Culex tarsalis Is a Competent Host of the Insect-Specific Alphavirus Eilat Virus (EILV)

Abstract

Eilat virus (EILV) is an insect-specific alphavirus that has the potential to be developed into a tool to combat mosquito-borne pathogens. However, its mosquito host range and transmission routes are not well understood. Here, we fill this gap by investigating EILV's host competence and tissue tropism in five mosquito species: Aedes aegypti, Culex tarsalis, Anopheles gambiae, Anopheles stephensi, and Anopheles albimanus. Of the tested species, C. tarsalis was the most competent host for EILV. The virus was found in C. tarsalis ovaries, but no vertical or venereal transmission was observed. Culex tarsalis also transmitted EILV via saliva, suggesting the potential for horizontal transmission between an unknown vertebrate or invertebrate host. We found that reptile (turtle and snake) cell lines were not competent for EILV infection. We tested a potential invertebrate host (Manduca sexta caterpillars) but found they were not susceptible to EILV infection. Together, our results suggest that EILV could be developed as a tool to target pathogenic viruses that use Culex tarsalis as a vector. Our work sheds light on the infection and transmission dynamics of a poorly understood insect-specific virus and reveals it may infect a broader range of mosquito species than previously recognized. IMPORTANCE The recent discovery of insect-specific alphaviruses presents opportunities both to study the biology of virus host range and to develop them into tools against pathogenic arboviruses. Here, we characterize the host range and transmission of Eilat virus in five mosquito species. We find that Culex tarsalis-a vector of harmful human pathogens, including West Nile virus-is a competent host of Eilat virus. However, how this virus is transmitted between mosquitoes remains unclear. We find that Eilat virus infects the tissues necessary for both vertical and horizontal transmission-a crucial step in discerning how Eilat virus maintains itself in nature.

Keywords: Alphavirus; Culex tarsalis; Eilat virus; insect-specific virus.

FIG 1

Viral titers of body, leg, and saliva samples from five mosquito species orally challenged with infectious EILV-eGFP. (A to C) Titers are plotted at 7 and 14 dpi for (A) body, (B) leg, and (C) saliva samples collected from mosquitoes orally challenged with EILV-eGFP (10⁷ FFU/mL). Each point represents a single mosquito sample, while horizontal bars depict the group medians. Significance was evaluated using Mann-Whitney U tests. ****, P < 0.0001.

FIG 2

Viral titers of body, leg, and saliva samples from three C. tarsalis strains orally challenged with EILV-eGFP. (A to C) Titers are plotted at 7 and 14 dpi for (A) body, (B) leg, and (C) saliva samples from mosquitoes orally challenged with EILV-eGFP (10⁷ FFU/mL). Each point represents a single mosquito sample, while horizontal bars depict the group medians. Note that data for C. tarsalis (YOLO) are replotted from Fig. 1. Statistical significance was evaluated using Mann-Whitney U tests. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

Viral titers of body, leg, and saliva samples from five mosquito species intrathoracically injected with EILV-eGFP. (A and B) Titers are plotted at 7 and 14 dpi for (A) body and (B) saliva samples following IT injection with EILV-eGFP (10⁷ FFU/mL). Each point represents a single mosquito sample, and horizontal bars depict group medians. Statistical significance was evaluated using the Mann-Whitney U test. ****, P < 0.0001.

FIG 4

EILV tissue tropism at 7 and 14 dpi in orally challenged mosquitoes. Representative images show eGFP fluorescence (or its absence) in midguts (MG), salivary glands (SG), and ovaries (OV) of mosquitoes fed an infectious bloodmeal containing EILV-eGFP (10⁷ FFU/mL). The bright-field and FITC images have been merged. All scale bars equal 100 μm.

FIG 5

EILV tissue tropism at 7 and 14 dpi in IT-injected mosquitoes. Representative images show eGFP fluorescence (or its absence) in midguts (MG), salivary glands (SG), and ovaries (OV) of mosquitoes injected with EILV-eGFP (10⁷ FFU/mL). The bright-field and FITC images have been merged. All scale bars equal 100 μm.