Sindbis virus infects specific gut cells for replication and dissemination from the posterior midgut of mosquitoes

Abstract Sindbis virus (SINV), a member of the genus Alphavirus, is the protype virus used to gain insight into other disease-causing Alphaviruses. As a mosquito-borne-virus (arbovirus), SINV transit in adult female mosquitoes includes attachment to the gut lumen and entry into the midgut cells, followed by replication and dissemination into the hemolymph through yet unknown specific mechanisms. Free-mated adult females, aged day 5-7, were fed a viremic bovine blood suspension via blood sausage at a final SINV titer at 107 PFU/ml. Midguts from fully engorged mosquitoes were dissected on day 5 and 7 post-bloodmeal and further examined by immunolabeling using FMRFamide antibody against enteroendocrine cells (EC). The results were investigated via confocal microscopy and distribution of SINV and ECs were documented. Oral infection of mosquitoes with SINV-TaV-eGFP led to GFP expression along the basal aspect of the posterior midgut (PMG) epithelial monolayer as early as day 5 p.i., persistent infection and dissemination of the virus was observed on day 30 p.i. following viremic blood feeding. ECs were observed along the entire length of the midgut with majority of ECs concentrated in the posterior midgut region. Additionally, our results demonstrated that SINV could indeed infect ECs and the accumulations of SINV associated GFP fluorescence coincided with these cells. Here we propose that ECs, positioned along the basal plasma membrane of the PMG, are involved in SINV dissemination pathway. Due to unique roles that ECs have in the exocytosis of secretory granules from the MG, these cells might serve as a gateway for virus entry into the host hemolymph. These findings suggest that midgut ECs are integral to arbovirus infection, dissemination, and availability for transmission

The alphavirus sindbis infects enteroendocrine cells in the midgut of Aedes aegypti

Transit of the arthropod-borne-virus (arbovirus) Sindbis (SINV) throughout adult female mosquitoes initiates with its attachment to the gut lumen, entry and amplification in midgut cells, followed by dissemination into the hemolymph. Free-mated adult females, aged day 5-7, were proffered a viremic blood suspension via sausage casings containing SINV-TaV-Green Fluorescent Protein (GFP) at a final titer of 106 PFU/mL. Midguts (MGs) from fully engorged mosquitoes were resected on days 5 and 7 post-bloodmeal, and immunolabeled using FMRFamide antibody against enteroendocrine cells (ECs) with a TX-Red secondary antibody. Following immunolabeling, the organs were investigated via laser confocal microscopy to identify the distribution of GFP and TX-Red. Infection using this reporter virus was observed as multiple GFP expression foci along the posterior midgut (PMG) epithelium and ECs were observed as TX-Red labeled cells scattered along the entire length of the MG. Our results demonstrated that SINVGFP did infect ECs, as indicated by the overlapping GFP and TX-Red channels shown as yellow in merged images. We propose that ECs may be involved in the SINV infection pathway in the mosquito MG. Due to the unique role that ECs have in the exocytosis of secretory granules from the MG and the apical-basolateral position of ECs in the PMG monolayer, we speculate that these cells may assist as a mechanism for arboviruses to cross the gut barriers. These findings suggest that MG ECs are involved in arbovirus infection of the invertebrate host