A gradient-free method for the purification of infective dengue virus for protein-level investigations

Jensen, Stephanie M.; Nguyen, Celina T.; Jewett, John C.

doi:10.1016/j.jviromet.2016.05.017

Cited by 9 publications

(8 citation statements)

References 29 publications

(27 reference statements)

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“…Virus was purified using a modified version of a previously published protocol ( 64 ). In brief, virus-containing media (9 mL) was layered on top of 3 mL of sterile-filtered 20% (wt/vol) sucrose and spun at 167,000 × g (Sorvall Surespin 630) for 3 h at 4 °C to pellet virus.…”

Section: Methodsmentioning

confidence: 99%

Small-molecule endoplasmic reticulum proteostasis regulator acts as a broad-spectrum inhibitor of dengue and Zika virus infections

Almasy

Davies

Lisy

et al. 2021

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

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Flaviviruses, including dengue and Zika, are widespread human pathogens; however, no broadly active therapeutics exist to fight infection. Recently, remodeling of endoplasmic reticulum (ER) proteostasis by pharmacologic regulators, such as compound 147, was shown to correct pathologic ER imbalances associated with protein misfolding diseases. Here, we establish an additional activity of compound 147 as an effective host-centered antiviral agent against flaviviruses. Compound 147 reduces infection by attenuating the infectivity of secreted virions without causing toxicity in host cells. Compound 147 is a preferential activator of the ATF6 pathway of the ER unfolded protein response, which requires targeting of cysteine residues primarily on protein disulfide isomerases (PDIs). We find that the antiviral activity of 147 is independent of ATF6 induction but does require modification of reactive thiols on protein targets. Targeting PDIs and additional non-PDI targets using RNAi and other small-molecule inhibitors was unable to recapitulate the antiviral effects, suggesting a unique polypharmacology may mediate the activity. Importantly, 147 can impair infection of multiple strains of dengue and Zika virus, indicating that it is suitable as a broad-spectrum antiviral agent.

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Section: Methodsmentioning

confidence: 99%

Small-molecule endoplasmic reticulum proteostasis regulator acts as a broad-spectrum inhibitor of dengue and Zika virus infections

Almasy

Davies

Lisy

et al. 2021

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

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“…Cells were lysed with RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8,0, 1× protease inhibitor cocktail) and centrifuged at 13,000 g for 15 min at 4 ℃. For the analysis of S proteins loaded onto pseudoviruses, the MLV-containing culture supernatant was subjected to ultracentrifugation (150,000 g for 2 h at 4 ℃) over 20% (w/v) sucrose cushion in a TL-100 centrifuge using the TLA100.3 rotor to collect pseudovirus pellets, as described 45 . Subsequently, both pseudovirus samples and cell lysates were subjected to SDS-PAGE, and the proteins were transferred to a PVDF membrane (GE Healthcare Life Sciences, Chicago, IL, USA; cat.…”

Section: Methodsmentioning

confidence: 99%

Enhanced Omicron subvariant cross-neutralization efficacy of a monovalent SARS-CoV-2 BA.4/5 mRNA vaccine encoding a noncleaved, nonfusogenic spike antigen

Seo,

Jung,

Woo

et al. 2023

Preprint

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The rapid emergence of diverse SARS-CoV-2 variants, notably the Omicron variant, poses challenges to vaccine development. Here, we present a noncleaved, nonfusogenic spike (S) protein eliciting robust B- and T-cell immune responses against Omicron BA.5. The antigen incorporates the R685S and R815A mutations, effectively preventing the shedding of the S1 subunit and eliminating fusogenic activity of the resulting S antigen, termed S(SA). Through reverse genetic analysis, we found that the noncleaved form S protein with the R685S mutation enhances ACE2-dependent viral entry in vitro compared to the wild-type S protein, without increasing the virulence of the mutant virus in mice. The mRNA vaccine encoding the Omicron BA.4/5 S(SA) antigen conferred protective immunity in mice following two doses of 1 μg Ψ-UTP- or UTP-incorporated mRNA vaccines. Despite a roughly 6-fold reduction in neutralizing potency, both mRNA vaccines exhibited broad neutralizing efficacy against Omicron subvariants, including the XBB lineage variants XBB.1.5 and XBB.1.16.

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“…The virus obtained from the sample was isolated and propagated in C6/36 cells, sequenced by the Sanger platform, and identified with the following access number in Genbank: KM279427.1. Viruses were propagated on C6/36 cells in Leibovitz L-15 media, supplemented with fetal bovine serum (5–10%), incubated at room temperature (25–28 °C) without a CO 2 atmosphere in closed culture bottles, and purified by a traditional gradient-free ultracentrifugation protocol in a sucrose solution followed by selective virion precipitation as previously reported [ 24 ]. Viral isolates were titrated using the protocol for the formation of lytic foci or plaques in Huh-7 cells, stained with Naphtol blue-black dye and Median Tissue Culture Infectious Dose (TCID 50 ), and the plaque-forming unit per mL (PFU/mL) was calculated by viral titration [ 25 ]; Equations and calculations can be viewed in Supplementary Information S1.…”

Section: Methodsmentioning

confidence: 99%

Development of a Rapid Gold Nanoparticle-Based Lateral Flow Immunoassay for the Detection of Dengue Virus

et al. 2022

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Flavivirus detection in humans and mosquito reservoirs has been an important issue since it can cause a variety of illnesses and could represent a health problem in geographical zones where the vector is endemic. In this work, we designed and characterized a biosensor based on gold nanoparticles (AuNPs) and antibody 4G2 for the detection of dengue virus (DENV) in vitro, obtaining different conjugates (with different antibody concentrations). The AuNP–4G2 conjugates at concentrations of 1, 3, and 6 µg/mL presented an increase in the average hydrodynamic diameter compared to the naked AuNPs. Also, as part of the characterization, differences in the UV-Vis absorbance spectrum and electrophoretic migration were observed between the conjugated AuNPs (with BSA or antibody) and naked AuNPs. Additionally, we used this biosensor (AuNP–4G2 conjugate with 3 µg/mL antibody) in the assembly of a competitive lateral flow assay (LFA) for the development of an alternative test to detect the flavivirus envelope protein in isolated DENV samples as a future tool for dengue detection (and other flaviviruses) in the mosquito vector (Aedesaegypti) for the identification of epidemic risk regions. Functionality tests were performed using Dengue virus 2 isolated solution (TCID50/mL = 4.58 × 103) as a positive sample and PBS buffer as a negative control. The results showed that it is possible to detect Dengue virus in vitro with this gold nanoparticle-based lateral flow assay with an estimated detection limit of 5.12 × 102 PFU. We suggest that this biosensor could be used as an additional detection tool by coupling it to different point-of-care tests (POCT) for the easy detection of other flaviviruses.

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A gradient-free method for the purification of infective dengue virus for protein-level investigations

Cited by 9 publications

References 29 publications

Small-molecule endoplasmic reticulum proteostasis regulator acts as a broad-spectrum inhibitor of dengue and Zika virus infections

Small-molecule endoplasmic reticulum proteostasis regulator acts as a broad-spectrum inhibitor of dengue and Zika virus infections

Enhanced Omicron subvariant cross-neutralization efficacy of a monovalent SARS-CoV-2 BA.4/5 mRNA vaccine encoding a noncleaved, nonfusogenic spike antigen

Development of a Rapid Gold Nanoparticle-Based Lateral Flow Immunoassay for the Detection of Dengue Virus

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