The Cranberry Extract Oximacro® Prevents Hazara Virus Infection by Inhibiting the Attachment to Target Cells Author Mattia Mirandola1+, Maria Vittoria Salvati1+, Carola Rodigari1, Ali Mirazimi2,3, Massimo E. Maffei4, Giorgio Gribaudo4 and Cristiano Salata1,* Affiliation 1Department of Molecular Medicine, University of Padova, 35121 Padova, Italy. 2Department of Laboratory Medicine, Karolinska Institutet, 17177 Stockholm, Sweden. 3National Veterinary Institute, 75189 Uppsala, Sweden (A.M.) 4Department of Life Sciences and Systems Biology, University of Turin, 10123 Turin, Italy. +Equally contributions Background and Aims removed from infected cells cultures, and cell Hazara Virus (HAZV) belongs to the Nairoviridae monolayers were fixed (using acetone:methanol family and is included in the same serogroup of the (1:1) solution). Subsequently, an immunostaining Crimean-Congo haemorrhagic fever virus was performed with an in house-developed primary (CCHFV) [1]. CCHFV is the most widespread tick- anti-nucleoprotein antibody (α-NP), and after this borne arbovirus responsible for a serious incubation cells were incubated with Alexa haemorrhagic disease for which specific and FluorTM 488 goat anti-rabbit IgG (Thermo Fisher effective treatment or preventive system are Scientific, Italy). missing, thus is classified in the group risk-4 Antiviral Assays: Full treatment, Vero cells were human pathogen [2,3]. Bioactive compounds treated with different concentration of cranberry derived from several natural products may provide extract (0-0.2-0.4-0.8-0.15-3.125-6.25-12.5-25-50- a natural source of broad-spectrum antiviral agents 100 μg/mL) for 1 h before the infection, during the and, in addition, provided by a good tolerability and HAZV adhesion and after the infection until the minimal side effects. Previous in vitro studies end of the experiment. Time-Of-Addiction Assays showed that a cranberry (Vaccinium macrocarpon (TOA) briefly, Vero cells were seeded in 96-wells Ait.) extract containing a high content of A-type plate. The following day, different extract proanthocyanidins (PAC-A), inhibits the concentrations (0-3.125-6.25-12.5-25-50-100 replication of herpes simplex and influenza viruses μg/mL) were added 1 h before infection (cells “Pre- by hampering their attachment to target cells [4,5]. Treatment”, PreT) or 1 h after infection (cells Given the broad-spectrum antimicrobial activity of “Post-Treatment”, PosT). In the “Co-Treatment” of polyphenols and the urgency to develop therapies cells and HAZV with cranberry extract experiment for the treatment of CCHF, we investigated the (CoT), cells were exposed during virus adsorption antiviral activity of cranberry extract against to different concentrations of extract (0-0.008- HAZV, a surrogate nairovirus model of CCHFV 0.04-0.2-1-5 μg/mL). Viral Attachment Assay that can be handled in Level 2 Biosecurity (VAA) Vero cells were seeded as for TOA. The Laboratories (BSL-2) [6]. Results indicates that the following day, cultures were cooled for 20 min at 4 cranberry extract exerts an antiviral activity against °C and then washed three times with cold PBS 1X. HAZV by targeting early stages of the viral After that, precooled Vero cells were infected at 4 replication cycle, such as the initial adsorption to °C for 2 h in presence of different concentrations of target cells. extract (0-0.008-0.04-0.2-1-5 μg/mL). After viral adsorption, cells were washed twice with PBS 1X Methodology at RT and incubated with DMEM medium Immunofluorescence Assay (IFA) was performed supplemented with 10% FBSi for 24 h at 37 °C. on Vero Cells. Briefly, the supernatant was The following day, medium was removed, and IFA was performed as described above. In all the determine the stage of HAZV replication cycle at assays, the cells were infected with serial 10-fold which the cranberry extract carried out the dilutions prepared starting from non-diluted virus inhibitory activity, we performed three different (105 FFU/well). treatments, as summarized in Figure 1a. As shown Data Analysis: GraphPad Prism 8 in Figure 1c and 1d, both PreT and CoT shows a concentration-dependent inhibitory activity of Results HAZV infection rate, with a EC50 of 11.08 µg/mL To evaluate the effect of the cranberry extract on and 0.02 µg/mL respectively. On the contrary PosT HAZV replication, Vero cells were pre-treated with show a weakly reduction in HAZV infectivity (data the extract for one hour before infection, then not shown). Starting from the results obtained from infected with HAZV. In addition to the pre- the “time-of-addition” experiments, next we treatment, cells were continuously exposed to the investigated in depth the cranberry extract's cranberry extract (full treatment) during all the antiviral activity at the early stages of HAZV steps (Figure 1a). Thus, the rate of infection was infection. To this end, a selective Viral Attachment evaluated by immunofluorescence (IFA) at 24 h Assay (VAA) was adopted to determine whether post infection (p.i.). Cranberry extract inhibited the cranberry extract could interfere with the initial HAZV infection in a dose-dependent manner adsorption of viral particles to the surface of target (Figure 1b) with an EC50 value of 0.6 μg/mL. To cells (Figure 1e). Conclusions • Full-Treatment Assay shows that the cranberry extract inhibits HAZV infectivity. • Time-of-addition assays demonstrate that the cranberry extract acts on the early steps of the HAZV replication cycle. • Viral Attachment Assay results suggest that the (a) (b) cranberry extract inhibits HAZV attachment to the cell surface. In conclusion, although further investigations are required to validate cranberry extract activity against CCHFV and to confirm the bioactive (c) (d) components responsible for the Figure 1. Evaluation of the antiviral activity of the Cranberry extract against HAZV. (a) Vero observed anti-HAZV activity, cells were treated with the cranberry extract at different concentrations throughout all the the results of this study suggest experiment. (b) Vero cells were treated with the cranberry extract 1 h before infection (PreT). (c) the cranberry extract as a In the CoT experiment, cells were exposed to the cranberry extract only during infection. (d) In the VAA, precooled Vero cells were infected with HAZV in presence of different concentrations promising candidate for the of extract for 2 h at 4 °C. ** p<0.01; **** p<0.0001. development of antivirals against CCHFV. References 1. Garrison, Aura R et al. “ICTV Virus Taxonomy Profile: Nairoviridae.” The Journal of general virology vol. 101,8 (2020): 798-799. doi:10.1099/jgv.0.001485 2. Hartlaub, Julia et al. “Sheep and Cattle Are Not Susceptible to Experimental Inoculation with Hazara Orthonairovirus, a Tick-Borne Arbovirus Closely Related to CCHFV.” Microorganisms vol. 8,12 1927. 4 Dec. 2020, doi:10.3390/microorganisms8121927 3. Dowall, Stuart D et al. “Hazara virus infection is lethal for adult type I interferon receptor-knockout mice and may act as a surrogate for infection with the human-pathogenic Crimean-Congo hemorrhagic fever virus.” The Journal of general virology vol. 93,Pt 3 (2012): 560-564. doi:10.1099/vir.0.038455-0 4. 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