Novel quantitative and specific RT-qPCR assay for UK subtypes of European strain of Tick-borne encephalitis virus

Two closely related tick-borne Orthoflaviviruses have been detected in the UK; tick-borne encephalitis virus (TBEV) and louping ill virus (LIV). Diagnosis of tick-borne encephalitis is achieved by detection of IgM/IgG antibodies using ELISA, Immunofluorescence, or by RT-PCR. High sequence and structural similarities between these viruses means they are hard to distinguish due to high cross-reactivity. We have developed a novel RT-qPCR assay using recently identified subtypes of TBEV in the UK and a probe incorporating locked nucleic acids, strengthening hybridisation between the probe and target RNA/DNA, resulting in greater discrimination between thermodynamically similar sequences. Cell-cultured virus extracts and an in vitro transcript of the NS1 gene were used for assay validation. TBEV was specifically detected to a sensitivity of 13 copies per reaction at 95 % confidence with 94 % efficiency. The assay showed no cross-reactivity to 10 Flavivirus RNA extracts tested. Of 43 clinical tick bite samples tested, 23 were TBEV seropositive and all tested negative by the currently used qPCR assay, matching our assay results. A sensitive, highly specific RT-qPCR assay that distinguishes between UK subtypes of TBEV and LIV. This distinction is vital for both accurate clinical diagnosis and vector surveillance, especially in the UK where both viruses co-circulate and cross-reactivity remains a significant diagnostic challenge.