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。埃及伊蚊登革热病毒的主要蚊媒，在美国佛罗里达州已广泛传播。自 2009 年以来，佛罗里达州向人类本土传播的登革热病毒一直在增加，同时不断输入登革热病例。然而，大多数首次感染登革热的病例是无症状的，并且该病毒可以在蚊子种群中维持，使监测复杂化并导致疾病风险被低估。宏基因组测序A. aegypti佛罗里达州海牛县的蚊子在没有人类 DENV-4 指示病例的情况下，即使本地获得的 DENV 病例，在 2 年内多个诱捕地点的蚊子中都存在登革热病毒血清型 4 (DENV-4) 基因组佛罗里达州从未报告过-4。这一发现表明： (i) DENV-4 可能在未被发现的人群中传播；(ii) 病毒一直存在于蚊子种群中，或 (iii) 检测到的完整基因组序列可能不代表活病毒。本研究表明，从 Manatee County DENV-4 (DENV-4M) 序列产生的感染性克隆能够感染哺乳动物和昆虫组织培养系统以及成年雌性A. aegypti用血粉喂食蚊子。然而，该病毒在蚊子中受到剂量依赖性感染屏障的影响，与来自有症状儿童的系统发育相关的野生型 (WT) 对照病毒 DENV-4H (菌株智人/Haiti-0075/2015，GenBank 加入 MK514144.1)。DENV-4M 在感染后 14 天从中肠传播到卵巢和唾液。在 DENV-4M 感染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，可以通过叮咬传播，并且可以垂直传播，这表明该病毒具有在没有人-蚊子传播的情况下在环境中维持的机制。这些发现表明佛罗里达州正在发生未发现的人-蚊子传播和/或病毒在蚊子种群中的长期维持，并强调了对蚊子中病毒进行主动监测的重要性。

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