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. 2022 Dec 21;10(6):e0394922.
doi: 10.1128/spectrum.03949-22. Epub 2022 Nov 29.

Defective Interfering Particles with Broad-Acting Antiviral Activity for Dengue, Zika, Yellow Fever, Respiratory Syncytial and SARS-CoV-2 Virus Infection

Affiliations

Defective Interfering Particles with Broad-Acting Antiviral Activity for Dengue, Zika, Yellow Fever, Respiratory Syncytial and SARS-CoV-2 Virus Infection

Min-Hsuan Lin et al. Microbiol Spectr. .

Abstract

More than 100 arboviruses, almost all of which have an RNA genome, cause disease in humans. RNA viruses are causing unprecedented health system challenges worldwide, many with little or no specific therapies or vaccines available. Certain species of mosquito can carry dengue virus (DENV), Zika virus (ZIKV) and yellow fever virus (YFV), where co-infection of these viruses has occurred. Here, we found that purified synthetic defective interfering particles (DIPs) derived from DENV type 2 (DENV-2) strongly suppressed replication of the aforementioned viruses, respiratory syncytial virus (RSV) and also the novel emerging virus SARS-CoV-2 in human cells. DENV DIPs produced in bioreactors, purified by column chromatography, and concentrated are virus-like particles that are about half the diameter of a typical DENV particle, but with similar ratios of the viral structural proteins envelope and capsid. Overall, DIP-treated cells inhibited DENV, ZIKV, YFV, RSV, and SARS-CoV-2 by at least 98% by mechanisms which included interferon (IFN)-dependent cellular antiviral responses. IMPORTANCE DIPs are spontaneously derived virus mutants with deletions in genes that block viral replication. DIPs play important roles in modulation of viral disease, innate immune responses, virus persistence and virus evolution. Here, we investigated the antiviral activity of highly purified synthetic DIPs derived from DENV, which were produced in bioreactors. DENV DIPs purified by column chromatography strongly inhibited five different RNA viruses, including DENV, ZIKV, YFV, RSV, and SARS-CoV-2 in human cells. DENV DIPs inhibited virus replication via delivery of a small, noninfectious viral RNA that activated cellular innate immunity, resulting in robust type 1 interferon responses. The work here presents a pathway for DIP production which is adaptable to Good Manufacturing Practice, so that their preclinical testing should be suitable for evaluation in subjects.

Keywords: RNA; SARS-CoV-2; Zika virus; antiviral therapy; chromatography; coronavirus; defective interfering particle; defective viral genome; dengue virus; purification; respiratory syncytial virus; yellow fever virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Enhancement of dengue virus (DENV) defective interfering particle (DIP) production in HEK-DI-290-ORF cells by the PKC/NF-κB activating agents. HEK-DI-290-ORF cells were treated with PEP005 (50 nM) or prostratin (2 μM) for 2 days. (A) Cell density in no-treatment control, PEP005-treated, and prostratin-treated groups on day 0 and day 2. (B) Levels of DI-290 RNA in culture supernatants were measured by reverse transcription-quantitative PCR (RT-qPCR). Data are presented as fold change relative to the no-treatment control group. Data are expressed as the mean ± standard deviation (SD) from three experiments. P values were calculated using a two-tailed Student’s t test.
FIG 2
FIG 2
Three-step chromatographic purification of DENV DIPs. (A) Chromatogram of the multimodal Capto Core 400 (CC400) column step of DENV DIP purification. The flowthrough fraction collected is indicated. (B) Chromatogram of the capture step for DENV DIPs using ceramic hydroxyapatite (CHT) medium, which was washed with 10 mM sodium phosphate buffer (pH 7.2) and eluted with an isocratic gradient using 0.6 M sodium phosphate buffer (pH 7.2). The fractions collected in the elute phase are indicated. (C) Samples from each fraction underwent ultracentrifugation. The pelleted material was collected and assayed for DI-290 RNA by RT-qPCR. The mean numbers of DI-290 RNA copies/mL measured in each fraction in triplicate assays are shown. Independent experiments were conducted where DI-290 RNA was always associated with the milli-absorbance unit (mAU) peak absorbance (ABS) at 280 nm. (D) The CHT elution peak as applied to a Cytiva G-25 desalting column equilibrated in 1× PBS and operated in flowthrough mode.
FIG 3
FIG 3
Size and morphology of purified DENV DIP particles. (A) Samples containing unpurified DENV DIPs, purified DIPs, or infectious DENV-2 underwent ultracentrifugation. The pelleted material was solubilized and used for Western blot analysis using anti-DENV antibodies directed to the viral envelope (E) or capsid (CA) proteins. (B) Dynamic light scattering (DLS) analysis of purified DENV DIP particle size distribution or (C) DLS of the same DENV DIP preparation after an additional 7 days of storage at 4°C. (D) Transmission electron microscopy (TEM) images (scale bar = 100 nM) of purified DENV DIPs.
FIG 4
FIG 4
DENV DIPs stimulate host innate immune responses. (A) Huh7 cells were incubated with DENV-2 (MOI [multiplicity of infection] = 0.01 CCID50 [50% cell culture infective dose]/cell) alone, or DENV-2 mixed with DENV DIP (equivalent to 1,000 DI-290 RNA copies/cell) for 15 h, and then the culture medium was replaced. Culture supernatants were collected at 72 h postinfection (hpi). Viral titers in culture supernatants were measured by a CCID50 assay. (B to F) To examine host innate immune response, Huh7 cells were treated with DENV-2 (MOI = 0.01 CCID50/cell, red line), DENV DIP (equivalent to 1,000 DI-290 RNA copies/cell, black line), or DENV-2 mixed with DENV DIP (green line) for 2, 24, and 72 h. Total RNA was extracted from the cells and the levels of interferon (IFN)-α, IFN-β, ISG15, protein kinase R (PKR), and 2′–5′ oligoadenylate synthetase 1 (OAS1) mRNA were quantified by RT-qPCR. The fold change relative to the untreated control cells was calculated (Ctrl, blue line). Data are shown as means ± SD from three replicate experiments. The P values were calculated using a two-tailed Student’s t test.
FIG 5
FIG 5
DENV DIP inhibits Zika virus (ZIKV), yellow fever virus (YFV), respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Huh7 or HEp-2 cells were incubated with ZIKV (MOI = 0.01 CCID50/cell), ZIKV mixed with DENV DIP (equivalent to 1,000 DI-290 RNA copies/cell), YFV (MOI = 0.01 CCID50/cell), YFV mixed with DENV DIP, RSV (MOI = 0.5 CCID50/cell), RSV mixed with DENV DIP, SARS-CoV-2 (MOI = 0.01 CCID50/cell), or SARS-CoV-2 mixed with DENV DIP for 15 h, after which the culture medium was replaced. Culture supernatants were collected at 72 hpi. Viral titers in culture supernatant samples were measured by CCID50 assay (A to D). (E) Level of virus replication inhibition by DIPs. Data are shown as means ± SD from at least three replicate experiments. P values were calculated using a two-tailed Student’s t test.

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