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°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.Dengue virus is a mosquito-borne flavivirus and the most widely prevalent arbovirus in tropical and subtropical regions of Asia, Africa, and Central and South America (4). There are four distinct serotypes, DEN-1, DEN-2, DEN-3, and DEN-4, which produce a spectrum of illness ranging from inapparent infection to moderate febrile illness and severe and fatal hemorrhagic disease. In recent years, dengue fever (DF) and its more serious forms, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), have emerged as a major public health problems, with expanded geographic distributions and increased epidemic activities (20).Dengue virus infection induces a life-long protective immunity to the homologous serotype but confers only partial and transient protection against subsequent infection by the other three serotypes. Therefore, multiple and sequential infections with the four dengue virus serotypes would be expected for people living in a region where dengue is hyperendemic due to the lack of cross-protective neutralizing antibodies. Seroepidemiological studies have shown that secondary infection is a major risk factor for DHF and DSS through antibody-dependent enhancement (6, 16). Therefore, rapid detection and differentiation between primary and secondary dengue virus infections and determination of the dengue virus serotypes of past and current ...

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.

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