Shu-Fen Chang

Development of Group- and Serotype-Specific One-Step SYBR Green I-Based Real-Time Reverse Transcription-PCR Assay for Dengue Virus

ABSTRACT 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 107 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/ml for DEN-4. Comparisons between the one-step SYBR Green-based RT-PCR assay and the conventional cell culture method in the clinical diagnosis of dengue virus infection from acute-phase serum samples of confirmed dengue patients were performed. The results showed that 83 and 67% of 193 acute-phase serum samples tested were positive by the one-step SYBR Green-based RT-PCR method and cell culture method, respectively. Further analysis showed that the one-step SYBR Green-based RT-PCR method could detect twice as many acute-phase serum samples with positive dengue-specific immunoglobulin M (IgM) and/or IgG antibodies than cell culture method. Our results demonstrate the potential clinical application of the one-step SYBR Green I-based RT-PCR assay for the detection and differentiation of dengue virus RNA.

Comparison of Capture Immunoglobulin M (IgM) and IgG Enzyme-Linked Immunosorbent Assay (ELISA) and Nonstructural Protein NS1 Serotype-Specific IgG ELISA for Differentiation of Primary and Secondary Dengue Virus Infections

ABSTRACT 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.

Fever Screening at Airports and Imported Dengue

Airport fever screening in Taiwan, July 2003–June 2004, identified 40 confirmed dengue cases. Results obtained by capture immunoglobulin (Ig) M and IgG enzyme-linked immunoassay, real time 1-step polymerase chain reaction, and virus isolation showed that 33 (82.5%) of 40 patients were viremic. Airport fever screening can thus quickly identify imported dengue cases.

Dengue NS1-specific antibody responses: isotype distribution and serotyping in patients with Dengue fever and Dengue hemorrhagic fever.

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. J. Med. Virol. 62:224–232, 2000.

Potential application of nonstructural protein NS1 serotype-specific immunoglobulin G enzyme-linked immunosorbent assay in the seroepidemiologic study of dengue virus infection: correlation of results with those of the plaque reduction neutralization test.

ABSTRACT 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.

Application of the Dengue Virus NS1 Antigen Rapid Test for On-Site Detection of Imported Dengue Cases at Airports

ABSTRACT We used the dengue virus NS1 antigen (Ag) rapid test for on-site detection of imported dengue cases at airports. Among 22 positive cases of dengue identified from 850 patients with a fever suspected to have dengue, 17 were NS1 Ag test positive. These findings demonstrate the usefulness of the NS1 Ag rapid test in screening imported dengue cases at airports.

Concurrent Isolation of Chikungunya Virus and Dengue Virus from a Patient with Coinfection Resulting from a Trip to Singapore

ABSTRACT We report two cases of imported infection in patients who had returned to Taiwan from Singapore: one was coinfected with chikungunya virus and dengue virus type 2, and the other was infected with the same dengue virus. Both viruses were successfully isolated from the coinfected case by using antibody neutralization and a plaque purification technique.

Antibody to the nonstructural protein NS1 of Japanese encephalitis virus: Potential application of MAB based indirect ELISA to differentiate infection from vaccination

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect and differentiate the antibody responses to Japanese encephalitis (JE) virus nonstructural protein NS1 between infected and vaccinated individuals. The results showed that all convalescent sera from JE patients contained NS1-specific IgG antibodies, while 65 and 40% of these sera showed detectable NS1-specific IgM and IgA antibodies, respectively. Specificity analysis showed that NS1-specific IgM and IgA antibodies from JE patients do not cross-react to dengue virus NS1 glycoprotein, while IgG antibodies from 10% of JE patients showed significant cross-reaction to dengue virus NS1 glycoprotein. To differentiate infection from vaccination, the immune sera from 24 children vaccinated with inactivated JE vaccine were analyzed. The data showed that none of these immune sera had detectable NS1-specific IgG antibodies. The results demonstrated the potential application of JE NS1-specific indirect ELISA to differentiate infection from vaccination.

Dengue Virus Serotyping Based on Envelope and Membrane and Nonstructural Protein NS1 Serotype-Specific Capture Immunoglobulin M Enzyme-Linked Immunosorbent Assays

ABSTRACT Envelope and membrane (E/M) and nonstructural protein NS1 serotype-specific capture Immunoglobulin M (IgM) enzyme-linked immunosorbent assays (ELISAs) were developed to differentiate four dengue virus serotypes. A total of 93 anti-dengue virus IgM-positive serum samples collected between days 5 and 45 of illness from 59 confirmed dengue patients were analyzed. The results showed that positive serotype specificity could be identified for 86.1 and 47.6% of serum samples tested for E/M-specific IgM antibodies versus 83.3 and 42.9% of serum samples tested for NS1-specific IgM antibodies from patients with primary and secondary dengue virus infections, respectively. Dual analyses with both E/M and NS1 serotype-specific capture IgM ELISAs showed that positive serotype specificity could be correctly identified for 98.6 and 61.9% of all of the primary and secondary serum samples tested, respectively. These findings suggested that E/M and NS1 serotype-specific capture IgM ELISAs have the potential to be of use in dengue virus serotyping.

Characteristics of dengue epidemics in Taiwan

Dengue fever is caused by dengue viruses (DENVs) and is the most common arboviral disease in tropical and subtropical regions of the world, with more than 50 million cases recorded every year. Dengue viruses (serotypes 1e4) are mosquito-borne members of the genus Flavivirus in the family Flaviviridae. In recent decades, the number of dengue cases reported worldwide and the number of countries with endemic dengue has increased dramatically because of the enlarging habitat of the mosquito vectors Aedes sp, growing numbers of susceptible human hosts, and increasing spread of DENVs through rapid and frequent global travel. Dengue disease can manifest in the form of the mild dengue fever or the more severe and potentially fatal dengue hemorrhagic fever/dengue shock syndrome, which has a fatality rate as high as 10e15% depending on the availability of health care. Currently there is no vaccine or therapeutic agent available against dengue fever. Dengue is not considered endemic in Taiwan and the constant importation of DENVs from the neighboring Southeast Asian countries through close commercial links and air travel is responsible for the local outbreaks each year. The dengue hemorrhagic fever cases in Taiwan are highly correlated with advanced age and secondary DENV infection. Studies on molecular epidemiology and phylogenetic analyses of dengue genome sequences reveal that each local outbreak was caused by a single imported DENV strain that disappears with the ending of each outbreak. Table 1 shows the serotype and genotype of DENVs isolated from major dengue epidemics (with more than 10