Beyond the Bite

Dengue has been a thorn in the side of global health efforts for decades now, causing an estimated 390 million infections each year. Spread by the Aedes aegypti mosquito, dengue fever can be caused by any of the four dengue virus (DENV) serotypes – all of which are transmitted in the same way: through the bite of an infected mosquito.

Julien Pompon, a scientist at the Research Institute for Development, France, and an international team of researchers have been investigating the role of subgenomic flaviviral RNA (sfRNA) in DENV transmission via mosquito saliva (1). sfRNA – a viral RNA fragment that corresponds to a non-coding sequence of the RNA genome of flaviviruses – is a key player in the complex interaction between viruses and their host cells, and scientists are still trying to fully understand its role in viral infections.

“We had previously studied the function of sfRNA in mosquitoes and observed that sfRNA inhibits the immune response in mosquitoes to increase viral multiplication and subsequent transmission (2),” says Pompon. “We also found that sfRNA was highly concentrated in salivary glands, which produce infectious saliva.”

This discovery led the team to go one step further. “Because sfRNA was also identified as a strong immune inhibitor in mammals, we hypothesized that sfRNA is secreted in saliva to provide an advantage to the virus in infecting skin cells,” he says. “Mechanistically, we discovered that sfRNA is secreted in extracellular vesicles in mosquito saliva. The secretion in extracellular vesicles protects sfRNA from RNAse and most probably enables the transfer of sfRNA into skin cells at the bite site. The immune inhibitory capacity of sfRNA then reduces the immune response in skin cells, thereby facilitating simultaneous infection by viruses also present in saliva.”

They also found that saliva with higher levels of sfRNA was associated with enhanced virus infectivity. But the research wasn’t without its challenges. “Salivary sfRNA could be secreted through different means: i) combined with RNA-binding proteins (RBP), ii) within the virions, or iii) in extracellular vesicles. We tested the hypotheses by indirect chemical modification of extracellular vesicles and RBP; however, we had trouble finding a chemical treatment that selectively degraded extracellular vesicles but not virions, because they both are made of a lipid membrane,” says Pompon. “We serendipitously observed that dimethyl sulfoxide, which was initially used as a control for the vehicle for proteinase K, only degraded extracellular vesicles and not virions. Based on this finding, we were able to support the hypothesis that sfRNA is secreted in extracellular vesicles and not virions.”

Notably, as a collaborative effort between multiple institutions, Pompon says they were able to reproduce the findings “in three different teams in three continents,” which boosts the reliability of their findings. 

Where does this work fit in the bigger research picture? Pompon says there is a lack of understanding of the salivary factors that increase skin infection to promote transmission, so the findings really do bring something new to the table: “We discovered a novel salivary component that enhances skin infection and therefore transmission. In doing so, we have improved our understanding of flaviviral transmission and the complex triangular interactions between the mosquito, virus, and humans that determine transmission success.”

Next up, the team aims to characterize the salivary extracellular vesicles that carry sfRNA and also plans to determine whether this transmission-enhancing mechanism is conserved across flaviviruses.