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 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 [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.