Host Responses to Sandfly Salivary Factors Enhance the Severity of Toscana Virus Infection

Arbovirus infections have increasingly become a profound burden on public health. Inflammatory response to mosquito bites and the saliva deposited, aids the replication of arboviruses. In this thesis, we developed a new in vivo model that mimics natural transmission by sandfly vectors, to define whether saliva from sandflies enhances vertebrate host susceptibility to Toscana virus (TOSV), an important and growing cause of infectious neurological disease. We established new quantitative PCR assays, and plaque assays to define TOSV titre. New flow cytometry panels and genetically modified TOSV were utilised to interrogate the cellular tropism of TOSV in skin, and how this was modified by sandfly saliva/bite. Our results show a statistically significant increase in virus RNA in mouse tissues, such as skin, when TOSV is co-inoculated with sandfly salivary factors, compared to inoculation with TOSV alone. Higher quantity of virus RNA was also associated with an increase in the number of clinical signs, including atypical neurological signs and inflamed foot joints. These findings suggest that the enhancement effect of sandfly saliva is a general pro-viral mechanism, not limited to specific vector species. We found virus infection by sandfly saliva/bite was more inflammatory with increased cytokine expression. We identified dermal fibroblasts as crucial for TOSV replication and our flow cytometry analysis suggested that sandfly saliva/bites alter fibroblast biology, promoting a more primitive state. We suggest that while this is an aspect of normal wound healing, it simultaneously increases their capacity for TOSV replication. Salivary microbiota was dispensable for its ability to enhance TOSV infection, suggesting that the sandfly encoded salivary factors are instead responsible. A better understanding of how sandfly saliva increases host susceptibility to arbovirus infection will provide new insights into this crucial intersection of virus, vector and host. Such insights can aid development of an urgently needed vaccine.