2021.08.30.458191.Full.Pdf

bioRxiv preprint doi: https://doi.org/10.1101/2021.08.30.458191; this version posted August 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Title: Intrinsic variation in the vertically transmitted insect-specific core virome of Aedes aegypti

2 Author names: Coatsworth, H.1,2.3, Bozic, J3,4,5, Carrillo, J.3,6,8, Buckner, E. A.3,4,6, Rivers, A. 3 R.3,7, Dinglasan, R. R.1,2,3, and Mathias, D. K.3,4

4 Author affiliations:

5 1Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USA

6 2Department of Infectious Diseases & Immunology, College of Veterinary Medicine, University 7 of Florida, Gainesville, Florida, USA

8 3CDC Southeastern Center of Excellence in Vector Borne Diseases, Gainesville, Florida, USA

9 4Entomology & Nematology Department, Florida Medical Entomology Laboratory, Institute of 10 Food and Agricultural Sciences, University of Florida, Vero Beach, Florida, USA

11 5Department of Entomology, the Center for Infectious Disease Dynamics, and the Huck 12 Institutes of the Life Sciences, the Pennsylvania State University, University Park, PA, USA.

13 6Manatee County Mosquito Control District, Palmetto, Florida, USA

14 7Genomics and Bioinformatics Research Unit, Agricultural Research Service, United States 15 Department of Agriculture, Gainesville, Florida, USA

16 8Lacerta Therapeutics, Production and Development, Alachua Florida, USA

17 Corresponding Author: D. K. Mathias, University of Florida, [email protected]

19 Abstract

20 Since 2009, local outbreaks of dengue (serotypes 1-3) mediated by Aedes aegypti mosquitoes have 21 occurred in the United States, particularly in Florida (FL). In 2016 and 2017, dengue virus serotype 22 4 was found alongside several insect-specific viruses (ISVs) in pools of A. aegypti from sites in 23 Manatee County, FL, in the absence of an index case. Although ISVs have been characterized in 24 A. aegypti globally, the constitution of a core virome in natural populations remains unclear. Using 25 mosquitoes sampled from the same area in 2018, we compared baseline ovary viromes of field 26 (G0) and lab (Orlando) A. aegypti via metagenomic RNA sequencing. Across all samples, virome 27 composition varied by sample type (field- or colony-derived). Four ISVs comprised >97% of virus 28 sequences: a novel partiti-like virus (Partitiviridae), a previously described toti-like virus 29 (Totiviridae), unclassified Riboviria, and four previously described orthomyxo-like viruses 30 (Orthormyxoviridae). Whole or partial genomes for the toti-like virus, partiti-like virus, and one 31 orthomyxo-like virus were assembled and analyzed phylogenetically. Multigenerational 32 maintenance of these ISVs was confirmed orthogonally by RT-PCR in G0 and G7 mosquitoes, 33 indicating vertical transmission as the mechanism for ISV sustentation. This study provides 34 fundamental information regarding ISV ecology, persistence, and variation in A. aegypti in nature.

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37 Introduction

38 Mosquito-borne viral diseases such as dengue, chikungunya, and Zika have spread to new areas, 39 putting over half the world’s population at risk of infection (Weaver 2014, Kraemer et al. 2015). 40 This increase is largely correlated with the global expansion of Aedes aegypti and Aedes 41 albopictus mosquitoes, the primary vectors for these arboviruses (Messina et al. 2019, Brady and 42 Hay 2020). In Florida, USA, both vector species are present, and A. aegypti populations thrive in 43 most of the state’s large population centers due to their urban niche (Britch et al. 2008, Reiskind 44 and Lounibos 2013, Wilke et al. 2019) and widespread insecticide resistance (Estep et al. 2018, 45 Mundis et al. 2020).

46 Autochthonous dengue virus (DENV, serotypes 1-3) infections have occurred in 11 of the last 12 47 years in urban centers in South Florida (CDC 2010, Rey 2014, 48 https://www.cdc.gov/dengue/statistics-maps/index.html), while in 2014 and 2016 outbreaks of 49 chikungunya (Kendrick et al. 2014) and Zika (Likos et al. 2016, Grubaugh et al. 2017), 50 respectively, occurred exclusively in counties with recent permanent population records of A. 51 aegypti (Hahn et al. 2017). Since the re-emergence of DENV in the Florida Keys in 2009, local 52 transmission of at least one serotype has occurred in the state each year except in 2017. 53 Moreover, despite the absence of a local human index case, dengue virus serotype 4 (DENV-4) 54 was detected and fully sequenced alongside numerous insect-specific viruses (ISVs) in the 55 abdomens of A. aegypti adult G0 females from Manatee County, Florida, in 2016 and 2017 56 (Boyles et al. 2020).

57 The ability of a mosquito to transmit DENV (i.e., its vector competence) varies based on external 58 environmental variables, the genetic backgrounds of the vector and virus, and the composition of 59 the mosquito’s microbiome (Tabachnick 2013). The latter is complex and includes ISVs, which 60 can only replicate in the cells of insect hosts (Sang et al. 2003, Nasar et al. 2012, Junglen et al., 61 2017). ISVs are pervasive in both wild-caught and laboratory-reared mosquitoes, and co- 62 infection of ISVs from the same viral family as DENV are known to influence the vector 63 competence of A. aegypti (Bolling et al. 2012, Zhang et al. 2017, Atoni et al. 2019, Baidaliuk et 64 al. 2019, Öhlund et al. 2019). However, most recently discovered ISVs belong to other virus 65 families, and given their pervasiveness, a better understanding of their ecology and impact on 66 mosquito biology is needed. The term “core virome” was recently coined to describe the set of 67 ISVs common to the majority of individuals in a mosquito population (Shi et al. 2019). The term 68 has since been divided into two categories: vertical (passed from mother to offspring) and 69 environmental (acquired from the environment) (Shi et al. 2020). To date, research on Aedes 70 mosquitoes suggests that at the population level core viromes are maintained across 71 developmental stages (Shi et al. 2020) and over short time scales (~ 1 year) (Boyles et al. 2020, 72 Shi et al. 2020). However, it is unknown how well vertical core viromes are sustained over 73 longer periods of time, or the degree to which environmental core viromes are transient. 74 Moreover, most colonies commonly used in arbovirus research have yet to be examined, and 75 these could be useful models to address questions about the impact of core viromes on mosquito 76 biology.

