatants of PSFV-116

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infected cells. 117

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Virus genome sequencing 119

Whole genome sequences of flaviviruses were determined by Illumina dye sequencing and rapid 120

amplification of cDNA ends (RACE) analyses. Double-stranded cDNAs were transcribed from RNA 121

extracted from virus isolates or from the mosquito homogenates. These were used for library 122

preparation using Nextera XT DNA Library Prep (Illumina, San Diego, CA, USA), followed by 123

sequencing with a MiSeq Reagent Kit v3 (600 cycles) and an Illumina MiSeq System (Illumina). 124

Programs used for the de novo assembly of virus genomes included the CLC Genomics Workbench 125

10 (CLC bio, Qiagen, Hilden, Germany), SPADes [16], and Trinity [17]. Virus-associated contigs 126

longer than 500 nucleotides were identified by BLASTN searches against viral genomes in the 127

National Center for Biotechnology Information (NCBI) database 128

(https://www.ncbi.nlm.nih.gov/genome/viruses/). 129

The 5'- and 3'-sequences at the ends of the flavivirus RNA genomes were determined using RACE 130

analyses; these studies were performed using a 5'-Full RACE Core Set (Takara) or SMARTer RACE 131

5'/3' kit (Takara) according the manufacturer’s instructions. A poly-A tail was ligated to the isolated 132

RNA using E. coli poly (A) polymerase prior to cDNA synthesis in order to amplify 3’-ends using the 133

SMARTer RACE system. Identified terminal sequences were confirmed by Sanger sequencing. 134

Unfortunately, we were unsuccessful in the amplification of the 3'-end of the MAFV or the 3'- or 5'-135

ends of OSFV untranslated region (UTR) sequences using these RACE systems. 136

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Analyses of genetics and molecular evolution 138

Deduced amino acid sequences of the novel flaviviruses were evaluated by BLAST search, and 139

homologies with previously-characterized viral structural and non-structural proteins were analyzed 140

by the fastp program using GENETYX Ver.15. 141

For the phylogenetic analysis, amino acid sequence alignment of flavivirus polyproteins were 142

generated using L-INS-i program in the MAFFT suite version 7.467 [18]; gaps were removed using 143

TrimAl program with a gappyout option [19]. The amino acid replacement model of 144

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Le_Gascuel_2008 [20] was selected using ProtTest3 [21]. Maximum likelihood-based phylogenetic 145

tree search was performed using RAxML-NG with 1000 bootstrap replicates [22]. 146

The amino acid sequences of flavivirus capsid proteins were aligned using ClustalW [23] and 147

MEGA7 [24]. To predict cleavage sites for each flavivirus protein, SignalP 5.0 and ProP 1.0 were 148

used to identify potential cleavage sites [25, 26], and the TMHMM Server v.2.0 or SOSUI were used 149

to predict transmembrane helices in proteins [27, 28]. Secondary structure of the Mansonia flavivirus 150

(MAFV) capsid protein sequence was predicted using the PSIPRED and DISOPRED server [29-31]. 151

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Virus growth in cells 153

Mosquito Aedes albopictus C6/36 cells, Aedes aegypti CCL-125 cells, Culex quinquefasciatus Hsu 154

cells, Culex tarsalis Chao Ball cells and mammalian Vero cells and BHK-21 cells were employed in 155

flavivirus growth assays. CCL-125 cells from the ATCC, Hsu and Chao Ball cells (provided from the 156

University of Queensland, originally from the University of Texas Medical Branch) were maintained 157

in Leibovitz’s L15 medium supplemented with 2% FBS, 10% tryptose phosphate broth, penicillin, 158

streptomycin, and 2 mM L-glutamine at 28 °C during virus infection assays. Cells in a 12-well plate 159

were infected with 108 copies of PSFV for 1 hour at 28 °C or 37 °C; cell supernatants were then 160

removed, and new medium was added after washing. Cell supernatants were collected at 0, 24, 48 and 161

72 hours later. The genome copy number of PSFV in the supernatant collected at each time point was 162

quantified by a quantitative RT-PCR assay using PSFV-specific primer sets (PSFV F 5’-163

GGTTATCACGTGGCAACAGTC and PSFV R 5’-TGCGGTACGCTAAGTCCAGAACG) and the 164

One Step TB Green II kit (Takara) in a StepOnePlus real time PCR system (Applied Biosystems). The 165

copy numbers were estimated by a standard curve method with serially diluted 109 copies of RT-PCR 166

product of PSFV genome (5,415 – 9,466 nt). 167

Immunostaining of the PSFV non-structural protein (NS)1 in C6/36, CCL-125 and Hsu cells at 72 168

hours after virus infection was performed with an anti-flavivirus NS1 monoclonal antibody (4G4) [32, 169

