A dengue type 2 reporter virus assay amenable to high-throughput screening.

Dengue virus (DV) is an important mosquito-borne flavivirus threatening almost half of the world's population. Prophylaxis and potent anti-DV drugs are urgently needed. Here, we developed a high content imaging-based (HCI) assay with DV type 2 expressing the fluorescent protein mCherry (DV2/mCherry) to improve the efficiency and robustness of the drug discovery process. For the construction of the reporter virus, the mCherry gene followed by the ribosome-skipping 2A sequence of the Thosea asigna virus (T2A) was placed upstream of the full DV2 open reading frame. The biological characteristics including mCherry expression, virus replication rate, and plaque phenotype was examined and validated in BHK-21, Vero and C6/36 cells. A robust image-based antiviral assay combined with an automated robotic system was then developed, with a Z' factor of 0.73. To validate the image-based antiviral assay, a panel of reference compounds with different molecular mechanisms of anti-DV activity was assessed: (i) the glycosylation inhibitor, Celgosivir, (ii) two NS4b-targeting compounds: a 3-Acyl-indole derivative and NITD618, and (iii) two nucleoside viral polymerase inhibitors, 2'CMC and 7DMA. The inhibition profiles were quantified and obtained by means of HCI and RT-qPCR. Both methods resulted in very comparable inhibition profiles. In conclusion, a powerful and robust assay was developed with a fully automated data generation and processing pipeline. It makes the new reporter virus assay amenable to high-throughput screening of large libraries of small molecules.

Mitigating the risk of Zika virus contamination of raw materials and cell lines in the manufacture of biologicals.

Ensuring the virological safety of biologicals is challenging due to the risk of viral contamination of raw materials and cell banks, and exposure during in-process handling to known and/or emerging viral pathogens. Viruses may contaminate raw materials and biologicals intended for human or veterinary use and remain undetected until appropriate testing measures are employed. The outbreak and expansive spread of the mosquito-borne flavivirus Zika virus (ZIKV) poses challenges to screening human- and animal -derived products used in the manufacture of biologicals. Here, we report the results of an in vitro study where detector cell lines were challenged with African and Asian lineages of ZIKV. We demonstrate that this pathogen is robustly detectable by in vitro assay, thereby providing assurance of detection of ZIKV, and in turn underpinning the robustness of in vitro virology assays in safety testing of biologicals.

The Viral Polymerase Inhibitor 7-Deaza-2'-C-Methyladenosine Is a Potent Inhibitor of In Vitro Zika Virus Replication and Delays Disease Progression in a Robust Mouse Infection Model.

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 in vitro assays to allow the identification of ZIKV inhibitors and demonstrate that the viral polymerase inhibitor 7-deaza-2'-C-methyladenosine (7DMA) efficiently inhibits replication. Infection of AG129 (IFN-α/ß 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 in vitro 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 in vivo 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. .

Synthetic strategy and antiviral evaluation of diamide containing heterocycles targeting dengue and yellow fever virus.

High-throughput screening of a subset of the CD3 chemical library (Centre for Drug Design and Discovery; KU Leuven) provided us with a lead compound 1, displaying low micromolar potency against dengue virus and yellow fever virus. Within a project aimed at discovering new inhibitors of flaviviruses, substitution of its central imidazole ring led to synthesis of variably substituted pyrazine dicarboxylamides and phthalic diamides, which were evaluated in cell-based assays for cytotoxicity and antiviral activity against the dengue virus (DENV) and yellow fever virus (YFV). Fourteen compounds inhibited DENV replication (EC50 ranging between 0.5 and 3.4 µM), with compounds 6b and 6d being the most potent inhibitors (EC50 0.5 µM) with selectivity indices (SI) > 235. Compound 7a likewise exhibited anti-DENV activity with an EC50 of 0.5 µM and an SI of >235. In addition, good antiviral activity of seven compounds in the series was also noted against the YFV with EC50 values ranging between 0.4 and 3.3 µM, with compound 6n being the most potent for this series with an EC50 0.4 µM and a selectivity index of >34. Finally, reversal of one of the central amide bonds as in series 13 proved deleterious to the inhibitory activity.

Flaviviral NS4b, chameleon and jack-in-the-box roles in viral replication and pathogenesis, and a molecular target for antiviral intervention.

