bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
1 The conservation of a core virome in Aedes 2 mosquitoes across different developmental stages 3 and continents 4
5 Chenyan Shi1,#, Lu Zhao2,3,#, Evans Atoni2,3, Weifeng Zeng4, Xiaomin Hu5, Jelle Matthijnssens1, 6 Zhiming Yuan2,3,*, Han Xia2,3,*
7 8 1. KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute, Laboratory of 9 Clinical and Epidemiological Virology, Laboratory of Viral Metagenomics, Leuven, Belgium 10 2. Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of 11 Sciences, Wuhan 430071, Hubei, China 12 3. University of Chinese Academy of Sciences, Beijing 10049, China 13 4. Liwan Center for Disease Control and Prevention, Guangzhou 510176, Guangdong, China 14 5. College of Life Science, South-Central University for Nationalities, Wuhan 430074, China 15 16 17
18 # These authors contributed equally to this work.
19 *Correspondence: Zhiming Yuan (Email: [email protected]) and Han Xia (Email: [email protected]) 20
21
22
23
24
25
26
27
28
29
30
31 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
32 Abstract
33 Mosquitoes belonging to the genus Aedes can efficiently transmit many pathogenic
34 arboviruses, placing a great burden on public health worldwide. In addition, they also carry a
35 number of insect specific viruses (ISVs), and it was recently suggested that some of these
36 ISVs might form a stable species-specific “core virome” in mosquito populations. However,
37 little is known about such a core virome in laboratory colonies and if it is present across
38 different developmental stages. In this study, we compared the viromes in eggs, larvae, pupae
39 and adults of Aedes albopictus mosquitoes collected from the field as well as from a lab
40 colony. The virome in lab-derived Ae. albopictus is very stable across all stages, consistent
41 with a vertical transmission route of these viruses, forming a “vertically transmitted core
42 virome”. The different stages of field collected Ae. albopictus mosquitoes also contains this
43 stable vertically transmitted core virome as well as another set of viruses shared by
44 mosquitoes across different stages, which might be an “environment derived core virome”.
45 Both these vertically and environmentally transmitted core viromes in Ae. albopictus deserve
46 more attention with respect to their effects on vector competence for important medically
47 relevant arboviruses. To further study this core set of ISVs, we screened 46 publically
48 available SRA viral metagenomic dataset of mosquitoes belonging to the genus Aedes. Some
49 of the identified core ISVs are identified in the majority of SRAs. In addition, a novel virus,
50 Aedes phasmavirus, is found to be distantly related to Yongsan bunyavirus 1, and the
51 genomes of the core virus Phasi Charoen-like phasivirus is highly prevalent in the majority of
52 the tested samples, with nucleotide identities ranging from 94% to 99%. Finally, Guadeloupe
53 mosquito virus, and some related viruses formed three separated phylogenetic clades. How
54 these core ISVs influence the biology of mosquito host, arboviruses infection and evolution
55 deserve to be further explored.
2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
56 1. Introduction
57 Mosquitoes are highly diverse and widely disseminated. They occupy numerous biotopes and
58 are potential vectors for several pathogenic arboviruses. Specifically, Aedes sp. impose a
59 great threat to global public health. This is due to their ecological plasticity traits that include
60 egg diapause, versatility in using natural and/or urban breeding spots [1] and opportunistic
61 feeding patterns [2, 3], which might have promoted their dispersion and adaptation to new
62 uncolonized territories that range from tropical to temperate regions [4].
63 Moreover, a large number of pathogenic arboviruses, such as Dengue virus (DENV), Yellow
64 fever virus (YFV), Zika virus (ZIKV) and Chikungunya virus (CHIKV), are efficiently
65 transmitted by Aedes (Ae.) mosquitoes, especially Ae. aegypti and Ae. Albopictus [5]. DENV
66 and CHIKV are the most common arboviral diseases, with more than 40% of the world’s
67 population living in areas with transmission of either of these viruses [6, 7]. Particularly,
68 DENV epidemics are becoming a major public health concern in Guangdong Province of
69 China. It experienced an unexpectedly explosive outbreak in 2014 with 45,224 reported
70 dengue fever cases [8], which is more than the total number of cases in the past 30 years in
71 this region [9]. YFV remains endemic in tropical regions in Africa as well as South and
72 Central America, and despite the presence of a good vaccine and large vaccination campaigns
73 in the past, Angola and neighbouring Democratic Republic of the Congo experienced a large
74 outbreak in recent years [10]. ZIKV is the most recent emergent arbovirus, receiving
75 attention worldwide due to its explosive spread in South America, and its association with
76 foetal microcephaly and Guillain-Barré syndrome [11, 12].
77 Due to the lack of therapeutic treatments and widely available vaccines, control of mosquito
78 population is the most efficient way to prevent arboviral diseases [13]. Mosquitoes are
79 holometabolous insects, whose life cycle goes through four separate stages, including eggs,
80 larva, and pupa in an aquatic habitat and a subaerial adult stage. Although only the females
3 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
81 with anthropophilic blood feeding habit can transmit arboviruses, reducing the population of
82 mosquito on the juvenile stages will decrease the number of adults and subsequently lower
83 the transmission chances. The conventional control strategies, including the use of insecticide
84 and environmental sanitation, are facing challenges due to their sustainability and
85 organizational complexity [13, 14]. In light of the holobiont concept, mosquito-associated
86 microbial communities play an important role in host biology, which may provide new
87 strategies for mosquito control [15].
88 To date, most of the conducted mosquito microbiota studies have solely focused on the
89 bacterial component in mosquitoes and their possible intrinsic roles in mosquito biology [16].
90 For example, the nutrient produced by symbiotic bacteria is very important for larvae
91 development [17], and the endosymbiotic alpha-proteobacteria Wolbachia is a natural
92 endosymbiont in several mosquito species and can induce cytoplasmic incompatibility [18].
93 When this bacterium is introduced into its not-natural host Ae. aegypti, it has strong
94 suppressive effect on DENV infection [19, 20]. More interestingly, some newly discovered
95 mosquito specific viruses have been proposed as future biological control agents against
96 arboviruses [21-23], as novel vaccine platforms [23] or used in diagnostic assays in chimeric
97 virus formation with structural protein genes from arboviruses [23], However, little is known
98 about the virome community dynamics in mosquitoes during its holometabolous
99 development. Some studies have performed viral metagenomics on field mosquitoes
100 collected from different countries (including the United States, Puerto Rico, China, Kenya,
101 Australia, Sweden, etc.), mainly focusing on novel virus discovery [24-32]. In a recent study
102 we showed the presence of a relatively stable “core eukaryotic virome” in field Ae. aegypti
103 and Culex quinquefasciatus mosquitoes from Guadeloupe [33]. However, the presence of
104 insect specific viruses (ISVs) in mosquito lab colonies is barely known, which limit our
105 understanding in their role in the development and physiology of mosquitoes. On the other
4 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
106 hand, knowing the normal healthy background virome as a reference, will allow us to
107 distinguish between inherent vertically transmitted components and components acquired
108 from the environment of the field mosquitoes, which will benefit the identification of
109 potential viral pathogens and improve the reliability of risk assessment using lab mosquitoes.