Mozzy Mabs - https://eshop.uniquest.com.au/mozzy-mabs/] and Alexa 488-labeled anti-mouse IgG. 170

Cell nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI). Fluorescent images 171

were evaluated using the Olympus IX-73 microscope (Olympus, Tokyo, Japan). 172

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Electron microscopy 174

Culture supernatants of PSFV-infected C6/36 cells were fixed with 0.25% glutaraldehyde and 175

concentrated using an Amicon Ultra 100K filter (Merck Millipore, Darmstadt, Germany). The virus 176

particles were then negatively stained in a 2% phosphotungstic acid solution (pH 5.8) on a collodion-177

carbon-coated copper grids (Nisshin EM Corporation, Tokyo, Japan). Virions were analyzed using a 178

H-7650 electron microscope at 80 kV (Hitachi, Kyoto, Japan). 179

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Results 181

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Mosquitoes were captured in a forested area of Trinidad in two different seasons, in October 2018, 183

and in August 2019. In October, which is the beginning of rainy season, the main mosquito species 184

collected was Psorophora (Ps.) albigenu. A total of 3,942 adult female mosquitoes were examined in 185

133 pools comprising each species for detection and isolation of both flaviviruses and alphaviruses 186

(Table 1). 187

The pan-flavivirus RT-PCR assay detected two different sequences with some similarity to 188

previously-identified flavivirus NS5 genes. These sequences were identified from RNAs extracted from 189

Ps. albigenu (1/47 pools) and Ochlerotatus (Och.) scapularis (1/6 pools) collected in October 2018. 190

Notably no alphavirus genes were detected in any of mosquitoes collected using the pan-alphavirus RT-191

PCR assay. 192

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A flavivirus was isolated from Ps. albigenu homogenate used to inoculate Aedes albopictus-derived 194

C6/36 cells and this was tentatively named Psorophora flavivirus (PSFV). This virus replicated in 195

C6/36, Aedes aegypti-derived CCL-125, and Culex tarsalis-derived Chao Ball cells without obvious 196

cytopathic effects; in contrast no replication was detected in Culex quinquefasciatus-derived Hsu cells 197

or in the mammalian Vero or BHK-21 cells (Figure 1A). Immunostaining for flavivirus NS1 protein 198

revealed that almost all C6/36 cells were infected with PSFV at 72 hours post-inoculation. In contrast, 199

focal infections in CCL-125 cells, and weak positive signals in Chao Ball cells were observed. Very 200

few NS1-positive foci were detected in Hsu cells (Figures 1B). Typical flavivirus particles of ~40 nm 201

in size were detected in supernatants of PSFV-infected C6/36 cells by electron microscopy (Figure 202

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1C). 203

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In efforts to identify the whole genome sequence of PSFV, RNA was extracted from the 205

supernatants of PSFV-infected cells and analyzed by RNA sequencing, followed by 5'- and 3'-RACE 206

assays. We confirmed that the supernatants containing PSFV was not contaminated by other viruses 207

by total RNA sequencing. The complete RNA genome sequence of PSFV (10,831 nt) was determined 208

and the viral polyprotein coding sequence (CDS; 3,448 aa) and those of specific viral proteins were 209

deduced (Table 2). The polyprotein of flaviviruses is proteolytically cleaved by both viral and host 210

proteases into three structural proteins (capsid, membrane, and envelope) and seven non-structural 211

proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). 212

The flavivirus identified in Och. Scapularis was named Ochlerotatus scapularis flavivirus (OSFV). 213

This virus was not amplified in any of the cell cultures examined; as such, total RNAs extracted from 214

the OSFV-positive mosquito pool were used for generation of the whole genome sequence by 215

Illumina sequencing and RACE assays. The complete polyprotein CDS of OSFV was identified 216

(3,411 aa), although we have not yet succeeded in identifying the ends of 5'-and 3'-terminal 217

untranslated regions (UTR) sequences (Table 2). 218

Finally, RNA sequencing analyses of total RNAs extracted from representative other species of 219

mosquito pools resulted in the identification of another novel flavivirus genome sequence from a pool 220

derived from Mansonia (Ma.) titillans. This flavivirus has tentatively been named Mansonia flavivirus 221

(MAFV). The almost complete genome sequence of MAFV, including the complete polyprotein CDS 222

(3,523 aa) and the 5'-UTR was identified by RACE assays (Table 2). The 5'-terminal nucleotide 223

sequence of MAFV, including the UTR and the capsid protein coding region was confirmed by 224

