Ting-Hsiang Lin

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.

Study of Sequence Variation of Dengue Type 3 Virus in Naturally Infected Mosquitoes and Human Hosts: Implications for Transmission and Evolution

ABSTRACT Dengue virus is an arbovirus that replicates alternately in the mosquito vector and human host. We investigated sequences of dengue type 3 virus in naturally infected Aedes aegypti mosquitoes and in eight patients from the same outbreak and reported that the extent of sequence variation seen with the mosquitoes was generally lower than that seen with the patients (mean diversity, 0.21 versus 0.38% and 0.09 versus 0.23% for the envelope [E] and capsid [C] genes, respectively). This was further verified with five experimentally infected mosquitoes (mean diversity, 0.09 and 0.10% for the E and C genes, respectively). Examination of the quasispecies structures of the E sequences of the mosquitoes and of the patients revealed that the sequences of the major variants were the same, suggesting that the major variant was transmitted. These findings support our hypothesis that mosquitoes contribute to the evolutionary conservation of dengue virus by maintaining a more homogenous viral population and a dominant variant during transmission.

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.

Comparative analysis of full genomic sequences among different genotypes of dengue virus type 3.

BackgroundAlthough 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.ResultsUsing maximum likelihood and Bayesian approaches, our phylogenetic analysis revealed that the Taiwans indigenous DENV-3 isolated from 1994 and 1998 dengue/DHF epidemics and one 1999 sporadic case were of the three different genotypes – I, II, and III, each associated with DENV-3 circulating in Indonesia, Thailand and Sri Lanka, respectively. Sequence diversity and selection pressure of different genomic regions among DENV-3 different genotypes was further examined to understand the global DENV-3 evolution. The highest nucleotide sequence diversity among the fully sequenced DENV-3 strains was found in the nonstructural protein 2A (mean ± SD: 5.84 ± 0.54) and envelope protein gene regions (mean ± SD: 5.04 ± 0.32). Further analysis found that positive selection pressure of DENV-3 may occur in the non-structural protein 1 gene region and the positive selection site was detected at position 178 of the NS1 gene.ConclusionOur study confirmed that the envelope protein is under purifying selection pressure although it presented higher sequence diversity. The detection of positive selection pressure in the non-structural protein along genotype II indicated that DENV-3 originated from Southeast Asia needs to monitor the emergence of DENV strains with epidemic potential for better epidemic prevention and vaccine development.

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.