110 The aim of this study was to analyze the core virome structure of Ae. albopictus mosquitoes
111 during distinct developmental stages, in a laboratory-derived colony originally obtained from
112 Jiangsu Province, China, and to compare these findings to the virome of field Ae. albopictus
113 mosquitoes collected in Guangzhou (Guangdong Province, China), around 1300 kilometer
114 apart. Furthermore, we conducted a comparative meta-analysis between the viruses identified
115 in this study and 46 publicly available virome SRA datasets of Aedes mosquitoes. Finally,
116 phylogenetic analyses were performed on three selected viruses: Aedes phasmavirus which is
117 highly present in both field and lab Ae. albopictus from China; Guadeloupe mosquito related
118 virus and Phasi Charoen-like phasivirus, which were found in the majority of investigated
119 virome data sets.
120 2. Materials and methods
121 2.1 Sample collection
122 Field larva, pupa and adult samples of Ae. albopictus were trapped in Guangzhou (Liwan
123 District), Guangdong Province of China in July-November of 2017 and 2018. In Kenya, adult
124 mosquitoes of Ae. aegypti were trapped during July and August of the year 2018 in Ukunda
125 and Kisumu. All samples were collected from residential quarter in urban area, transported to
126 the laboratory using an appropriate cold chain and stored at -80 ℃. Mosquito species were
127 determined by morphological identification and samples were assigned into pools
128 according to date of collection (Table 1). An Ae. albopictus colony was established in our
129 laboratory in 2017 (Wuhan, China), which originated from the stable colony from the
5 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
130 National Institute for Communicable Disease Control and Prevention, China CDC (Beijing,
131 China). The adult mosquitoes were held at (27–30 °C), with 60–85% relative humidity, and a
132 photoperiod of 12:12 h light-dark cycle. The larvae were fed with a mixture of yeast powder
133 and wheat mill. Adult mosquitoes were placed into cages (30 cm × 30 cm×30 cm) and the
134 males were fed with 10% sucrose solution, while the females were fed with blood from mice.
135 The egg, larva, pupa, male and female adult samples of lab colonies mosquitoes were
136 collected respectively in August of 2017 (Table 1).
137 Table 1 – Aedes spp. from China and Kenya used for viral metagenomic sequencing Sample names Mosquito species Collection Habitats Locations Mosquito Obtained years numbers used trimmed reads for sequencing number 17-Lab-egg Ae. albopictus 2017 Lab Wuhan, Hubei, China 200 23,801,510 17-Lab-larva Ae. albopictus 2017 Lab Wuhan, Hubei, China 250 22,990,930 17-Lab-pupa Ae. albopictus 2017 Lab Wuhan, Hubei, China 250 18,066,298 17-Lab-adultF Ae. albopictus 2017 Lab Wuhan, Hubei, China 250 20,109,482 17-Lab-adultM Ae. albopictus 2017 Lab Wuhan, Hubei, China 250 19,227,638 17-GZ-larva Ae. albopictus 2017 Field Liwan district, Guangzhou, China 2000 29,564,646 17-GZ-adult Ae. albopictus 2017 Field Liwan district, Guangzhou, China 1000 37,766,072 18-GZ-larva Ae. albopictus 2018 Field Liwan district, Guangzhou, China 2000 35,580,984 18-GZ-pupa Ae. albopictus 2018 Field Liwan district, Guangzhou, China 500 25,744,126 18-GZ-adult Ae. albopictus 2018 Field Liwan district, Guangzhou, China 1100 29,644,914 18-Kenya-1 Ae. aegypti 2018 Field Ukunda, Kwale, Kenya 1100 17,758,834 18-Kenya-2 Ae. aegypti 2018 Field Kisumu, Kisumu, Kenya 840 26,678,724 138
139 2.2 Sample preparation for NGS
140 Pooled mosquito samples were triturated by a Tgrinder electric tissue grinder OSE-Y30
141 (TIANGEN,China) on ice using sterile pestles with 200 µl of Roswell Park Memorial
142 Institute (RPMI) medium. Mosquito homogenates were clarified by centrifugation at
143 20,000×g (4 °C for 30 min) and filtered through a 0.45 µm membrane filter (Millipore,
144 Billerica, USA). Supernatants were collected and stored at -80 °C. RNA was extracted from
145 the supernatants per the manufacturer’s instructions by using RNeasy mini kit (Qiagen,
146 Germany). Then strand-specific libraries were prepared using the TruSeq® Stranded Total
6 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
147 RNA Sample Preparation kit (Illumina, USA). TruSeq PE (paired-end) Cluster Kit v3
148 (Illumina, USA) and TruSeq SBS Kit v3 - HS (300 Cycles) (Illumina, USA) were used for
149 sequencing with 150 bp per read (PE 2×150 bp), which was performed at Shanghai
150 Biotechnology Corporation with the Illumina HiSeq 2500 platform (Illumina, USA).
151 2.3 Bioinformatic analysis
152 The obtained raw paired-end reads were trimmed for quality and adapters using
153 Trimmomatic [34] and the remaining reads were de novo assembled into contigs using
154 metaSPAdes [35]. Contigs from all pools longer than 500bp were filtered for redundancy at
155 95% nucleotide identity over 80% of the length using ClusterGenomes [36]. The
156 representative contigs collection was classified using DIAMOND [37] against the nr database
157 (updated on 29th Sep 2019) on sensitive mode for taxonomic annotation. KronaTools [38]
158 were used to parse the output file of DIAMOND, which found the least common ancestor of
159 the best 25 DIAMOND hits (based on BLASTx score) for each contig. All contigs annotated
160 as eukaryotic virus were extracted using an in-house python script. The abundance and length
161 coverage of eukaryotic virus contigs in individual pool were obtained by aligning trimmed
162 and decontaminated reads of each samples to the representative contigs collection using
163 BBMap [39]. Abundance tables from eukaryotic virus were extracted and further used for
164 making heatmaps in R with the ComplexHeatmap [40], ggplot2 [41], phyloseq [42] packages.
165 The length coverage of each viral species per sample was calculated by dividing the contigs
166 length covered (by at least one read) by the total length of the contigs.
167 2.4 Eukaryotic virus screening of Aedes mosquito virome data in SRA
168 To investigate the level of conservation of the eukaryotic core viruses identified in Chinese
169 samples from this study with those of other Aedes mosquitoes worldwide, we retrieved 46
170 published SRA datasets (including 25 datasets of Aedes sp. from Guadeloupe [33], eight from
7 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
171 Puerto Rico [24], four from USA [24], seven from Australia [43, 44], one from Thailand [44]
172 and one from China [45]) (Additional file 1 and Fig. 1). Two more viral metagenomic data of
173 Ae. aegypti collected in Kenya were also used (Table 1 and Fig. 1). The world map
174 displaying the geographic origin of all Aedes virome datasets used in this study were drawn
175 with ArcGIS (ArcMap 10.5). The trimmed reads of the two datasets of Ae. aegypti from
176 Kenya were de novo assembled using metaSPAdes [35]. The raw reads of SRA data sets
177 were de novo assembled by SKESA [46] with default settings. These obtained contigs were
178 taxonomically annotated using DIAMOND against the nr database (updated on 29th Sep
179 2019) on sensitive mode. KronaTools [38] and an in-house python script were used to parse
180 the output file of DIAMOND and extracted eukaryotic virus contigs.