Sanger sequencing using specific primers. The genome sequences of these three novel flaviviruses 225

have been submitted to the DDBJ/EMBL/GenBank databases and have been assigned accession 226

numbers LC567151 (PSFV), LC567152 (OSFV), and LC567153 (MAFV). 227

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We performed a BLAST analysis, and found that the PSFV CDS shared 60% amino acid sequence 229

identity with the CDS of the lineage II ISF Marisma virus (accession number MF139576). Likewise, 230

the CDSs of OSFV and MAFV share 60% and 40% identity with previously-identified lineage I ISFs, 231

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including Xishuangbanna aedes flavivirus (accession number KU201526) and Sabethes flavivirus 232

(accession number MH899446), respectively. When the amino acid homologies of the structural and 233

non-structural proteins were evaluated separately, we found that PSFV structural proteins shared 45-234

48% identity and non-structural proteins shared 62-64% identity with those of lineage II ISFs. Of 235

note, these flaviviruses are primarily those that were originally isolated from Aedes spp. mosquitoes, 236

which are included within the proposed new lineage IIa (Table 3 and supplemental Table 1) [33-39]. 237

The results of pairwise comparisons and evolutionary distances among OSFV, MAFV and related 238

lineage I ISFs revealed that MAFV is a highly divergent flavivirus with sequence identities with the 239

closest known relative being 30% with the structural proteins of Calbertado virus, and 42% with the 240

non-structural proteins of Nienokoue virus (Table 4 and supplemental Table 2). The relationships 241

between OSFV and MAFV, which were identified from the same area but from different species of 242

mosquitoes, suggest that they are divergent ISF species in the lineage I in that their non-structural 243

proteins share approximately 40% amino acid sequence identity (Table 4). 244

245

A phylogenetic tree built using the full-length flavivirus polyprotein CDSs revealed that the 246

separation of PSFV is deepest within the clade of lineage IIa ISFs (Figure 2). Likewise, MAFV is 247

deeply separate in lineage Id ISFs consisting of Sabethes flavivirus (MH899446), Culiseta flavivirus 248

(KT599442), Mercadeo virus (NC 027819), and Calbertado virus (KX669682) (Figure 3). In addition 249

MAFV was frequently located basal to all three Culicinae-associated lineages (i.e. Ia, Ic, and Id 250

[39]) in the bootstrap trees (data not shown). These results suggest that MAFV may retain ancestral 251

characteristics of the current known Culicinae-associated lineage I ISFs. Meanwhile, OSFV clearly 252

belonged to a clade of Aedes-associated lineage Ia; these results were consistent with the fact that 253

OSFV was isolated from Ochlerotatus mosquitoes, a species that is closely related to mosquitoes of 254

the genus Aedes in the tribe Aedini [40-42]. 255

256

Multiple alignments and predictions of individual viral protein sequences revealed that MAFV 257

CDS is larger than those of any of the other known flaviviruses due to the addition of more than 100 258

amino acid residues at the N-terminus end of the polyprotein (Figure 4). A putative cleavage site for 259

the viral protease NS2B/NS3 was predicted at a site 249 aa where upstream of the alpha-helices that 260

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comprise the anchored capsid region. Typical helical structures and positively-charged residues were 261

identified at the C-terminus of the MAFV capsid protein (Figure 4), although the disordered N-262

terminal region shares no specific similarity with any other viruses or any known sequences in a 263

BLAST database search. As such, the characteristic capsid protein of MAFV can be deduced only 264

from the genomic sequence. 265

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Discussion 267

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Some arboviruses, including ILHV and MAYV which have natural enzootic cycles involving 269

forest-dwelling mosquitoes and animals in the Amazon basin have the potential to emerge as human 270

pathogens as a result of increased human travel, deforestation and urbanization in tropical areas [43]. 271

Climate change also modifies the distribution of vector mosquitoes and increases the possibility of 272

new arbovirus disease outbreaks in areas of temperate climate [44]. In late October, the forested area 273

where we collected mosquitoes in Trinidad was inhabited with the mosquito species Ps. albigenu, at 274

high density. Ps. albigenu is distributed widely in South America, with high population densities in 275

areas of high rainfall [45]. Arboviruses, including ILHV, MAYV, EEVs, DENV have all been 276

identified in Psorophora spp. which are present in numerous South American countries [46-48]. The 277

feeding habitats of the Psorophora spp. indicated that it could probably serve as a vector for sylvatic 278

transmission of other arboviruses [49]. 279

The present study has explored the possibility that medically-significant arboviruses might be 280

identified in forested areas of the Bolivian Amazon basin region and our overall goal was to collect 281

information which could facilitate preemptive measures against emerging mosquito-borne diseases. 282