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77 To begin filling these gaps in knowledge, we used tissue-specific RNA metagenomics and 78 reverse-transcription PCR to follow up on the previous virome profiles characterized for 79 Floridian A. aegypti (Boyles et al. 2020). Our objectives were to i) define the vertically 80 transmitted core virome in ovary pools from G0 field- and lab-derived A. aegypti, ii) examine the 81 persistence of specific core ISVs over time in ovaries of G7 descendants of field-derived females, 82 iii) assess variability among the sexes for core ISVs maintained in a field-derived colony, and iv) 83 use phylogenetics to investigate evolutionary relationships between core virome members and 84 previously described ISVs. We hypothesized that elements of the vertical core virome would be 85 present in both field- and lab-derived “Orlando” (ORL) strain A. aegypti due to both originating 86 from Florida, but that field-derived samples would have higher ISV diversity due to greater 87 environmental variability. We further predicted that male ISV infection status would mimic that 88 of their female counterparts because of efficient vertical transmission and that A. aegypti ISVs 89 would cluster phylogenetically with ISVs from other mosquito taxa.

90 Results

91 Partiti- and toti-like insect-specific viruses dominate the Aedes aegypti virome

92 To identify core virome ISV members as well as compare field versus lab A. aegypti virome 93 diversity, viral reads from each ovary pool were assigned to their lowest common ancestor 94 (LCA) and summed based on read count. Partitiviridae reads comprised between 50-76% of all 95 viral reads in the field-derived Manatee County (Palmetto, P) samples, while unclassified 96 Riboviria reads made up >57% of the viral reads in the ORL sample and between 5-11% of the P 97 samples (Figure 1). Viral reads aligning best with Atrato partiti-like virus 3 were the most 98 prevalent across all the P samples, making up 70, 72, 51, 67 and 76 % of the viral reads (P1 – 5 99 ovary pools respectively), but were completely absent from the ORL sample. Reads aligning to 100 A. aegypti toti-like virus accounted for 19, 17, 33, 10, and 11% of the reads for the P samples, 101 and represented 42% of the ORL sequences. Together, 75-90% of all viral reads from field- 102 derived samples aligned to these two groups (Partitiviridae and Totiviridae). In addition, 42% of 103 the ORL reads and ~5% of reads in each of the P samples aligned with an unclassified Riboviria 104 virus, followed by 13% matching to a dsRNA virus environmental sample in ORL and ~1.5% in 105 all P samples. Sequences matching to four viruses in Orthomyxoviridae (Guadeloupe mosquito 106 quaranja-like virus 1, Whidbey virus, unclassified Orthomyxoviridae, and Aedes alboannulatus 107 orthomyxo-like virus) were restricted to field-derived samples and collectively accounted for 1.7, 108 2.4, 2.0, 11.4, and 7.2% of the reads from P1 – P5, respectively. The remaining viral sequences 109 in these samples each represented less than one percent of the total viral sequence pool, and in 110 order of prevalence, matched to A. aegypti virga-like virus, A. aegypti anphevirus, unclassified 111 viruses, Chuvirus Mos8Chu0, Liao ning virus, and an unclassified flavivirus (Figure 1). The 112 field-derived P samples were more diverse than the ORL sample, as the P samples had an 113 average of 12 different viral assignments, while the ORL sample only contained 5.

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114 115 Figure 1. Proportion of aligned viral reads across ovary pools of field-derived Palmetto (P, Manatee Country, FL, USA) and lab- 116 reared Orlando (ORL) A. aegypti. The samples are ranked in descending order of viral read prevalence, grouped, and colored by viral .

117 classification. Fuchsia: Partitiviridae, purple: unclassified Totiviridae, blue: unclassified Riboviria, green: Orthomyxoviridae, yellow: The copyrightholderforthispreprint 118 Xinmoviridae, red: unclassified viruses, brown: Reovirdiae, grey: Flaviviridae.

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119 Genome assembly of Palmetto toti-, partiti- and orthomyxo-like viruses

120 Whole or partial genome assembly was completed for our Palmetto toti-, partiti- and orthomyxo- 121 like viruses to compare the overall similarity between the viruses found here and those reported 122 previously. Eight open reading frames (ORFs) were predicted based on the assembled Palmetto 123 toti-like virus contigs. Only 2 of these ORFs (ORF 2 [1,033aa] and ORF 3 [1,021aa]) assembled 124 in a linear format akin to the typical capsid-RdRp Totiviridae structure (Figures 2, 3) and created 125 products with known sequence similarity. The 1,033aa long ORF of the Palmetto toti-like virus 126 showed high amino acid similarity to other A. aegypti RNA-dependent RNA polymerase (RdRp) 127 genes in toti-like viruses (>98% identity), with high query coverage (~90%) to 926aa RdRp 128 sequences isolated from individual Aedes aegypti mosquitoes from Guadeloupe, a Caribbean 129 island (QEM39131.1 and QEM39133.1) (Shi et al., 2019). The 1,021aa sequence showed high 130 similarity and query coverage to the 1,008aa long Guadeloupe Aedes aegypti toti-like capsid 131 sequences (>99% and 98%, respectively) isolated from the samples. Together, these sequences 132 represent the RdRp and capsid genes of the Palmetto toti-like virus identified herein.

133 In contrast, only three ORFs were predicted for the Palmetto partiti-like virus, which likely has a 134 simple two-segmented genomic structure characteristic of the Partitiviridae (Figure 4). Only one 135 of these ORFs (ORF 1 [446aa]) had sequence similarity to known sequences, with 78% identity 136 and 95% query coverage to a 472aa RdRp Atrato partiti-like virus sequence (QHA33899.1) 137 isolated from Psorophora albipes mosquitoes in Colombia, and 79% identity and 86% query 138 coverage to another RdRp Atrato partiti-like virus sequence (QHA33901.1, 387aa) isolated from 139 Anopheles darlingi mosquitoes in Colombia. Therefore, this sequence was classified as the RdRp 140 for the Palmetto partiti-like virus.

