Serotyping dengue virus (DENV) from suspect human specimens is crucial for developing sound epidemiological control measurements early in the transmission season and for effective patient management. We modified DENV consensus D1 (mD1) and serotype-specific TS2 (mTS2) and redesigned serotype-specific TS1 (rTS1) and TS4 (rTS4) as described previously in the conventional capsid and premembrane gene ( ). In addition, we designed two new sets of amplimers and probes, located at nonstructural protein 5 (NS5) and the 3 noncoding region (3NC) of DENV. The NS5 protocol utilizes two flaviviral consensus outer amplimers (mFU1 and CFD2) and four dengue virus serotype-specific TaqMan fluorogenic probes. The 3NC protocol uses two DENV consensus amplimers, DC10418 and CDC10564. The conventional gel-based, heminested detection method was adapted for the C-prM protocol for detecting and serotyping dengue viruses. In addition, we developed the real-time SYBR green I and postamplification melting temperature curve analysis for the mD1/TS and 3NC protocols using identical amplification conditions. The NS5 amplimer/probe set was formulated as a one-tube, multiplex, real-time reverse transcriptase PCR for serotype identification. Three sets of amplimers and probes were verified for their specificity in tests with yellow fever, Japanese encephalitis, St. Louis encephalitis, and West Nile viruses; optimized against 109 DENV strains; and validated for detection of the virus in sera from two different panels of acute-phase human dengue serum specimens and one panel of virus isolates from dengue patients' serum specimens. Clinical evaluation by two separate laboratories indicated that the C-prM was more sensitive (100%) than the NS5 (91%) or the 3NC (91%) protocol.

We have found that NS1 serotype-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) can be used to differentiate primary and secondary dengue virus infections. This is due to the fact that the NS1-specific IgG antibody cannot be detected before day 9 of illness for primary infection, so the NS1-specific IgG antibodies measured in acute-phase sera must come from previous infection. Comparison of NS1 serotype-specific IgG ELISA with envelope-and membrane-specific capture IgM and IgG ELISA in the differentiation of primary and secondary dengue virus infections showed good correlation (95.90% agreement). Most important, we have found that the serotype of the dengue virus from the majority of patients with primary infection could be correctly identified when convalescent-phase or postinfection sera were analyzed by NS1 serotype-specific IgG ELISA. These findings suggested that NS1 serotype-specific IgG ELISA could be reliably applied for serodiagnosis and seroepidemiological study of dengue virus infection.

A quantitative one-step SYBR Green I-based reverse transcription (RT)-PCR system was developed for the detection and differentiation of four different dengue virus serotypes in acute-phase serum samples. A set of group-and serotype-specific primer pairs was designed against conserved sequences in the core region and evaluated for clinical diagnosis. A linear relationship was obtained between the amount of input RNA and cycle threshold (Ct) value over a range of 10 to 10 7 PFU per ml of cell culture-derived dengue viruses. The detection limit of the group-specific primer pair was between 4.1 and 43.5 PFU/ml for four dengue serotypes. The detection limit of each of the serotype-specific primer pairs was calculated to be 10 PFU/ml for dengue virus serotype 1 (DEN-1), 4.6 PFU/ml for DEN-2, 4.1 PFU/ml for DEN-3, and 5 PFU Dengue virus is a mosquito-borne flavivirus and the most prevalent arbovirus in tropical and subtropical regions of Asia, Africa, and Central and South America (7). Dengue virus belongs to the family Flaviviridae. It produces a spectrum of illness ranging from inapparent infection to moderate febrile illness to severe and fatal hemorrhagic disease (2, 18). There are four distinct serotypes-designated DEN-1, DEN-2, DEN-3, and DEN-4-which can be distinguished by serological and molecular methods. The flavivirus genome is approximately 11 kb in length, and the complete genome sequence is known for isolates of all four dengue virus serotypes. The genome is composed of three structural protein genes, encoding the nucleocapsid or core protein (C), a membrane-associated protein (M), an envelope protein (E), and seven nonstructural (NS) protein genes (4). The order of proteins encoded is

Abstract. Japanese encephalitis (JE) is an endemic disease in Taiwan. A mass vaccination program of children against JE was first implemented in 1968. Along with general improvements in various aspects of living conditions over the years, the program has brought JE well under control. The main characteristics of JE epidemiology in Taiwan in the past 3 decades are as follows. The transmission mode remains unchanged-that is, the amplification stage of the virus in pigs is followed by a human epidemic each year. The frequency of JE incidence has dropped significantly.

