In vitro anti‐foot‐and‐mouth disease virus activity of magnesium oxide nanoparticles
2015; Institution of Engineering and Technology; Volume: 9; Issue: 5 Linguagem: Inglês
10.1049/iet-nbt.2014.0028
ISSN1751-875X
AutoresSolmaz Rafiei, Seyedeh Elham Rezatofighi, Mohammad Roayaei Ardakani, Omid Madadgar,
Tópico(s)Vector-Borne Animal Diseases
ResumoIET NanobiotechnologyVolume 9, Issue 5 p. 247-251 Research ArticleFree Access In vitro anti-foot-and-mouth disease virus activity of magnesium oxide nanoparticles Solmaz Rafiei, Solmaz Rafiei Department of Biology, Faculty of Science, University of Shahid Chamran, Ahvaz, IranSearch for more papers by this authorSeyedeh Elham Rezatofighi, Corresponding Author Seyedeh Elham Rezatofighi e.tofighi@yahoo.com Department of Biology, Faculty of Science, University of Shahid Chamran, Ahvaz, IranSearch for more papers by this authorMohammad Roayaei Ardakani, Mohammad Roayaei Ardakani Department of Biology, Faculty of Science, University of Shahid Chamran, Ahvaz, IranSearch for more papers by this authorOmid Madadgar, Omid Madadgar Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, IranSearch for more papers by this author Solmaz Rafiei, Solmaz Rafiei Department of Biology, Faculty of Science, University of Shahid Chamran, Ahvaz, IranSearch for more papers by this authorSeyedeh Elham Rezatofighi, Corresponding Author Seyedeh Elham Rezatofighi e.tofighi@yahoo.com Department of Biology, Faculty of Science, University of Shahid Chamran, Ahvaz, IranSearch for more papers by this authorMohammad Roayaei Ardakani, Mohammad Roayaei Ardakani Department of Biology, Faculty of Science, University of Shahid Chamran, Ahvaz, IranSearch for more papers by this authorOmid Madadgar, Omid Madadgar Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, IranSearch for more papers by this author First published: 01 October 2015 https://doi.org/10.1049/iet-nbt.2014.0028Citations: 21AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Foot-and-mouth disease (FMD) is an extremely contagious viral disease of cloven-hoofed animals that can lead to huge economic losses in the livestock production. No antiviral therapies are available for treating FMD virus (FMDV) infections in animals. The antiviral effects of magnesium oxide nanoparticles (MgO NPs) on the FMDV were investigated in cell culture. The viability of the cells after MgO NP treatment was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The direct effects of MgO NPs on the FMDV in extracellular (virucidal assay) and also different stages of virus replication (antiviral assay) were evaluated by plaque reduction assay. The results showed that MgO NPs were safe at concentrations up to 250 µg/ml in the Razi Bovine kidney cell line. The treatments with NPs indicated that the MgO NPs exerted in vitro virucidal and antiviral activities. Plaque reduction assay revealed that MgO NPs can inhibit FMDV by more than 90% at the early stages of infection such as attachment and penetration but not after penetration. The results of this study suggested that NPs might be applied locally as an antiviral agent in early stages of infection in susceptible animals. 1 Introduction Nanomedicine refers to the nanoscale strategies that are mainly applicable in diagnostic and therapeutic fields and consists of nanoparticles (NPs) and nanoconstructs [1]. Nanosized materials have interesting physicochemical properties compared with larger materials because of their high surface-area-to-volume ratio [2]. Metal NPs provide a new opportunity for the development of novel antiviral therapies [3]. Currently, metallic NPs are being explored extensively as potential antibacterial and antiviral agents [4]. Metal NPs are promising antiviral candidates because they generally attack a broad range of targets in viruses resulting in their broad antiviral activity and the reduced risk of antiviral resistance. Attachment and entry of viruses into host cells need multivalent interplay between viral surface ligands and cell membrane receptors. Interfering with these processes inhibits virus entry into the target cells, which is one of the most desirable strategies being followed in the development of new antiviral drugs [3]. Some studies have shown the antiviral activity of silver NPs (Ag NPs) and gold NPs (Au NPs) [4]. Different NPs such as magnesium oxide (MgO), copper oxide, zinc oxide (ZnO), titanium dioxide (TiO2), gold (Au) and silver (Ag) have antimicrobial activities and can be used as bactericides alone or in combination with antibiotics [5]. These novel antibacterial NPs are harmless to mammalian cells and the environment at low concentrations [6, 7]. MgO NPs alone or in combination with other antimicrobial agents have been proposed as a bactericide to improve microbiological food safety by treating the food products [8, 9]. In addition, these NPs have advantages such as low cost, biocompatibility and economically possible production [6, 