Two TaqMan real-time quantitative PCR assays for the detection of Alongshan virus, a new member of the tick-borne Flaviviridae family

<b>Introduction.</b> Alongshan virus (ALSV) is a tick-borne <i>Flaviviridae</i>. It has been detected in <i>Ixodes</i> ricinus and <i>Ixodes persulcatus</i> across China, Russia, Finland, Switzerland and Germany. <b>Hypothesis/Gap Statement.</b> However, the clinical relevance and the pathogenicity of ALSV in humans remain 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. <b>Aim.</b> We aimed to develop, validate and integrate two ALSV-specific TaqMan real-time quantitative PCR (qPCR) assays on our open, high-throughput molecular diagnostic platform. <b>Methodology.</b> 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 modifications where needed to accommodate documented viral diversity and to harmonize the annealing temperature with TaqMan probe-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. <b>Results.</b> Detection frequencies for NS3 PCR were 100%, 100%, 92%, 72%, 20%, 28 and 0% at 1,000, 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 <i>a priori</i> definition of limit of detection (LoD) as ≥95% positive replicates, LoD was 100 copies per reaction for both real-time qPCRs. However, as the PCRs are performed in triplicate in our platform, the LoD can be estimated at five copies per reaction for the NS3 real-time qPCR and two 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 cycle thresholds for NS5. <b>Conclusions.</b> We validated two ALSV real-time 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 tick surveillance and future clinical studies of potential ALSV pathogenic role.