Two real-time TaqMan RT-qPCRs assays for the detection of Alongshan Virus (ALSV), a new member of the tick-borne Flaviviridae family

Introduction: Alongshan virus (ALSV) is a tick-borne Flaviviridae. It has been detected in Ixodes ricinus and Ixodes persulcatus across China, Russia, Finland, Switzerland, and Germany. Hypothesis/Gap Statement: However, the clinical relevance and the pathogenicity of ALSV in humans remains unclear. Sensitive and specific molecular tools are needed to support surveillance and to enable clinical investigations of ALSV in suspected cases of tick‑borne meningoencephalitis. Aim: We aimed to develop, validate and integrate two ALSV‑specific real‑time TaqMan RT‑qPCR assays on our open, high‑throughput molecular diagnostic platform. Methodology: We designed assays targeting conserved regions of the NS3 (helicase-protease) and NS5 (RNA‑dependent RNA polymerase) genes, incorporating degenerate bases and Locked Nucleic Acid (LNA) modifications where needed to accommodate documented viral diversity and to harmonise the annealing temperature with TaqMan probes-related technologies and our platform. Analytical sensitivity and reproducibility were assessed using synthetic plasmids carrying the targets; specificity was evaluated against 41 cerebrospinal‑fluid (CSF) pathogens and 30 winter CSF specimens from patients with suspected central nervous system infection. ALSV‑positive Swiss tick extracts served as biological positives. Results: Detection frequencies for NS3 PCR were 100%, 100%, 92%, 72%, 20%, 28, and 0% at 1000, 100, 10, 5, 2, 1 and 0.1 copies per reaction respectively. For NS5, the detection frequencies were 100%, 100%, 92%, 88%, 40%, 20% and 0% at the same concentrations. Using a priori definition of limit of detection (LoD) as ≥95% positive replicates, LoD was 100 copies per reaction for both RT-qPCRs. However, as the PCR are performed in triplicate in our platform, the LoD can be estimated at 5 copies per reaction for the NS3 RT-qPCR and 2 copies per reaction for the NS5 PCR. Intra‑ and inter‑run reproducibility across five independent runs met diagnostic standards. Specificity was 100% (71/71). ALSV‑positive tick samples were detected by both assays, with lower Cts for NS5. Conclusions: We validated two ALSV RT-qPCR assays suitable for integration into open molecular diagnostic platforms. These assays enable syndromic testing alongside other encephalitis-associated viruses (e.g., Tick-borne encephalitis virus and West Nile virus) and will facilitate, timely clinical management of suspected cases, high-throughput ticks surveillance and future clinical studies of potential ALSV pathogenic role.