The mechanism of replication of the flavivirus Japanese encephalitis virus (JEV) is not well known. The structures at the 3 end of the viral genome are highly conserved among divergent flaviviruses, suggesting that they may function as cis-acting signals for RNA replication and, as such, might specifically bind to cellular or viral proteins. UV cross-linking experiments were performed to identify the proteins that bind with the JEV plus-strand 3 noncoding region (NCR). Two proteins, p71 and p110, from JEV-infected but not from uninfected cell extracts were shown to bind specifically to the plus-strand 3 NCR. The quantities of these binding proteins increased during the course of JEV infection and correlated with the levels of JEV RNA synthesis in cell extracts. UV cross-linking coupled with Western blot and immunoprecipitation analysis showed that the p110 and p71 proteins were JEV NS5 and NS3, respectively, which are proposed as components of the RNA replicase. The putative stem-loop structure present within the plus-strand 3 NCR was required for the binding of these proteins. Furthermore, both proteins could interact with each other and form a protein-protein complex in vivo. These findings suggest that the 3 NCR of JEV genomic RNA may form a replication complex together with NS3 and NS5; this complex may be involved in JEV minus-strand RNA synthesis.

To understand the antibody responses to dengue (DEN) nonstructural 1 (NS1) glycoprotein and their roles in protective immunity or pathogenesis of dengue fever (DF) and dengue hemorrhagic fever (DHF), we have analyzed the NS1-speccific IgM, IgA and IgG antibodies from patients with DF and DHF. An isotype-specific, indirect enzyme-linked immunosorbent assay (ELISA) was established by coating a NS1-specific monoclonal antibody (MAb), D2/8-1, to capture soluble NS1 antigens secreted in the culture supernatants of Vero cells infected with DEN virus. We observed strong anti-NS1 antibody responses in all of the convalescent sera of patients with DF and DHF. Similar NS1-specific isotypic and serotypic antibody responses were found in the sera from DF and DHF patients. The results showed that all DEN infections induced significant NS1-specific IgG, whereas 75% and 60% of primary DF patients vs. 40% and 90% of secondary DF patients produced IgM and IgA antibodies, respectively. Specificity analysis showed that DEN NS1-specific IgG and IgA antibodies cross-react strongly to Japanese encephalitis (JE) virus NS1 glycoprotein, whereas DEN NS1-specific IgM antibodies do not cross-react to JE virus NS1 glycoprotein at all. The serotype specificity of NS1-specific IgM, IgA and IgG were found to be 80%, 67% and 75% for primary infections, and 50%, 22% and 30% for secondary infections in positive samples of DF patients. Similar pattern was found in DHF patients. The results showed that all of the DF and DHF patients produced significant NS1-specific antibodies. We did not observe direct correlation between the anti-NS1 antibody responses and DHF because sera from patients with DF and DHF showed similar anti-NS1 antibody responses.

An NS1 serotype-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed to differentiate primary and secondary dengue virus infections and serotypes of primary dengue virus infection. For this report, we carried out retrospective seroepidemiologic studies on serum samples collected from residents of Liuchiu Hsiang, Pingtung County, an isolated island in southern Taiwan during 1997-1998. The results demonstrated that good correlation existed between dengue virus NS1 serotype-specific immunoglobulin G (IgG) ELISA and dengue virus plaque reduction neutralization test (PRNT). Our data suggested that NS1 serotype-specific IgG ELISA could replace PRNT for seroepidemiologic studies to differentiate Japanese encephalitis and dengue virus infections and for dengue virus serotyping.Dengue virus (DEN) is a mosquito-borne flavivirus and the most prevalent arbovirus in tropical and subtropical regions of the world. There are four distinct serotypes, DEN-1, DEN-2, DEN-3, and DEN-4. Infection induces life-long protective immunity to the homologous serotype, but there is no crossreactive immunity to the heterologous serotypes. The global prevalence of dengue has grown dramatically in recent decades. The disease is now endemic in more than 100 countries in Africa, the Americas, the Eastern Mediterranean, Southeast Asia, and the Western Pacific (5).There have been a number of historical dengue epidemics (either regional or island-wide) over the last century

Background: Although the previous study demonstrated the envelope protein of dengue viruses is under purifying selection pressure, little is known about the genetic differences of full-length viral genomes of DENV-3. In our study, complete genomic sequencing of DENV-3 strains collected from different geographical locations and isolation years were determined and the sequence diversity as well as selection pressure sites in the DENV genome other than within the E gene were also analyzed.