78 research outputs found
Image1_Combination of the parent analogue of remdesivir (GS-441524) and molnupiravir results in a markedly potent antiviral effect in SARS-CoV-2 infected Syrian hamsters.pdf
Remdesivir was the first antiviral drug to be approved for the treatment of severe COVID-19; followed by molnupiravir (another prodrug of a nucleoside analogue) and the protease inhibitor nirmatrelvir. Combination of antiviral drugs may result in improved potency and help to avoid or delay the development of resistant variants. We set out to explore the combined antiviral potency of GS-441524 (the parent nucleoside of remdesivir) and molnupiravir against SARS-CoV-2. In SARS-CoV-2 (BA.5) infected A549-Dual™ hACE2-TMPRSS2 cells, the combination resulted in an overall additive antiviral effect with a synergism at certain concentrations. Next, the combined effect was explored in Syrian hamsters infected with SARS-CoV-2 (Beta, B.1.351); treatment was started at the time of infection and continued twice daily for four consecutive days. At day 4 post-infection, GS-441524 (50 mg/kg, oral BID) and molnupiravir (150 mg/kg, oral BID) as monotherapy reduced infectious viral loads by 0.5 and 1.6 log10, respectively, compared to the vehicle control. When GS-441524 (50 mg/kg, BID) and molnupiravir (150 mg/kg, BID) were combined, infectious virus was no longer detectable in the lungs of 7 out of 10 of the treated hamsters (4.0 log10 reduction) and titers in the other animals were reduced by ∼2 log10. The combined antiviral activity of molnupiravir which acts by inducing lethal mutagenesis and GS-441524, which acts as a chain termination appears to be highly effective in reducing SARS-CoV-2 replication/infectivity. The unexpected potent antiviral effect of the combination warrants further exploration as a potential treatment for COVID-19.</p
The Viral Polymerase Inhibitor 7-Deaza-2’-<i>C</i>-Methyladenosine Is a Potent Inhibitor of <i>In Vitro</i> Zika Virus Replication and Delays Disease Progression in a Robust Mouse Infection Model
<div><p>Zika virus (ZIKV) is an emerging flavivirus typically causing a dengue-like febrile illness, but neurological complications, such as microcephaly in newborns, have potentially been linked to this viral infection. We established a panel of <i>in vitro</i> assays to allow the identification of ZIKV inhibitors and demonstrate that the viral polymerase inhibitor 7-deaza-2’-<i>C</i>-methyladenosine (7DMA) efficiently inhibits replication. Infection of AG129 (IFN-α/β and IFN-γ receptor knock-out) mice with ZIKV resulted in acute neutrophilic encephalitis with viral antigens accumulating in neurons of the brain and spinal cord. Additionally, high levels of viral RNA were detected in the spleen, liver and kidney, and levels of IFN-γ and IL-18 were systematically increased in serum of ZIKV-infected mice. Interestingly, the virus was also detected in testicles of infected mice. In line with its <i>in vitro</i> anti-ZIKV activity, 7DMA reduced viremia and delayed virus-induced morbidity and mortality in infected mice, which also validates this small animal model to assess the <i>in vivo</i> efficacy of novel ZIKV inhibitors. Since AG129 mice can generate an antibody response, and have been used in dengue vaccine studies, the model can also be used to assess the efficacy of ZIKV vaccines. </p></div
Viral replication kinetics of ZIKV and time-of-drug-addition studies.
<p>In viral kinetics studies, Vero cells were infected with ZIKV at an MOI~1.0 and harvested at the indicated time points pi. Data are expressed as percentage viral replication compared to viral RNA replication in infected cells at 24 hours pi (white circles). In time-of-drug-addition studies, ZIKV-infected cells were treated with 7DMA (178 μM; black bars) or ribavirin (205 μM; grey bars) at different time points pi. Cells were harvested at 24 hours pi and viral RNA was extracted and quantified by RT-qPCR. Data are expressed as percentage inhibition of viral replication compared to viral RNA replication in untreated, infected cells at 24 hours pi.</p
Establishment and characterization of an animal model for ZIKV infection.
<p>Male (8–14 weeks of age) 129/Sv mice deficient in both IFN-alpha/beta (IFN-α/β) and IFN-gamma (IFN-γ) receptors (AG129) were inoculated intraperitoneally with 200 μL of different inoculums (ranging from 1×10<sup>1</sup>–1×10<sup>5</sup> PFU/ml) of ZIKV. Mice were observed daily for body weight loss and the development of virus-induced disease. (<b>A</b>) Median day of euthanasia (MDE) is as follows: day 13.5, 15.0, 14.0, 14.5 and 18.5 pi for mice inoculated with 1×10<sup>5</sup> (n = 6), 1×10<sup>4</sup> (n = 6), 1×10<sup>3</sup> (n = 5), 1×10<sup>2</sup> (n = 2) and 1×10<sup>1</sup> (n = 4) PFU/mL, respectively. (<b>B</b>) Viral RNA load in brain (n = 7), spleen (n = 5), kidney (n = 5) and liver (n = 6) from ZIKV-infected mice as determined by RT-qPCR. Levels of IFN-γ (<b>C</b>) and IL-18 (<b>D</b>) were significantly increased throughout the course of infection in sera of AG129 mice (grey boxes) compared to those in sera of uninfected AG129 mice (white boxes). Statistical analysis was performed using the unpaired, two-tailed t-test. *, p<0.05.</p
Presence of ZIKV antigens in the brain (A), spinal cord (D) and liver (E) of ZIKV-infected AG129 mice, whereas ZIKV antigens were absent in tissues of uninfected mice (brain, B), as shown by histopathological analysis.
