Transmission Potential of Floridian Aedes aegypti Mosquitoes for Dengue Virus Serotype 4: Implications for Estimating Local Dengue Risk

Abstract

DENV-4 was found circulating in Florida (FL) A. aegypti mosquitoes in the absence of human index cases in the state (2016 to 2017). How DENV-4 was maintained locally is unclear, presenting a major gap in our understanding of DENV-4 public health risk.

Geographic Partitioning of Dengue Virus Transmission Risk in Florida

“…Mammalian cell culture and virus propagation methods for this experimental design were conducted similarly to previously published methods [17]. Briefly, for the preparation of virus stocks, Vero E6 cells grown as monolayers were seeded onto the 75 cm 2 growing surfaces of filter-cap T75 cell culture flasks and incubated until they attained 80% confluency.…”

“…The cells were then individually inoculated with DENV-1 through DENV-4 laboratory stock viruses (DENV-1 L (VR-1856™), DENV-2 L (VR-1584™), DENV-3 L (VR-1256_FD™), DENV-4 L (VR-1490™)) obtained from the American Type Culture Collection (ATCC.org) as well as a low passage (second passage) DENV-1 (DENV-1 H) isolate from a child in Haiti (strain: Haiti/1207/2014: KT279761.2) and a low passage (second passage) DENV-4 isolate, also from a child in Haiti (strain: Haiti/0075/2015: MK514144.1) (Table A1). All virus stocks were collected between 7-and 9-days post-inoculation when approximately 50% of the cells showed virus-specific cytopathic effects (CPE) and were tittered, stabilized with 10% w/v molecular grade trehalose and cryopreserved as previously described [17]. All virus titers and infectious doses for these experiments are listed in Table A1.…”

“…We maintained the mosquitoes for 14 days on a 10% sucrose solution. On day 14, we cold anesthetized the mosquitoes, removed their legs and wings, and manually inserted their proboscis into a capillary tube (Drummond, Broomall, PA, USA) filled with 3 µL of 1:1 hematocrit: HIHS [17]. Each proboscis was withdrawn after 45 min or after 2 µL of hematocrit was imbibed.…”

“…A standard curve of each virus was run for each assay to determine the virus-specific regression analysis for estimating PFUe/mL. to provide a complete analysis and comparison between all virus groups and mosquito populations [17]. In all tables and figures, numbers/bars/scatter plots bearing at least one of the same alphabetical letters are statistically similar in their values, whereas numbers/bars/scatter plots that do not have an alphabetical letter in common are statistically different.…”

“…aegypti exhibit considerable variation in vector competence for DENV. This variation is influenced by virus serotype and strain, as well as mosquito genetics, mosquito metagenome, and environmental factors [12][13][14][15][16][17][18][19][20][21][22]. As such, the overwhelming number of estimates of Ae.…”