Paragraph: Word Count: 204 (Excludes title and key words) Text Word Count: 4,662 (Main text; excludes "Abstract" and "Importance") Disclaimers Not applicable The incidence of locally acquired dengue infections increased during the last decade in the United States, compelling a sustained research effort on the dengue mosquito vector, Aedes aegypti, and its microbiome, which has been shown to influence virus transmission success. We examined the 'metavirome' of four populations of Ae. aegypti mosquitoes collected in 2016-2017 from Manatee County, Florida. Unexpectedly, we discovered that dengue virus serotype 4 (DENV4) was circulating in these mosquito populations, representing the first documented case of such a phenomenon in the absence of a local DENV4 human case in this county over a two-year period. We confirmed that all of the mosquito populations carried the same DENV4 strain, assembled its full genome, validated infection orthogonally by reverse transcriptase PCR, traced the virus origin, estimated the time period of its introduction to the Caribbean region, as well as explored the viral genetic signatures and mosquito-specific virome associations that potentially mediated DENV4 persistence in mosquitoes. We discuss the significance of prolonged maintenance of these DENV4 infections in Ae. aegypti that occurred in the absence of a DENV4 human index case in Manatee County with respect to the inability of current surveillance paradigms to detect mosquito vector infections prior to a potential local outbreak. Since 1999, dengue outbreaks in the continental United States (U.S.) involving local transmission have occurred episodically and only in Florida and Texas. In Florida, these episodes appear to be coincident with increased introductions of dengue virus into the region through human travel and migration from endemic countries. To date, the U.S. public health author/funder. All rights reserved. No reuse allowed without permission. : bioRxiv preprint 5 response to dengue outbreaks is largely reactive, and implementation of comprehensive arbovirus surveillance in advance of predictable transmission seasons, which would enable proactive preventative efforts, remains unsupported. The significance of our finding is that it is the first documented report of non-outbreak DENV4 transmission and maintenance within a local mosquito vector population in the continental U.S.in the absence of a human case during a two-year time period. Our data suggest that molecular surveillance of mosquito populations in high-risk, high tourism areas of the U.S., may allow for proactive, targeted vector control before potential arbovirus outbreaks. : bioRxiv preprint 7 have been maintained vertically for at least one generation (but potentially more) in these Ae. 24 aegypti mosquito populations along Florida's Gulf Coast. We followed up this unexpected 25 finding with genetic analyses to determine the DENV4 strain's likely location of origin, assess 26 the timeframe of virus introduction, and investigate strain-specific mutations that may have 27 enabled adaptation to and/or persistence within local mosquito populations. 28 29 Methods 30 Mosquito sample preparation and viral RNASeq 31 Ae. aegypti eggs were collected in ovitraps in the summers of 2016-2017 (May 15, 2016, and 32 June 19, 2017) from four Manatee County sites (Fig. 1a) . To avoid cross contamination of 33 mosquito viromes, each year eggs from each site were hatched independently in distilled 34 water, reared to adulthood, speciated and then frozen. Female abdomens were pooled (N= 35 20/pool) separately for the four collection sites for a total of eight individual pools. Total RNA 36 was extracted using the AllPrep DNA/RNA Mini Kit (Qiagen), and rRNA depleted with the 37 NEBNext rRNA Depletion Kit (New England BioLabs). The NEBNext Ultra II Directional RNA 38 Library Prep Kit (New England BioLabs) was used to prepare shotgun metagenomics libraries. 39 Reverse-transcribed RNA libraries were sequenced using a HiSeq 3000 (Illumina) instrument 40 in 2x101 run mode. The data were deposited into the NCBI Sequence Read Archive and 41 Biosample archive under BioProject PRJNA547758. 42 43 Initial assembly and metavirome analysis 44 BBduk (version 37.75; https://sourceforge.net/projects/bbmap/) was used to trim adaptor 45 sequences and remove contaminants. Ae. aegypti sequences were removed using BBsplit 46 author/funder. All rights reserved. No reuse allowed without permission. : bioRxiv preprint 9 assembly with Spades in meta mode [8] and classification using Diamond [9] for a complete 71 DENV4 genome. Read-mapping with Bowtie [10] revealed incorrect bases near the 3' end, 72 which were manually corrected. The genome was annotated using the Genome Annotation 73 Transfer Utility [16] from the Virus Pathogen Database and Analysis Resource (ViPR) [17]. 74 75 Phylogenetic and Molecular Clock analyses 76 Two hundred thirty-four DENV4 genome sequences from GenBank (Table S1) were aligned 77 using MAFFT version 7.407 [18] with the L-INS-I method [19]. IQ-TREE software [20] was 78 used to evaluate phylogenetic signal in the genomes by likelihood mapping [21] and to infer 79 maximum likelihood (ML) phylogeny based on the best-fit model according to the Bayesian 80 Information Criterion (BIC) [20, 22]. Statistical robustness for internal branching order was 81 assessed by Ultrafast Bootstrap (BB) Approximation (2,000 replicates), and strong statistical 82 support was defined as BB>90% [23]. 83 84 To estimate when DENV4 entered Florida, we used 145 strains including all isolates from the 85 Americas, related Asian and African isolates, and randomly reduced oversampled Brazilian 86 isolates. The strains in this dataset were not recombinant, as assessed by scanning the 87 alignments for possible recombination points using the RDP, GENECONV, MaxChi, 88 CHIMAERA, and 3Seq algorithms implemented in the RDP4 software (available from 89 http://web.cbio.uct.ac.za/~darren/rdp.html) [24]. Correlation between root-to-tip genetic 90 divergence and date of sampling was conducted [25] to assess clock signal before Bayesian 91 phylodynamic analysis. Time-scaled trees were reconstructed using the Bayesian 92 phylodynamic inference framework in BEAST v.1.8.4 [26,27]. Markov Chain Monte Carlo 93 (MCMC) samplers were run until 200/250 million generations to ensure Markov chain mixing, 94 author/funder. All rights reserved. No reuse allowed without permission. 
 Title: Article type: Research Article Title: Under-the-radar dengue virus infections in natural populations of Aedes aegypti mosquitoes Running title: Dengue virus maintenance in mosquito vectors
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