Flow Cytometry-Based Assay for Titrating Dengue Virus

Lambeth, Cassandra; White, Laura; Johnston, Robert E.; Silva, A. M. de

doi:10.1128/jcm.43.7.3267-3272.2005

Cited by 117 publications

(106 citation statements)

References 18 publications

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“…Wells that expressed at least one E-expressing cell were counted as positive, and the median tissue culture infective dose (TCID 50 ) was calculated according to the method of Reed and Muench (37). For DENV-GFP, infectious unit titers were also determined by limiting dilution and quantitation of GFP positivity by FACS, as described previously (38).…”

Section: Methodsmentioning

confidence: 99%

Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

Schoggins

Dorner

Feulner

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

166

171

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Dengue virus (DENV) is a global disease threat for which there are no approved antivirals or vaccines. Establishing state-of-the-art screening systems that rely on fluorescent or luminescent reporters may accelerate the development of anti-DENV therapeutics. However, relatively few reporter DENV platforms exist. Here, we show that DENV can be genetically engineered to express a green fluorescent protein or firefly luciferase. Reporter viruses are infectious in vitro and in vivo and are sensitive to antiviral compounds, neutralizing antibodies, and interferons. Bioluminescence imaging was used to follow the dynamics of DENV infection in mice and revealed that the virus localized predominantly to lymphoid and gut-associated tissues. The high-throughput potential of reporter DENV was demonstrated by screening a library of more than 350 IFN-stimulated genes for antiviral activity. Several antiviral effectors were identified, and they targeted DENV at two distinct life cycle steps. These viruses provide a powerful platform for applications ranging from validation of vaccine candidates to antiviral discovery. Flaviviridae family that causes significant morbidity and mortality worldwide. Each year, over 50 million people are affected by dengue fever, and ∼500,000 are hospitalized with the more severe dengue hemorrhagic fever (1). The virus is endemic to tropical environments in Southeast Asia, the Pacific, and the Americas, and has recently reemerged as far north as southern Florida (2). Currently, no vaccines or antivirals have been approved for prevention or treatment of DENV infection.Four genetically and antigenically distinct DENV serotypes circulate, and each can be isolated from infected human sera and propagated in cell culture. The pathogenesis and immune response to patient-derived virus can be studied in vitro and in vivo by quantifying viral genomes or antigens. These detection methods are also used to study the molecular virology of DENV with reagents such as virus-like particles (VLPs) and subgenomic replicons. In some cases, VLPs and subgenomic replicons have been engineered to take advantage of reporter proteins, such as luciferase, which can be used in high-throughput screening platforms for discovery of inhibitors of viral entry or replication (3). A caveat to these tools is the inability to fully recapitulate the entire viral life cycle. This can be overcome by launching virus production from full-length infectious molecular clones, which have been generated for DENV serotypes 1, 2, and 4 (4-6).Full-length flavivirus infectious clones are often difficult to work with, largely due to instability in various bacterial cell lines and the inability to rescue sufficient quantities of DNA for downstream applications (7). Additionally, mutagenesis of viral sequences may result in disruption of the various long-range interactions required for establishment of a productive replication complex (8-10). Thus, strategies to generate infectious viruses expressing heterologous sequences have to contend with both sub...

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Section: Methodsmentioning

confidence: 99%

Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

Schoggins

Dorner

Feulner

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

166

171

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“…Mouse serum samples collected at days 9, 12, and 25 were tested for neutralization activity in a fluorescenceactivated cell sorting (FACS) neutralization test (FNT) in C6/36 cells (27). Briefly, 96-well plates were seeded at a density of 1.3 ϫ 10 5 cell/well and incubated overnight at 28°C.…”

Section: Generation Of Blt-nod/scid Mice Nodcg-prkdcmentioning

confidence: 99%

Utility of Humanized BLT Mice for Analysis of Dengue Virus Infection and Antiviral Drug Testing

Frias-Stäheli

Dorner

Marukian

et al. 2014

J Virol

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Dengue virus (DENV) is the cause of a potentially life-threatening disease that affects millions of people worldwide. The lack of a small animal model that mimics the symptoms of DENV infection in humans has slowed the understanding of viral pathogenesis and the development of therapies and vaccines. Here, we investigated the use of humanized "bone marrow liver thymus" (BLT) mice as a model for immunological studies and assayed their applicability for preclinical testing of antiviral compounds. Human immune system (HIS) BLT-NOD/SCID mice were inoculated intravenously with a lowpassage, clinical isolate of DENV-2, and this resulted in sustained viremia and infection of leukocytes in lymphoid and nonlymphoid organs. In addition, DENV infection increased serum cytokine levels and elicited DENV-2-neutralizing human IgM antibodies. Following restimulation with DENV-infected dendritic cells, in vivo-primed T cells became activated and acquired effector function. An adenosine nucleoside inhibitor of DENV decreased the circulating viral RNA when administered simultaneously or 2 days postinfection, simulating a potential treatment protocol for DENV infection in humans. In summary, we demonstrate that BLT mice are susceptible to infection with clinical DENV isolates, mount virusspecific adaptive immune responses, and respond to antiviral drug treatment. Although additional refinements to the model are required, BLT mice are a suitable platform to study aspects of DENV infection and pathogenesis and for preclinical testing of drug and vaccine candidates. IMPORTANCEInfection with dengue virus remains a major medical problem. Progress in our understanding of the disease and development of therapeutics has been hampered by the scarcity of small animal models. Here, we show that humanized mice, i.e., animals engrafted with components of a human immune system, that were infected with a patient-derived dengue virus strain developed clinical symptoms of the disease and mounted virus-specific immune responses. We further show that this mouse model can be used to test preclinically the efficacy of antiviral drugs.

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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%

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-Based Assay for Titrating Dengue Virus

Cited by 117 publications

References 18 publications

Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

Utility of Humanized BLT Mice for Analysis of Dengue Virus Infection and Antiviral Drug Testing

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

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