181 182 Fig 1. Global distribution of Aedes mosquitoes virome datasets used in this study
183 2.5 The presence of eukaryotic virus in all Aedes virome datasets
184 Eukaryotic virus contigs longer than 500 bp from all 58 Aedes virome datasets were merged
185 according to their taxonomical annotation. A viral species was considered as presence in the
186 sample as long as the sample had one contig (>500 bp) assigned to the species. The viral
8 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
187 species are shown in the heatmap (Figure 3) only if they were present in samples from at least
188 three countries and contain at least one contig that was longer than 1500bp.
189 2.6 Phylogenetic analysis
190 To obtain the complete genomes related to Yongsan bunyavirus 1 (YBV1), Phasi Charoen-
191 like phasivirus (PCLPV) and Guadeloupe mosquito virus (GMV) from all Aedes virome
192 datasets, the trimmed reads of each sample were individually mapped (with BWA [47])
193 against the selected reference genomes: 17-Lab-egg-L (MT361040), 17-Lab-egg-M
194 (MT361040) and 17-Lab-pupa-S (MT361044) as the reference for YBV1, PCLPV isolate Rio
195 (KR003786.1, KR003784.1 and KR003785.1) for PCLPV and 18-GZ-larva-seg1
196 (MT361057) and 18-GZ-pupa-seg2 (MT361060) for GMV. The consensus sequences of
197 these viruses were generated from the bam files using samtools and bcftools [48]. Accession
198 numbers of these viruses obtained from our dataset are in Additional file 2 and viral genome
199 sequences recovered from SRA datasets can be found in
200 https://github.com/Matthijnssenslab/AedesVirome.
201 The nucleotide sequence of the complete genome or complete coding region of each genome
202 (segment) of these viruses were used to determine the evolutionary history of the discovered
203 viruses together with appropriate reference strains from GenBank. Alignments of sequences
204 were performed with MAFFT v7.222 [49] using the most accurate algorithm L-INS-I with
205 1000 cycles of iterative refinement. Ambiguously aligned regions were removed by trimAl
206 v1.2 [50] using automated trimming heuristic, which is optimized for Maximum-Likelihood
207 (ML) phylogenetic tree reconstruction. Phylogenetic trees for each segment were
208 reconstructed from 1,000 ultrafast bootstrap maximum likelihood (ML) tree replicates using
209 IQ-TREE v1.6.11 [51] with best-fit model selection by ModelFinder [52]. FigTree v1.4.3
210 [53] was used for phylogenetic trees visualization.
9 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
211 3 Results
212 3.1 Comparison of the eukaryotic virome in field and lab Ae. albopictus
213 The egg, larva, pupa and adult (male and female) pools from a lab colony in Wuhan, as well
214 as the larva, pupa and adult pools from the field in Guangzhou underwent the metagenomic
215 sequencing and an average of 27 million trimmed reads were obtained per pool (Table 1),
216 which were de novo assembled into 1,657,229 contigs in total. The clustering of 71,303
217 contigs longer than 500 bp from all samples at 95% nucleotide identity over 80% of the
218 length results in 56,419 representative contigs. According to BLASTx annotation results
219 using Diamond, the majority of the representative contigs (93%) belonged to Eukaryota
220 (mainly derived from the mosquito host genome) and 179 contigs were assigned as
221 eukaryotic viruses. Eukaryotic viral reads in each pool occupied 0.2% to 1.8% of trimmed
222 reads, except for pool 17-GZ-larva which contained 15.8% viral reads. Most of the
223 eukaryotic viral contigs were annotated as belonging to 80 different viruses (including
224 several viruses with segmented genomes), although some of these had very low similarities to
225 known viruses in the database (Fig. 2). No known pathogenic arboviruses were identified,
226 and the closest relatives of the identified viruses were generally found in insects or plant.
227 Only 20 of the 80 viral species belonged to an established viral genus or family (e.g.
228 Flavivirus, Iflavirus, Phasivirus, Quaranjavirus, Rhabdoviridae, Virgaviridae, Totiviridae,
229 Nodaviridae), whereas the remaining ones are currently unclassified.
230 The heatmap in Fig. 2 displays the mapped read numbers against each of the representative
231 (partial) viral genome as well as the length coverage of assembled contigs of each virus
232 (Additional File 3 and 4). The virome in field Ae. albopictus collected from both 2017 and
233 2018 showed significantly higher richness and diversity compared to that in lab-derived
234 mosquito pools. The adjusted p-value of Wilcoxon test on Chao1 and Shannon index were
235 0.012 and 0.008, respectively (Additional File 5). The virome profile of the different pools of
10 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
236 lab colony are relatively stable, except that several viral species are absent or low abundant in
237 the larvae pool (Fig. 2). The wild larvae, pupa and adult pools collected in GZ in 2018 and
238 the adult pool in 2017 also display a very similar viral community. However, the larvae pool
239 collected in GZ 2017 contained more than 30 additional unique viral species with almost
240 100% length coverage, which are almost completely absent in the adults collected at the same
241 time and in the same place. Furthermore, the lab and field Ae. albopictus have 20 viral
242 species in common. Among them, some contigs only distantly related (47% BLASTx
243 identity) to YBV1, are present in all lab and field derived mosquito pools with a high
244 abundance and length coverage. This is also true for Phasi Charoen-like phasivirus, and
245 contigs annotated as Yongsan tombus-like virus 1, except in field mosquitoes collected in
246 2018. Some viruses such as Phasi Charoen-like phasivirus appear to be present in in higher
247 levels in the lab-derived Ae. albopictus samples. In contrast, reads of some viruses appear to
248 be present in higher number in the field samples, as is the case for Guato virus and
249 Guadeloupe mosquito mononega-like virus. Furthermore, some viruses are only present in
250 field mosquitoes among all pools with high abundance, such as Hubei partiti−like virus 22,
251 Hubei mosquito virus 2 and Wenzhou sobemo−like virus 4.
11 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
r P rasilia r r P ovir P avir avir P avir r ke vir r avir r r avir r r avir r r ke vir W r e ke vir r W r r r r ke vir r ke vir W r r v r Beihai picor ke vir far avir log10 reads number r r Beihai picor ke vir r ke vir r r r r v r v r Beihai picor ke vir W r ke vir e ke vir r ke vir coverage perctage r r r Picor avirales sp. r ra vir P r Nora vir P r r r W r ke vir al species r r ke vir ovir vi ra r r r T r BLASTx Identity r r Y avir ke vir Per r ke vir Nephila clavipes vir Sara ak vir r ke vir r e r ke vir r W ke vir Collect location e e ke vir W r e ke vir e T r ke vir Y yavir (Aedes phasmavirus) Y ke vir r pyr r r ke vir r r A r r Kai a vir avivir ke phasivir Tr r r r Riv r ra vir r a a a a a a v v v v v v r r r r r r 252 253 Fig. 2 Abundance and length coverage of eukaryotic viral species in wild and lab-derived Aedes albopictus 254 pools. The heatmaps show the total mapped reads number on a log10 scale (left) and length coverage (right) 255 of assembled contigs of each virus. The hierarchical clustering is based on the Bray–Curtis distance matrix 256 calculated from the log10 reads number. The viral species names shown in the heatmap are from the 257 taxonomic annotation by DIAMOND and KronaTools. For each of the contigs assigned to a particular species, 258 the ORF with the highest BLASTx identity to a reference sequence was taken, and the median identity of these 259 different ORFs is shown in the shaded green bar.