141 Twenty ORFs were predicted from the Orthomyxoviridae contigs, and 6 of these ORFs had 142 known sequence similarity based on non-redundant protein blast searches. Four of these ORFs 143 appeared to represent one virus (Palmetto orthomyxo-like virus), while the remaining two ORFs 144 are likely from two other orthomyxoviruses: ORF 4 (229aa) with 33% identity and 65% query 145 coverage to an Atrato Chu-like virus 5 hypothetical protein 2 (QHA33674.1) identified in 146 Psorophora albipes from Colombia, and ORF 5 (294aa) with 66% identity and 87% query 147 coverage with the nucleoprotein of Wuhan Mosquito Virus 6 (QRW42410.1) isolated from 148 Culex tarsalis mosquitoes from California. The 4 Palmetto orthomyxo-like virus ORFs had 149 highest sequence similarity to Guadeloupe mosquito quaranja-like virus 1 (GMQLV1), isolated 150 from A. aegypti collected in San Diego County, California (Batson et al. 2021). Like other 151 viruses in the Orthomyxoviridae, quaranjaviruses have a segmented genome, and GMQLV1 was 152 originally described as likely having 6 or 7 segments (Shi et al.2019). However, recently 153 reported data (Batson et al. 2021) indicate that 8 segments are more likely (Figures 5-8). ORF7 154 (800aa) had 100% identity and 98% query coverage to GMQLV1 PB2 (QRW42587.1); ORF 10 155 (797aa) had 99.75% identity and 98% query coverage to GMQLV1 PB1 (QRW42591.1); ORF 156 11 (716aa) had 99.86% identity and 98% query coverage to GMQLV1 PA (QRW42581.1); and 157 ORF 16 (584aa) had 99.64% identity and 95% query coverage to GMQLV1 NP (QRW42580.1). 158 These 4 ORFs likely represent the nucleoprotein (NP - ORF 16), and the heterotrimeric RdRp

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159 complex (PB2 – ORF7, PB1 – ORF10, PA – ORF11) for our Palmetto orthomyxo-like virus, 160 which is likely identical to GMQLV1.

161 Reverse transcription PCR confirmation supports virus vertical transmission

162 To assess vertical transmission success of these three ISVs, reverse transcription PCR with 163 primers designed from metagenomic data for the Palmetto toti-, partiti- and orthomyxo-like 164 viruses (Table S1) was used to re-screen RNA remaining from the original G0 pools, as well as 165 RNA from ovaries of G7 females and from G7 males. This approach yielded amplicons for the 166 toti-like virus capsid gene, the partiti-like virus RdRp gene, and the orthomyxo-like virus PA 167 gene (Figure 9). Amplicons were sequenced and the presence of all three viruses was confirmed 168 in both G0 and G7 ovary pools (Figure 9 A, C, E). The Palmetto orthomyxo-like virus was 169 present in all G7 adult-male pools (Figure 9 B, D, F), while the Palmetto partiti-like virus was 170 found only in a subset of these samples, occurring in 3 of 5 pools. Intriguingly, no males were 171 positive for Palmetto toti-like virus. Although the occurrence and frequency of environmental 172 acquisition and venereal transmission of these viruses remain open questions, the ovary-positive 173 data and presence across multiple generations suggests viral persistence in the population by 174 vertical transmission.

175 Viral gene phylogenetics

176 Phylogenetics on virus gene segments was completed to investigate the evolutionary 177 relationships of each of ISVs to infer their ecological history. Database mining and protein 178 alignments resulted in 25 sequences spanning 1008 sites for the toti-like capsid tree, 101 179 sequences (1032 sites) for the toti-like RdRp tree, 101 sequences (447 sites) for the partiti-like 180 RdRp tree, 78 sequences (1668 sites) for the orthomyxo-like PB2 tree, 101 sequences (1041 181 sites) for the orthomyxo-like PB1 tree, 101 sequences (1388 sites) for the orthomyxo-like PA 182 tree, and 63 sequences (932 sites) for the orthomyxo-like NP tree. After Smart Model Selection 183 (SMS), five trees resulted in an LG optimal model with Gamma rates (G), invariable sites (I), 184 and empirical frequencies (F) (LG+G+I+F). Log-likelihood ratios for the toti-like trees were - 185 53142.30 and -23019.63 (RdRp and capsid, respectively), -44320.2 for the partiti-like RdRp tree, 186 and -77638.40 and -94785.91 for the orthomyxo-like PB1 and PA trees, respectively. The 187 orthomyxo-like PB2 and NP trees resulted in a LG+G+F optimal model with log-likelihood 188 ratios of -86514.82 and -49892.72, respectively.

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(which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade bioRxiv preprint doi: https://doi.org/10.1101/2021.08.30.458191 available undera CC-BY-NC-ND 4.0Internationallicense ; this versionpostedAugust31,2021. . The copyrightholderforthispreprint 189 190 Figure 2. Maximum likelihood tree of toti-like virus capsid sequences (using an LG+G+I+F model) based on amino acid sequences. 191 The tree is representative of 25 sequences spanning 1008 sites and 47 branches. Mosquito-derived virus sequences are colored in blue, 192 while the Palmetto sequence is in red. Virus names are appended to all sequences derived from Diptera. The generalized viral genomic 193 structure is to the right of the tree, with the segment analyzed in color (green polygon represents capsid/nucleoprotein sequence). The 194 numbers on each branch within the inset denote branch support lengths. Scale bar is representative of 0.5 amino acid substitutions per 195 site.

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(which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade bioRxiv preprint doi: https://doi.org/10.1101/2021.08.30.458191 available undera CC-BY-NC-ND 4.0Internationallicense ; this versionpostedAugust31,2021. . The copyrightholderforthispreprint 196 197 Figure 3. Maximum likelihood tree of toti-like virus RdRp sequences (using an LG+G+I+F model) based on amino acid sequences. 198 The tree is representative of 101 sequences spanning 1032 sites and 199 branches. Mosquito-derived virus sequences are colored in 199 blue, while the Palmetto sequence is in red. Virus names are appended to all sequences derived from Diptera. The generalized viral 200 genomic structure is to the right of the tree, with the segment analyzed in color (purple polygon represents polymerase sequence). The 201 numbers on each branch within the inset denote branch support lengths. Scale bar is representative of 0.9 amino acid substitutions per 202 site.

