The Tick&Mosq-Enceph RT-qPCR Multiplex comprises a series of specific targeted reagents designed for Usutu virus, Louping ill virus, Tick-borne encephalitis virus (related) and West Nile virus detection by using multiplexed qPCR. Usutu virus, Louping ill virus, Tick-borne encephalitis virus, and West Nile virus are enveloped viruses with a single-stranded RNA genomes, that belong to the family Flaviviridae, genus Orthoflavivirus. West Nile virus and Usutu virus primarily infect birds, which act as the main reservoir hosts, but can also infect humans and other mammals. Louping ill virus primarily infects sheep and other livestock, as well as wild animals, and can occasionally infect humans. Tick-borne encephalitis virus primarily infects small mammals such as rodents, which act as reservoir hosts, and can also infect humans and other mammals. In humans, these viruses cause diseases that range from asymptomatic or mild febrile illnesses to severe neurological disorders, such as encephalitis and meningoencephalitis. Transmission occurs through arthropod vectors: mosquitoes (particularly species of the Culex genus) are responsible for spreading Usutu virus and West Nile virus, while ticks (especially Ixodes species) transmit Louping ill virus and Tick-borne encephalitis virus. These viruses are widely distributed across Africa, Europe and Asia. In addition to Tick-borne encephalitis virus, the TBEV-rel qPCR assay detects Louping ill virus and Omsk hemorrhagic fever virus. These viruses share a high degree of sequence homology and are therefore considered by some authors to be the same species. Louping ill virus exhibits high genetic variability and presents taxonomic difficulties.