Development and validation of a novel enzyme-linked immunosorbent assay for the differentiation of tick-borne encephalitis infections caused by different virus subtypes

Abstract

Objectives: Tick-borne encephalitis (TBE) is an infection caused by the tick-borne encephalitis virus (TBEV) that can lead to symptoms of central nervous system inflammation. There are five subtypes of TBEV, three of which - European, Siberian and Far Eastern - occur in Europe. As it is thought that different subtype infections exhibit varying clinical courses and outcomes, serological differentiation of the virus subtypes is clearly important. However, to date, this has proved difficult to achieve.

Methods: An ELISA format was developed based on TBE virus NS1 antigen against the European, Siberian and Far Eastern subtype. The three NS1 antigens were biotechnologically produced in a human cell line and used for ELISA coating. Sera from German (European subtype) and Russian (Siberian and/or Far Eastern subtypes) TBE patients with positive TBEV IgG were used to test the reactivity against these three NS1 antigens.

Results: Testing of 23 German and 32 Russian TBEV IgG-positive sera showed that the ELISA was able to differentiate between TBEV European subtype and TBEV Siberian and Far Eastern subtype infections.

Conclusions: In geographical areas where two or more TBEV subtype infections can occur, the NS1-IgG ELISA developed here constitutes an important diagnostic tool to differentiate between European subtype infections and Siberian/Far Eastern subtype infections and to use the new assay for epidemiological studies to clarify the importance of particular subtype infections in an area. Consequently, it may help to better describe and anticipate the clinical courses and outcomes of particular TBEV subtype infections.

Keywords: ELISA; Flavivirus; NS1 antibodies; NS1 antigen; TBEV subtypes.

Fig. 1

A: Mean ODs of TBEV-EU sera (group 1, n = 23) tested against TBEV-EU NS1, TBEV-Sib NS1, and TBEV-FE NS1 (*** highly significant; Mann–Whitney test, p < 0.0001). B: ODs of Russian sera (group 2, n = 32) tested against TBEV-EU NS1, TBEV-Sib NS1, and TBEV-FE NS1 (* significant; Mann–Whitney test, p < 0.05). The test has a dynamic cut-off, the absolute value of which can vary depending on the ELISA plate. The cut-off line shown in the figure is for orientation and represents an “average cut-off” between “borderline” and “positive”. The boxplot diagrams show the median (line in the box), upper and lower quartiles (upper and lower limits of the box) as well as the minimum and maximum (ends of the whiskers)

Fig. 2

A: Comparison of individual serum ODs of TBEV-EU sera (group 1, n = 23) against NS1 antigen of TBEV-EU, TBEV-Sib and TBEV-FE. B: Comparison of individual serum ODs of Russian sera (group 2, n = 32) against NS1 antigen of TBEV-EU, TBEV-Sib and TBEV-FE. Green lines: OD drop between TBEV-EU and Sib and between Sib and FE; Red lines: OD drop between EU and Sib, with increase between Sib and FE; Blue lines: Increase in OD between EU and Sib and drop between Sib and FE; Yellow lines: Increase in OD between EU and Sib and increase between Sib and FE.

Fig. 3

Mean ODs of sera of all groups tested against NS1 antigen of TBEV-EU, TBEV-Sib, and TBEV-FE. The boxplot diagrams show the median (line in the box), upper and lower quartiles (upper and lower limits of the box) as well as the minimum and maximum (ends of the whiskers)