Detection of Phleboviruses in sandflies from Northern Italy: Insights into toscana, fermo and other arboviruses

Numerous arboviruses transmitted by hematophagous arthropods are actively monitored due to their widespread distribution and public health relevance. Here are reported the results of regional surveillance for Phleboviruses in sandflies captured in northern Italy, including Toscana virus (TOSV) and Fermo virus (FERV). The captures were conducted during 2022 and 2023 using CDC miniature light traps placed in the hilly regions of Emilia-Romagna. The female sandflies were grouped into pools of 50 insects and stored at -80°C until further analysis. RNA was extracted using the BioSprint® 96 One-For-All Vet kit (Qiagen) in the KingFisherTM workstation (Thermo ScientificTM) reverse transcribed using RNase(H-)M-MLV reverse transcriptase (Promega) along with random primers (Roche)and then tested for Toscana virus (TOSV) and Fermo virus (FERV) using specific PCR assays, in addition to a Pan-phlebovirus PCR. Amplicons were sequenced using the Sanger technique. A total of 98,886 sandflies were captured. The species identified include Phlebotomus perfiliewii (87.2%) and Phlebotomus perniciosus (12.2%). A total of 70,398 sandflies were analyzed, divided into 1,712 pools tested for the presence of TOSV, revealing 74 positive pools (4.32%), and for the presence of FERV, with 337 positive pools (19.68%). Three sites recorded the highest number of positive pools for TOSV, all located in the province of Forlì-Cesena. Several sandfly pools (36) also tested positive for both viruses, indicating co-presence of TOSV and FERV. Additionally, positivity for other Phleboviruses was detected, specifically 35 sequences related to the Ponticelli virus (21 from the province of Forlì-Cesena), 12 sequences from the Corfou virus (8 of which are from the province of Parma), and 2 sequences related to the Punique virus (from the province of Forlì-Cesena). The presence of significant captures in certain lowland sites, an environment commonly considered unfavorable for the presence of sandflies, has been showing increasing numbers in recent years. The detection of at least 7 different Phleboviruses is surprising and is likely partly related to the high mutation rate observed in these viruses (Daoudi et al., 2023), thus confirming the abundant presence and circulation of these viruses in the study area (Calzolari et al., 2018). Furthermore, the use of specific real-time PCR protocols for TOSV and FERV has effectively allowed for the detection of a higher number of positive pools than what was evidenced by Pan-phlebovirus PCR. The epidemiology and potential pathogenic capability of these viruses for humans and animals remain to be investigated, both for those already known to cause disease in humans and for those recently described with a less understood cycle. Daoudi et al., 2023 Viruses 15(2):422 Calzolari et al., 2018 Infect. Genet. Evol. 64, 131-134