SummaryPreviously, a system for packaging tick-borne encephalitis virus (TBEV) subgenomic replicon RNAs into single-round infectious virus-like particles (VLPs) was developed. In the present study, VLPs were applied to measuring the levels of neutralizing antibodies against TBEV as an alternative to performing neutralization tests with live virus. As markers of VLP infection, the genes for GFP and luciferase were inserted into the TBEV replicon, which was then packaged into VLPs. The reporter genes were expressed in cells that were infected with the VLPs, and this infection was inhibited by neutralizing antibodies to TBEV. Serum samples from wild rodents were used to evaluate the neutralization test using VLPs. All the sera that were positive in the conventional neutralization test were also found to be positive in the neutralization test using VLPs, and there were highly significant correlations between the neutralization titres obtained using the native virus and those using VLPs.These results indicate that VLPs that express reporter genes represent a useful and safe alternative to conventional neutralization testing using live virus.Keywords: tick-borne encephalitis, virus-like particles, and neutralization test 3 TextTick-borne encephalitis virus (TBEV), which belongs to the family Flaviviridae genus Flavivirus, causes fatal encephalitis in humans with serious sequelae (Dumpis et al., 1999). TBE occurs widely across Europe, Russia, and Far-Eastern Asia, including Japan (Blaskovic et al., 1967; Korenberg and Kovalevskii, 1999; Lindgren and Gustafson, 2001;Ormaasen et al., 2001;Roggendorf et al., 1981), and more than 10,000 cases of the disease are reported annually. TBEV can be divided into three subtypes: 1) the Far-Eastern subtype, known as Russian spring summer encephalitis (RSSE) virus; 2) the European subtype, known as Central European encephalitis (CEE) virus; and 3) the Siberian subtype (Bakhvalova et al., 2000;Ecker et al., 1999). TBEV is transmitted by tick bites and is maintained in the zoonotic transmission cycle between ticks and wild vertebrate hosts, with humans acting as accidental hosts. The major tick vector of the European subtype is Ixodes ricinus, whereas I. persulcatus is the major tick vector for the other two viral subtypes (Ecker et al., 1999;Gaunt et al., 2001;Hayasaka et al., 2001;Lundkvist et al., 2001).Effective diagnosis of TBE infection relies on the detection of specific antibodies. However, the presence of cross-reactive antigenic structures among flaviviruses makes it difficult to differentiate between TBEV and other flavivirus infections/vaccinations using IgG-ELISA and the HI test (Holzmann et al., 1996). For cases that involve contact with other flaviviruses or vaccinations, a neutralizing test, which is the most specific serological test, is required to confirm the diagnosis of TBE infection. However, since TBEV is classified as a biosafety level 3 or 4 virus, a high-level biocontainment facility is required to handle the live virus required for neutralization testing. V...