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(which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade bioRxiv preprint doi: https://doi.org/10.1101/2021.08.30.458191 available undera CC-BY-NC-ND 4.0Internationallicense ; this versionpostedAugust31,2021. . The copyrightholderforthispreprint 203 204 Figure 4. Maximum likelihood tree of partiti-like RdRp virus sequences (using an LG+G+I+F model) based on amino acid sequences. 205 The tree is representative of 101 sequences spanning 447 sites and 199 branches. Mosquito-derived virus sequences are colored in 206 blue, while the Palmetto sequence is in red. Virus names are appended to all sequences derived from Diptera. The generalized viral 207 genomic structure is to the right of the tree, with the segment analyzed in color (purple polygon represents polymerase sequence). The 208 numbers on each branch within the inset denote branch support lengths. Scale bar is representative of 0.5 amino acid substitutions per 209 site.

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210 The copyrightholderforthispreprint 211 Figure 5. Maximum likelihood tree of orthomyxo-like of the PB2 virus sequences (using an LG+G+F model) based on amino acid 212 sequences. The tree is representative of 78 sequences spanning 1668 sites and 153 branches. Mosquito-derived virus sequences are 213 colored in blue, while the Palmetto sequence is in red. Virus names are appended to all sequences derived from Diptera or Ixodida. 214 The generalized viral genomic structure is to the right of the tree, with the segment analyzed in color (purple polygon represents 215 polymerase sequence). The numbers on each branch within the inset denote branch support lengths. Scale bar is representative of 0.7 216 amino acid substitutions per site.

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(which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade bioRxiv preprint doi: https://doi.org/10.1101/2021.08.30.458191 available undera CC-BY-NC-ND 4.0Internationallicense ; this versionpostedAugust31,2021. . The copyrightholderforthispreprint 217 218 Figure 6. Maximum likelihood tree of orthomyxo-like of the PB1 virus sequences (using an LG+G+I+F model) based on amino acid 219 sequences. The tree is representative of 101 sequences spanning 1041 sites and 199 branches. Mosquito-derived virus sequences are 220 colored in blue, while the Palmetto sequence is in red. Virus names are appended to all sequences derived from Diptera or Ixodida. 221 The generalized viral genomic structure is to the right of the tree, with the segment analyzed in color (purple polygon represents 222 polymerase sequence). The numbers on each branch within the inset denote branch support lengths. Scale bar is representative of 0.4 223 amino acid substitutions per site.

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(which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade bioRxiv preprint doi: https://doi.org/10.1101/2021.08.30.458191 available undera CC-BY-NC-ND 4.0Internationallicense ; this versionpostedAugust31,2021. . The copyrightholderforthispreprint 224 225 Figure 7. Maximum likelihood tree of orthomyxo-like of the PA virus sequences (using an LG+G+I+F model) based on amino acid 226 sequences. The tree is representative of 101 sequences spanning 1388 sites and 199 branches. Mosquito-derived virus sequences are 227 colored in blue, while the Palmetto sequence is in red. Virus names are appended to all sequences derived from Diptera or Ixodida. 228 The generalized viral genomic structure is to the right of the tree, with the segment analyzed in color (purple polygon represents 229 polymerase sequence). The numbers on each branch within the inset denote branch support lengths. Scale bar is representative of 0.6 230 amino acid substitutions per site.

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(which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade bioRxiv preprint doi: https://doi.org/10.1101/2021.08.30.458191 available undera CC-BY-NC-ND 4.0Internationallicense ; this versionpostedAugust31,2021. . The copyrightholderforthispreprint 231 232 Figure 8. Maximum likelihood tree of orthomyxo-like nucleoprotein (NP) virus sequences (using an LG+G+F model) based on amino 233 acid sequences. The tree is representative of 63 sequences spanning 932 sites and 123 branches. Mosquito-derived virus sequences are 234 colored in blue, while the Palmetto sequence is in red. Virus names are appended to all sequences derived from Diptera or Ixodida. 235 The generalized viral genomic structure is to the right of the tree, with the segment analyzed in color (green polygon represents 236 capsid/nucleoprotein sequence). The numbers on each branch within the inset denote branch support lengths. Scale bar is 237 representative of 0.6 amino acid substitutions per site.

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238 239 Figure 9. Agarose gel images showing results of RT-PCR screens for Palmetto toti-like virus 240 (PTLV) in ovaries (A) and males (B), Palmetto partiti-like virus (PPLV) in ovaries (C) and males 241 (D), and Palmetto orthomyxo-like virus (POLV) in ovaries (E) and males (F). For each primer 242 set (Table S1), 1 – 5 denote P1 – P5 ovary pools from G0 females, 6 – 10 denote ovary pools 243 from G7 females, and 11 – 15 denote pools of males. For each set of reactions, NTC is the no 244 template control, L denotes 100-bp ladder, and +Ctl is the positive control for male assays, which 245 consisted of a previously positive ovary pool.

246 Both the toti-like capsid and RdRp gene trees showed distinct mosquito clusters (Figures 2 and 247 3) with strong branch support. The Palmetto toti-like virus identified herein clustered with other 248 Aedes aegypti toti-like viruses. Culex derived toti-like viruses made up a distinct clade in the 249 RdRp tree, while in the capsid tree two of the Culex-derived viruses appeared more closely 250 related to viruses from Ochlerotatus, a subgenus of Aedes according to traditional Culicidae 251 systematics (Wilkerson et al. 2015). Because of better representation of RdRp sequences in 252 databases, we were able to analyze a broader range of totivirus sequences in the RdRp virus tree 253 revealing distant relatedness to totiviruses from numerous non-dipteran arthropods, fungi and 254 oomycota.

255 In contrast, the partiti-like RdRp phylogenetic tree showed no obvious mosquito clustering, as 256 mosquito-derived partiti-like virus sequences were dispersed throughout the tree (Figure 4). Our 257 Palmetto partiti-like virus formed a small but well-supported clade with other mosquito partiti- 258 like viruses (Atrato partiti-like virus 3 and Broome partiti-like virus), as well as partiti-like 259 viruses from other non-dipteran arthropods, plants, and molluscs. Virus sequences from non- 260 dipteran arthropods, dipterans, and insects made up most of the remaining branches in the tree.

