Vector surveillance programs require rapid and accurate methods to screen large quantities of trapped insect samples for arbovirus. This study aims to develop a new method to screen large pools of mosquitoes for arboviruses, while also determining their species composition. DNA barcoding is a molecular approach to vector species identification that when combined with next-generation sequencing, has the potential to process large pools of vector samples simultaneously.
Initially, pools of 100 mosquitoes will be used for DNA and RNA extraction, consisting of 94 x Aedes camptorhynchus, 5 x Anopheles annulipes and 1 x Ross River virus (RRV)-infected Aedes notoscriptus. The E2 gene will be used to identify RRV, while Cytochrome Oxidase I (COI) will be used for mosquito species determination. These amplicons will be used for library preparation and then sequenced on an Illumina MiSeq. Reads will be blasted (BLASTN) against a virus sequence library to check for the presence of RRV, and then against the ‘Mosquitoes of Australia- Victoria – Australia’ dataset on the Barcode of Life Database (BOLD) to determine species identities and allowing comparison with both domestic and exotic species that have been previously barcoded DNA barcode. A DNA barcoding project, contributing to the international “Mosquitoes of the World” program on Barcode of Life Database (BOLD), has been established by our laboratory for endemic mosquito species of temperate Australia, and included 2736 species detected in Victoria.
In the future, this method will be further developed by increasing the targets screened to a wider variety of arboviruses, and larger pool sizes to determine the level of sensitivity for both arbovirus and species detection. Once established, this approach will significantly improve diagnostic efficiency in surveillance programs by decreasing the cost, time and specialist skill requirements associated with current identification and screening techniques.