Although we identified no mosquito-borne arboviruses, we did instead discover a diverse group of novel 283

flaviviruses from several species of local mosquitoes. 284

PSFV appears to be an Aedes-associated lineage IIa ISF that is closely-related to the MBFVs. This 285

is consistent with the fact that PSFV was isolated from the genus Psorophora which belongs in the 286

mosquito tribe Aedini, as is the mosquito genus, Aedes. The results of PSFV growth assays indicated 287

that not only Aedes-derived C6/36 and CCL-125 cells but also Culex-derived Chao Ball cells were 288

susceptible to PSFV infection (Figures 1B). Further research is required to understand the host 289

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species-specificity of linage II ISFs. Although PSFV did not replicate in mammalian BHK-21 or Vero 290

cells, there remains the possibility that PSFV may have one or more other vertebrate hosts that inhabit 291

this region. 292

Recent reports indicated that ISFs have the potential to inhibit arboviruses via a mechanism known 293

as superinfection exclusion [50]. As such, there are ongoing studies that utilize a lineage II ISF to 294

generate chimeric flaviviruses with MBFVs as candidate vaccine antigens [51]. The differences 295

between lineage II ISFs and mosquito-borne arboviruses involved in host restriction factors are now 296

under investigation to develop strategies to combat arbovirus-associated diseases. 297

Two novel lineage I (classical) ISFs were also identified in this study. These flaviviruses could not 298

be isolated from the mosquito pools, most likely due to low virus copy numbers, a low susceptibility 299

of C6/36 and Hsu cells, or the presence of other competing mosquito-specific viruses which 300

proliferate at a higher rate. The Illumina sequencing of total RNAs from mosquitoes included an 301

average coverage of OSFV sequence at 27 with that of MAFV at 64. In contrast, the average coverage 302

of other insect-specific RNA viruses such as negevirus and picorna-like virus were more than 10,000 303

(data not shown). However, the complete polyprotein CDS and almost the entire genome sequences of 304

OSFV and MAFV were successfully identified in RNA extracted directly from mosquitoes. 305

The OSFV and especially the MAFV display sequences that are evolutionarily diverse are not 306

closely related to those in previously-characterized flaviviruses. A phylogenetic analysis suggested 307

that the MAFV may be one of the oldest ISF that was diverged in the lineage of the Culicinae-308

associated lineage I ISFs (Figure 3). The low sequence identity shared by the MAFV genome with 309

known flaviviruses may be why this virus was not detected by the pan-flavivirus RT-PCR assay. 310

Investigations on the structure of the flavivirus capsid protein have revealed that both the structure 311

and charge distribution are well conserved among flaviviruses [52-55]. The MAFV capsid proteins 312

contain five α-helices; a host signal peptidase cleaves at the junction between capsid helix α5 and prM 313

at the ER lumen, leaving helix α5 anchored within the ER membrane (anchor C). The viral protease 314

NS2B/NS3 cleaves at the junction between helix α4 with α5 at the protein cytoplasmic face to release 315

the mature capsid protein. The capsid protein generates a dimer at the highly positively-charged α4 316

helices that have been implicated in interactions with viral RNA [56, 57]. As noted above, the 317

deduced amino acid sequence of the MAFV capsid protein is almost 2-times longer than those of 318

12

other flaviviruses. The predicted viral protease cleavage site and the α-helix sequence of anchor 319

capsid regions have been identified in the MAFV capsid protein. The highly charged helix α4 domain 320

appears to be conserved among all the lineage II ISFs, including PSFV. The lineage I ISFs, including 321

OSFV and MAFV, also maintain α-helical structures at the C-terminal region of the capsid proteins 322

that include positively charged residues (Figure 4). The uncharacteristically long N-terminal region of 323

the MAFV capsid protein indicates that it most likely includes one or more regions of disordered 324

structure which have the potential to interact with host and/or viral proteins. Further investigations 325

should reveal novel functions of the MAFV capsid protein that might have been lost during virus 326

evolution. 327

328

Author statements 329

Investigation: Y.O., Y.S., K.M., R.N., H.S., S.N., K.K., T.W., T.M., W.W.H., Supervision: Y.O., S.N., 330

J.A.P., H.S., Resources: Y.O., K.M., R.N., F.K., A.L., T.W., T.M., R.A.H., J.A.P., M.S., K.K., S.N., 331

Project Administration: J.A.P., H.S., Writing – Original Draft Preparation: Y.O., S.N., K.M., R.A.H., 332

W.W.H., Writing – Review and Editing: all 333

334

Conflict of Interest 335

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

13

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

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487

488

17

489

Table 1. Mosquito species analyzed in this study 490 Season Place Species No.

mosquito No. pool

Alphavirus (+) pool

Flavivirus (+) pool

Identified virus

2018 Oct Trinidad Psorophora albigenu 1,779 47 0 1a Psorophora flavivirus (PSFV) bvmq18-51

Ochlerotatus scapularis 188 6 0 1a Ochlerotatus scapularis flavivirus (OSFV) bvmq18-25