12 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
260 3.2 Prevalence of viruses in public SRA data sets derived from Aedes mosquitoes
261 All 46 available viral metagenomic data of Aedes sp. from public database (SRA) together
262 with two samples from Kenya (Additional file 1) were further analyzed to determine the
263 prevalence of conserved ISVs in various Aedes mosquitoes. These samples were collected
264 from locations in different continents including the USA, Puerto Rico, Australia, Thailand,
265 Guadeloupe, China and Kenya. The mosquito species in the majority of samples are Ae.
266 aegypti, except the one from China (Ae. Albopictus) and five from Southwestern Australia
267 (Ae. alboannulatus or Ae. Camptorhynchus). The heatmap in Fig. 3 shows the presence and
268 absence of 42 viruses in the 58 analyzed virome data sets, grouped per collection locations
269 and viral families. Only the viral species present in minimum three locations and containing
270 at least one contig longer than 1.5 kb are displayed in the figure. Notably, the total number of
271 viral species present in samples from the USA and Puerto Rico are much less than in other
272 locations, which may due to the low sequencing depths, , or different viruses present in these
273 samples.
274 Totiviridae is the most prevalent viral family, containing two highly abundant species (Aedes
275 aegypti totivirus and Australian Anopheles totivirus) with positivity rates of 63.8% and
276 60.3%, respectively. Phasi Charoen-like phasivirus in the Phenuiviridae is present in all four
277 Aedes species and all locations (except USA, which only has three viruses presented). In
278 addition, the families Flaviviridae, Orthomyxoviridae, Rhabdoviridae and Xinmoviridae also
279 contain viral species present in 5 or more locations and at least Ae. aegypti and Ae. albopictus
280 mosquitoes. For the unclassified viral species, Chuvirus Mos8Chu0 and Kaiowa virus are
281 present in 40 and 35 out of 58 samples, respectively, and were found in all four Aedes
282 species. Guadeloupe mosquito virus is detected in field Ae. aegypti and Ae. albopictus
283 mosquitoes from Puerto Rico, Thailand, Guadeloupe, Kenya and China. Humaita-Tubiacanga
284 virus (absent in lab-derived and wild Ae. albopictus from this study) are found in field Ae.
13 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
285 albopictus from Yunnan (China) and field Ae. aegypti from 5 locations. Some of the viruses
286 that are highly prevalent in both lab and field Ae. albopictus from China, including Culex
287 mononega-like virus 2, Guadeloupe mosquito mononega-like virus and YBV1, are absent in
288 most countries except in Ae. camptorhynchus from Australia.
USA Puerto Rico Australia TH Guadeloupe KE China Guadeloupe mosquito virus W ke virus 4 Hubei mosquito virus 2 Chuvirus Mos8Chu0 Kaiowa virus Tubiacanga virus ke virus 21 Cule ke virus 2 ke virus Yongsan bunyavirus 1(Aedes phasmavirus) unclassified ke virus 1 Hubei odonate virus 15 Culex nege ke virus 1 Culex Buny ke virus Shayang Fly Virus 1 Cule ke virus Salarivirus Mos8CM0 Beihai par ke virus 2 Chaq virus Hubei tetragnatha maxillosa virus 8 ke virus 1 Xishuangbanna aedes flavivirus Menghai flavivirus Flaviviridae Cell fusing agent virus Kamiti River virus Wuhan Mosquito Virus 6 Whidbey virus Orthomyxoviridae Guadeloupe mosquito quar ke virus 1 Wuhan Mosquito Virus 4 Aedes aegypti totivirus Totiviridae Australian Anopheles totivirus ke virus Ohlsdorf virus Rhabdoviridae Wuhan Ant Virus Aedes anphevirus Xinmoviridae Aedes aegypti anphevirus Phenuiviridae ke phasivirus Metaviridae Trichoplusia ni TED virus Phasmaviridae Wuhan mosquito orthophasmavirus 2 Reoviridae Liao ning virus Tombusviridae Yongsan tomb ke virus 1 Virgaviridae Bomb rus Vir2 0 20 40 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2015 2015 2015 2015 2015 2015 2016 2016 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2017 2018 2018 2016 2017 2017 2018 2018 2018 2017 2017 2017 2017 2017 8 a a a v v v 3 r r r y y n n e e total_pre K K SRR3168918 SRR3168919 SRR3168926 SRR3168927 SRR3168924 SRR3168925 SRR3168916 SRR3168922 SRR3168921 SRR3168920 SRR3168917 SRR3168923 SRR6155880 SRR6155881 SRR5665575 SRR5665565 SRR5665568 SRR5665570 SRR5665573 SRR6155879 SRR8455260 SRR8455259 SRR8455239 SRR8455240 SRR8455263 SRR8455243 SRR8455252 SRR8455255 SRR8455247 SRR8455248 SRR8455253 SRR8455264 SRR8455249 SRR8455256 SRR8455236 SRR84552 SRR8455242 SRR8455244 SRR8455237 SRR8455235 SRR8455241 SRR8455258 SRR8455251 SRR8455254 SRR8455250 SRR7204303
Absence Aedes aegypti Aedes alboannulatus 289 Presence Aedes camptorhynchus Aedes albopictus 290 Fig. 3 Presence and Absence of viral species in Aedes sp. virome datasets. The viral species shown in the 291 heatmap present in samples from at least three locations and contain at least one contig that is longer than 292 1500 bp. The specific viral species was considered as presence in the sample, as long as the sample had one 293 contigs (>500 bp) assigned to the species.
294 3.3 Phylogeny of three selected viruses
295 Phylogenetic analysis was conducted on three selected viruses related to YBV1, which
296 presented in both field and lab Ae. albopictus from China with high abundance, as well as the
297 highly prevalent PCLPV and newly described GMV. Recovered coding complete viral
298 genomes related to YBV1, PCLPV and GMV from analyzed viral metagenomic data of
299 Aedes mosquitoes as well as corresponding reference genomes from GenBank were used for
300 phylogeny. YBV1 is a newly identified virus reported in 2019, which has a high prevalence
14 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
301 in Ae. vexans nipponii from Korea [54], and classified in the family Phasmaviridae. A novel
302 virus of this family (tentatively named as Aedes phasmavirus in this study) distantly related
303 to YBV1 (vide infra) was presented in lab Ae. albopictus from China and filed Aedes
304 mosquitoes collected from three countries, including China, Korea, and Australia (Table 2).
