Evaluation of Detection Methods Using Sugar Dipped FTA-Filters for Collection of Mosquito Borne Viruses

Arboviruses, transmitted to humans and animals through the bites of infected mosquitoes, are a major public health concern worldwide, as they can cause a wide range of diseases. Honey-baited filter cards (also called FTA cards) in mosquito traps have been used for sampling of arboviruses, but the sensitivity of the method is still not clear. The aims of this project was to develop a sensitive and cost-effective method for ribonucleic acid (RNA) isolation from FTA cards to detect low amounts of viral RNA. To identify viruses, metagenomic sequencing, reverse transcription quantitative real-time (RT-qPCR), and family primers with Sanger sequencing were tested as alternative strategies. Honey-dipped FTA cards inoculated with Sindbis virus were used to evaluate two RNA extraction methods and analysed with RT-qPCR. To evaluate RNA stability and for metagenomic sequencing, inoculated Sindbis and Usutu virus on honey-dipped FTA cards were extracted and analysed with RT-qPCR. Sindbis, Usutu, Inkoo, and O’nyong’nyong viruses were used for the evaluation of family primers with reverse transcription PCR and Sanger sequencing. Both extraction methods could extract low levels of RNA, and Sindbis and Usutu viruses were observed to be stable for 2 weeks. Only the Usutu virus could be identified with the correct genus after metagenomic sequencing. When using family primers, the viruses analysed could be identified after sequencing. Our results indicate that FTA cards can be used to detect Sindbis and Usutu virus without losing RNA if stored at room temperature and can thus be implemented when a cold chain isn’t practicable. For virus detection, several methods can be used depending on the scientific question. This study contributes to the development of a sensitive method to detect mosquito-borne viruses and, thereby, observing and improving public health.