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Dengue virus is the most prevalent mosquito-borne virus, causing approximately 390 million infections and 25,000 deaths per year.  Aedes aegypti , the primary mosquito vector of dengue virus, is well-established throughout the state of Florida, United States. Autochthonous transmission of dengue virus to humans in Florida has been increasing since 2009, alongside consistent importation of dengue cases. However, most cases of first infection with dengue are asymptomatic and the virus can be maintained in mosquito populations, complicating surveillance and leading to an underestimation of disease risk. Metagenomic sequencing of  A. aegypti  mosquitoes in Manatee County, Florida revealed the presence of dengue virus serotype 4 (DENV-4) genomes in mosquitoes from multiple trapping sites over 2years, in the absence of a human DENV-4 index case, and even though a locally acquired case of DENV-4 has never been reported in Florida. This finding suggested that: (i) DENV-4 may circulate among humans undetected; (ii) the virus was being maintained in the mosquito population, or (iii) the detected complete genome sequence may not represent a viable virus. This study demonstrates that an infectious clone generated from the Manatee County DENV-4 (DENV-4M) sequence is capable of infecting mammalian and insect tissue culture systems, as well as adult female  A. aegypti  mosquitoes when fed in a blood meal. However, the virus is subject to a dose dependent infection barrier in mosquitoes, and has a kinetic delay compared to a phylogenetically related wild-type (WT) control virus from a symptomatic child, DENV-4H (strain  Homo sapiens /Haiti-0075/2015, GenBank accession MK514144.1). DENV-4M disseminates from the midgut to the ovary and saliva at 14days post-infection. Viral RNA was also detectable in the adult female offspring of DENV-4M infected mosquitoes. These results demonstrate that the virus is capable of infecting vector mosquitoes, is transmissible by bite, and is vertically transmitted, indicating a mechanism for maintenance in the environment without human-mosquito transmission. These findings suggest undetected human-mosquito transmission and/or long-term maintenance of the virus in the mosquito population is occurring in Florida, and underscore the importance of proactive surveillance for viruses in mosquitoes.    GRAPHICAL ABSTRACT   In order to better assess the public health risk posed by a detection of DENV-4 RNA in Manatee County, FL  Aedes aegypti , we produced an infectious clone using the sequence from the wild-caught mosquitoes and characterized it  via  laboratory infections of mosquitoes and mosquito tissues.

Infection Kinetics and Transmissibility of a Reanimated Dengue Virus Serotype 4 Identified Originally in Wild Aedes aegypti From Florida