305 PCLPV, a widely distributed mosquito viruses, has been identified from lab colonies,
306 mosquito cell lines and wild Aedes mosquitoes in eight counties or regions (China, Puerto
307 Rico, Thailand, Kenya, USA, Australia, Guadeloupe, Brazil) (Table 2). It has been suggested
308 that is maintained in nature through vertical transmission [55]. A recent study has reported
309 that GMV is closely related to Wenzhou sobemo-like virus 4 and Hubei mosquito virus 2
310 [56]. GMV related virus has been detected only in mosquito sample collected from the field,
311 indicating it is likely acquired horizontally from the environment.
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
15 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
327 Table 2 The distribution of three selected viruses
region origin Aedes PCLPV GMV related virus phasmavirus (relate to YBV1)
lab China 2017-Wuhan-lab + +
Belgium 2018-Belgium-lab +
UK 2017-Bristol-lab +
field China 2016-Zhejiang + +
2017-Guangzhou + + +
2018-Guanghozu + + +
Puerto Rico 2014-Puerto Rico + +
Thailand 2008-Thailand +
2015-Thailand + +
Kenya 2018-Kenya1 + +
2018-Kenya2 + +
USA 2014-USA
2015-USA +
Australia 2014-Australia +
2015-Australia + +
2016-Australia +
Korea 2016-Korea +
Guadeloupe 2016-Guadeloupe + +
2017-Guadeloupe + +
Brazil 2012-Brazil +
328
329 330
331
332
333
16 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
334 3.3.1 Phylogeny of Aedes phasmavirus
335 In both lab and field derived Ae. albopictus sequence data from China in 2017 and 2018,
336 three contigs of Aedes phasmavirus (APV) in each sample were found to share highest amino
337 acid identities of 50.8%, 48.1%, and 43.6% with S-, M- and L-segments of YBV1
338 (MH703047.1, MH703046.1, MH703045.1), respectively. In total, 10 viral genomes of APV
339 with three segments could been recovered from all lab and field Ae. albopictus pools of this
340 study in China (17-lab-egg, 17-lab-larva, 17-lab-pupa, 17-lab-adultM, 17-lab-adultF, 17-GZ-
341 larva, 17-GZ-adult, 18-GZ-larva, 18-GZ-pupa, 18-GZ-adult) with the longest lengths of 1185,
342 2022, and 6468 nt corresponding for the complete cds (coding sequence) of the nucleocapsid,
343 glycoprotein and RNA-dependent RNA polymerase (RdRp) genes, respectively. For all three
344 segments, the nucleotide identity among the 10 assembled viral genomes ranged between
345 96% and 100%, which indicated they represent closely related variants. In the phylogenetic
346 analysis of the three segments separately, the 10 viral genomes of APV clustered within the
347 genus Orthophasmavirus in the family of Phasmaviridae (Fig. 4). They closest relative was
348 YBV1 identified in Ae. vexans mosquitoes collected in South Korea in 2016, but with low
349 nucleotide similarities (ranging from 52% to 57%) in the coding regions of the S-, M-, and L-
350 segments. Thus, the APVs presented in the lab and field Ae. albopictus in China appears to
351 represent a novel species in the genus Orthophasmavirus.
352
17 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
353 Fig. 4. Maximum likelihood phylogeny on the nucleotide level of complete cds region in S, M, and L segment of 354 the newly found Orthophasmaviruses in Aedes mosquitoes from China (highlighted with green square) against 355 other representative members in family Phasmaviriade. Representatives in Orthobunyavirus belonged to the 356 Peribunyaviridae were used as the outgroup.
357
18 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
358 3.3.2 Phylogeny of Phasi Charoen-like phasivirus
359 Since the abundance of Phasi Charoen-like phasivirus (PCLPV) reads in the Ae. albopictus
360 from Guangzhou and lab-derived are relatively low, no complete genome was recovered from
361 these pools. All three segments of PCLPV with a complete coding region were successfully
362 recovered in one Ae. aegypti sample from Puerto Rico collected in 2014 and two Ae. aegypti
363 samples from Kenya collected in 2018. The phylogenetic analysis was performed on these
364 newly identified PCLPVs, all other PCLPV genomes in GenBank (including the ones
365 identified in Ae. aegypti mosquitoes from Thailand in 2008, from US in 2015, from Australia
366 in 2016, from Zhejiang Province of China in 2016, from Guadeloupe in 2017 and in Aag2
367 cells from UK) and two more genomes obtained from lab Aag2 cell lines in Belgium (Fig. 5).
368 It is interesting to note that all these PCLPVs originating from distant geographic locations,
369 different years and field mosquitoes vs. lab cell lines, all cluster very closely in the three
370 Maximum-likelihood trees of each segment. The nucleotide similarities among the genomes
371 of PCLPVs were very high, ranging from 94-98%, 95-99% and 94-99%, for the S, M and L
372 segments, respectively.
373
19 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
S segment
M segment
L segment
374 375 Fig. 5 Maximum-likelihood phylogenetic tree based on nucleotide sequence of complete coding regions of the 376 S, M, and L segment of PCLPV identified from Aedes mosquitoes and cells, together with other representative 377 members of Phenuiviriade. The strains from Guadeloupe are highlighted with red squares, strains derived from 378 Belgian Aag2 cells with black diamonds, strains from Kenya with brown square, and from Puerto Rico with blue 379 square. Representative Orthonairovirus strains belonging to the Nairoviridae are used as outgroup.
20 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
380
381 3.3.3 Phylogeny of Guadeloupe mosquito virus related viruses
382 Twelve viral genomes with complete coding regions and similar to GMV, Wenzhou sobemo-
383 like virus 4 and Hubei mosquito virus 2 were obtained from all the analyzed virome datasets,
384 including six pools of field Ae. albopictus from China (17-GZ-larvae, 17-GZ-adult, 18-GZ-
385 larva, 18-GZ-pupa, 18-GZ-adult, SRR7204303-CN-2016), three pools of Ae. aegypti and Cx.
386 quinquefasciatus mixture from Puerto Rico (SRR3168916-PR-2014, SRR3168921-PR-2014,
387 SRR3168924-PR-2014), two Ae. aegypti pools from Kenya (18-Kenya1 and 18-Kenya2) and
388 one from Thailand (SRR6155879-TH-2015). The viral genomes contain two segments,
389 encoding an RdRp and one hypothetical protein on segment 1 (1500 - 1600 nt), and a capsid
390 and one hypothetical protein encoded by segment 2 (3000 - 3200 nt). GMV, Wenzhou
391 sobemo-like virus 4 and Hubei mosquito virus 2 are all newly described and currently
392 unclassified viruses with distant relationship to the family Luteoviridae and Sobemoviridae
393 [56].