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261 All the orthomyxo-like trees (PB2, PB1, PA, and NP) displayed similar trends to one another, 262 with two distinct mosquito clusters, loosely organized into Culex- and Aedes-derived viruses, 263 separated by ISVs from primarily hematophagous Dipterans (Figures 5-8). In all the orthomyxo- 264 like gene segment trees, our Palmetto orthomyxo-like virus was part of the well-supported 265 Aedes-based mosquito clade, directly clustering with Guadeloupe mosquito quaranja-like virus 1, 266 Whidbey virus, and Aedes detritus orthomyxo-like virus.

267 Discussion

268 Conservation of a vertically transmitted Floridian Aedes aegypti ‘core virome’

269 Many of the viruses detected through our sequence analysis were present in both the field- and 270 lab-derived samples. Of the sequences common to all samples, the most abundant corresponded 271 to Aedes aegypti toti-like virus, although a dsRNA virus environmental sample and an 272 unclassified Riboviria virus were also prevalent (Figure 1). The Palmetto toti-like virus identified 273 here across all A. aegypti samples showed striking similarity to Aedes aegypti toti-like viruses 274 from Guadaloupe and an unnamed virus identified in A. aegypti from Thailand, which was first 275 described as dsRNA environmental virus sample. Totiviridae and toti-like virus samples have 276 been found in several mosquito genera with widespread global prevalence, including Anopheles 277 in Liberia (Fauver et al. 2016), Armigeres in China (Zhai et al. 2010), Culex in Belgium (Wang 278 et al. 2020), Culex in California (Batson et al. 2021), Culex in Australia (Williams et al. 2020), 279 Culex in Japan (Isawa et al. 2011), and Mansonia in Brazil (de Lara Pinto et al. 2017). 280 Metagenomic analyses of G0 Manatee County mosquitoes in 2016 and 2017 also identified a toti- 281 like virus (Anopheles totivirus), as well as a dsRNA virus from an environmental sample (Boyles 282 et al. 2020). This is further support that A. aegypti mosquitoes with similar genetic backgrounds 283 (here, representative of Florida-based populations) share the same virus families (i.e., ‘core 284 virome’ components), as previously reported (Öhlund et al. 2019, Shi et al. 2019, Wang et al. 285 2020, Konstantinidis et al. 2021).

286 The field A. aegypti insect-specific viral landscape is more diverse

287 Despite the pooled nature of our samples, there were clear differences in the general diversity of 288 the virus communities, as 12 eukaryotic virus taxa were found in the field-derived samples 289 compared to only five in the ORL sample. This greater virome diversity of field-derived samples 290 was also noted in the 2016-2017 Manatee County G0 samples (Boyles et al. 2020), as were 291 numerous other partiti-like viruses (Hubei partiti-like viruses 29, 32, 33, 34, Wenling partiti-like 292 virus 2) and two viruses in Orthomyxoviridae (Whidbey virus, Aedes alboanulatus orthomyxo- 293 like virus). The Palmetto partiti-like virus (Partitiviridae) and the four taxa belonging to the 294 family Orthomyxoviridae (Guadeloupe mosquito quaranja-like virus 1, Whidbey virus, Aedes 295 alboannulatus orthomyxo-like virus, and an unclassified Orthomyxoviridae virus) (Figure 1) 296 were solely found in the field samples. These differences likely stem from variation between the 297 field population and lab colony. While the field populations were exposed to diverse 298 environments, various feeding sources, and dynamic larval habitats, the lab ORL A. aegypti have 299 been raised for decades under stable, standardized conditions. As such, the contrast in ecological 300 conditions experienced by mosquitoes in the lab versus the field may reflect the viral diversity of

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301 their respective environments. Although non-vertical routes of ISV acquisition in mosquitoes 302 (i.e., horizontal transmission) are not well studied, to date the data suggest that these viruses may 303 establish new infections through plant or detritus feeding by larvae, directly from water in the 304 larval environment, from the meconium upon adult eclosion, from fungal or parasitic infection, 305 via parasitoids, through nectar feeding by adults, and from one mosquito to another during 306 mating (Vasilakis and Tesh 2015, Dolja and Koonin 2018, Agboli et al. 2019, Xu et al. 2020). As 307 the ORL strain A. aegypti have been raised in laboratory settings for over 60 years (Kuno 2010), 308 these diverse viral acquisition routes would be limited.

309 Totiviridae and Partiviridae phylogenies point to shared plant- and fungal-based lineages

310 The clear mosquito-associated clade present in the toti-virus like sequences (Figures 2 and 3) 311 could represent widespread concurrent viral movement or long host co-evolution (Wang et al. 312 2020), as is thought to be the case for Bunyaviridae, Flaviviridae, and Rhabdoviridae ISVs, 313 which are assumed to have evolved and diversified alongside their host (Vasilakis and Tesh 314 2015). Historically, totiviruses were primarily fungal associated, and likely represent a viral 315 taxon with an ancient origin (Dolja and Koonin 2018). This historic basis is mirrored in our toti- 316 like virus RdRp tree (Figure 3), as mosquito-associated sequences matched to dipteran and non- 317 dipteran arthropod virus RdRps, followed by distant clades of fungal toti-like virus RdRps. The 318 jump of these toti-like viruses into invertebrate systems is likely due to horizontal virus transfer 319 (Dolja and Koonin 2018), and may have occurred within the mosquito itself, as fungi are 320 common inhabitants of the mosquito microbiome. Similarly, the Partitiviridae and partiti-like 321 viruses were historically associated with plants and fungi but have increased in prevalence 322 among arthropods (Faizah et al. 2020). Partitiviridae and partiti-like viruses have been found 323 across numerous mosquito genera (Culex, Culiseta, Coquilettidia, Anopheles, Aedes) with a 324 nearly global distribution (North and South America, Africa, Asia, and Europe) (Öhlund et al. 325 2019, Shi et al. 2019, Wang et al. 2020, Konstantinidis et al. 2021). The low congruence between 326 viral phylogeny and host-range for these partiti-like viruses may suggest a recent host-switching 327 event (Grubaugh et al. 2016, Dolja and Koonin 2018), as the Palmetto partiti-like virus was 328 highly divergent (<80% similarity) from other known partiti-like viruses and likely represents a 329 novel virus. Cross-species transmission (i.e., horizontal virus transfer) is clearly common 330 throughout the ISV landscape (Shi et al. 2018) and has likely been a major factor in the 331 evolutionary history of the partiti-like virus described herein (Figure 4).

