Flow Cytometry Compared with Indirect Immunofluorescence for Rapid Detection of Dengue Virus Type 1 after Amplification in Tissue Culture

Kao, Chuan-Liang; Wu, Meng-Chan; Chiu, Yi-Jui; Lin, Jing-Lin; Wu, Yin-Chang; Y, Yueh; Chen, Li‐Kuang; Shaio, Men‐Fang; King, Chwan-Chuen

doi:10.1128/jcm.39.10.3672-3677.2001

Cited by 34 publications

(25 citation statements)

References 31 publications

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“…Sera that have been collected from suspected dengue cases in the first 3-5 days of fever (the viraemic phase) can be used for virus isolation. After an incubation period permitting virus replication, viral identification is performed using dengue-specific monoclonal antibodies in immunofluorescence and PCR assays 63,64,72,73 . Serum is often used for virus isolation but plasma, leukocytes, whole blood and tissues obtained at autopsy can also be used 63,74,75 .…”

Section: Virus Isolationmentioning

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: Virus Isolationmentioning

confidence: 99%

Dengue: a continuing global threat

et al. 2010

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“…We decided to harvest cells at 24 hpi for the FACS titration assay, because the first round of infection peaks at this time point and also because 24 hpi is too early to detect cell-to-cell spread of virus. Previous studies have also indicated that there is minimal cell-to-cell spread of DENV in C6/36 cells at 24 hpi (8). Next, we determined if the FACS assay was able to detect quantitative differences in the number of virus particles in samples.…”

Section: Resultsmentioning

confidence: 94%

“…Within the past 10 years, fluorescence-activated cell sorter (FACS)-based methods have been developed to follow infection and determine titers of viruses such as human immunodeficiency virus, herpesvirus, measles virus, influenza virus, Epstein-Barr virus, and rabies virus (1,4,12,13,16). FACS has also been used to detect DENV in clinical samples and to measure the ability of the virus to infect a variety of cells (2,3,7,8,11,17). Here we report on a FACS-based assay for titrating DENVs and for characterizing the ability of antisera to neutralize the virus.…”

mentioning

confidence: 99%

Flow Cytometry-Based Assay for Titrating Dengue Virus

et al. 2005

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Plaque assays for titrating dengue virus (DENV) are time-consuming and not suitable for strains that do not plaque. Fluorescence-activated cell sorting (FACS) has been used to detect DENV-infected cells. Here we describe a FACS-based assay for titrating DENV. We determined that at 24 h postinfection, the number of infected cells detected by FACS represented the first round of infection and therefore could be used as a readout of the number of infectious particles in the inoculum. When the titers of different laboratory and clinical strains of DENV were compared using FACS, plaque, and endpoint dilution assays, for most strains the FACS titers were comparable to titers obtained by plaque or endpoint dilution assays. The FACS assay is an improvement over the plaque assay because the infection period is reduced from 5 to 7 days to 24 h and the assay can be used to titrate clinical isolates that frequently do not form clear plaques on cell monolayers. The novel FACS-based methods described here will facilitate laboratory studies of dengue.Dengue is a mosquito-borne viral disease of global public health significance. Throughout the world there are more than 2.5 billion people at risk of dengue virus (DENV) infection. Each year there are an estimated 100 million cases of dengue viral infection worldwide, with 250,000 people developing the more severe dengue hemorrhagic fever/dengue shock syndrome, which is often fatal (5). DENV is a positive-polarity RNA virus in the family Flaviviridae, and the DENV complex consists of four distinct serotypes designated DENV1 through DENV4 (10).Laboratory studies of dengue virus are difficult because the virus does not grow to high titers in cell culture and assays for titrating virus and measuring virus neutralization are timeconsuming. These problems are exacerbated when working with primary clinical isolates of virus. Standard methods for titrating DENV and measuring the ability of antisera to neutralize the virus are based on plaque assays which require 5 to 7 days to complete. Some isolates, especially among primary clinical isolates, do not form clear plaques on cell monolayers. Better methods for titrating the virus and measuring the ability of antisera to neutralize dengue need to be developed. Within the past 10 years, fluorescence-activated cell sorter (FACS)-based methods have been developed to follow infection and determine titers of viruses such as human immunodeficiency virus, herpesvirus, measles virus, influenza virus, Epstein-Barr virus, and rabies virus (1,4,12,13,16). FACS has also been used to detect DENV in clinical samples and to measure the ability of the virus to infect a variety of cells (2,3,7,8,11,17). Here we report on a FACS-based assay for titrating DENVs and for characterizing the ability of antisera to neutralize the virus. MATERIALS AND METHODSCells, viruses, and antisera. Aedes albopictus C6/36 mosquito cells were obtained from the Centers for Disease Control and Prevention, Fort Collins. Vero 76 (African Green monkey kidney) cells were purchased fro...

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“…Additionally, this approach has been used to diagnose and titer flaviviruses of importance in veterinary and human public health, such as bovine viral diarrhea virus and DENV [18][19][20] . In this study, we showed that the DENV in clinical samples of patients with acute febrile symptoms can be rapidly detected using PBMCs by FACS, as demonstrated by the observed 29.1% DENV positivity that includes two patients with DENV-1 and 42 patients with DENV-2.…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, the FACS approach diagnosed a lower number of DENV-positive patients compared to the NS1 assay. This could be probably due to the multiple antigenic determinants that are expressed, both intracellularly and on the cell surface, in the NS1 protein, which is involved in enhancing viral RNA replication and also in the release of infectious viral particles 19 . Additionally, the temporal increase in antiviral immunity, including the appearance of neutralizing antibodies, causes reduced DENV viremia and probably decreases the number of infected cells in blood 33 .…”

Section: Discussionmentioning

confidence: 99%

Evaluating the use of fluorescence-based flow cytometry assay for dengue diagnosis using peripheral blood mononuclear cells

Barreira

Scheucher

Romeiro

et al. 2018

Rev. Soc. Bras. Med. Trop.

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Introduction: Dengue virus (DENV) is the most important arthropod-borne viral disease worldwide with an estimated 50 million infections occurring each year. Methods: In this study, we present a flow cytometry assay (FACS) for diagnosing DENV, and compare its results with those of the non-structural protein 1 (NS1) immunochromatographic assay and reverse transcriptase polymerase chain reaction (RT-PCR). Results: All three assays identified 29.1% (39/134) of the patients as denguepositive. The FACS approach and real-time RT-PCR detected the DENV in 39 and 44 samples, respectively. On the other hand, the immunochromatographic assay detected the NS1 protein in 40.1% (56/134) of the patients. The Cohen's kappa coefficient analysis revealed a substantial agreement among the three methods. Conclusions: The FACS approach may be a useful alternative for dengue diagnosis and can be implemented in public and private laboratories.

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Flow Cytometry Compared with Indirect Immunofluorescence for Rapid Detection of Dengue Virus Type 1 after Amplification in Tissue Culture

Abstract: Dengue virus (DV) was detected early in infected mosquito

Cited by 34 publications

References 31 publications

Dengue: a continuing global threat

Dengue: a continuing global threat

Flow Cytometry-Based Assay for Titrating Dengue Virus

Evaluating the use of fluorescence-based flow cytometry assay for dengue diagnosis using peripheral blood mononuclear cells

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