One health investigation of emerging mosquito-borne alphaviruses and orthobunyaviruses.

Background: Mosquito-borne viruses are becoming a large threat globally. Autochthonous viruses (West Nile virus, Usutu virus, tick-borne encephalitis virus, Toscana virus) and Aedes-borne viruses at risk of introduction and transmission in Italy (i.e., dengue virus, Zika virus, and chikungunya virus) have established surveillance systems already in place, in the framework of “One Health” approach. Although other emerging vector‑borne viruses, such as Tahyna, Batai, and Sindbis, have been detected in vectors and animals and linked to sporadic cases of febrile illness or encephalitis in humans across some European countries, their circulation in Italy and their impact on human infection and disease remain largely unknown. Additionally, the recent and continued spread of invasive mosquito species into Europe may facilitate the establishment of competent populations of mosquitoes leading to increased transmission of these arboviruses. Therefore, there is a need to collect more data to understand the potential impact of these viruses on human health in order to prevent possible outbreaks. Aim of the study: To investigate the presence of the emerging mosquito-borne neurotropic viral pathogens, including TAHV, BATV and other orthobunyavirales and the alphavirus SINV, in the Veneto region, Italy, by testing Culex pipiens mosquitoes, collected for WNV/USUV surveillance from 2021 to 2024. Materials and methods: A multiplex real-time RT-PCR assay was designed and optimized for detection of TAHV, BATV and SINV in high-throughput screening. A pan-orthobunyavirus real-time RT-PCR assay was also developed for high-throughput screening. For high-throughput screening, nucleic acid samples purified from mosquito pools were pooled in groups of four samples into 96-well plates. Then, four 96-well plates were combined into one 384-well plate, using a manual pipetting system (Liquidator 96, Mettler Toledo). For real-time RT-PCR set-up in 384-well plates, nucleic acid pools in the 384-well plates were dispensed by an automatic system (Echo 525 Liquid Handler, Beckman Coulter) to a final reaction volume of 5 microliters to increase throughput and run on a QS5 real-time PCR instrument (ThermoFisher Scientific). SINV, TAHV, and BATV positive controls were generated by cloning amplicons into plasmids using the TOPO TA cloning system (Invitrogen Thermo Fisher). The samples with positive results from multiplex real-time RT-PCR on pools were tested individually by singleplex real-time RT-PCR in order to identify the positive samples and to confirm the obtained data. Metagenomics sequencing using a MinION Nanopore instrument (Oxford Nanopore Technologies) was carried out to validate the presence of these viruses in the samples. Results: The developed multiplex real-time RT-PCR in high-throughput 384-well format successfully detected RNA controls of TAHV, BATV and SINV with sensitivity and specificity similar to the singleplex assays run in 96-well format. Optimal reaction conditions were established, enabling simultaneous amplification without significant primer-dimer formation or competition between reactions. A total of n = 10375 mosquito pools were tested (n = 2511 in 2021, n = 2449 in 2022, n = 2546 in 2023, n = 2869 in 2024). Positive samples were found for all the viruses. The largest amount of positive results were obtained for TAHV (n = 95), followed by BATV (n = 13). Unfortunately, we were unable to confirm the positivity of the pools with SINV. Conclusions: This study was the first to demonstrate the widespread presence of the neurotropic viruses TAHV, BATV, and SINV in Culex mosquitoes in the Veneto Region. In addition, this study led to the development of a multiplex PCR assay for the detection of TAHV, BATV, and SINV. The assay is scalable, high-throughput, and adaptable for the inclusion of additional viral targets.