Jyh-Hsiung Huang

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.

The Dual-Specific Binding of Dengue Virus and Target Cells for the Antibody-Dependent Enhancement of Dengue Virus Infection

Using flow cytometric assay and monoclonal anti-dengue Ab, we observed that both anti-E and anti-prM Abs could enhance dengue virus infection in a concentration-dependent but serotype-independent manner. Increases were found in both the percentage of dengue-infected cells and the expression of dengue E and NS1 protein per cell. Dengue virion binding and infection were enhanced on FcR-bearing cells via the Fc-FcγRII pathway. Furthermore, anti-prM Ab also enhanced dengue virion binding and infection on cells lacking FcR, such as BHK-21 or A549 cells, by the mechanism of peptide (CPFLKQNEPEDIDCW)-specific binding. Anti-prM Ab cross-reacted with BHK-21 or A549 cells and recognized self-Ags such as heat shock protein 60. In summary, a novel mechanism of anti-prM Ab-mediated enhancement on dengue virus infection was found to be mediated by dual specific binding to dengue virion and to target cells, in addition to the traditional enhancement on FcR-bearing cells.

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.

Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay

Background The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. Methodology/Principal Findings We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. Conclusions/Significance Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level.

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.

Antibody responses to an immunodominant nonstructural 1 synthetic peptide in patients with dengue fever and dengue hemorrhagic fever

Two flaviviruses, dengue (DEN) virus and Japanese encephalitis (JE) virus, are important because of their global distribution and the frequency of epidemics in tropical and subtropical areas. To study the B‐cell epitopes of nonstructural 1 (NS1) glycoprotein and anti‐NS1 antibody response in DEN infection, a series of 15‐mer synthetic peptides from the predicted B‐cell linear epitopes of DEN‐2 NS1 protein were prepared. Enzyme‐linked immunosorbent assay (ELISA) was performed to analyze antibody responses to these peptides from sera of both DEN and JE patients. One peptide derived from DEN‐2 NS1, D2 NS1‐P1 (amino acids 1–15), was identified as the immunodominant epitope that reacted with sera from dengue fever (DF) patients but not JE patients. The isotype of D2 NS1‐P1‐specific antibodies was mainly immunoglobulin M (IgM) in all sera that tested positive. A specificity study demonstrated that sera from all four DEN types reacted with D2 NS1‐P1. A dynamics study showed that specific antibodies to this peptide could be detected as early as 2 days after the onset of symptoms. We observed significant anti‐D2 NS1‐P1 antibody responses in 45% of patients with primary and secondary infections with DF or with dengue hemorrhagic fever. This is the first report demonstrating that significant anti‐DEN NS1 antibodies can be induced in the sera of patients with primary DEN infection. J. Med. Virol. 57:1–8, 1999.

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.