A rapid fluorescent focus inhibition test for detection of neutralizing antibodies to tick-borne encephalitis virus
Introduction
Tick borne encephalitis (TBE), is endemic in several countries in central and northern Europe, in the Baltic states and in Russia, with thousands of cases recorded each year (Kunz, 1992). The causative virus, TBE virus, is a member of the flaviviridae (Westaway et al., 1985), a virus family which comprises several viruses pathogenic for man, e.g. yellow fever virus (YF) virus, dengue viruses, and Japanese encephalitis (JE) virus. Two subtypes of TBE virus have been identified, the European and the Far Eastern subtypes, which are associated with their respective tick vectors Ixodes ricinus and Ixodes persulcatus (Monath and Heinz, 1996). The envelope (E) protein of TBE virus induces the formation of neutralizing antibodies (Heinz et al., 1981), and has been shown to exhibit a very high degree of homogeneity between the European strains (Heinz and Kunz, 1981, Guirakhoo et al., 1987). Immunization studies have shown that a vaccine based on the European subtype TBE virus will elicit protection against both subtypes of TBE (Holzmann et al., 1992). In addition to the acute disease, a high proportion of TBE-patients (35.7%) suffer from a post-encephalitic syndrome with neurological sequelae (Haglund et al., 1996).
Serological cross-reactions within the flaviviridae are common, and antibody patterns following flavivirus infections or immunization are often difficult to interpret (Calisher and Monath, 1988). Vaccination has been shown to lower efficiently and safely the number of TBE cases (Kunz et al., 1991), but an accurate and rapid assessment of the neutralizing antibody response after clinical disease or vaccination has been hampered by lack of reliable and standardized tests.
We developed a rapid fluorescent focus inhibition test (RFFIT) and compared it with a standard plaque reduction neutralization test (PRNT) and a hemagglutination inhibition test (HI) for antibody detection in late convalescent sera from 18 patients with a previous clinical and serological diagnosis of TBE.
Section snippets
Patient sera
Late convalescent sera from 18 patients with a previous clinical and serological diagnosis of TBE (Günther et al., 1997) and drawn approximately one year post onset of disease were examined by three serological methods for antibodies to TBE virus. Sera were stored at −20°C. Ten convalescent sera from patients diagnosed previously with dengue fever, confirmed by the presence of neutralizing antibodies (Vene et al., 1995), were assayed for neutralizing antibodies to TBE virus in order to assess
Results
A comparison of assay performance stability between RFFIT and PRNT is shown in Table 1. The desired virus dose (30–90 FFD50) was obtained in each of the five RFFITs at the one virus dilution (1:200) used, while in only 3/10 PRNTs performed an acceptable virus dose (30–70 PFU) was achieved. The virus dilutions containing the necessary amount of PFUs for an acceptable PRNT varied between 1:1.600 and 1:34.000. In 3/10 assays no visible plaques were observed.
Table 2 shows the antibody titres for
Discussion
The immune responses after TBE-vaccination as well as clinical disease have been studied extensively (Guirakhoo et al., 1990, Klockmann et al., 1991, Kunz et al., 1991, Holzmann et al., 1996, Günther et al., 1997), mainly by use of assays (e.g. HI and ELISA) which, in addition to TBE-virus specific antibodies, also will detect flavivirus cross-reactive antibodies. The use of neutralization assays has been relatively rare (Klockmann et al., 1991, Holzmann et al., 1996), a fact that probably
Acknowledgements
Mari Gilljam is gratefully acknowledged for excellent technical assistance.
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