Japanese encephalitis virus (JEV) belongs to the genus Flavivirus within the family Flaviviridae. Members of the genus Flavivirus are predominantly arthropodborne viruses and frequently cause significant morbidity and mortality in mammals and birds (6). JEV is distributed in the south and southeast regions of Asia and kept in a zoonotic transmission cycle between pigs or birds and mosquitoes (6,50,57). JEV spreads to dead-end hosts, including humans, through the bite of JEVinfected mosquitoes and causes infection of the central nervous system, with a high mortality rate (6, 57). JEV has a single-stranded positive-strand RNA genome approximately 11 kb in length, which is capped at the 5Ј end but lacks modification of the 3Ј terminus by polyadenylation (34). The genomic RNA encodes a single large open reading frame, and a polyprotein translated from the genome is cleaved co-and posttranslationally by host and viral proteases to yield three structural proteins, the core, precursor membrane (prM), and envelope (E) proteins, and seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (53). Although the core protein has very little amino acid homology to other flaviviruses-for example, the core protein of JEV has only 25% homology to that of tick-borne encephalitis virus (TBEV)-the structural properties, such as the hydrophobicity profile, abundances of basic amino acid residues, and secondary structures, are very similar (11,20,36). The flavivirus core proteins commonly contain two hydrophobic sequences in the center and a carboxyl-terminal end, and the carboxyl-terminal hydrophobic region serves as a signal sequence of prM. The signal-anchor sequence is cleaved off by the viral protease NS2B-3, and this cleavage is required for the subsequent liberation of the amino terminus of prM by the host signal peptidase (35,52,63). The mature core protein, released from the endoplasmic reticulum (ER) membrane, is believed to bind to the genomic RNA via the basic amino acid clusters at the amino and carboxyl termini and forms nucleocapsids (23). The central hydrophobic region of the core protein may be associated with the ER membrane, and this interaction is believed to facilitate the assembly of nucleocapsid and two membrane proteins, prM and E, and to bud into the ER lumen as virions (39). The removal of the central hydrophobic region of the TBEV core protein increased the production of the subviral particles that consist of (pr)M and E proteins but that lack a core protein and genomic RNA (26,27).In addition to their role as structural proteins, core proteins of dengue virus (DEN) and Kunjin virus (KUN) are localized not only in the cytoplasm but also in the nucleus, especially in the nucleoli of several infected cell lines (4,38,55,59,61). Transport from the cytoplasm to the nucleus occurs through nuclear pore complexes that penetrate the double lipid layers of the nuclear envelope. Small molecules up to 9 nm in diameter (Ͻ50 kDa) can freely diffuse through the nuclear pore complexes, while most macromolecu...

Acute-and convalescent-phase sera from patients with dengue (DEN) hemorrhagic fever (DHF) and Japanese encephalitis (JE) that contained pre-existing flavivirus antibodies were tested for cross-reacting antibodies to DEN, JE and yellow fever (YF) viruses by a neutralization (N) test . A fourfold or greater rise in N antibody titer in the convalescent-phase was considered significant . Of 39 DHF cases, obtained at Chiang Mai University Hospital, Thailand, 15 (38.5%) showed a rise in DEN antibody titer, while another 15 (38.5%) showed a significant rise in both DEN and JE N antibody titers. On the other hand, eight (61.5%) of 13 JE cases obtained at the same Hospital , showed a significant rise in JE antibody titer, while two (15.4%) showed a significant rise in both DEN and JE antibody titers. Sucrose gradient centrifugation and fractionation of these two crossreactive JE sera revealed that IgM class antibody was specific for JE , while IgG class antibody was cross-reactive. Of three JE cases with pre-existing YF antibody obtained in Okinawa , Japan, two showed a significant rise in YF and JE antibodies. Both IgM and IgG class antibodies to YF virus were elevated. These results indicate that the cross-reactivity among flaviviruses in different subgroups (complexes), was observed quite often, even by the N test , in sequential flavivirus infection.Key words: Flaviviruses, Sequential infections, Cross-neutralization testThere are more than 60 flaviviruses , including Japanese encephalitis (JE), dengue (DEN) and yellow fever (YF) viruses, and contain crossreactive antigens which make serodiagnosis difficult (11 for review). This is especially true in the regions where two or more flaviviruses are prevalent . Among the serological tests, the neutralization (N) test is considered to be more specific than complement-fixation (CF), hemagglutination-inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) (16 for review). On the basis of close relationship in cross-neutralization tests using hyperimmune polyclonal antisera, flaviviruses are divided into at least eight antigenic subgroups (complexes), where JE and DEN viruses are classified into different subgroups, while YF virus remains unassigned (I).In Japan, only one flavivirus , i.e. JE virus, has been prevailing and HI test or ELISA has been employed for serodiagnosis without problems. While in the Southeast Asian countries such as Thailand and Vietnam, JE and DEN viruses have been coexisting and the outbreaks caused by these viruses have taken place simultaneously in the rainy season (6). It has been reported that in the secondary DEN virus infection, the serotype of the causative virus of the recent infection could not be identified by N test because of the cross-reactions . It was possible only by virus isolation or detection of specific viral genomic sequence (13). Secondary DEN infection in previously immune individuals with another serotype of DEN virus elicits broadly

The antigenic site of dengue type 2 virus (DEN2)-neutralizing monoclonal antibody (mab) 3H5 was investigated by mutational analysis. Sequence comparisons indicated that much of the 12-amino-acid sequence extending from position 386 to 397 of the DEN2 envelope glycoprotein (E) previously thought to represent the DEN2-specific mab 3H5 binding site was also present in some dengue type 1, 3, or 4 virus strains. However, the region occupied by the Glu-Pro-Gly sequence at upstream positions 383 to 385 was completely conserved among DEN2 strains, but divergent in other serotype viruses, suggesting that this sequence might be part of the antigenic site of mab 3H5. We investigated this possibility by employing the previously constructed chimeric DEN2(PreM-E)/DEN4 cDNA clone to produce viable mutants bearing DEN2 PreM and E sequences that could be analyzed for binding to and neutralization by mab 3H5. We constructed 13 such DEN2 mutants that contained a single amino acid substitution in the region between positions 383 and 393 of DEN2 E. Each single substitution in the region spanning positions 386 through 393 of DEN2 yielded a virus that was as reactive with mab 3H5 as the parental chimeric virus. These results are consistent with the extent of sequence conservation in the region. In contrast, 5 of 6 mutants that sustained an amino acid substitution at position 383, 384, or 385 failed to react with mab 3H5 as detected by immunofluorescence assay and failed to be neutralized by the mab. Interestingly, each of the 5 mab-resistant DEN2 mutants also exhibited reduced mouse neurovirulence compared to parental chimeric DEN2 when inoculated intracerebrally. These observations suggest that the Glu-Pro-Gly sequence at positions 383-386 of the DEN2 E is a component of the site against which mab 3H5 is directed. In the recently determined three-dimensional structure of the related tick-borne encephalitis virus E, the Glu-Pro-Gly sequence would be located on the lateral surface of the immunoglobulin-like domain that is proposed to bind to the host cell receptor.

An improved rapid focus reduction neutralization test for Japanese encephalitis (JE) virus in microtiter system was developed. 96-well tissue culture plates were used for preparation of cell monolayers and PAP (peroxidase-antiperoxidase) staining technique was used for visualization of foci of infected cells. As the test has many advantages over other methods, it might be applied widely for rapid diagnosis and seroepidemiological studies.