Culex tatoi 141 4 0 0

Mansonia titillans 25 1 0 1b Mansonia flavivirus (MAFV) bvmq18-1

Coquillettidia nigricans 5 1 0 0

Others 2 2 0 0

Buena Vista /Amboro NP

Psorophora albigenu 2 2 0 0

Ochlerotatus scapularis 3 1 0 0

Culex quinquefasciatus 57 3 0 0

Others 2 2 0 0

2019 Aug Trinidad Psorophora sp. 8 3 0 0

Ochlerotatus serratus 111 4 0 0

Culex quinquefasciatus 24 2 0 0

Culex maxi 356 8 0 0

Culex spp. 31 5 0 0

Mansonia titillans 879 28 0 0

Coquillettidia nigricans 3 2 0 0

Anopheles oswaldoi 280 7 0 0

Uranotaenia sp. 38 2 0 0

Others 8 3 0 0

Total 3,942 133 0 3

a The viral gene was identified by pan-flavivirus RT-PCR. 491

b The viral gene was identified by total RNA sequencing. 492

493

18

Table 2. Predicted structure of the viral genomes and encoded proteins 494

Genome (nt)

5'-UTR (nt)

3'-UTR (nt)

CDS (aa)

C/ ancC (aa)

pre/ M (aa)

E (aa)

NS1 (aa)

NS2A (aa)

NS2B (aa)

NS3 (aa)

NS4A (aa)

2K/ NS4B (aa)

NS5 (aa)

PSFV 10,831 114 373 3,448 105/ 22 91/ 74 501 355 227 131 621 128 23/ 263 807

OSFV 10,449~a a~33 183~a 3,411 121/ 23 86/ 62 437 403 195 166 599 142 23/ 266 888

MAFV 10,982~b 88 325~b 3,523 249/ 20 85/ 63 426 394 228 148 593 147 23/ 251 896

aThe 5'- and 3'-terminal of UTR sequences have not been determined. 495

bThe 3'-terminal of UTR sequence has not been determined. 496

UTR, untranslated region; CDS, cording sequence; C, capsid protein; ancC, anchor capsid; 497

pre, pre-membrane protein; M, membrane protein; E, envelope protein; NS, non-structural protein. 498

499

19

Table 3. Comparison of the deduced amino acid sequences of structural and non-structural proteins 500

of PSFV with those of other flaviviruses 501 PSFV Structural Non-structural

Identity (%)

Similarity (%)

Identity (%)

Similarity (%)

Lineage IIa ISFs

Panmunjeom 47 84 64 90 Donggang 46 85 64 91 Marisma 46 85 64 91 Ilomantsi 45 84 64 91 Long Pine Key 45 83 63 90 Lammi 48 85 62 90 Chaoyang 48 85 62 90

Lineage IIb ISFs

Barkedji 41 81 49 83 Nhumirim 38 80 49 83 Nanay 38 78 49 83

Mosquito-borne

flaviviruses

West Nile 42 81 50 84 Zika 37 81 52 84 Dengue 1 37 77 48 82 Yellow fever 34 79 48 82

502

Table 4. Comparison of the deduced amino acid sequences of structural and non-structural proteins 503

of OSFV and MAFV to those of other lineage I ISFs. 504 OSFV MAFV Structural Non-structural Structural Non-structural

Identity (%)

Similarity (%)

Identity (%)

Similarity (%)

Identity (%)

Similarity (%)

Identity (%)

Similarity (%)

Xishuangbanna 52 84 61 88 24 72 40 76 Menghai 51 84 59 87 24 74 39 75 Cell-fusing agent 36 77 46 80 28 74 40 76 Parramatta River 37 76 45 79 27 74 41 76 Hanko 35 74 44 79 26 73 40 76 Palm Creek 37 79 42 75 24 79 41 77 Culex flavi 36 78 41 75 28 71 41 76 Nienokoue 35 75 41 75 28 71 42 77 Mercadeo 28 74 38 73 29 75 41 75 Culiseta 27 70 40 75 28 75 43 77 Calbertado 27 70 40 73 30 71 39 73 Sabethes flavi 25 68 39 74 29 71 40 75 Anopheles flavi 26 69 40 74 28 70 40 76 MAFV 24 71 40 75 100 100 100 100 PSFV 21 60 29 66 18 58 29 67

505

20

Figure legends 506

Figure 1. Characterization of the isolated Psorophora flavivirus (PSFV) 507

A. Viral RNA copy number of PSFV in supernatants from infected C6/36, CCL-125, Hsu, Chao Ball, 508

Vero or BHK-21 cells at time points indicated as hours post-infection (h.p.i). Data represented the 509

mean (±SD) of three independent experiments. B. C6/36, CCL-125, Chao Ball and Hsu cells infected 510

with PSFV or mock for 72 hrs were stained with anti-NS1 antibody and Alexa-488 conjugated 511

secondary antibody; cell nuclei were counterstained with DAPI. Scale bars: 20 µm. C. Negatively-512

stained enveloped virus particles ~40 nm in size of were detected in the supernatant of PSFV-infected 513