394 The phylogenetic analysis based on segment 1 and 2 indicated that these sequences could be
395 separated into three clades (Fig. 6). The genomes identified from Thailand in 2015, Puerto
396 Rico in 2014 and 1 strain from Kenya clustered together with the Guadeloupe mosquito
397 viruses found in Guadeloupe in 2016 and 2017 to form clade-1, with the nucleotide identity
398 within this clade ranging from 90% - 99% for both segments. The genomes recovered from
399 Ae. albopictus collected from Guangzhou in 2017 and 2018 cluster together with the one
400 recovered from the same host of Yunnan in 2016 and Wenzhou sobemo-like virus 4 in clade-
401 2, with the nucleotide identities among them ranging from 94% to 98%. The third clade
402 consisted of the Hubei mosquito virus 2 and the sequences from 2018-Kenya2, with
403 nucleotide identities ranging from 70% - 100%. The nucleotide similarities for both segments
404 among these three clades ranged from 54% to 69%.
21 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Segment 1 Segment 2
405 406 Fig. 6 Maximum-likelihood phylogenetic tree based on nucleotide sequence of the complete coding regions in 407 segment 1 and 2 of Guadeloupe mosquito virus related viruses. The red, blue, yellow, brown and green 408 squares after the sequence names highlight the strains from Guadeloupe, Puerto Rico, Thailand, Kenya and 409 China, respectively. Motts Mill virus identified from tick is used as the outgroup.
410 4. Discussion
411 In this study, we firstly characterized the eukaryotic virome across different developmental
412 stages of lab-derived and field collected Ae. albopictus from China. As could be expected, the
413 virome diversity in lab Ae. albopictus is lower than that in field mosquitoes (Fig. 2), probably
414 due to the less complex environment and clean water and food resources. Unlike bacterial
415 communities in mosquitoes [57-62], it is interesting to note that the virome in lab-derived Ae.
416 albopictus is very stable across all stages, consistent with a vertical transmission of these
417 viruses. Only for the larvae, relatively few viral reads were identified. This finding suggests
418 that the virus might remain dormant at a very low concentration without or with very low
419 rates of replication. The fact that these viruses are also present in field mosquitoes suggest
420 that Ae. albopictus contains a “vertically transmitted core virome” as was described before
421 for Ae. aegypti and Culex quinquefasciatus from Guadeloupe [56]. In addition, another set of
422 viruses was identified to be shared by the field collected Ae. albopictus mosquitoes across
423 different stages, suggesting that they have a richer “core virome”. Whether or not these
424 additional viruses were acquired from the environment, or that the lab derived mosquitoes
425 lost these viruses in captivity, remains to be determined. All these vertically transmitted core
426 viromes in Ae. albopictus deserve more attention with respect to their effects on vector
22 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
427 competence for important medically relevant arboviruses. The larva pool collected in the wild
428 in 2017 is an outlier, as it contained a lot of unique viruses, which probably originated from
429 the particular water environment they lived in, which could be contaminated by other co-
430 inhabiting insects or larvae. Alternatively, it could be that a number of mosquitoes, other than
431 Ae. albopictus were included in the pool by accident. These results further suggest a
432 significant influence of the breeding site ecology on the mosquito viral community.
433 Since some viruses (e.g. PCLPV, GMV and Aedes aegypti totivirus) identified in Ae.
434 albopictus from China were also identified as part of the core virome in Ae. aegypti from
435 Guadeloupe [33], we were interested to find out how these core viruses in Aedes mosquitoes
436 were further distributed around the world. Therefore, we explored 46 viral metagenomic
437 datasets of Aedes sp. from public database (SRA) and two of Ae. aegypti from Kenya.
438 Although the samples are from different countries in different continents (China, US,
439 Australia, Kenya, Thailand, Puerto Rico, Guadeloupe), from different mosquito species (Ae.
440 aegypti, Ae. albopictus, Ae. alboannulatus, Ae. camptorhynchus), were treated using different
441 wet-lab and sequencing procedures, used different amounts of pooled mosquitoes and
442 sequencing depths, a highly prevalent set of widely distributed viruses could be identified on
443 species or family level, such as PCLPV, GMV and Totiviridae (Fig. 3). How these conserved
444 viruses in Aedes mosquitoes interact with an arbovirus infection are interesting points for
445 further studies. A previous study explored the effect of PCLPV together with Cell-fusing
446 agent virus (CFAV) on the replication of arboviruses in cell line Aa23 derived from Ae.
447 albopictus [63]. The Aa23 cell lines persistently infected with CFAV was further inoculated
448 with PCLPV. CFAV-PCLPV positive Aa23 cells strongly inhibit the growth of two
449 flaviviruses (ZIKV and DENV) and completely blocked the infection of bunyavirus La
450 Crosse virus. Although the exact blocking mechanism is not known, this results on one hand
451 suggest that the data generated from laboratory cell lines persistently infected by mosquito-
23 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
452 specific viruses (MSVs) should be interpreted with caution. On the other hand, the intra-
453 MSVs interaction need to be considered when the influence of MSVs on vector competence
454 is explored.
455 Although many viruses are very prevalent in Aedes mosquitoes, such as Chuvirus Mos8Chu0,
456 Kaiowa virus, Wuhan mosquito virus 6, Whidbey virus, Aedes aegypti totivirus and
457 Australian Anopheles totivirus, phylogenetic analysis was further performed on GMV and
458 PCLPV which had the greatest number of coding complete genomes, and APV that is highly
459 abundant in both field and lab Ae. albopictus from China. APV was distantly relates to the
460 YBV1 identified from Ae. vexans from South Korea forming a separate clade. All the
461 Chinese strains from both lab and field collected mosquitoes were very similar (Fig. 4). For
462 PCLPV, the genomes found in samples from different years, locations and habitats are very
463 similar for all three segments (Fig. 5). Interactions between bunyaviruses and arthropods has
464 been demonstrated over 20 million years [64-68]. The findings that both APV and PCLPV
465 seems to be very closely related is puzzling and might suggest a rather relatively recent
466 spread of this virus, or a very slow evolutionary rate, which might be unexpected for RNA
467 viruses. However, the effects of these viruses on their host is very poorly understood. A
468 recent study revealed that Aag2 cells with pre-existing infection with PCLPV do not affect
469 the infection and growth of the mosquito-borne viruses in genus Flavivirus, Alphavirus and
470 Rhabdovirus in cell culture [69]. GMV is a recently described two-segmented, currently
471 unclassified virus. Three separate clades are observed for GMVs and GMV-related viruses,
472 largely clustering according to locations. This group of viruses should be proposed as a new
473 family and their role in arboviruses infection need to be further studied.
474 In summary, our results reveal that the virome is stable across different stages in both lab and
475 field Aedes albopictus, and that a number of core viruses exists in Aedes mosquitoes of at
476 least four species from seven countries. Since the number of the available Aedes virome data
24 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
477 sets is still rather limited, and wet-lab procedures, sequencing depths and pool size differ
478 strongly between the analyzed data sets, the core viruses need to be further confirmed by
479 NGS or PCR. To fully characterize and understand the genetic and phenotypic diversity of
480 mosquito-specific viruses, samples from individual Aedes mosquitoes from more locations,
481 species and time points, processed and sequenced with the same method should be analyzed.
482
483 Funding
484 This work was supported by the Ministry of Science and Technology of the People’s Republic of 485 China [2018ZX10101004]; the National Health Commission of the People’s Republic of China 486 [2018ZX10711001-006]; the Chinese Academy of Sciences [153211KYSB20160001]; the Wuhan 487 Institute of Virology, China [WIV-135-PY2]; the Health Commission of Hubei Province [WJ2019Q060].
