Investigation of Saliva Proteins from Different Culicoides Species and their Interaction with the Bluetongue Virus Particle

Abstract

The transmission, infectivity and virulence of many arthropod transmitted pathogens can be influenced by components of saliva from their arthropod vector. Culicoides saliva is therefore considered likely to play an important role in orbivirus trans­mission mechanisms. Difficulties in collecting saliva from such small insects have until recently limited our ability to analyse and identify Culicoides saliva proteins, or determine their func­tions and effects on the transmission efficiency, infectivity or virulence of these viruses. However, we have recently devel­oped an efficient method to collect relatively large quantities of uncontaminated Culicoides saliva proteins, using protein bind­ing filters. Saliva proteins were collected from C. nubeculosus [an inefficient bluetongue virus (BTV) vector from Europe] and C. sonorensis (a North American vector of BTV and epizootic haemorrhagic disease virus) for direct protein sequencing by electrophoresis and mass spectrometry. These analyses have identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Previous studies have shown that the larger of the BTV sero­type 1 (BTV-1) outer-capsid components (the cell attachment protein ‘VP2’) can be cleaved by proteases, forming infectious subviral particles (ISVP) that have an enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sono­rensis). We show that treatment with saliva from adult Culicoides also cleaved VP2 from several different BTV strains. This cleavage appears to be a step-wise process, with intermediate cleavage products being generated at lower temperature or lower saliva protein concentrations. Incubating purified BTV-1 particles with C. sonorensis saliva also increased their infectivity for KC cells about 10 fold, while infectivity for baby hamster kidney (BHK) cells was reduced by 2-4 fold. The saliva proteins from C. sonorensis cleaved BTV-VP2 more efficiently than those from C. nubeculosus, suggesting that the level of protease activity in the insect saliva could help determine the efficiency of BTV infection in the insect. The saliva protein collection method is now being applied to wild caught C. imicola (the South African BTV vector) to investigate whether the composition of the saliva protein is involved in the determination of vector competence.