C6/36 cells by the electron microscopy; scale bar indicates 50 nm. 514

515

Figure 2. Molecular phylogenetic analysis of flavivirus polyproteins 516

The amino acid sequences of flavivirus polyproteins were aligned using the MAFFT program; 517

Maximum likelihood-based phylogenetic tree was generated using RAxML-NG with 1000 bootstrap 518

replicates; bootstrap values are shown adjacent to the tree branches. The tree is drawn to scale with 519

branch lengths representing the number of substitutions per site. MBFV, mosquito-borne flaviviruses; 520

ISFs, insect-specific flaviviruses; TBFV, tick-borne flaviviruses; NKV, no known vector flaviviruses. 521

Each flavivirus sequence has an appended GenBank accession number. 522

523

Figure 3. Molecular phylogenetic analysis of lineage I ISFs polyprotein 524

The phylogenetic tree focuses on the lineage I ISFs from the phylogenetic analysis shown in Figure 2. 525

Sublineages Ia, Ib, Ic, and Id within the lineage I ISFs are indicated by bar lines. The tree is drawn to 526

scale with branch lengths representing the number of substitutions per site. MBFV, mosquito-borne 527

flaviviruses; ISFs, insect-specific flaviviruses; TBFV, tick-borne flaviviruses; NKV, no known vector 528

flaviviruses. Each flavivirus sequence has an appended GenBank accession number. 529

530

Figure 4. Sequence of the capsid protein of MAFV 531

A. Schematic representation of the flavivirus genome and predicted coding sequences. The deduced 532

amino acid sequence of the MAFV capsid protein is shown with the predicted helical and disordered 533

secondary structure. Amino acid residues forming the helices are highlighted in yellow, and the 534

21

amino-terminal disordered residues are highlighted in gray. Hydrophobic, positively- and negatively-535

charged amino acids are indicated in green, blue and red, respectively. B. Multiple alignment was 536

performed using the deduced amino acid sequences of the capsid proteins of lineage I ISFs, including 537

OSFV and MAFV, lineage II ISFs, and MBFVs. The predicted viral protease NS2B/NS3 cleavage 538

sites are indicated by arrows. The yellow bars represent conserved helices α3, α4 and α5 as were 539

reported previously in capsid proteins of ZIKV and DENV. The gray bar represents the membrane-540

anchor regions of the capsid proteins. 541

BC6/36 CCL‐125

50 um

Hsu

50 nm

Figure 1

C

RN

A c

opie

s /1

ul s

up

A

Mock 

PSFV 

Chao Ball

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

0 24 48 72

C6/36

CCL-125

Hsu

ChaoBall

Vero

BHK-21

h.p.i.

PSFV RNA

0.5

MG587038: Binjari virusMN954647: Hidden Valley virus

100

93

52

8768

100

60 100

100

100

10073

100

100

100

100

100

100

100

100

84

100

100

95

54

100

100

100

100

100

100

100100

100

100

100

89

100

100

45

60

100

100

100

100

KC734551: Bagaza virus

DQ211652: West Nile virusEU082199: Yaounde virus

M18370: Japanese encephalitis virusAY453411: Usutu virus

AF161266: Murray Valley encephalitis virusDQ525916: St. Louis encephalitis virus

AY632539: Ilheus virus

JF895923: Tembusu virusJX236040: Ntaya virus

AY632536: Aroa virusAY632541: Kokobera virus

DQ859059: Zika virusAF326573: Dengue virus 4

M19197: Dengue virus 2

M93130: Dengue virus 3U88536: Dengue virus 1

MF139575: Nanay virusNC 024017: Nhumirim virusLC497470: Barkedji virusLC497469: Barkedji-like virus

JQ308185: Chaoyang virusFJ606789: Lammi virus

KY290254: Long Pine Key virus

JQ086551: Donggang virusMF139576: Marisma mosquito virus

KC692067: Ilomantsi virusKY072986: Panmunjeom flavivirus

Psorophora flavivirus

AB114858: Yokose virusDQ837641: Entebbe bat virus

DQ859063: Sepik virusDQ859058: Wesselsbron virus

JN620362: Yellow fever virusDQ859060: Edge Hill virus

DQ859057: Bouboui virusDQ859062: Saboya virusDQ859065: Uganda S virusDQ859056: Banzi virus

