P. progresses to the second phase in 20 to 30% of patients. In this second phase, central nervous system manifestations (meningitis, encephalitis, and meningoencephalitis) occur (4, 20). About one-third of patients who experience the second phase have sequelae (4, 9, 10, 19). IgM antibodies are detectable in the serum during the second stage of the illness, but reverse transcription-PCR is almost always negative (31). The reported case fatality rates vary between 0 and 3% in Europe (19, 36) to up to 30% in Asian parts of Russia, which may reflect differences in the virulence of TBEV strains or differences in clinical alert or hospitalization rates in western European countries and in Asia (5). The methods used to detect TBEV antibodies in serum include hemagglutination inhibition, neutralization, immunofluorescence, complement fixation, and enzyme immunoassay (EIA). The serology is complicated by the wide cross-reactivity between different flaviviruses (32). There are some commercially available immunoglobulin M (IgM) and IgG tests (30) mainly based on purified and inactivated TBEV antigen. Although the use of commercially available EIA tests in a diagnostic laboratory does not require any special safety precautions, the production and purification of TBEV PROM1 antigen requires biosafety level 3 facilities and specially trained staff. The TBEV M and E proteins are translocated to the endoplasmic reticulum and acquire their mature conformations in the secretory pathway, where prM is Oxoadipic acid cleaved by furin into its mature form, M (3, 24, 35). These proteins have also been expressed in recombinant expression systems in mammalian cells, where they have been shown to form secreted virus-like particles (1, 14, 33). The structure of the particles has been characterized (6). They are about 30 nm in diameter, compared to about 50 nm for virions. Virus-like particles are apparently also formed normally during flavivirus infection (32). Oxoadipic acid The aims of this study were, first, to express optimally folded, secreted, and antigenic TBEV M and E proteins or virus-like particles in insect cells, not reported before for this arthropod-borne biohazardous virus, and subsequently to assess the applicability of the antigen in diagnostic tests for tick-borne encephalitis. MATERIALS AND METHODS Construction of recombinant baculoviruses. RNA was isolated from mouse brains infected with TBEV strain Kumlinge A52 (2) with TriPure reagent (Roche Molecular Biochemicals, Basel, Switzerland). The segment coding for the TBEV prM and E proteins was reverse transcribed to cDNA with forward primer 5-TTTTCTAGATCTAATGGTTGGCTTGCAAAAACG and reverse primer 5-TTTCTGCAGCTAGTCATACCATTCTGAGACCTC. The reverse transcription reaction was performed with the Moloney murine leukemia virus reverse transcriptase enzyme (Fermentas, Vilnius, Lithuania) at 37C for 1.5 h. The resulting cDNA was amplified by PCR with the same primers as in the reverse transcription reaction and the enzymes DNA polymerase (Fermentas) and extender (Stratagene, La Jolla, Calif.) with six cycles of 95C for 50 s, 50C for 50 s, and 72C for 4 min, followed by 31 cycles of 95C for 50 s, 55C for 50 s, and 72C for 3 min. The amplified DNA was then extracted from the 1% agarose gel with the QIAquick gel extraction kit protocol (Qiagen, Hilden, Germany) and cloned into the pGEM-T vector (Promega, Madison, Wis.) according to the manufacturer’s instructions. From the plasmid vector, the insert was cut with = 48) to positive results in reference Progen tick-borne encephalitis IgM test (= 48) and false-negative results (none). The two serum samples positive for rheumatoid factor and negative in our IgM test were not included in these calculations because they were apparently false-positives in the reference test. Thus, the sensitivity for this panel was 100%. We also included acute-phase serum samples from patients infected with another flavivirus, dengue Oxoadipic acid virus. Out of seven serum samples positive for dengue virus IgM antibodies in a commercial -capture IgM test (Focus Technologies), six were negative in our TBEV IgM test and one gave a low positive result. When those were included in the calculation of the specificity, when calculated as the ratio of the negative results from our test (= 106) to the correct negative results (= 106) and the false-positive results (= 1), the specificity was 99%. If the cross-reacting flavivirus serum samples were excluded, the specificity was 100%. IgG-IFA results. IgG antibodies to TBEV in serum samples were detected by IFA with baculovirus TBEV ME-infected acetone-fixed insect cells as the antigen (Fig. ?(Fig.5).5). Of 88 serum samples, 84 (95%) that were negative (titer 10) in the hemagglutination inhibition test were also negative (titer 10) in the IgG-IFA. All acute-phase tick-borne encephalitis serum samples (=.