Visualisation of blue-tongue virus infection and replication in the midgut of Culicoides biting midges

Culicoides biting midges are biological vectors of several arboviruses of veterinary importance, including bluetongue virus (BTV), an orbivirus that causes bluetongue disease in domestic and wild ruminants leading to significant morbidity and mortality. Susceptibility to BTV infection in Culicoides is thought to be limited by several barriers. These may consist of a mesenteron infection barrier (MIB) that controls virus entry into the midgut epithelium and a mesenteron escape barrier (MEB), which viruses must overcome to disseminate to secondary tissues and infect the salivary apparatus prior to transmission to ruminant hosts. A limited number of studies have investigated the nature of these barriers and the dynamics of BTV infection in Culicoides over time. Here, we used immunofluorescence microscopy techniques to visualise BTV infection and replication within the midgut of Culicoides to characterise the factors that determine susceptibility to BTV infection.

Dissected midguts and salivary glands of Culicoides sonorensis orally infected with BTV serotype 4 were fixed in 4% paraformaldehyde prior to immunolabelling against DNA (DAPI), tubulin and BTV structural and non-structural proteins. Using 3D confocal imaging, BTV infection in the midgut of Culicoides was observed from 4 days post infection (dpi) and at 8dpi in the salivary apparatus. Immunofluorescence images and qPCR of the rest of the body were used to determine the location of potential barriers to BTV dissemination in the dissected individuals. Further, these techniques are used to assess the impact of blood feeding and rearing temperature on midgut permeability to virus dissemination as determinants of vector competence.