Development of Real-Time Reverse Transcriptase PCR Assays To Detect and Serotype Dengue Viruses

Chien, Li‐Jung; Liao, Tsai‐Ling; Shu, Pei-Yun; Huang, Jiansheng; Gubler, Duane J.; Chang, Gwong‐Jen J.

doi:10.1128/jcm.44.4.1295-1304.2006

Cited by 193 publications

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“…The detection of the amplified target by fluorescent probes replaces the need for post-amplification electrophoresis. Many real-time RT-PCR assays have been developed that are either 'singleplex', detecting one single serotype per reaction, or 'multiplex', identifying all four serotypes from a single sample [99][100][101] . One advantage of this assay is the ability to determine viral titre early in dengue illness, which is believed to be an important predictor of disease severity 102 .…”

Section: Real-time Rt-pcrmentioning

confidence: 99%

Dengue: a continuing global threat

et al. 2010

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Dengue fever and dengue haemorrhagic fever are important arthropod-borne viral diseases. Each year, there are ~50 million dengue infections and ~500,000 individuals are hospitalized with dengue haemorrhagic fever, mainly in Southeast Asia, the Pacific and the Americas. Illness is produced by any of the four dengue virus serotypes. A global strategy aimed at increasing the capacity for surveillance and outbreak response, changing behaviours and reducing the disease burden using integrated vector management in conjunction with early and accurate diagnosis has been advocated. Antiviral drugs and vaccines that are currently under development could also make an important contribution to dengue control in the future.Dengue is the most important arthropod-borne viral infection of humans. Worldwide, an estimated 2.5 billion people are at risk of infection, approximately 975 million of whom live in urban areas in tropical and sub-tropical countries in Southeast Asia, the Pacific and the Americas 1 . Transmission also occurs in Africa and the Eastern Mediterranean, and rural Europe PMC Funders GroupAuthor Manuscript Nat Rev Microbiol. Author manuscript; available in PMC 2015 February 19. Published in final edited form as:Nat Rev Microbiol. 2010 December ; 8(12 0): S7-16. doi:10.1038/nrmicro2460. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts communities are increasingly being affected. It is estimated that more than 50 million infections occur each year, including 500,000 hospitalizations for dengue haemorrhagic fever, mainly among children, with the case fatality rate exceeding 5% in some areas [1][2][3][4] . The geographical areas in which dengue transmission occurs have expanded in recent years (FIG. 1), and all four dengue virus serotypes (DENV-1-4) are now circulating in Asia, Africa and the Americas, a dramatically different scenario from that which prevailed 20 or 30 years ago (FIG. 2). The molecular epidemiology of these serotypes has been studied in an attempt to understand their evolutionary relationships 11 .This Review will provide an update on our understanding of the pathogenesis of this successful pathogen, how we diagnose and control infection and the progress that has been made in vaccine development. Dengue virus pathogenesisDengue viruses belong to the genus flavivirus within the Flaviviridae family. DENV-1-4 evolved in non-human primates from a common ancestor and each entered the urban cycle independently an estimated 500-1,000 years ago 12 . The virion comprises a spherical particle, 40-50 nm in diameter, with a lipopolysaccharide envelope. The positive singlestrand RNA genome (FIG. 3), which is approximately 11 kb in length, has a single open reading frame that encodes three structural proteins -the capsid (C), membrane (M) and envelope (E) glycoproteins -and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 [18][19][20] . ADE occurs when mononuclear phagocytes are infected through their Fc receptors by immune complexes that form between DE...

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Section: Real-time Rt-pcrmentioning

confidence: 99%

Dengue: a continuing global threat

et al. 2010

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“…The gold standard for diagnosis of acute DENV infection is viral isolation, but the procedure is costly, time-consuming, and technically difficult to perform (8). Reverse transcriptase PCR (RT-PCR) has been widely adopted as an alternative to viral isolation for the diagnosis of acute infection, but PCR is technically intensive and expensive, and sensitivity varies from 80 to 90% based on primer sets (4,14). An IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) is useful primarily for diagnosing dengue infection in the late acute or early convalescent phase of the illness but is often insensitive for early-acute-phase infections (19).…”

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Utility of a Commercial Nonstructural Protein 1 Antigen Capture Kit as a Dengue Virus Diagnostic Tool