1998 DENGUE HEMORRHAGIC FEVER EPIDEMIC IN TAIWAN

To the Editor: The rapid spreading of dengue viruses has led to increasing incidence rates of dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS) worldwide in the past 20 years. The global pandemic of DF and DHF in 1998 was associated with the largest DF epidemics many tropical or subtropical countries had ever experienced (1,2). Here we report the unique epidemiologic characteristics of DF and DHF caused by dengue virus type 3 (DEN-3) in Taiwan, where dengue is not endemic. The recent epidemics of dengue in Taiwan started when dengue virus type 2 (DEN-2) was first introduced into the southern off-islet of Hsiao-Liu-Chiu in 1981 after an absence of 38 years since World War II. Tainan City in southern Taiwan had not had a dengue epidemic since l942–1943 until three dengue outbreaks occurred there in the last decade. The first outbreak of DEN-1 in 1994 and the second of DEN-2 in 1997 involved few confirmed cases. The third epidemic of dengue, which was attributed to DEN-3, began in October 1998 and continued into January 1999. From August 1, 1998, to January 31, 1999, physicians in all the hospitals and clinics in Tainan City were required to report any suspected dengue cases who met the criteria of fever (>38oC) and two or more of the following symptoms and signs: headache, retroorbital pain, myalgia, arthralgia, rash, and hemorrhagic manifestations. Patients who met the criteria were invited to participate in the study; informed consent was given by the patients, and plasma or serum samples were collected for laboratory confirmation. When a physician reported a suspected dengue case, a minimum of 100 blood samples would be collected from the patient’s neighbors by the Tainan City Health Bureau staff. The blood specimens were transported to the laboratory at the National Institute of Preventive Medicine for confirmation. A confirmed dengue case was required to be positive by either reverse transcription–polymerase reaction (3), or demonstrate seroconversion by dengue-specific immunoglobulin (Ig) M and seronegativity for Japanese encephalitis virus (JEV)-specific IgM by IgM-enzyme-linked immunosorbent assay (IgM-ELISA) (4). Dengue viruses were isolated in C6/36 cell cultures and identified to serotype with monoclonal antibodies (5). The clinical diagnosis of DHF was based on the World Health Organization’s (WHO) criteria that were revised in 1997. Those confirmed dengue cases were classified as primary, secondary, or indeterminate infections, depending on the ratio of dengue-specific IgM/IgG as measured by the capture IgM and IgG ELISA (6). Of 225 case-patients with suspected dengue, 142 patients, of which 74 (52.5%) were female and 68 (48.2%) were male (62.7%), had their cases confirmed by laboratory diagnosis during the study period. Their ages ranged from 7 to 79 years (mean: 39 years). Of the 23 DHF case-patients meeting the WHO’s case definition, 17 were male. The ages of the DHF case-patients ranged from 13 to 73 years, with a mean age of 42 years. The epidemic began in October and peaked in November. In the Central District, where the earliest and majority of DHF cases occurred (52.2%), the DHF/DF ratio increased with time, from 11% during the first interval, to 20% and 30% during the second and third intervals, respectively. Although this increase was not statistically significant by Fisher’s exact test because of the small sample size, similar results were reported in Cuba’s DHF epidemic in both 1981 and 1997 (7). Many RNA viruses, such as influenza, increase in virulence through transmission; possible virulence mechanisms of dengue viruses are now under investigation. In other words, the duration of epidemic has to be as short as possible to avoid the emergence of DHF cases in that region. In our study, 88 dengue cases (62%) were classified as primary infections, 32 (22.5%) as secondary infections, and 22 (15.5%) as undetermined because of the lack of paired acute- and convalescent-phase samples. DHF cases showed no significant association with secondary infections (odds ratio = 1.92 [95% CI 0.64 to 5.76], p = 0.19). Because the last documented transmission of dengue viruses in Taiwan occurred during the island-wide dengue epidemic in 1943, both DF and DHF cases were stratified into two birth cohort groups. Of DF case-patients born after 1943, 94% (83/88) had primary infection, and 11 (92%) of 12 DHF case-patients had primary infection. By contrast, 84% (27/32) of all the dengue case-patients born before and during 1943 had secondary infection, and 7 (78%) of 9 DHF case-patients had secondary infections. After the cohorts were stratified by age, DHF cases were not associated with secondary infection (Mantel-Haenszel) weighted odds ratio: 0.84 [95% CI: 0.11 to 5.62]) (p = 0.6). Therefore, age was not a confounding factor or effect modifier for DHF case-patients in Tainans 1998 epidemic. DHF cases were mostly associated with primary infection in Tainan, where no large-scale epidemic of dengue had been reported from 1944 to 1997. Our observation of DHF in adult case-patients was different from that in dengue hyperendemic Asian countries where 80% of DHF case-patients are children (8). Dengue virus type 3 was the only serotype isolated during the epidemic in Tainan in 1998. However, this situation was similar to that in Tonga in 1974, where a dengue virus was also newly introduced (9), and to recent epidemics in south and central America (10). More adults may have been affected because fewer dengue epidemics, and therefore fewer exposures to dengue viruses during childhood, had occurred; subsequently, the immune status in adults had changed. Presumably, previous observations of severe dengue in children in Southeast Asia were the result of immunity to infection in the older population, rather than a particular susceptibility to DHF among children. Our results were not influenced by the age structure of DF and DHF case-patients in cases of indeterminate infection or in cases of sub-clinical infection in children (0.4%, unpub. data). The dengue virus with epidemic potential replicated to higher viremia titer and was associated with disease severity without consideration of immune status (11). The investigation on molecular evolution of DEN-3 virus during the 1998 epidemic in Taiwan, currently in progress, will elucidate the possible role of virus variation in the pathogenesis of DHF.

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.