Dengue virus and other flaviviruses such as the yellow fever, West Nile, and Japanese encephalitis viruses are emerging vector-borne human pathogens that affect annually more than 100 million individuals and that may cause debilitating and potentially fatal hemorrhagic and encephalitic diseases. Currently, there are no specific antiviral drugs for the treatment of flavivirus-associated disease. A better understanding of the flavivirus-host interactions during the different events of the flaviviral life cycle may be essential when developing novel antiviral strategies. The flaviviral non-structural protein 4b (NS4b) appears to play an important role in flaviviral replication by facilitating the formation of the viral replication complexes and in counteracting innate immune responses such as the following: (i) type I IFN signaling; (ii) RNA interference; (iii) formation of stress granules; and (iv) the unfolded protein response. Intriguingly, NS4b has recently been shown to constitute an excellent target for the selective inhibition of flavivirus replication. We here review the current knowledge on NS4b.

Synthesis and evaluation of imidazole-4,5- and pyrazine-2,3-dicarboxamides targeting dengue and yellow fever virus.

The results of a high-throughput screening assay using the dengue virus-2 replicon showed that the imidazole 4,5-dicarboxamide (I45DC) derivative (15a) has a high dengue virus inhibitory activity. Based on 15a as a lead compound, a novel class of both disubstituted I45DCs and the resembling pyrazine 2,3-dicarboxamides (P23DCs) were synthesized. Here, we report on their in vitro inhibitory activity against dengue virus (DENV) and yellow fever virus (YFV). Some of these first generation compounds have shown activity against both viruses in the micromolar range. Within this series, compound 15b was observed to display the highest antiviral potency against YFV with an EC50 = 1.85 µM. In addition, compounds 20a and 20b both potently inhibited replication of DENV (EC50 = 0.93 µM) in Vero cells.

In search of Flavivirus inhibitors part 2: tritylated, diphenylmethylated and other alkylated nucleoside analogues.

Several flaviviruses, such as the yellow fever virus and the dengue virus cause severe and potentially lethal infection in man. Following up on our initial hit 3',5'-bistritylated uridine 1, a series of alkylated nucleoside analogues were synthesized and evaluated for their in vitro antiviral activities against dengue fever virus and yellow fever virus. Hereto, alkyl and aryl groups were attached at various positions of the sugar ring combined with subtle variation of the heterocyclic base. Among the new series of derivatives, 3',5'-di-O-trityl-5-fluoro-2'-deoxyuridine (39) was the most efficient in this series and inhibited both yellow fever virus and dengue virus replication with a 50% effective concentration (EC50) of ∼1 µg/mL without considerable cytotoxicity. The other fluorinated derivatives proved more toxic. Almost all diphenylmethylated pyrimidine nucleosides with 3',5'-di-O-benzhydryl-2'-deoxyuridine (50) as the example were endowed with strong cytotoxic effects down to 1 µg/mL.

Zebavidin--an avidin-like protein from zebrafish.

The avidin protein family members are well known for their high affinity towards D-biotin and high structural stability. These properties make avidins valuable tools for a wide range of biotechnology applications. We have identified a new member of the avidin family in the zebrafish (Danio rerio) genome, hereafter called zebavidin. The protein is highly expressed in the gonads of both male and female zebrafish and in the gills of male fish, but our data suggest that zebavidin is not crucial for the developing embryo. Biophysical and structural characterisation of zebavidin revealed distinct properties not found in any previously characterised avidins. Gel filtration chromatography and native mass spectrometry suggest that the protein forms dimers in the absence of biotin at low ionic strength, but assembles into tetramers upon binding biotin. Ligand binding was analysed using radioactive and fluorescently labelled biotin and isothermal titration calorimetry. Moreover, the crystal structure of zebavidin in complex with biotin was solved at 2.4 Å resolution and unveiled unique ligand binding and subunit interface architectures; the atomic-level details support our physicochemical observations.

In search of flavivirus inhibitors: evaluation of different tritylated nucleoside analogues.

Following up on a hit that was identified in a large scale cell-based antiviral screening effort, a series of triphenylmethyl alkylated nucleoside analogues were synthesized and evaluated for their in vitro antiviral activities against the dengue virus (DENV) and the yellow fever virus (YFV). Hereto, trityl moieties were attached at various positions of the sugar ring combined with subtle variations of the heterocyclic base. Several triphenylmethyl modified nucleosides were uncovered being endowed with submicromolar in vitro antiviral activity against the YFV. The most selective inhibitor in this series was 3',5'-bis-O-tritylated-5-chlorouridine (1b) affording a selectivity index of over 90, whereas the 3',5'-bis-O-tritylated inosine congener (5b) displayed the highest activity, but proved more toxic. The finding of these lipophilic structures being endowed with high antiviral activity for flaviviruses, should stimulate the interest for further structure-activity research.