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. We present our surveillance results on imported dengue cases in Taiwan during [2008][2009][2010]. A total of 734 imported dengue patients were identified. The travelers were arriving from 18 countries, including Southeast Asia, the Indian subcontinent, South Pacific islands, and Latin America. Gene sequences from 358 dengue virus (DENV) isolates were used to perform phylogenetic analyses, thus, providing an updated view of the geographic distribution and dynamic transmission of DENV strains circulating in these countries. Our results suggest that the DENV-1 genotype I and DENV-2 Cosmopolitan genotype comprise the predominant DENV strains circulating in Southeast Asian countries. The DENV-3 Genotype III strain was found to be newly emerging in several Southeast Asian countries, however, the Asian genotype 2 and the Asian/American genotype of DENV-2 strains appeared to be less prevalent in Southeast Asia. Furthermore, imported dengue viruses are representative of the overall patterns of serotype/genotype frequencies of dengue outbreaks that occurred in Taiwan.

No abstract

Japanese encephalitis (JE) is a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV). Pigs and water birds are the main amplifying and maintenance hosts of the virus. In this study, we conducted a JEV survey in mosquitoes captured in pig farms and water bird wetland habitats in Taiwan during 2005 to 2012. A total of 102,633 mosquitoes were collected. Culex tritaeniorhynchus was the most common mosquito species found in the pig farms and wetlands. Among the 26 mosquito species collected, 11 tested positive for JEV by RT-PCR, including Cx. tritaeniorhynchus, Cx. annulus, Anopheles sinensis, Armigeres subalbatus, and Cx. fuscocephala. Among those testing positive, Cx. tritaeniorhynchus was the predominant vector species for the transmission of JEV genotypes I and III in Taiwan. The JEV infection rate was significantly higher in the mosquitoes from the pig farms than those from the wetlands. A phylogenetic analysis of the JEV envelope gene sequences isolated from the captured mosquitoes demonstrated that the predominant JEV genotype has shifted from genotype III to genotype I (GI), providing evidence for transmission cycle maintenance and multiple introductions of the GI strains in Taiwan during 2008 to 2012. This study demonstrates the intense JEV transmission activity in Taiwan, highlights the importance of JE vaccination for controlling the epidemic, and provides valuable information for the assessment of the vaccine's efficacy.

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

Recurrent event data are frequently encountered in studies with longitudinal designs. Let the recurrence time be the time between two successive recurrent events. Recurrence times can be treated as a type of correlated survival data in statistical analysis. In general, because of the ordinal nature of recurrence times, statistical methods that are appropriate for standard correlated survival data in marginal models may not be applicable to recurrence time data. Specifically, for estimating the marginal survival function, the Kaplan-Meier estimator derived from the pooled recurrence times serves as a consistent estimator for standard correlated survival data but not for recurrence time data. In this article we consider the problem of how to estimate the marginal survival function in nonparametric models. A class of nonparametric estimators is introduced. The appropriateness of the estimators is confirmed by statistical theory and simulations. Simulation and analysis from schizophrenia data are presented to illustrate the estimators' performance.