JX498940: Tick-borne encephalitis virus

AY323489: Omsk hemorrhagic fever virusAY323490: Kyasanur forest disease virus

AF160193: Apoi virusAJ242984: Modoc virus

AJ299445: Montana myotis leukoencephalitis virusAF144692: Rio Bravo virus

100

100

Lineage I ISFs

NKV

TBFV

Lineage IIb ISFs

MBFV

MBFV

NKV

Lineage IIaISFs

Figure 2

Figure 3

0.5

100

46

100

100

100

100

93

100

100

98

100

93

100

42

100

99

33

100

61

KT599442: Culiseta flavivirus

Mansonia flavivirus

KX148547: Anopheles flavivirus

KX669682: Calbertado virusNC 027819: Mercadeo virus

MH899446: Sabethes flavivirus

KU201526: Xishuangbanna aedes flavivirusKX907452: Menghai flavivirus Ochlerotatus scapularis flavivirus

M91671: Cell-fusing agent virus

NC 027817: Parramatta River virusNC 030401: Hanko virus

KX652378: Mosquito flavivirus

GQ165808: Culex flavivirus

655454924: Nienokoue virus

NC 030400: Nakiwogo virusKC505248: Palm Creek virus

MF352616 Mac Peak virusKY460522 Karumba virus

MF352617 Haslams Creek virus

Psorophora flavivirus

NKVTBFV

Lineage IIb ISFs

MBFV

MBFV

NKV

Lineage IIa ISFs

Lineage Ib

Lineage Id

Lineage Ia

Lineage Ic

MAFVOSFVMenghai flavivirusHanko virusMercadeo virusCFAVCulex flavivirusNienokoue virusPSFVMarisma virusLammi virusBarkedji virusZika virusWest Nile virusDengue virus 1

Figure 4

5’ UTR 3’ UTR

NS3 NS4BNS2AE NS4A2BNS1C prM NS5

1 MMFWNNGNEVSNLPGGKNPFKKPHEAGENASNRSKGAYGAGKLTNLKNSQGGRALATGGGWGSGLGGSSGTSRTHAPLSPTSFRTRINNF 90

91 GWFGGSGSGGVNNPPKAHATKAKLNQPDGYAHGWGSNSGSGARTMSQATHTTWKNKANLNSASEYGQVGNSKAMIPYLAGNQPPMLGGHP 180

181 DERQGRKKGQLKTRKLGQAQVKDRMKQFFNLSITEALEACMLVMVQLFSLLTLMIQRLIWQATRERKTRSVGFYFPLIVWMAFMATCCA 269

Helix

Disordered

HydrophobicPositively ChargedNegatively Charged

A

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

Line

age

ILi

neag

e II

MB

FV

Line

age

ILi

neag

e II

MB

FV

Line

age

ILi

neag

e II

MB

FV

MAFVOSFVMenghai flavivirusHanko virusMercadeo virusCFAVCulex flavivirusNienokoue virusPSFVMarisma virusLammi virusBarkedji virusZika virusWest Nile virusDengue virus 1

MAFVOSFVMenghai flavivirusHanko virusMercadeo virusCFAVCulex flavivirusNienokoue virusPSFVMarisma virusLammi virusBarkedji virusZika virusWest Nile virusDengue virus 1