332 Orthomyxoviridae phylogenies show a distinct hematophagous arthropod clade with 333 known human pathogenic members

334 The phylogenies for the Orthomyxoviridae genome segments displayed similar trends, with 335 viruses from non-hematophagous, non-dipteran arthropods as ancestral to those in mosquito- 336 enriched clades in all four trees (Figures 5-8). Known vertebrate pathogenic viruses formed 337 distinct and separate sub-clades from the mosquito and hematophagous dipteran viruses. The 338 former largely included tick-vectored vertebrate viruses such as Bourbon virus, Johnston Atoll 339 quaranjavirus, and Batken virus, among others. The latter was sub-differentiated loosely into 340 Aedes and Culex ISV sub-clades, with our Palmetto orthomyxo-like virus falling into the Aedes 341 clade. Our Palmetto orthomyxo-like virus appears to be nearly identical to the Guadeloupe

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342 mosquito quaranja-like virus 1 (GMQLV1) first identified in A. aegypti from the Caribbean by 343 Shi et al. (2019), and as such, is likely a member of the genus Quaranjavirus. This genus was 344 only recently described (Presti et al. 2009), and genomes of its viruses likely consist of eight 345 negative-sense, single-stranded RNA segments (Batson et al. 2021) (Figures 5-8). Genome 346 fragments recovered from our data matched to genes on four segments. Most known 347 Quaranjavirus members differ from their influenza relatives via their surface glycoprotein 348 (gp64), which has similarity to Baculoviridae members, and is hypothesized to have been the 349 catalyst for virus entry and fusion in ticks (Allison et al. 2015). These quaranjaviruses have 350 demonstrated horizontal transmission cycles akin to arboviruses between ticks and tropical and 351 subtropical birds (Presti et al. 2009), which is likely why viruses from birds and ticks appear as 352 distant relatives to the Palmetto orthomyxo-like virus in our gene trees (Figures 5-8). 353 Interestingly, these tick-vectored quaranjaviruses showed a lack of replication in Aedes 354 albopictus C6/36 cells (Presti et al. 2009, Allison et al. 2015), suggesting that host specificity 355 may be more strictly defined for quaranjaviruses (i.e., less amenable to host switching). In turn, 356 this supports the notion that ISVs vary in their efficiency of vertical transmission, and hence, 357 their capacity to be maintained in a host population by this mechanism over time. The 358 phylogenies based on gene segments for the orthomyxo-like viruses suggest high transmission 359 fidelity, while the partiti-like virus tree suggests greater amenability to host switching. Such 360 patterns may provide clues to ISV ecology, including their degree of specialization and relative 361 ability to use environmental routes to infect mosquitoes horizontally.

362 Palmetto toti-like, partiti-like and orthomyxo-like insect specific viruses are vertically 363 maintained in A. aegypti

364 Vertical maintenance of all three Palmetto insect specific viruses identified herein is likely 365 occurring, as we were able to detect each virus across A. aegypti generations post-field collection 366 through multiple generations (G0 and G7) via RT-PCR (Figure 9), a trend noted with numerous 367 ISVs (Lutomiah et al. 2007, Bolling et al. 2011, Haddow et al. 2013, Contreras-Gutierrez et al. 368 2017, Frangeul et al. 2020). As all ovary pools were positive in both G0 and G7 generations for 369 all three viruses (Figure 9A, C, E), transovarial transmission was likely the primary route of 370 vertical transmission. As males are known to carry a wealth of ISVs (Frangeul et al. 2020), we 371 also tested pools of adult males for each of our three viruses. Male pools were consistently 372 positive for Palmetto orthomyxo-like virus, positive in the majority of pools for Palmetto partiti- 373 like virus, and notably negative for Palmetto toti-like virus (Figure 9B, D, F). These 374 discrepancies in positivity may suggest differences in transstadial transmission between the sexes 375 during development, which could arise from sex-specific tissue tropisms as adult structures form 376 in the pupal stage. Moreover, testes might display specificity for certain viruses in their small 377 RNA machinery, leading to a more efficient antiviral response (Frangeul et al. 2020). 378 Alternatively, since the toti-like virus was also discovered in our lab-adapted colony (ORL), it 379 may have been acquired during blood feeding. Both G0 and G7 females were fed on chickens to 380 enable egg production and oviposition prior to ovary dissection. This is the only environmental 381 factor in our study that differs between the sexes, but because totiviruses are not common in 382 domestic birds, we find this route unlikely. For the two viruses present in males, venereal 383 transmission may complement vertical transmission as a secondary maintenance mechanism for

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384 ISV persistence, as has been described for bunyaviruses and alphaviruses in other mosquito 385 species (Ovenden and Mahon 1984, Schopen et al. 1991).

386 Metagenomic sequencing utility and relevance

387 Many of the virus assignments for our A. aegypti (Palmetto) mosquitoes matched to unclassified 388 Riboviria lacking further taxonomic resolution, a trend noted in other publications (Öhlund et al. 389 2019, Shi et al. 2019, Williams et al. 2020). This is likely due to limitations imposed by 390 incomplete virus databases and augmented by the difficulty of recovering segmented virus 391 genomes from pooled samples with high virus similarity (Shi et al. 2019, Batson et al. 2021). As 392 more studies examine viral communities and increase the overall database size, taxonomic 393 classification of this metagenomic ‘viral dark matter’ should drastically improve (Batson et al. 394 2021).

395 Despite the presence of DENV-4 in the abdomens of Manatee County mosquitoes in 2016 and 396 2017 (Boyles et al. 2020), we did not identify any circulating human pathogenic viruses in the 397 ovaries of our field-derived 2018 Manatee County mosquitoes from Palmetto. The effects of the 398 virome, particularly from the presence of various combinations of viruses, are largely still 399 unknown. Studies have shown variation in vector competence with ISV infection (Bolling et al. 400 2012, Zhang et al. 2017, Baidaliuk et al. 2019) and a positive association between ISV infection 401 and human pathogen infection in mosquitoes (Newman et al. 2011). However, these results do 402 not hold true in all systems (Kent et al. 2010, Crockett et al. 2012). Furthermore, although 403 potential ISV-arbovirus interaction modes, such as competitive inhibition and superinfection 404 exclusion, have been proposed (Vasilakis and Tesh 2015, Roundy et al. 2016,), mechanistic data 405 regarding these interactions, especially on a community scale (i.e., the whole microbiome within 406 a mosquito), are severely lacking. ISV infection in mosquitoes is widely assumed to be 407 commensal (Hall et al. 2016), although examples of ISV-insect interactions with outcomes that 408 depend on the biological context can be found in the literature. For example, a partiti-like virus 409 infection in the fall armyworm (Spodoptera frugiperda) causes negative fitness effects in S. 410 frugiperda but renders the caterpillar more resistant to a pathogenic nucleopolyhedrovirus (Xu et 411 al. 2020). Furthermore, the low abundance of some of these presumed ISVs and high co- 412 appearance with fungal pathogens may mean that some ISVs are more likely associated with 413 infecting fungi than the mosquito itself (Shi et al. 2017). It remains unclear if similar symbioses 414 are occurring or could occur with the three ISVs studied here.

