A reporter virus particle seroneutralization assay for tick-borne encephalitis virus overcomes ELISA limitations

Background Tick-borne encephalitis (TBE) virus is the most common tick-transmitted Orthoflavivirus in Europe. Due to its non-specific symptoms, TBE is primarily diagnosed by ELISA-based detection of specific antibodies in the patient serum. However, cross-reactivity between orthoflaviviruses complicates the diagnosis. Specificity problems may be overcome by serum neutralization assays (SNT), however clinically relevant orthoflaviviruses require handling in biosafety level 3 (BSL-3) and they have highly divergent viral kinetics and cell tropisms. Methods We present a reporter viral particle (RVP) based SNT in which the infectivity is measured by luminescence and that can be performed under BSL-2 conditions. Findings The RVP-based SNT for TBEV exhibited a remarkable correlation with the traditional virus-based SNT (R2=0.8614, p<0.0001). Notably, the RVP-based assay demonstrated a sensitivity of 91.7% (95% CI: 87.2-97.1%) and specificity of 100% (95% CI: 79.6-100%). We also tested the cross-reactivity of serum samples in RVP-based assays against other orthoflaviviruses (yellow fever virus, dengue virus type 2, Zika virus, West Nile virus and Japanese encephalitis virus). Interestingly, in 90% of cases where a serum sample had tested TBEV-positive by ELISA but negative by RVP-based SNT, we identified antibodies against other orthoflaviviruses. Interpretations The RVP-based seroneutralization assay show clinical relevance and broad-applicability. Funding This study was supported by Bavarian Nordic grant to R.A. and V.C. ### Competing Interest Statement The project was financed by a research grant from Bavarian Nordic, but the authors were completely independent from the funding source regarding the study plan, analysis, writing and submission. ### Funding Statement The study was funded by Bavarian Nordic. The salary of V.C is supported by the Swiss National Science Foundation [PZ00P3_193,289] ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee of CER-VD gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors