A reporter virus particle seroneutralization assay for tick-borne encephalitis virus overcomes ELISA limitations
Authors: Ackermann-Gäumann Rahel , Dentand Alexis , Lienhard Reto , Saeed Mohsan , MacDonald Margaret R. , Coste Alix T , Cagno Valeria 
Published: January 2024

ABSTRACTBackgroundTick-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.MethodsWe 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.FindingsThe 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.InterpretationsThe RVP-based seroneutralization assay show clinical relevance and broad- applicability.FundingThis study was supported by Bavarian Nordic grant to R.A. and V.C.RESEARCH IN CONTEXTEvidence before this studyELISA tests for orthoflavivirus serology are the method of choice in all diagnostic laboratories despite the cross-reactivity issues. Although seroneutralization testing (SNT) provides more reliable results, it requires BSL-3 conditions and approximately a week to obtain the results. However, developing tests with a broader applicability could overcome the problem of cross-reactivity of antibodies against flaviviruses could be overcome leading to a more accurate diagnosis and fewer non-useful results. Although alternative serological tests for other orthoflaviviruses have been investigated they have limitations, including lack of uniformity for different orthoflaviviruses, the need for a BSL-3 laboratory to perform them, and results taking 4-5 days. The reporter viral particle system (RVP) we used in this study has been reported for all orthoflaviviruses, except for YFV. However, its applicability has not been tested in comparison to traditional methods with clinical samples.Added value of this studyWe tested the RVP system uniformly for different orthoflaviviruses and evaluated the sensitivity and specificity of SNT based on RVP compared to virus-based and to ELISA. Additionally, we found that false positives in ELISA in our clinical samples are frequently related to YFV positive samples.Implications of all the available evidenceThis study demonstrates the reliability and broad applicability of implementing RVP-based SNT in a clinical setting. This test can overcome the issues of false positive results from ELISA tests. Additionally, our data suggest that it is important to consider YFV exposure or vaccination anamnesis in patient’s medical history. This is consistent with the phylogenetic similarity between YFV and TBEV if compared to other flaviviruses.