488
489 Competing interests
490 The authors declare that they have no competing interests.
491
492 Additional files
493 Additional file 1: SRA datasets used in this study
494 Additional file 2: Accession number of YBV1, PCLPV and GMV related viruses identified in this 495 study.
496 Additional file 3: Reads number of each eukaryotic viral species in each sequencing sample
497 Additional file 4: Coverage of each eukaryotic viral species in each sequencing sample
498 Additional file 5: Alpha diversity of virome in field and lab Aedes albopictus 499
500
25 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
501 Reference
502 1. Paupy, C., et al., Aedes albopictus, an arbovirus vector: From the darkness to the light. 503 Microbes and Infection, 2009. 11(14-15): p. 1177-1185. 504 2. Delatte, H., et al., Blood-Feeding Behavior of Aedes albopictus, a Vector of Chikungunya on 505 La Reunion. Vector-Borne and Zoonotic Diseases, 2010. 10(3): p. 249-258. 506 3. Kamgang, B., et al., Notes on the blood-feeding behavior of Aedes albopictus (Diptera: 507 Culicidae) in Cameroon. Parasites & Vectors, 2012. 5. 508 4. Bonizzoni, M., et al., The invasive mosquito species Aedes albopictus: current knowledge 509 and future perspectives. Trends in Parasitology, 2013. 29(9): p. 460-468. 510 5. Organization, W.H. Vector-borne diseases [internet]. Available from: 511 https://www.who.int/news-room/fact-sheets/detail/vector-borne-diseases. 512 6. Weaver, S.C., et al., Zika, Chikungunya, and Other Emerging Vector-Borne Viral Diseases. 513 Annu Rev Med, 2018. 69: p. 395-408. 514 7. Mirzaian, E., et al., Mosquito-borne illnesses in travelers: a review of risk and prevention. 515 Pharmacotherapy, 2010. 30(10): p. 1031-43. 516 8. Xiao, J.P., et al., Characterizing a large outbreak of dengue fever in Guangdong Province, 517 China. Infect Dis Poverty, 2016. 5: p. 44. 518 9. Zhu, G., et al., Spatiotemporal analysis of the dengue outbreak in Guangdong Province, 519 China. BMC Infect Dis, 2019. 19(1): p. 493. 520 10. Burki, T., Yellow fever in Africa: a disaster waiting to happen. Lancet Infect Dis, 2016. 16(8): p. 521 896-7. 522 11. Baud, D., et al., An update on Zika virus infection. Lancet, 2017. 390(10107): p. 2099-2109. 523 12. Musso, D. and D.J. Gubler, Zika Virus. Clin Microbiol Rev, 2016. 29(3): p. 487-524. 524 13. Scolari, F., M. Casiraghi, and M. Bonizzoni, Aedes spp. and Their Microbiota: A Review. 525 Frontiers in Microbiology, 2019. 10. 526 14. McGraw, E.A. and S.L. O'Neill, Beyond insecticides: new thinking on an ancient problem. Nat 527 Rev Microbiol, 2013. 11(3): p. 181-93. 528 15. Guegan, M., et al., The mosquito holobiont: fresh insight into mosquito-microbiota 529 interactions. Microbiome, 2018. 6(1): p. 49. 530 16. Guegan, M., et al., The mosquito holobiont: fresh insight into mosquito-microbiota 531 interactions. Microbiome, 2018. 6. 532 17. Correa, M.A., et al., Generation of axenic Aedes aegypti demonstrate live bacteria are not 533 required for mosquito development. Nature Communications, 2018. 9. 534 18. Ma, W.J., F. Vavre, and L.W. Beukeboom, Manipulation of arthropod sex determination by 535 endosymbionts: diversity and molecular mechanisms. Sex Dev, 2014. 8(1-3): p. 59-73. 536 19. Walker, T., et al., The wMel Wolbachia strain blocks dengue and invades caged Aedes 537 aegypti populations. Nature, 2011. 476(7361): p. 450-U101. 538 20. Moreira, L.A., et al., A Wolbachia symbiont in Aedes aegypti limits infection with dengue, 539 Chikungunya, and Plasmodium. Cell, 2009. 139(7): p. 1268-78. 540 21. Adelman, Z.N., et al., Sindbis virus-induced silencing of dengue viruses in mosquitoes. Insect 541 Molecular Biology, 2001. 10(3): p. 265-273. 542 22. Bolling, B.G., et al., Insect-Specific Virus Discovery: Significance for the Arbovirus Community. 543 Viruses-Basel, 2015. 7(9): p. 4911-4928. 544 23. Bolling, B.G., et al., Insect-Specific Viruses Detected in Laboratory Mosquito Colonies and 545 Their Potential Implications for Experiments Evaluating Arbovirus Vector Competence. 546 American Journal of Tropical Medicine and Hygiene, 2015. 92(2): p. 422-428. 547 24. Frey, K.G., et al., Bioinformatic Characterization of Mosquito Viromes within the Eastern 548 United States and Puerto Rico: Discovery of Novel Viruses. Evol Bioinform Online, 2016. 549 12(Suppl 2): p. 1-12.