viral protease α5α4α3

anchor capsid

249

Structural MAFV OSFV Menghai Xishuan-gbanna

Calber-tado

Anophel-es flavi CFAV Hanko Culiseta Merca-

deoParrama-tta river

Palm Creek

Culex flavi

Mosquito flavi

Nakiwo-go

Nieno-koue PSFV Non-structural

MAFV 0 0.591 0.603 0.594 0.596 0.597 0.594 0.592 0.590 0.589 0.586 0.587 0.583 0.583 0.580 0.575 0.714 MAFVOSFV 0.774 0.398 0.380 0.589 0.594 0.529 0.548 0.587 0.585 0.540 0.579 0.584 0.587 0.581 0.580 0.723 OSFVMenghai 0.788 0.482 0.220 0.597 0.600 0.528 0.540 0.601 0.591 0.531 0.583 0.592 0.584 0.588 0.582 0.729 MenghaiXishuangbanna 0.784 0.467 0.318 0.588 0.590 0.518 0.526 0.591 0.578 0.523 0.570 0.584 0.586 0.581 0.578 0.718 Xishuangbanna Calbertado 0.720 0.741 0.754 0.760 0.603 0.597 0.596 0.471 0.456 0.591 0.575 0.576 0.585 0.575 0.577 0.730 Calbertado Anopheles flavi 0.733 0.748 0.758 0.755 0.722 0.591 0.604 0.604 0.596 0.598 0.560 0.566 0.570 0.567 0.542 0.714 Anopheles flaviCFAV 0.740 0.639 0.642 0.621 0.716 0.737 0.551 0.591 0.587 0.545 0.584 0.585 0.588 0.588 0.585 0.721 CFAVHanko 0.757 0.654 0.644 0.651 0.734 0.718 0.593 0.597 0.593 0.256 0.586 0.582 0.585 0.581 0.579 0.720 HankoCuliseta 0.733 0.747 0.747 0.754 0.585 0.710 0.685 0.707 0.272 0.600 0.571 0.569 0.566 0.573 0.570 0.731 CulisetaMercadeo 0.722 0.738 0.720 0.742 0.532 0.705 0.676 0.676 0.348 0.591 0.573 0.561 0.572 0.574 0.571 0.731 MercadeoParramatta river 0.755 0.648 0.644 0.655 0.723 0.717 0.595 0.350 0.701 0.667 0.587 0.579 0.585 0.582 0.580 0.724 Parramatta riverPalm Creek 0.765 0.640 0.638 0.651 0.724 0.737 0.490 0.607 0.703 0.694 0.615 0.437 0.442 0.294 0.402 0.721 Palm CreekCulex flavi 0.736 0.647 0.640 0.633 0.706 0.741 0.324 0.618 0.675 0.686 0.598 0.493 0.282 0.451 0.439 0.719 Culex flaviMosquito flavi 0.747 0.639 0.635 0.627 0.703 0.750 0.256 0.620 0.699 0.687 0.617 0.491 0.298 0.458 0.452 0.720 Mosquito flaviNakiwogo 0.752 0.672 0.674 0.669 0.739 0.757 0.539 0.630 0.703 0.717 0.644 0.449 0.555 0.540 0.411 0.720 NakiwogoNienokoue 0.746 0.641 0.643 0.636 0.688 0.726 0.479 0.617 0.686 0.672 0.608 0.507 0.472 0.480 0.527 0.715 NienokouePSFV 0.867 0.868 0.861 0.854 0.886 0.879 0.854 0.861 0.864 0.857 0.860 0.869 0.854 0.854 0.866 0.861 PSFV

Structural PSFV Dong-gang Marisma Ilomantsi Panmun-

jeomLong Pine Key Lammi Chaoyang Barkedji Nhumirim Nanay Zika West Nile Dengue 1 Yellow

fever Non-structural

PSFV 0 0.349 0.351 0.360 0.360 0.367 0.369 0.371 0.508 0.509 0.503 0.469 0.494 0.514 0.515 PSFVDonggang 0.539 0.093 0.262 0.260 0.278 0.318 0.308 0.497 0.490 0.499 0.455 0.486 0.507 0.507 DonggangMarisma 0.535 0.144 0.267 0.262 0.283 0.320 0.312 0.501 0.495 0.499 0.461 0.486 0.504 0.508 MarismaIlomantsi 0.539 0.378 0.369 0.084 0.298 0.318 0.315 0.501 0.501 0.504 0.465 0.482 0.516 0.518 IlomantsiPanmunjeom 0.526 0.363 0.372 0.136 0.301 0.313 0.312 0.503 0.502 0.509 0.471 0.485 0.512 0.523 PanmunjeomLong Pine Key 0.551 0.363 0.366 0.425 0.434 0.335 0.336 0.499 0.505 0.499 0.467 0.484 0.520 0.516 Long Pine Key Lammi 0.518 0.454 0.458 0.483 0.471 0.479 0.110 0.494 0.495 0.501 0.473 0.479 0.509 0.508 LammiChaoyang 0.517 0.440 0.453 0.463 0.461 0.471 0.255 0.492 0.493 0.497 0.473 0.478 0.504 0.506 ChaoyangBarkedji 0.579 0.557 0.553 0.565 0.571 0.576 0.587 0.572 0.272 0.450 0.469 0.490 0.509 0.528 BarkedjiNhumirim 0.606 0.553 0.557 0.583 0.578 0.587 0.603 0.593 0.337 0.455 0.471 0.488 0.512 0.522 NhumirimNanay 0.623 0.628 0.631 0.619 0.626 0.631 0.633 0.635 0.549 0.550 0.496 0.505 0.516 0.526 NanayZika 0.619 0.569 0.582 0.608 0.613 0.574 0.578 0.578 0.592 0.574 0.605 0.404 0.438 0.514 Zika West Nile 0.569 0.554 0.556 0.554 0.549 0.583 0.572 0.559 0.557 0.551 0.612 0.496 0.459 0.533 West Nile Dengue 1 0.620 0.608 0.611 0.612 0.615 0.611 0.589 0.599 0.576 0.604 0.613 0.461 0.549 0.532 Dengue 1Yellow fever 0.668 0.637 0.634 0.625 0.635 0.645 0.636 0.623 0.633 0.652 0.662 0.614 0.607 0.616 Yellow fever

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.