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Joanna Cymerys,1,* Michalina Bartak,1,* Anna Słońska,1 Agata Lange,2 Sławomir Jaworski,2 Marcin Chodkowski,3 Agnieszka Ostrowska,2 Mateusz Wierzbicki,2 Ewa Sawosz,2 Marcin W Bańbura1 1Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland; 2Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, Poland; 3Military Institute of Hygiene and Epidemiology, Warsaw, Poland*These authors contributed equally to this workCorrespondence: Joanna Cymerys, Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences, 02-786, Warsaw, Poland, Email joanna_cymerys@sggw.edu.plBackground: The high infectivity of coronaviruses has led to increased interest in developing new strategies to prevent virus spread. Silver nanoparticles (AgNPs) and graphene oxide (GO) have attracted much attention in the antiviral field. We investigated the potential antiviral activity of GO and AgNPs combined in the nanocomposite GO-Ag against murine betacoronavirus MHV using an in vitro model.Methods: GO, AgNPs, and GO-Ag characterization (size distribution, zeta potential, TEM visualization, FT-IR, and EDX analysis) and XTT assay were performed. The antiviral activity of GO-Ag nanocomposites was evaluated by RT-qPCR and TCID50 assays. The results were compared with free AgNPs and pure GO. Cell growth and morphology of MHV-infected hepatocytes treated with GO-Ag composites were analyzed by JuLI™Br. Immunofluorescence was used to visualize the cell receptor used by MHV. Ultrastructural SEM analysis was performed to examine cell morphology after MHV infection and GO-Ag composite treatment.Results: A significant reduction in virus titer was observed for all nanocomposites tested, ranging from 3.2 to 7.3 log10 TCID50. The highest titer reduction was obtained for GO 5 μg/mL - Ag 25 μg/mL in the post-treatment method. These results were confirmed by RT-qPCR analysis. The results indicate that GO-Ag nanocomposites exhibited better antiviral activity compared to AgNPs and GO. Moreover, the attachment of AgNPs to the GO flake platform reduced their cytotoxicity. In addition, the GO-Ag composite modulates the distribution of the Ceacam1 cell receptor and can modulate cell morphology.Conclusion: Graphene oxide sheets act as a stabilizing agent, inhibiting the accumulation of AgNPs and reducing their cellular toxicity. The GO-Ag composite can physically bind and inhibit murine betacoronavirus from entering cells. Furthermore, the constant presence of GO-Ag can inhibit MHV replication and significantly limit its extracellular release. In conclusion, GO-Ag shows promise as an antiviral coating on solid surfaces to minimize virus transmission and spread. Keywords: graphene oxide, silver nanoparticles, GO-Ag composite, antiviral, coronavirus, MHV

Piotr Orlowski,1 Magdalena Zmigrodzka,2 Emilia Tomaszewska,3 Katarzyna Ranoszek-Soliwoda,3 Beata Pajak,4 Anna Slonska,5 Joanna Cymerys,5 Grzegorz Celichowski,3 Jaroslaw Grobelny,3 Malgorzata Krzyzowska1 1Laboratory of Nanobiology and Biomaterials, Military Institute of Hygiene and Epidemiology, Warsaw, Poland; 2Department of Pathology and Veterinary Diagnostics, Institute of Veterinary Medicine, Warsaw University of Life Sciences (WULS-SGGW), Warsaw, Poland; 3Department of Materials Technology and Chemistry, Faculty of Chemistry, University of Lodz, Lodz, Poland; 4Laboratory of Genetics and Molecular Biology, Military Institute of Hygiene and Epidemiology, Warsaw, Poland; 5Division of Microbiology, Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw 02-786, PolandCorrespondence: Malgorzata Krzyzowska Email krzyzowskam@yahoo.comBackground: Polyphenols possess antioxidant, anti-inflammatory and antimicrobial properties and have been used in the treatment of skin wounds and burns. We previously showed that tannic acid-modified AgNPs sized > 26 nm promote wound healing, while tannic acid-modified AgNPs sized 13 nm can elicit strong local inflammatory response. In this study, we tested bimetallic Au@AgNPs sized 30 nm modified with selected flavonoid and non-flavonoid compounds for wound healing applications.Methods: Bimetallic Au@AgNPs were obtained by growing an Ag layer on AuNPs and further modified with selected polyphenols. After toxicity tests and in vitro scratch assay in HaCaT cells, modified lymph node assay as well as the mouse splint wound model were further used to access the wound healing potential of selected non-toxic modifications.Results: Tannic acid, gallic acid, polydatin, resveratrol, catechin, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate and procyanidin B2 used to modify Au@AgNPs exhibited good toxicological profiles in HaCaT cells. Au@AgNPs modified with 15 μM tannic acid, 200 μM resveratrol, 200 μM epicatechin gallate, 1000 μM gallic acid and 200 μM procyanidin B2 induced wound healing in vivo and did not lead to the local irritation or inflammation. Tannic acid-modified Au@AgNPs induced epithelial-to-mesenchymal transition (EMT) – like re-epithelialization, while other polyphenol modifications of Au@AgNPs acted through proliferation and wound closure.Conclusion: Bimetallic Au@AgNPs can be used as a basis for modification with selected polyphenols for topical uses. In addition, we have demonstrated that particular polyphenols used to modify bimetallic nanoparticles may show different effects upon different stages of wound healing.Keywords: polyphenols, bimetallic nanoparticles, wound healing, mouse model

Viruses replicating in the nucleus need to cross the nuclear membrane barrier during infection, therefore disruption of specific nuclear transport pathways is crucial for their replication cycle. In the present study we have investigated the influence of nucleo-cytoplasmic transport inhibitors -- ivermectin and leptomycin B, on EHV-1 replication in primary murine neurons. Obtained results suggest that the examined proteins -- exportin 1 and importin alpha/beta may participate, but are not required, during EHV-1 infection. Based on these results, it can be assumed that EHV-1 is able to use other receptors for nucleo-cytoplasmic transport.