Is it time to consider west Nile and Usutu viruses endemic in central Italy?

West Nile (WNV) and Usutu (USUV) viruses co-circulated in a region of Central Italy (Lazio) in 2018, as evidenced by the detection of WNV in the nervous tissues of symptomatic horses and USUV in blood donors and mosquito pools. To assess whether these viruses were endemic in the region, we analysed: 1) diapausing Culex pipiens mosquitoes collected during the winter seasons 2022-2023 and 2023-2024, 2) Cx. pipiens mosquitoes collected during the adult activity period from April to November in 2022 and 2023 across 4 provinces, and 3) sera from 52 horses and tissues from 537 birds. Field-collected Cx. pipiens, including both diapausing and non-diapausing individuals, were tested in pools for WNV and USUV using real-time RT-PCR. Serum samples from horses were tested with two WNV ELISA assays, IgM and IgG, while bird tissues were tested for both viruses via real-time RT-PCR. A total of 18,834 Cx. pipiens females were collected, including 9,812 mosquitoes during the winter seasons and 9,022 during the adult activity periods. Mosquitoes were tested in 623 pools, with all pools of diapausing mosquitoes testing negative for both viruses and 12 pools of non-diapausing mosquitoes positive to USUV. The WNV IgG positivity of 7 horse sera, which were negative at the beginning of the study period, was not confirmed by the virus neutralization test. All tissue samples were negative for WNV and USUV. Since WNV and USUV were not detected in diapausing mosquitoes, there was no evidence of the two viruses endemicity in the study area.