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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
1
Title: Diverse Mosquito Specific Flaviviruses in the Bolivian Amazon basin 1
2
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
6
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: [email protected] 27
28
Keywords: Flavivirus, Insect-specific flavivirus, Mosquito, Bolivia, Amazon 29
2
Repositories: LC567151, LC567152, LC567153 30
31
Abstract 32
33
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
50
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
3
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
75
Materials and Methods 76
77
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
4
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
5
infected cells. 117
118
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
137
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
6
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
152
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
173
7
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
180
Results 181
182
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
193
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
8
1C). 203
204
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
228
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
9
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
10
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
266
Discussion 267
268
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
11
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
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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.