26 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
550 25. Xia, H., et al., Comparative Metagenomic Profiling of Viromes Associated with Four Common 551 Mosquito Species in China. Virol Sin, 2018. 33(1): p. 59-66. 552 26. Shi, C., et al., A metagenomic survey of viral abundance and diversity in mosquitoes from 553 Hubei province. PLoS One, 2015. 10(6): p. e0129845. 554 27. Atoni, E., et al., Metagenomic Virome Analysis of Culex Mosquitoes from Kenya and China. 555 Viruses, 2018. 10(1). 556 28. Sadeghi, M., et al., Virome of >12 thousand Culex mosquitoes from throughout California. 557 Virology, 2018. 523: p. 74-88. 558 29. Fauver, J.R., et al., West African Anopheles gambiae mosquitoes harbor a taxonomically 559 diverse virome including new insect-specific flaviviruses, mononegaviruses, and totiviruses. 560 Virology, 2016. 498: p. 288-299. 561 30. Hameed, M., et al., A viral metagenomic analysis reveals rich viral abundance and diversity in 562 mosquitoes from pig farms. Transbound Emerg Dis, 2019. 563 31. Ohlund, P., et al., Viromics Reveal a Number of Novel RNA Viruses in Swedish Mosquitoes. 564 Viruses, 2019. 11(11). 565 32. Pettersson, J.H., et al., Meta-Transcriptomic Comparison of the RNA Viromes of the 566 Mosquito Vectors Culex pipiens and Culex torrentium in Northern Europe. Viruses, 2019. 567 11(11). 568 33. Shi, C., et al., Stable distinct core eukaryotic viromes in different mosquito species from 569 Guadeloupe, using single mosquito viral metagenomics. Microbiome, 2019. 7(1): p. 121. 570 34. Bolger, A.M., M. Lohse, and B. Usadel, Trimmomatic: a flexible trimmer for Illumina 571 sequence data. Bioinformatics, 2014. 30(15): p. 2114-20. 572 35. Bankevich, A., et al., SPAdes: a new genome assembly algorithm and its applications to 573 single-cell sequencing. J Comput Biol, 2012. 19(5): p. 455-77. 574 36. https://bitbucket.org/MAVERICLab/docker-clustergenomes. 575 37. Buchfink, B., C. Xie, and D.H. Huson, Fast and sensitive protein alignment using DIAMOND. 576 Nat Methods, 2015. 12(1): p. 59-60. 577 38. Ondov, B.D., N.H. Bergman, and A.M. Phillippy, Interactive metagenomic visualization in a 578 Web browser. BMC Bioinformatics, 2011. 12: p. 385. 579 39. https://github.com/BioInfoTools/BBMap. 580 40. Gu, Z., R. Eils, and M. Schlesner, Complex heatmaps reveal patterns and correlations in 581 multidimensional genomic data. Bioinformatics, 2016. 32(18): p. 2847-9. 582 41. Wickham, H., ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York, 2016. 583 42. McMurdie, P.J. and S. Holmes, phyloseq: an R package for reproducible interactive analysis 584 and graphics of microbiome census data. PLoS One, 2013. 8(4): p. e61217. 585 43. Shi, M., et al., High-Resolution Metatranscriptomics Reveals the Ecological Dynamics of 586 Mosquito-Associated RNA Viruses in Western Australia. J Virol, 2017. 91(17). 587 44. Zakrzewski, M., et al., Mapping the virome in wild-caught Aedes aegypti from Cairns and 588 Bangkok. Scientific Reports, 2018. 8. 589 45. Xiao, P.P., et al., Metagenomic Analysis of Flaviviridae in Mosquito Viromes Isolated From 590 Yunnan Province in China Reveals Genes From Dengue and Zika Viruses. Frontiers in Cellular 591 and Infection Microbiology, 2018. 8. 592 46. Souvorov, A., R. Agarwala, and D.J. Lipman, SKESA: strategic k-mer extension for scrupulous 593 assemblies. Genome Biology, 2018. 19. 594 47. Li, H. and R. Durbin, Fast and accurate short read alignment with Burrows-Wheeler 595 transform. Bioinformatics, 2009. 25(14): p. 1754-60. 596 48. Li, H., et al., The Sequence Alignment/Map format and SAMtools. Bioinformatics, 2009. 597 25(16): p. 2078-9. 598 49. Katoh, K., et al., MAFFT: a novel method for rapid multiple sequence alignment based on 599 fast Fourier transform. Nucleic Acids Res, 2002. 30(14): p. 3059-66.
27 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.058701; this version posted April 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
600 50. Capella-Gutierrez, S., J.M. Silla-Martinez, and T. Gabaldon, trimAl: a tool for automated 601 alignment trimming in large-scale phylogenetic analyses. Bioinformatics, 2009. 25(15): p. 602 1972-3. 603 51. Nguyen, L.T., et al., IQ-TREE: a fast and effective stochastic algorithm for estimating 604 maximum-likelihood phylogenies. Mol Biol Evol, 2015. 32(1): p. 268-74. 605 52. Kalyaanamoorthy, S., et al., ModelFinder: fast model selection for accurate phylogenetic 606 estimates. Nat Methods, 2017. 14(6): p. 587-589. 607 53. https://github.com/rambaut/figtree/releases. 608 54. Sanborn, M.A., et al., Metagenomic Analysis Reveals Three Novel and Prevalent Mosquito 609 Viruses from a Single Pool of Aedes vexans nipponii Collected in the Republic of Korea. 610 Viruses, 2019. 11(3). 611 55. Zhang, X., et al., Discovery and high prevalence of Phasi Charoen-like virus in field-captured 612 Aedes aegypti in South China. Virology, 2018. 523: p. 35-40. 613 56. Shi, C., et al., Stable distinct core eukaryotic viromes in different mosquito species from 614 Guadeloupe, using single mosquito viral metagenomics. Microbiome, 2019. 615 57. Chavshin, A.R., et al., Malpighian tubules are important determinants of Pseudomonas 616 transstadial transmission and longtime persistence in Anopheles stephensi. Parasit Vectors, 617 2015. 8: p. 36. 618 58. Coon, K.L., M.R. Brown, and M.R. Strand, Gut bacteria differentially affect egg production in 619 the anautogenous mosquito Aedes aegypti and facultatively autogenous mosquito Aedes 620 atropalpus (Diptera: Culicidae). Parasit Vectors, 2016. 9(1): p. 375. 621 59. Favia, G., et al., Bacteria of the genus Asaia stably associate with Anopheles stephensi, an 622 Asian malarial mosquito vector. Proc Natl Acad Sci U S A, 2007. 104(21): p. 9047-51. 623 60. Chen, S., M. Bagdasarian, and E.D. Walker, Elizabethkingia anophelis: molecular 624 manipulation and interactions with mosquito hosts. Appl Environ Microbiol, 2015. 81(6): p. 625 2233-43. 626 61. Coon, K.L., et al., Mosquitoes rely on their gut microbiota for development. Molecular 627 Ecology, 2014. 23(11): p. 2727-2739. 628 62. Moll, R.M., et al., Meconial peritrophic membranes and the fate of midgut bacteria during 629 mosquito (Diptera: Culicidae) metamorphosis. J Med Entomol, 2001. 38(1): p. 29-32. 630 63. Schultz, M.J., H.M. Frydman, and J.H. Connor, Dual Insect specific virus infection limits 631 Arbovirus replication in Aedes mosquito cells. Virology, 2018. 518: p. 406-413. 632 64. Ballinger, M.J., et al., Discovery and Evolution of Bunyavirids in Arctic Phantom Midges and 633 Ancient Bunyavirid-Like Sequences in Insect Genomes. Journal of Virology, 2014. 88(16): p. 634 8783-8794. 635 65. Li, C.X., et al., Unprecedented genomic diversity of RNA viruses in arthropods reveals the 636 ancestry of negative-sense RNA viruses. Elife, 2015. 4. 637 66. Qin, X.C., et al., A tick-borne segmented RNA virus contains genome segments derived from 638 unsegmented viral ancestors. Proceedings of the National Academy of Sciences of the 639 United States of America, 2014. 111(18): p. 6744-6749. 640 67. Tokarz, R., et al., Genome characterization of Long Island tick rhabdovirus, a new virus 641 identified in Amblyomma americanum ticks. Virology Journal, 2014. 11. 642 68. Tokarz, R., et al., Virome Analysis of Amblyomma americanum, Dermacentor variabilis, and 643 Ixodes scapularis Ticks Reveals Novel Highly Divergent Vertebrate and Invertebrate Viruses. 644 Journal of Virology, 2014. 88(19): p. 11480-11492. 645 69. Fredericks, A.C., et al., Aedes aegypti (Aag2)-derived clonal mosquito cell lines reveal the 646 effects of pre-existing persistent infection with the insect-specific bunyavirus Phasi Charoen- 647 like virus on arbovirus replication. PLoS Negl Trop Dis, 2019. 13(11): p. e0007346. 648
649
28