415 Conclusions

416 Our data suggest that there is a core set of vertically transmitted ISVs in Palmetto Aedes aegypti 417 representing at least three virus families: Partitiviridae, Totiviridae and Orthomyxoviridae. The 418 toti-like virus was also prevalent in the ovaries of A. aegypti derived from a colony long 419 maintained in the laboratory. We were able to confirm that vertical maintenance of three of these 420 viruses is likely occurring via transovarial transmission, as the Palmetto partiti-, toti-and 421 orthomyxo-like viruses were present in all ovary pools spanning seven generations. Although the 422 orthomyxo-like virus was also present in all male pools, occurrence of the partiti-like virus was 423 less consistent among males, and the toti-like virus was absent from male pools altogether,

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424 suggesting potential variation in sex-specificity among ISVs. The striking differences in distant 425 host taxa between the toti-like and partiti-like virus trees compared to the orthomyxo-like virus 426 tree may suggest disparate routes of viral evolution or acquisition (e.g., plant/fungi-based versus 427 vertebrate-based). However, because these data were obtained from pools of ovaries and males, 428 we lack the individual-level resolution to determine ISV prevalence in the population or colony, 429 assess the variability of ISV community composition among individuals, or assess within- 430 mosquito virus abundance. Furthermore, despite previous detection and full genome sequencing 431 of DENV-4 in G0 Manatee County A. aegypti collected in 2016 and 2017 (Boyles et al. 2020), 432 we did not detect human pathogenic viruses in our metagenomic screen. Nevertheless, this study 433 provides ecological baseline data for ISV occurrence and vertical transmission for a natural, 434 endemic vector species in a state with rising local DENV transmission. Further research on ISV 435 presence/persistence in mosquitoes and their impact on the suite of biological parameters that 436 determine vectorial capacity could help contextualize the role ISVs play in arboviral 437 transmission.

438 Materials and Methods

439 Mosquito Collections and Sample Preparation

440 Mosquito eggs were sampled from Palmetto, Florida, in July and August of 2018 in collaboration 441 with the Manatee County Mosquito Control District. Fifteen ovijars were placed throughout a 442 peri-urban area of approximately 1 square kilometer, and ovijar locations were the same as those 443 previously described to sample Aedes spp. eggs in 2016-2017 (Boyles et al. 2020) (Figure 10). 444 Egg papers were returned to the insectary at the Florida Medical Entomology Laboratory 445 (FMEL), hatched in distilled water, reared to adulthood, and sorted by species into separate 446 cages by ovijar. Eggs from four ovijars yielded sufficient A. aegypti adults for successful mating. 447 On days 5-7 post-emergence, females (G0) were fed on chickens (IACUC protocol 201807682) 448 and three days later transferred individually to 50 mL ovicages lined with moist seed germination 449 paper. Gravid mosquitoes were allowed to oviposit over a two-day period, and eggs were dried 450 and stored separately. Upon egg collection, each female was cold anesthetized on ice, surface 451 sterilized in 70% ethanol, rinsed twice with sterile PBS, and then dissected one at a time to 452 remove pairs of ovaries. Following dissection, ovaries were immediately transferred to RNAlater 453 solution (Invitrogen, Waltham, MA) and then combined into five pools from 24-42 females 454 depending on ovijar location and date of egg collection. These pools were then stored at -80°C 455 until processed for RNA. Eggs collected from these mosquitoes were used to establish a field- 456 derived colony from Palmetto. After seven generations of maintenance in the laboratory, ovaries 457 were dissected from 50 G7 A. aegypti females as described above, combined into five pools of 458 ten pairs, and stored at -80°C in RNAlater. Similarly, 15 G7 A. aegypti males were collected 459 from the same cage as the females, killed by freezing at -20°C for 15 minutes, and stored as five 460 pools of three individuals in RNAlater at -80°C. These same methods were used on the ORL A. 461 aegypti.

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462 463 Figure 10. Locations of ovitraps (green triangles) throughout Palmetto, Manatee County, FL. 464 Ovitraps covered approximately 1 square kilometer in a peri-urban (i.e., residential) 465 environment.

466 Prior to RNA extraction, ovary pools from G0 females were thawed on ice, and RNAlater was 467 removed. Pools were homogenized manually in 0.2 mL of TRIzol reagent (Invitrogen, Waltham, 468 MA) using a sterilized pestle. The volume of TRIzol was then brought up to 1.0 mL for each 469 pool, and RNA was extracted following the manufacturer’s instructions, followed by treatment 470 with TURBO DNase (Invitrogen, Waltham, MA). RNA samples were shipped on dry ice to 471 Novogene, where RNA metagenomic libraries were prepared using the NEBNext Ultra II 472 Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA). Libraries 473 were pair-end sequenced (2x150 bp) using the Illumina HiSeq 4000 platform (Figure 11A). RNA 474 from G7 males and from ovaries dissected from G7 females was extracted as described above and 475 used to investigate vertical transmission by reverse transcription PCR as described below (Figure 476 11B)

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477

478 Figure 11. Workflow diagram illustrating the overarching study design for the (A) metagenomic sequencing and (B) vertical . 479 transmission assays. In brief, an RNA metagenomic pipeline was employed to discover the insect specific viruses present in the The copyrightholderforthispreprint 480 ovaries of Aedes aegypti from Palmetto, in Manatee County, FL. Identified ISVs were confirmed via RT-PCR in the G0 and G7 481 generations. Figure created with BioRender.com.

