A Novel Multiplex RT-PCR Assay for Simultaneous Detection of Dengue and Chikungunya Viruses.

The goal of the study was to develop a specific, sensitive, and cost-effective molecular RT-PCR diagnostic assay for the rapid and simultaneous detection of the serotypes of dengue virus (DENV) and Chikungunya virus (CHIKV) from sera of suspected febrile patients. A single-tube, single-step multiplex RT-PCR (mRT-PCR) assay was designed for the detection of viral genomes from clinical and field samples. Specificity and sensitivity of the mRT-PCR assay were evaluated against six different combinations using two reverse transcriptases (AMV-RT and RT-Ace) and three DNA polymerases (LA-Taq, rTaq, and Tth). Among the six combinations, the AMV-RT and LA-Taq combination was more specific and sensitive than other enzyme combinations for detecting viral genomes of DENV-1, DENV-2, DENV-3, and DENV-4 (p < 0.01), and for detecting viral genomes of CHIKV (p < 0.05). The detection limits of the mRT-PCR were 10 focus forming units (FFU) for CHIKV and 1 FFU, 20 FFU, 0.1 FFU, and 10 FFU for DENV-1, DENV-2, DENV-3, and DENV-4, respectively. The primers used for the mRT-PCR did not show any cross-reactivity among the serotypes of DENV or CHIKV. Specificity and sensitivity of the newly developed mRT-PCR were validated using serum samples collected from febrile patients during dengue outbreaks in Bangladesh. The sensitivity for serotype detection of DENV and CHIKV was superior to the virus isolation method and the antigen detection method using the Dengue NS1-Ag assay. This novel mRT-PCR method can be used for molecular epidemiological surveillance of DENV and CHIKV in epidemic and endemic countries.

Dengue virus strain DEN2 16681 utilizes a specific glycochain of syndecan-2 proteoglycan as a receptor.

Dengue virus (DENV) causes fever and severe haemorrhagic symptoms in humans. The DEN2 16681 strain, derived from a dengue haemorrhagic fever patient, has been widely used in studies related to DENV pathogenesis, such as mouse and non-human primate haemorrhagic models and human vascular endothelial-cell permeability. To clarify the entry mechanism of the 16681 strain, we characterized a novel cell receptor for this strain. Our two major findings were as follows: firstly, the SDC2 membrane protein was an effective DEN2 16681 receptor in a cloned K562 cell line. Secondly, a heparan sulfate (HS) glycochain (of four glycochains in SDC2) is the specific binding site of DENV and seems to be involved in tissue-culture adaptation. Our findings present an entry mechanism that could be implicated for DENV adaptation and HS-mediated DENV infection.

Molecular characterization and clinical evaluation of dengue outbreak in 2002 in Bangladesh.

During the febrile illness epidemic in Bangladesh in 2002, 58 people died out of the 6,132 affected. Two hundred hospitalized patients were analyzed clinically, serologically and virologically to determine the features of this dengue infection. Among the 10- to 70-year-old age group of the 200 clinically suspected dengue patients, 100 (50%) were confirmed as dengue cases by virus isolation and dengue IgM-capture ELISA. Of the 100 dengue-confirmed cases, the mean age was 29.0 (+/-12.4). The possible dengue secondary infection rate determined by Flavivirus IgG-indirect ELISA was 78% in 2002. Eight dengue virus strains were isolated, representing the first dengue virus isolation in the country, and all of the strains were dengue virus type-3 (DEN-3). Sequence data for the envelope gene of the DEN-3 Bangladeshi isolates were used in a phylogenetic comparison with DEN-3 from other countries. A phylogenetic analysis revealed that all 8 strains of DEN-3 were clustered within a well-supported independent sub-cluster of genotype II and were closely related to the Thai isolates from the 1990s. Therefore, it is likely that the currently circulating DEN-3 viruses entered Bangladesh from neighboring countries.

Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay.

The development and validation of a one-step, real-time, and quantitative dengue virus serotype-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the 3' noncoding region for the rapid detection and differentiation of dengue virus serotypes are reported. The RT-LAMP assay is very simple and rapid, wherein the amplification can be obtained in 30 min under isothermal conditions at 63 degrees C by employing a set of four serotype-specific primer mixtures through real-time monitoring in an inexpensive turbidimeter. The evaluation of the RT-LAMP assay for use for clinical diagnosis with a limited number of patient serum samples, confirmed to be infected with each serotype, revealed a higher sensitivity by picking up 100% samples as positive, whereas 87% and 81% of the samples were positive by reverse transcription-PCR and virus isolation, respectively. The sensitivity and specificity of the RT-LAMP assay for the detection of viral RNA in patient serum samples with reference to virus isolation were 100% and 93%, respectively. The optimal assay conditions with zero background and no cross-reaction with other closely related members of the Flavivirus family (Japanese encephalitis, West Nile, and St. Louis encephalitis viruses) as well as within the four serotypes of dengue virus were established. None of the serum samples from healthy individuals screened in this study showed any cross-reaction with the four dengue virus serotype-specific RT-LAMP assay primers. These findings demonstrate that RT-LAMP assay has the potential clinical application for detection and differentiation of dengue virus serotypes, especially in developing countries.