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

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

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

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 1×10¹–1×10⁵ 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 1×10⁵ (n = 6), 1×10⁴ (n = 6), 1×10³ (n = 5), 1×10² (n = 2) and 1×10¹ (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

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. Log₁₀ 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

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 1×10⁴ 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

A lethal disease model for New World hantaviruses using immunosuppressed Syrian hamsters

<div><p>Background</p><p>Hantavirus, the hemorrhagic causative agent of two clinical diseases, is found worldwide with variation in severity, incidence and mortality. The most lethal hantaviruses are found on the American continent where the most prevalent viruses like Andes virus and Sin Nombre virus are known to cause hantavirus pulmonary syndrome. New World hantavirus infection of immunocompetent hamsters results in an asymptomatic infection except for Andes virus and Maporal virus; the only hantaviruses causing a lethal disease in immunocompetent Syrian hamsters mimicking hantavirus pulmonary syndrome in humans.</p><p>Methodology/Principal findings</p><p>Hamsters, immunosuppressed with dexamethasone and cyclophosphamide, were infected intramuscularly with different New World hantavirus strains (Bayou virus, Black Creek Canal virus, Caño Delgadito virus, Choclo virus, Laguna Negra virus, and Maporal virus). In the present study, we show that immunosuppression of hamsters followed by infection with a New World hantavirus results in an acute disease that precisely mimics both hantavirus disease in humans and Andes virus infection of hamsters.</p><p>Conclusions/ Significance</p><p>Infected hamsters showed specific clinical signs of disease and moreover, histological analysis of lung tissue showed signs of pulmonary edema and inflammation within alveolar septa. In this study, we were able to infect immunosuppressed hamsters with different New World hantaviruses reaching a lethal outcome with signs of disease mimicking human disease.</p></div

Survival curves of golden Syrian hamsters infected with New World hantaviruses.

<p>A) Infection with New World hantaviruses (BAYV, BCCV, CDV, SNV and ANDV) leading to a mortality rate of 100%. B) Infection with New World hantaviruses leading to a mortality rate of 75% for CHOV and 50% for LNV and MAPV. BAYV, CDV, BCCV, ANDV and SNV, Log-rank: p value <0.0001, Gehan-Breslow-Wilcoxon: p value = 0.0002; LNV and MAPV, Log-rank: p value = 0.0251, Gehan-Breslow-Wilcoxon: p value = 0.0265; and CHOV; Log-rank: p value = 0.0024, Gehan-Breslow-Wilcoxon: p value = 0.0032.</p

Dexamethasone and cyclophosphamide dosing scheme.

<p>Dexamethasone and cyclophosphamide dosing scheme.</p