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482 Bioinformatic Analyses of Read level Metagenomic Data

483 Sequence contaminants and adapters were removed using BBduk v. 38.86 484 (https://sourceforge.net/projects/bbmap/). Aedes aegypti reads were mapped to the A. aegypti 485 genome (version AaegL5) and removed using BBmap. Local similarity searches were performed 486 against the National Center for Biotechnology Information’s non-redundant protein database 487 (NCBI nr) (downloaded 01/21) (NCBI Resource Coordinators 2018) using DIAMOND v 2.0.7 488 (Buchfink et al. 2015). MEGAN6 v 6.19.2 (Huson et al. 2007), was used to assign reads to each 489 lowest common ancestor (LCA) using the default naive LCA settings (in read count mode), 490 mapping against NCBI nr (megan-map-Jan2021.db.zip from the MEGAN6 website 491 (https://software-ab.informatik.uni-tuebingen.de/download/megan6)).

492 Viral Genome Assembly

493 All A. aegypti unmapped read files were merged, and contigs were assembled de novo using 494 SPAdes v 3.14.1 (Bankevich et al. 2012) in metagenomics mode using merged and unmerged 495 reads. Reads found to align to Atrato partiti-like virus 3 were separately parsed into contigs using 496 the same pipeline (sub-divided due to low similarity with known database matches). Local 497 similarity searches were performed against NCBI nr (NCBI Resource Coordinators, 2018) using 498 DIAMOND v 2.0.7 (Buchfink et al. 2015). MEGAN6 v 6.19.2 (Huson et al. 2007), was used to 499 assign each LCA (using the long-read import option), and to visualize and extract viral contigs. 500 NCBI’s open reading frame (ORF) finder (https://www.ncbi.nlm.nih.gov/orffinder/) was used to 501 identify ORFs in each assembled viral contig. ORFs >300 nucleotides in length were searched 502 using BLAST (Zhang et al. 2000) against the nr protein sequence database through NCBI.

503 Reverse Transcription PCR

504 For a subset of ISVs identified through metagenomics, mapped viral reads from each sample 505 were aligned to create consensus sequences using the Velvet Optimiser (v2.2.6) (Zerbino et al. 506 2008) to enable primer design for reverse transcription PCR to confirm viral RNA in the original 507 pools and screen tissues from subsequent generations. Consensus sequences were then searched 508 against the NCBI nr database (NCBI Resource Coordinators 2018) using Blastx to confirm the 509 virus and protein match. Primers were designed using Primer3 with default parameters to yield 510 amplicons ranging from 150 – 500 bp (Table S1).

511 For reverse transcription PCR, we performed a two-step procedure: cDNA first-strand synthesis 512 using an RNA template followed by traditional PCR. For cDNA synthesis, 5 µg of total RNA 513 from each ovary pool (field- and colony-derived) was used to generate cDNA with the RevertAid 514 First Strand cDNA Synthesis kit (Thermo Scientific, Waltham, MA) following the 515 manufacturer’s instructions for random hexamers. PCR amplifications were performed on each 516 pool with all primer sets in 25 µl reactions, each containing dNTPs at a concentration of 0.2 mM, 517 primers at a concentration of 0.2 µM, 1.0 units of Taq DNA polymerase (DreamTaq, Thermo 518 Scientific, Waltham, MA), 1x Taq polymerase buffer, and 1 µl of cDNA from the first-strand 519 synthesis reaction diluted 1:1 with ultrapure water. All PCR amplifications were performed with 520 an initial melt step of 95°C for 5 minutes followed by 35 cycles of 95°C for 30 seconds, 60°C for 521 30 seconds, and 72°C for 30 seconds, followed by a final extension step of 72°C for 10 minutes.

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522 Amplicons were electrophoresed on 2% agarose gels, stained with GelRed nucleic acid stain 523 (Biotium, Fremont, CA), and visualized on a gel documentation system (Azure Biosystems 524 c200). For samples yielding a PCR amplicon, products were purified using the GeneJET PCR 525 Purification Kit (Thermo Scientific, Waltham, MA). Purified amplicons were ligated into 526 plasmids using the CloneJET PCR Cloning Kit (Thermo Scientific, Waltham, MA) and then 527 incubated with chemically competent E. coli (DH5α) (New England Biolabs, Ipswich, MA ) 528 following the manufacturer’s instructions. Transformed E. coli were grown overnight on 529 selective LB agar plates with carbenicillin (100 µg/mL) at 37°C. Colonies carrying the plasmid 530 and amplicon were transferred to liquid LB media with carbenicillin (100 µg/mL) and grown 531 overnight shaking at 220 RPM at 37°C. Cultures were harvested by centrifugation in 532 microcentrifuge tubes at 17,000 x g for 5 minutes, and plasmid DNA was extracted using the 533 GeneJET plasmid miniprep kit (Thermo Scientific, Waltham, MA). Plasmid DNA was sent to 534 Eurofins Genomics for sequencing using a plasmid-specific primer included with the CloneJET 535 kit.

536 Viral Gene Phylogenetics

537 Alignments were created from resultant viral RdRp and capsid sequence BLAST results using 538 MUSCLE (Edgar, 2004) in MEGA X 10.0.3 (Kumar et al. 2018) (Supplementary Files 1-7). 539 PhylML v 3.0 (Guindon et al. 2010) and Smart Model Selection (SMS) (Lefort et al. 2017) were 540 used to create an optimized maximum likelihood (ML) phylogenetic tree for each alignment. 541 Branch supports were computed by an approximate likelihood ratio test (aLRT) with SH-like 542 support as implemented in PhyML. Trees were annotated and colored in FigTree v1.4.4 543 (https://github.com/rambaut/figtree/releases).

544 Data Availability

545 Cleaned and trimmed sequence files have been deposited in NCBI as a BioProject and will be 546 made publicly available upon publication.

547 Acknowledgments

548 We would like to thank X. Wang and T. Stenn for assistance with mosquito feeding and colony 549 maintenance and J. Crosby for animal husbandry. A fellowship for J. Bozic and an internship for 550 J. Carrillo were supported by funds through the CDC Southeastern Center of Excellence in 551 Vector-Borne Diseases (U01CK000510).

552 Competing Interests

553 The authors declare no competing (financial or non-financial) interests.

554 References

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