Multidisciplinary investigation on "An improved genome for the Asian tiger mosquito Aedes albopictus and its applications in studying endogenous viral sequences"

Mosquito-borne diseases, including arboviral diseases such as Dengue, Chikungunya and Zika, have increased their world-wide incidence in the past 50 years and currently account for about 17% of all infectious diseases globally. Urbanization, globalization, increased international mobility and the widespread distribution of the main arboviral vectors, the mosquitoes Aedes aegypti and Aedes albopictus are all factors that have contributed to the (re)-emergence of arboviral diseases. Effective therapies and vaccines are limited for most arboviruses. Accordingly, vector control is the primary way to prevent transmission of arboviruses to humans. A deep understanding of the interaction and co-evolution between viruses and mosquito vectors is expected to aid in the development of novel transmission control strategies. The capacity of mosquitoes to support viral replication and transmission is called vector competence and is a dynamic and variable trait affected by many factors, suggesting an “arms race” between mosquitoes and viruses. The main arboviral vector in Europe is the invasive species Ae. albopictus, which has increasingly received attention from the scientific community due to its quick worldwide spread from south East Asia in the past 50 years. Nonretroviral RNA endogenous viral elements (nrEVEs) with similarities to the non-retroviral RNA viruses of the Flaviviridae and Rhabdoviridae family have been found with high frequency in Ae. albopictus mosquitoes. These viral integrations often interact with the most-recently characterized of the three RNA interference (RNAi) pathways: the PIWI-interacting RNA (piRNA) pathway. Most, but not all, nrEVEs in the genome of Ae. albopictus map next to transposable elements (TEs) fragments in piRNA clusters and produce piRNAs, small molecules that associate with Argonaute proteins of the PIWI clade to silence TEs based on sequence complementarity to piRNAs. In addition to its canonical role in preserving genome integrity, the piRNA pathway has antiviral activity in Aedes spp. mosquitoes. Despite the abundance of nrEVEs the biology, functional role, and patterns of integration in wild mosquito populations are still relatively unexplored. nrEVEs could be expressed and influence the phenotype of mosquitoes acting as a form of host antiviral immunity. A difficulty in studying the Ae. albopictus genome was the absence of a high-quality reference genome. During my PhD, I focused my attention on Ae. albopictus to primarily improve knowledge of its genome. I contributed to the sequencing, assembly, and annotation of a new reference genome for Ae. albopictus based on long-reads sequencing technologies; I coordinated an international consortium to annotate and characterize genomic features and their expression and produce a physical map of the genome. The availability of this new assembly allowed me to ask more specific questions on the landscape of viral integrations. A newly developed bioinformatic pipeline was combined with molecular biology techniques to identify viral sequences integrated into the genomes of wild-collected mosquitoes from different geographical locations. I also used the new genome assembly to reconstruct and identify RNA viruses in mosquito small-RNA sequencing data. I correlated the viruses identified in the mosquitoes with their population-specific nrEVE landscape, under the hypothesis that the pattern of nrEVEs is shaped by exposure to viruses. Lastly, I applied the CRISPR-Cas9 genome editing technology on Ae. albopictus embryos to modify a viral integration and a piRNA cluster and test the hypothesis that viral integrations have a role as immunity effectors against cognate viral infections. Overall, results gained through my PhD activities will enhance our understanding on the genome structure of Ae. albopictus and the importance of repetitive elements like viral integrations in the context of its biology.