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et al. 2010

Clin Vaccine Immunol

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Annually, over 2.5 billion people are at risk for infection with dengue virus (DENV), while between 50 and 100 million people contract the infection. There is an urgent need for alternative diagnostic tools that can detect DENV during acute infection. Recent studies have shown that DENV nonstructural protein 1 (NS1) is detectable in the blood as early as the onset of symptoms and persists well into the convalescent phase of the infection. We evaluated the utility of the Bio-Rad Platelia DENV NS1 antigen capture kit in combination with real-time reverse transcriptase PCR (RT-PCR) and an IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for refining a new algorithm for the diagnosis of acute-or convalescent-phase DENV infection with a single clinical sample. We tested the Bio-Rad kit with three panels of sera. These panels were designed to evaluate the sensitivities of the NS1 kit for (i) early-convalescent-phase samples, (ii) acute-phase samples with false-negative PCR results, and (iii) IgM-negative convalescent-phase samples from patients with confirmed secondary DENV infections. Results show that NS1 can be detected in 22% of serum samples collected more than 10 days after the onset of illness and in 22% of samples that did not elicit an IgM response. Additionally, NS1 was detected in 37% of the tested acute-phase samples with false-negative PCR results, suggesting that NS1 detection may be valuable in increasing the sensitivity of current acute-phase diagnostics. These results will improve diagnosis with a single acute-phase or early-convalescent-phase sample for disease surveillance and clinical diagnosis.

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“…Antes del inicio de este trabajo, se confirmó que se trataba de un DENV2 mediante una reacción en cadena de la polimerasa (PCR) semianidada para los genes de cápside y premembrana (C-PreM), según lo descrito por Chien, et al (9). Para obtener el ARN viral, se infectaron células C6/36 (ATCC) con el virus INS-2009 y, a partir de los sobrenadantes de los cultivos, se hizo la extracción utilizando el estuche QIAamp Viral RNA ® (Qiagen).…”

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Polyclonal antibodies against recombinant dengue virus NS3 protein

et al. 2017

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Introducción. El dengue es una enfermedad causada por uno de los cuatro serotipos del virus del dengue (DENV) y es endémica en, aproximadamente, 130 países. Su incidencia ha aumentado notablemente en las últimas décadas, así como la frecuencia y la magnitud de los brotes. A pesar de los esfuerzos, no existen tratamientos profilácticos ni terapéuticos contra la enfermedad y, en ese contexto, el estudio de los procesos que gobiernan el ciclo de infección del DENV es esencial para desarrollar vacunas o terapias antivirales. Una de las moléculas del DENV más prometedoras es la proteína no estructural 3 (NS3), la cual es indispensable para la replicación viral y es uno de los principales blancos inmunológicos durante la infección. Objetivo. Producir anticuerpos policlonales para contribuir a los futuros estudios sobre las interacciones entre la proteína NS3 y otras proteínas celulares. Materiales y métodos. Se expresaron dos proteínas recombinantes del dominio helicasa de NS3 del DENV de serotipo 2, las cuales se emplearon para inmunizar ratas y producir anticuerpos policlonales. Resultados. Los anticuerpos producidos fueron útiles en ensayos deWestern blot e inmunofluorescencia y se reportó por primera vez un anticuerpo policlonal anti-NS3 que permitió la inmunoprecipitación de la proteína viral y la detecta con Western blot sin necesidad de inducir sobreexpresión de NS3 o de usar extractos de células marcados metabólicamente con radioisótopos. Conclusión. Las proteínas recombinantes expresadas y los anticuerpos producidos constituyen herramientas valiosas para estudiar procesos infecciosos del DENV que involucren a la proteína NS3 y evaluar pruebas dirigidas a interferir las funciones de esta proteína. Polyclonal antibodies against recombinant dengue virus NS3 proteinIntroduction: Dengue is a disease caused by one of four serotypes of the dengue virus (DENV) and is endemic in approximately 130 countries. The incidence of dengue has increased dramatically in recent decades, as well as the frequency and magnitude of outbreaks. Despite all efforts, there are no prophylactic or therapeutic treatments for the disease. Accordingly, research on the processes governing the DENV infection cycle is essential to develop vaccines or antiviral therapies. One of the most attractive DENV molecules to investigate is nonstructural protein 3 (NS3), which is essential for viral replication and a major immune target for infection. Objective: To produce antibodies to support future studies on NS3 and its cellular interactions with other proteins. Materials and methods: Two recombinant proteins of the helicase domain of DENV NS3 serotype 2 were expressed, and used to immunize mice and produce polyclonal antibodies. Results: The antibodies produced were useful in Western blot and immunofluorescence tests. We report an NS3 antibody that immunoprecipitates the viral protein and detects it in Western blot with no need to over-express it or use cell extracts with metabolic radiolabeling.

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Development of Real-Time Reverse Transcriptase PCR Assays To Detect and Serotype Dengue Viruses

Cited by 193 publications

References 29 publications

Dengue: a continuing global threat

Dengue: a continuing global threat

Utility of a Commercial Nonstructural Protein 1 Antigen Capture Kit as a Dengue Virus Diagnostic Tool

Polyclonal antibodies against recombinant dengue virus NS3 protein

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