Two real-time TaqMan PCRs 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. Ticks carrying ALSV have been identified in various regions, including China, Russia, Finland, Switzerland, and Germany, mainly within Ixodes ricinus and Ixodes persulcatus species. Hypothesis/Gap Statement: However, the clinical relevance and the pathogenicity of ALSV in humans is still unknown. Aim: We aimed to validate and integrate a real-time polymerase chain reaction (PCR) assay on our in-house molecular diagnostic platform to accurately detect ALSV and then further investigate its clinical relevance and its potential role as agent of meningoencephalitis in humans. Methodology: We customized two ALSV-specific Taqman RT-PCR assays targeting the NS3 and NS5 genes to align with the genetic diversity of ALSV sequences and the technical features of TaqMan probes-related technologies. Positive controls included ALSV RNA from infected Swiss ticks and synthetic plasmids including the target region of the RT-PCR assays. Results: To determine the limit of detection of the ALSV PCR assay, we used serial dilutions of the synthetic plasmid. Amplification rates of 100%, 92%, and 20% were respectively achieved for 1000, 100, and 10 copies per reaction for the NS3 PCR and 100%, 100%, 20% for the NS5 PCR. Intra- and inter-run reproducibility, evaluated over five independent runs with plasmid dilutions representing 100 and 10 DNA copies per reaction, met diagnostic standards. The assay exhibited 100% specificity (71/71) when tested against (i) a panel of cerebrospinal fluid pathogens (n=41), including bacteria, viruses, and fungi, and (ii) CSF specimens from our biobank (n=30), collected during winter when local tick exposure is unlikely due to low temperatures. In contrast specific amplification was obtained in ALSV-infected tick samples. Conclusions: Two ALSV real-time PCRs were validated for rapid, sensitive, and specific detection of ALSV RNA. Integration of ALSV PCR into open molecular diagnostic platform such as our, allows for syndromic testing alongside other encephalitis-associated viruses, such as Tick-Borne encephalitis virus or West Nile virus. This PCR will facilitate high-throughput screening of ticks for surveillance purposes. Moreover, this assay could be integrated into other in-house molecular diagnostic platform, in order to accurately detect ALSV to further explore ALSV pathogenic role.