Molecular determinants of arbovirus transmission by mosquitoes

Every year over 400 million people become infected with arthropod-borne (arbo)viruses such as Zika virus (ZIKV), West Nile virus (WNV), and chikungunya virus (CHIKV) via mosquito bites. ZIKV and CHIKV are predominantly transmitted by Aedes aegypti mosquitoes, while Culex pipiens is the primary vector mosquito for WNV. To complete their life cycle, arboviruses have to infect and replicate in both their vertebrate host and mosquito vector. A successful virus replication cycle requires the interaction with and recruitment of host factors and the evasion of antiviral responses. While research on arbovirus-mosquito interactions has established the significance of mosquito immune responses and physical barriers for arbovirus transmission, the molecular details of interactions between arboviruses and mosquitoes are still largely a ‘black box’. This thesis investigated the molecular mechanisms and virus-mosquito interactions that play an important role in the transmission of arboviruses by mosquitoes. First, the possibility of mosquitoes to simultaneously transmit multiple arboviruses was investigated during a co-infection of Ae. aegypti mosquitoes with ZIKV and CHIKV. The results demonstrate that Ae. aegypti can simultaneously transmit both viruses via a single bite, without significant interference. Second, the mosquito antiviral RNA-interference response, which is mediated by small RNAs, was investigated during WNV infection in both Culex and Aedes mosquito cell lines. Via de novo assembly of small RNAs, the presence of insect-specific viruses (ISVs) was uncovered in these cells. Both Aedes and Culex cells produced two types of small RNA responses to WNV and ISV infection: small-interfering RNAs and PIWI-interacting RNAs. The formation of these PIWI-interacting RNAs was strongly dependent on the virus family and cell origin, indicating that small RNA responses are less generic than was previously anticipated. Next, two viral molecules were investigated for their role in arbovirus transmission by mosquitoes: the non-structural protein 3 (nsP3) of CHIKV and the non-coding subgenomic flavivirus RNA (sfRNA) of ZIKV and WNV. The interaction of CHIKV nsP3 with the mammalian protein G3BP and its mosquito ortholog Rasputin is shown to play an important role in CHIKV transmission by Ae. aegypti mosquitoes to vertebrate hosts. SfRNA was demonstrated as a determinant of ZIKV transmission by Ae. aegypti and WNV transmission by Cx. pipiens. SfRNA mediates ZIKV and WNV in overcoming the mosquito midgut-barrier, an essential step in the arbovirus transmission cycle, and enhances virus accumulation in the mosquito saliva. Small RNA deep sequencing demonstrated that WNV sfRNA is processed by the antiviral small RNA machinery of Culex mosquitoes, however, sfRNA did not affect the magnitude of the small RNA response. Via RNA-affinity purification mosquito proteins were identified that interact with ZIKV and WNV sfRNA, including the DEAD-box helicase ME31B. Silencing of ME31B resulted in increased ZIKV and WNV virus replication in mosquito cells, implicating an antiviral function of ME31B. The uncovered interactions of sfRNA with specific mosquito proteins and antiviral pathways increase our fundamental understanding of how this non-coding RNA mediates virus transmission. In conclusion, the research described in this thesis significantly contributes to our understanding of arbovirus transmission by expanding the knowledge of arbovirus-vector interactions. Understanding arbovirus transmission at the molecular level may ultimately lead to the development of novel strategies to prevent the further spread of arboviral disease.