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Zika virus is a global health threat due to significantly elevated risk of fetus malformations in infected pregnant women. Currently, neither an effective therapy nor a prophylactic vaccination is available for clinical use, desperately necessitating novel therapeutics and approaches to obtain them. Here, we present a structural model of the Zika virus RNA-dependent RNA polymerase (ZIKV RdRp) in complex with template and nascent RNAs, Mg 2+ ions and accessing nucleoside triphosphate. The model allowed for docking studies aimed at effective pre-screening of potential inhibitors of ZIKV RdRp. Applicability of the structural model for docking studies was illustrated with the NITD008 artificial nucleotide that is known to effectively inhibit the function of the ZIKV RdRp. The ZIKV RdRp - RNA structural model is provided for all possible variations of the nascent RNA bases pairs to enhance its general utility in docking and modelling experiments. The developed model makes the rational design of novel nucleosides and nucleotide analogues feasible and thus provides a solid platform for the development of advanced antiviral therapy.

Background: The recent Zika virus (ZIKV) outbreak has linked ZIKV with microcephaly and other central nervous system pathologies in humans. Astrocytes are among the first cells to respond to ZIKV infection in the brain and are also targets for virus infection. In this study, we investigated the interaction between ZIKV and primary human brain cortical astrocytes (HBCA)., Results: HBCAs were highly sensitive to representatives of both Asian and African ZIKV lineages and produced high viral yields. The infection was associated with limited immune cytokine/chemokine response activation; the highest increase of expression, following infection, was seen in CXCL-10 (IP-10), interleukin-6, 8, 12, and CCL5 (RANTES). Ultrastructural changes in the ZIKV-infected HBCA were characterized by electron tomography (ET). ET reconstructions elucidated high-resolution 3D images of the proliferating and extensively rearranged endoplasmic reticulum (ER) containing viral particles and virus-induced vesicles, tightly juxtaposed to collapsed ER cisternae., Conclusions: The results confirm that human astrocytes are sensitive to ZIKV infection and could be a source of proinflammatory cytokines in the ZIKV-infected brain tissue.

We describe the expression and purification of an active recombinant Zika virus RNA-dependent RNA polymerase (RdRp). Next, we present the development and optimization of an in vitro assay to measure its activity. We then applied the assay to selected triphosphate analogs and discovered that 2'-C-methylated nucleosides exhibit strong inhibitory activity. Surprisingly, also carbocyclic derivatives with the carbohydrate locked in a North-like conformation as well as a ribonucleotide with a South conformation exhibited strong activity. Our results suggest that the traditional 2'-C-methylated nucleosides pursued in the race for anti-HCV treatment can be superseded by brand new scaffolds in the case of the Zika virus., (Copyright © 2016 Elsevier B.V. All rights reserved.)

To find an effective drug for Zika virus, it is important to understand how numerous proteins which are critical for the virus' structure and function interact with their counterparts. One approach to inhibiting the flavivirus is to deter its ability to bind onto glycoproteins; however, the crystal structures of envelope proteins of the ever-evolving viral strains that decipher glycosidic or drug-molecular interactions are not always available. To fill this gap, we are reporting a holistic, simulation-based approach to predict compounds that will inhibit ligand binding onto a structurally unresolved protein, in this case the Zika virus envelope protein (ZVEP), by developing a three-dimensional general structure and analyzing sites at which ligands and small drug-like molecules interact. By examining how glycan molecules and small-molecule probes interact with a freshly resolved ZVEP homology model, we report the susceptibility of ZVEP to inhibition via two small molecules, ZINC33683341 and ZINC49605556-by preferentially binding onto the primary receptor responsible for the virus' virulence. Antiviral activity was confirmed when ZINC33683341 was tested in cell culture. We anticipate the results to be a starting point for drug discovery targeting Zika virus and other emerging pathogens.

Michaela Vojnikova,1,2,* Martina Sukupova,1,3,* Michal Stefanik,1,4 Petra Strakova,3– 5 Jan Haviernik,4 Katerina Kapolkova,1 Eliska Gruberova,1 Klara Raskova,1 Hana Michalkova,1 Pavel Svec,1 Marie Peskova Kudlickova,1 Ivana Huvarova,4 Daniel Ruzek,3– 5 Jiri Salat,3– 5 Vladimir Pekarik,1 Ludek Eyer,3– 5 Zbynek Heger1 1Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic; 2Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic; 3Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic; 4Department of infectious Diseases and Preventive Medicine, Veterinary Research Institute, Brno, Czech Republic; 5Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Ceske Budejovice, Czech Republic*These authors contributed equally to this workCorrespondence: Zbynek Heger, Research Group for Molecular Biology and Nanomedicine, Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, CZ-613 00, Czech Republic, Tel +420-5-4513-3350, Fax +420-5-4521-2044, Email heger@mendelu.czBackground: As highlighted by recent pandemic outbreaks, antiviral drugs are crucial resources in the global battle against viral diseases. Unfortunately, most antiviral drugs are characterized by a plethora of side effects and low efficiency/poor bioavailability owing to their insolubility. This also applies to the arylnaphthalide lignin family member, diphyllin (Diph). Diph acts as a vacuolar ATPase inhibitor and has been previously identified as a promising candidate with broad-spectrum antiviral activity. However, its physicochemical properties preclude its efficient administration in vivo, complicating preclinical testing.Methods: We produced human recombinant H- ferritin (HsaFtH) and used it as a delivery vehicle for Diph encapsulation through pH-mediated reversible reassembly of HsaFtH. Diph nanoformulation was subsequently thoroughly characterized and tested for its non-target cytotoxicity and antiviral efficiency using a panel of pathogenic viral strain.Results: We revealed that loading into HsaFtH decreased the undesired cytotoxicity of Diph in mammalian host cells. We also confirmed that encapsulated Diph exhibited slightly lower antiviral activity than free Diph, which may be due to the differential uptake mechanism and kinetics of free Diph and Diph@HsaFtH. Furthermore, we confirmed that the antiviral effect was mediated solely by Diph with no contribution from HsaFtH.Conclusion: It was confirmed that HsaFtH is a suitable vehicle that allows easy loading of Diph and production of highly homogeneous nanoparticles dispersion with promising broad-spectrum antiviral activity.Keywords: drug delivery, SARS-CoV-2, TBEV, WNV, Zika virus