10]. Foot-and-mouth disease virus (FMDV) causes a highly contagious disease in cloven-hoofed animals, such as swine, cattle, sheep, goats and wild ruminants. Mortality rate of foot-and-mouth disease (FMD) is usually low; however, it causes severe economic losses for the livestock productivity. The FMDV belongs to the Picornaviridae family and Aphtovirus genus. Seven distinct serotypes (A, Asia-1, C, O, SAT-1, SAT-2 and SAT-3) and hundreds of strains are recognised worldwide [11, 12]. The disease is endemic in Iran and the most prevalent serotypes are O followed by Asia 1 and A [13]. Importantly, FMDV is a serious threat to the beef and dairy industries because it has high spreading capacity and could change its antigenic identity [14]. Immune responses against one serotype of FMDV could not confer protective immunity to other serotypes. In regions where FMD is endemic, vaccination is the primary method to control and eradicate the disease. It takes at least 7 days to develop a protective immunity in vaccinated animals, which can be a practical challenge in combating outbreaks. This issue indicates that the development of emergency antiviral agents is useful for preventing FMD outbreaks, suppressing of the virus at the early stages of infection, and decreasing the clinical signs of the disease [11]. To the best of our knowledge, the antiviral activity of MgO NPs has not been investigated. In this study, the antiviral effects of MgO NPs on the FMDV were studied. 2 Material and methods 2.1 Cell line, virus and test NP Iran-Razi-Khedmati Bovine Kidney (IRKHBK) cell line known as Razi Bovine kidney (RBK) was grown in Roswell Park Memorial Institute 1640 (RPMI-1640) (Gibco) medium, supplemented with 10% foetal bovine serum (FBS) (Gibco), 100 units/ml of penicillin G and 100 μg/ml of streptomycin (Gibco) at 37°C in a humidified 5% CO2 incubator. FMDV type O IRN/1/2010 was obtained from Razi Vaccine and Serum Research Center (Hessarak, Iran). MgO nanopowder (diameter: 40 nm) was obtained from IoLiTec company (Germany, Co.: NO-0012-HP). 2.2 X-ray diffraction (XRD) and transmission electron microscopy (TEM) image XRD pattern was recorded using a Philips Analytical X-Ray diffractometer operated at 40 kV and 30 mA by means of the Cu Kα radiation. The TEM specimen of MgO NP was prepared by casting 5 μl of water dispersion of the MgO NP onto a carbon-coated copper microgrid. The specimen was dried at room temperature. The morphology of NPs was investigated using a Zeiss Em10C Transmission Electronic Microscope (Germany). The zeta potential measurement was carried out on a ZetaSizer (Nano-Z, Malvern Instrument Ltd., UK). 2.3 Cytotoxicity assay The viability of the cells after MgO NPs treatment was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Briefly, cells were seeded (1 × 105 cells per well) in triplicate in 96-well plates (Nunc, Denmark) for 2 days at 37°C. Then, the medium was removed and 100 µl of different concentrations of MgO NP were prepared in free-serum medium (0, 15.62, 31.25, 62.5, 125, 250, 500 and 1000 μg/ml) and finally added to ∼90% confluent cells. After 72 h, the treated and untreated cells were washed with phosphate buffer saline (PBS) and incubated with 100 µl MTT (5 mg/ml) (Sigma, Germany) for 4 h at 37°C. To dissolve the blue formazan crystals, 150 μl of isopropanol was added to each well, and further incubated for 30 min at 37°C. The optical density was measured at 580 nm in a BioRad ELISA reader (USA). Percentage of inhibition was calculated using the following formula: inhibition per cent (%) = [1 − (At/As)] × 100, where As is the absorbance of the solvent and At is the absorbance of the test sample [15]. The 50% cytotoxic concentration (CC50) was calculated from dose–response curves. 2.4 Virus culture and plaque reduction assay RBK cells were grown to ∼90% confluent with RPMI containing 10% FBS in a 24-well cell culture plate (Nunc, Denmark). The culture medium was removed and ten-fold serial dilutions of FMDV in a serum-free medium were inoculated and incubated for 1 h at 37°C; then the inoculated viruses were removed and the cells were washed with PBS to remove unabsorbed viruses. The agar overlay was prepared as follows: 1% agarose (Cinnagen, Iran) in ultrapure water was prepared, autoclaved and added to RPMI medium at a 1:1 ratio, respectively; then before solidifying, 2 ml of overlay medium was added to each well. After incubation for 3–4 days at 37°C in 5% CO2, the cells were stained with 0.05% neutral red in PBS and viral plaques were counted under inverted microscope (Olympus, USA) [16]. To determine plaque reduction assay for each test, the MgO NP concentrations that reduced the number of plaques by 50 and 90% (50% inhibition concentration: IC50 and 90% inhibition concentration: IC90, respectively) in comparison to the untreated virus control were calculated by non-linear regression program. The selectivity index (SI) was derived by ratio of CC50 to IC50 to estimate the therapeutic window [15]. 