<p>Infiltration of neutrophils is shown in the brain of ZIKV-infected mice (as detected by hematoxylin-eosin staining; C), but not in the brain of uninfected mice (F).</p
In vivo efficacy of 7DMA against ZIKV. AG129 mice (male, 8–14 weeks of age; n = 9) were treated with 50 mg/kg/day 7DMA sodium carboxymethylcellulose (CMC-Na)] <i>via</i> oral gavage or with vehicle [0.5% or 0.2% CMC-Na; n = 9] for 10 days.
<p>Mice were infected intraperitoneally with 200 μL of a 1×10<sup>4</sup> PFU/mL stock of ZIKV 1 hour after the first treatment on day 0. (<b>A</b>) Percentage survival between ZIKV-infected mice treated with vehicle (● and ■) or 7DMA (○ and □) was compared using the Log-rank (Mantel-Cox) test. Data represent results from 2 independently performed studies. (<b>B</b>) Viral RNA load in serum on day 1, 2, 3, 5, 6, 7 and 8 pi of ZIKV-infected mice treated with vehicle (white boxes) or 7DMA (grey boxes), as determined by RT-qPCR. Statistical analysis was performed using the unpaired, two-tailed t-test. Data are representative of 2 independent experiments. (<b>C</b>) Viral RNA load in testicles of vehicle-treated, ZIKV-infected AG129 mice at day 5 pi, as determined by RT-qPCR. (<b>D</b>) Expression at different time points pi of IFN-γ in sera of ZIKV-infected mice treated with vehicle (white boxes) or 7DMA (grey boxes), as determined using the ProcartaPlex Mouse IFN- γ, IL-18, IL-6, IP-10, TNF-α Simplex kit (e-Bioscience). Data represent results from 2 independent experiments.</p
Dose-dependent inhibition of ZIKV RNA replication by 7DMA.
<p>(<b>A</b>) Vero cell cultures infected with ZIKV strain MR766 were treated with different concentrations of 7DMA. Viral RNA levels in the supernatant were quantified on day 4 pi by means of RT-qPCR and are expressed as percentage inhibition of untreated virus control (black bars). Mock-infected cells were treated with the same dilution series of 7DMA. Cell viability was determined by means of the MTS/PMS method and is expressed as percentage of cell growth of untreated control (white circles). Data represent mean values ± standard deviations (SD) for three independent experiments. Log<sub>10</sub> reduction values in viral RNA load are depicted in italics at the top of each bar. (<b>B</b>) Antiviral activity of 7DMA against ZIKV as determined in an immunofluorescence assay. At a concentration of 11 μM, 7DMA almost completely blocked viral antigen expression (left panel) compared to untreated, infected cells (right panel) and infected cells treated at a lower concentration (5.6 and 2.8 μM; two panels in the middle).</p
Antiviral and metabolic activity of a selection of compounds against ZIKV strain MR766.
<p>Antiviral and metabolic activity of a selection of compounds against ZIKV strain MR766.</p
Dose-dependent antiviral activity of HHA, GNA and UDA in DENV-infected MDDC.
<p>(A) MDDC were infected with DENV-2 in the absence (−) or presence of dose-dependent concentrations of HHA. The number of DENV-2 positive cells was determined by flow cytometry using 5 µg/ml anti-DENV antibody recognizing the E-protein of DENV-2 (clone 3H5). In each plot, the number of DENV positive cells is indicated. (B) MDDC were infected with the four serotypes of DENV in the presence or absence of various concentrations of HHA, GNA and UDA. DENV infection was analyzed by flow cytometry using an anti-PrM antibody recognizing all four DENV serotypes (clone 2H2). % of infected cells compared to the positive virus control (VC) ± SEM of 4 to 12 different blood donors is shown.</p
Antiviral assays with DENV-2 in Raji/DC-SIGN<sup>+</sup> cells and HHA.
<p>Raji/DC-SIGN<sup>+</sup> cells were infected with DENV-2 under four different experimental conditions. (•) Standard antiviral assay: cells were infected with DENV-2 together with HHA (2000-400-80-16-3.2 nM) and incubated at 37°C to allow internalization of the virus. (□) Prebinding assay: cells were pretreated with HHA (2000-400-80-16 nM) before binding to DENV-2. (▴) Postbinding assay: cells were infected with DENV-2 and after washing to remove unbound virus, HHA was added dose-dependently (2000-400-80-16 nM). (∇) Pre-exposure assay: cell-free DENV-2 was pre-exposed to HHA prior to exposure to Raji/DC-SIGN<sup>+</sup> cells. Raji/DC-SIGN<sup>+</sup> cells were infected with a 50-fold dilution of HHA-exposed DENV (final HHA concentration: 40-8-1.6-0.32 nM). For all four conditions, cells were washed twice to remove virus and/or compound and collected after 4 days. Viral infectivity was analyzed by flow cytometry. Data represent the mean % of inhibition of viral replication ± SEM of 3 independent experiments.</p
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