A Viral Polymerase Inhibitor Reduces Zika Virus Replication in the Reproductive Organs of Male Mice

In humans, Zika virus and viral RNA have been detected in semen up to 2.2 months and 6 months post infection (pi), respectively. Although the contribution of sexual transmission to the spread of ZIKV is too low to sustain an outbreak, it can increase the risk of infection and the epidemic size as well as prolong the duration of an outbreak. In this study, we explored the potential of antivirals to serve as an effective strategy to prevent sexual transmission. Male AG129 mice infected with a ZIKV isolate from Suriname were treated with the nucleoside analog, 7-deaza-2′-C-methyladenosine (7DMA), that was previously shown to be efficacious in reducing ZIKV viremia and delaying ZIKV-induced disease in mice. Following treatment, viral RNA and infectious virus titers were consistently reduced in the male reproductive organs compared to vehicle-treated mice. This reduction of ZIKV loads in the testis was confirmed by the detection of lower levels of ZIKV antigens. Our data illustrate the value of this mouse model to validate the efficacy of new potential ZIKV drugs at the level of the male reproductive system

Small-molecule inhibitors of TBK1 serve as an adjuvant for a plasmid-launched live-attenuated yellow fever vaccine

Plasmid-launched live-attenuated vaccines (PLLAV), also called infectious DNA (iDNA) vaccines, combine the assets of genetic immunization with the potency of replication-competent live viral vaccines. However, due to their origin as bacterial plasmid DNA, efficient delivery of PLLAV may be hampered by innate signaling pathways such as the cGAS-STING-mediated sensing of cytosolic DNA, resulting in an unfavorable proinflammatory and antiviral response locally at the site of immunization. Employing several complementary cell-based systems and using the yellow fever vaccine (YF17D) and the respective PLLAV-YF17D, we screened a panel of small molecules known to interfere with antiviral signaling for their proviral activity and identified two potent inhibitors of the TANK-binding kinase 1 (TBK1), BX795 and CYT387, to enhance YF17D replication and hence efficacy of PLLAV-YF17D transfection. In tissue culture, BX795 could fully revert the block that plasmid transfection poses on YF17D infection in a type I interferon dependent manner, as confirmed by (i) a marked change in gene expression signatures, (ii) a rescue of full YF17D replication, and (iii) a massively increased virus yield. Inhibitors of TBK1 may hence be considered an adjuvant to potentiate novel PLLAV vaccines, which might boost PLLAV delivery toward their use in vivo

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

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

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

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

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

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

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

Comparing infectivity and virulence of emerging SARS-CoV-2 variants in Syrian hamsters

Background: Within one year after its emergence, more than 108 million people acquired SARS-CoV-2 and almost 2·4 million succumbed to COVID-19. New SARS-CoV-2 variants of concern (VoC) are emerging all over the world, with the threat of being more readily transmitted, being more virulent, or escaping naturally acquired and vaccine-induced immunity. At least three major prototypic VoC have been identified, i.e. the United Kingdom, UK (B.1.1.7), South African (B.1.351) and Brazilian (B.1.1.28.1) variants. These are replacing formerly dominant strains and sparking new COVID-19 epidemics. Methods: We studied the effect of infection with prototypic VoC from both B.1.1.7 and B.1.351 variants in female Syrian golden hamsters to assess their relative infectivity and virulence in direct comparison to two basal SARS-CoV-2 strains isolated in early 2020. Findings: A very efficient infection of the lower respiratory tract of hamsters by these VoC is observed. In line with clinical evidence from patients infected with these VoC, no major differences in disease outcome were observed as compared to the original strains as was quantified by (i) histological scoring, (ii) micro-computed tomography, and (iii) analysis of the expression profiles of selected antiviral and pro-inflammatory cytokine genes. Noteworthy however, in hamsters infected with VoC B.1.1.7, a particularly strong elevation of proinflammatory cytokines was detected. Interpretation: We established relevant preclinical infection models that will be pivotal to assess the efficacy of current and future vaccine(s) (candidates) as well as therapeutics (small molecules and antibodies) against two important SARS-CoV-2 VoC. Funding: Stated in the acknowledgment