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.

登革热病毒是最流行的蚊媒病毒，每年造成约 3.9 亿人感染和 25,000 人死亡。埃及伊蚊是登革热病毒的主要蚊媒，在美国佛罗里达州广泛传播。自 2009 年以来，佛罗里达州登革热病毒向人类的本土传播不断增加，同时登革热病例持续输入。然而，大多数首次感染登革热的病例都是无症状的，并且病毒可以在蚊子种群中保留，从而使监测变得复杂并导致疾病风险的低估。对佛罗里达州海牛县埃及伊蚊的宏基因组测序显示，在没有人类 DENV-4 指示病例的情况下，两年多来来自多个诱捕地点的蚊子中存在登革热病毒血清型 4 (DENV-4) 基因组，尽管佛罗里达州从未报告过本地感染的 DENV-4 病例。这一发现表明：(i) DENV-4 可能在人类中传播而不被发现； (ii) 该病毒保留在蚊子种群中，或者 (iii) 检测到的完整基因组序列可能不代表可行的病毒。这项研究表明，从海牛县 DENV-4 (DENV-4M) 序列产生的传染性克隆能够感染哺乳动物和昆虫组织培养系统，以及在以血粉喂养的成年雌性埃及伊蚊中感染。然而，该病毒在蚊子中受到剂量依赖性感染屏障的影响，并且与来自有症状儿童的系统发育相关野生型 (WT) 对照病毒 DENV-4H（智人/海地-0075/ 2015，GenBank 登录号 MK514144.1)。 DENV-4M 在感染后 14 天从中肠传播到卵巢和唾液。 在感染 DENV-4M 的蚊子的成年雌性后代中也可检测到病毒 RNA。这些结果表明，该病毒能够感染媒介蚊子，可通过叮咬传播，并且可以垂直传播，表明该病毒存在一种在环境中维持的机制，而无需人与蚊子之间的传播。这些发现表明，佛罗里达州正在发生未检测到的人类蚊子传播和/或病毒在蚊子种群中长期维持，并强调了主动监测蚊子病毒的重要性。

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