Vector Competence of Florida Culicoides insignis (Diptera: Ceratopogonidae) for Epizootic Hemorrhagic Disease Virus Serotype-2

Abstract

Epizootic hemorrhagic disease virus (EHDV; family Reoviridae, genus Orbivirus) is an arthropod-borne virus of ungulates, primarily white-tailed deer in North America. Culicoides sonorensis, the only confirmed North American vector of EHDV, is rarely collected from Florida despite annual virus outbreaks. Culicoides insignis is an abundant species in Florida and is also a confirmed vector of the closely related Bluetongue virus. In this study, oral challenge of C. insignis was performed to determine vector competence for EHDV serotype-2. Field-collected female midges were provided bovine blood spiked with three different titers of EHDV-2 (5.05, 4.00, or 2.94 log₁₀PFUe/mL). After an incubation period of 10 days or after death, bodies and legs were collected. Saliva was collected daily from all females from 3 days post feeding until their death using honey card assays. All samples were tested for EHDV RNA using RT-qPCR. Our results suggest that C. insignis is a weakly competent vector of EHDV-2 that can support a transmissible infection when it ingests a high virus titer (29% of midges had virus positive saliva when infected at 5.05 log₁₀PFUe/mL), but not lower virus titers. Nevertheless, due to the high density of this species, particularly in peninsular Florida, it is likely that C. insignis plays a role in the transmission of EHDV-2.

Keywords: Orbivirus; Reoviridae; arboviruses.

Figure 1

Location of the two field sites in Florida from where Culicoides insignis midges were collected. Map generated using QGIS3 and shapefiles from the Florida Geographic Data Library database.

Figure 2

Infection, dissemination, and transmission rates for per os infection trials conducted with three infectious titers of EHDV-2. Fisher’s exact tests indicated significant differences in infection rates (p < 0.001). Pairwise significance is indicated above bars for infection rates. Infection rates were not significantly different between the 2.94 and 4.00 log₁₀PFUe/mL infections (A); however, infection rates were significantly higher when midges were fed an infectious titer of 5.05 log₁₀PFUe/mL (B) than when they were fed either of the lower titers tested. No significant difference was found for dissemination rates (p = 0.22) or transmission rates (p = 0.47), so pairwise significance was not tested. Numbers in parenthesis indicate the number of positive samples.

Figure 3

Viral titers of EHDV-2 in bodies, legs, and saliva of Culicoides insignis infected at three viral titers. Center lines of boxplots represent the median viral titer. Upper and lower extent of boxes represent the third and first quartile, respectively. Whiskers represent the estimated maximum and minimum values (1.5× the interquartile length). Points outside of whiskers represent outliers. Plots represented by a solid horizontal line represent one data point for that treatment. No significant difference in viral titer was detected between treatments for body titers (p = 0.30). Sample sizes for leg and saliva titers were low, excluding the possibility of meaningful statistical comparison.