2.5 Mode of antiviral activity To determine the mode of antiviral activity of MgO NPs, the NPs were added to the cells in each specific step of the viral infection cycle. For all the experiments of antiviral activity, MgO NPs were dissolved in distilled water and used at concentrations of 25, 50, 100, 150, 200 and 250 µg/ml. These processes were repeated independently three times and performed in triplicate. Percentage inhibition (PI) of infectivity was determined by plaque reduction assay. The standard deviation ± mean were calculated at each stage. To elucidate the effect of MgO NPs on the prevention of cell infection with the virus, pre-treatment assay was conducted. RBK cell monolayers were treated with different concentrations of MgO NPs for 2 h at 37°C. Then, unabsorbed MgO NPs were removed and the cells were washed with PBS. For each well, FMDV was inoculated at one multiplicity of infection (MOI). The plates were incubated for 1 h at 37°C, and then the cells were washed with PBS to remove unattached viruses. In co-treatment assay, different concentrations of MgO NPs were added to RBK cells along with one MOI of FMDV/well and incubated for 1 h at 37°C. Co-treatment assay was performed to assess the influence of NPs on the virus attachment to cell. In the next step, adsorption assay was done to determine the possible effect of NPs on the penetration step of virus. One MOI of FMDV was inoculated to each well and incubated for 1 h at 4°C. At this temperature, viral particles were attached to the cells, but did not enter it. After the attachment of the viruses, the cells were washed with PBS and treated with different concentrations of MgO NPs. To evaluate the antiviral activity of MgO NPs, the post-treatment assay was conducted. One MOI of FMDV was inoculated to each well and incubated for 1 h at 37°C. 3 and 6 h after the attachment period, the cells were treated with different concentrations of MgO NPs. To study the effect of NPs on the virus itself (virucidal assay), the cell-free viruses were treated with different concentrations of NPs or medium without NPs. Moreover, after incubation for 60 min at either 4 or 37°C, they were inoculated into the cell cultures. All treatment assays were overlaid with 2 ml of growth medium containing 1% agarose at 37°C. After 3 days, the viral plaques were counted and PI was calculated [17-20]. 2.6 Statistical analysis All statistical analyses were performed with SAS. All data were presented as mean ± SD. Multiple comparison between two means was conducted by the Duncan multiple range method. Probability of p < 0.05 was considered significant. 3 Results 3.1 NP characterisation The TEM image of MgO NPs showed that the size of NPs is <50 nm (Fig. 1) and also, as shown in Fig. 2, the intensity of the reflection in the XRD pattern is related to MgO NPs (JCPDS card no. 4–829). The zeta potential measurement of MgO NPs was 26 mV (Fig. 3). Hence, NPs exert incipient stability in water. Fig. 1Open in figure viewerPowerPoint TEM image of MgO NPs Fig. 2Open in figure viewerPowerPoint XRD patterns of MgO NPs Diffraction angle is between 30° and 90° Fig. 3Open in figure viewerPowerPoint Zeta potential measurement of the MgO NPs dispersed in deionised water 3.2 Cytotoxicity To examine the effect of MgO NPs on the cell toxicity, RBK cells were treated at different concentrations of MgO NPs. The MgO NPs did not affect RBK cell viability at concentrations ≤250 µg/ml 72 h post treatment by MTT assay. The dose–response curve showed that the CC50 value was above 400 μg/ml, indicating that the death of virus-infected cells at low concentrations of NPs was not due to MgO cytotoxicity (Fig. 4). Moreover, no changes in cell morphology compared with the control were observed at concentrations ≤250 µg/ml by microscopic examination of the cell monolayers. The SI for MgO NPs was determined as 5.3. SI is a ratio of the amount of a therapeutic agent that causes toxicity to the amount that causes the therapeutic effect and shows therapeutic window (Figs. 4 and 5). Fig. 4Open in figure viewerPowerPoint MgO NPs cytotoxicity Different concentrations of MgO NPs were added to the RBK cells and cytotoxicity was measured by MTT assay Fig. 5Open in figure viewerPowerPoint Cytotoxicity of MgO NPs on RBK cell line Impact of MgO NPs on cell morphology was visualised by an inverted microscope (Olympus) (40× magnification) a Untreated RBK cell line (cell control) b Treatment of RBK cells with 250 μg/ml MgO NPs for 72 h No cytotoxicity was observed c Treatment of RBK cells with 500 μg/ml MgO NPs for 72 h Changes in cell morphology and cell death were observed at this concentration 3.3 The antiviral activity The antiviral activity of the MgO NPs was investigated by the virus plaque reduction assay. The NPs were added to RBK cells at different times relative to the virus infection cycle, to identify the stage of the virus infection cycle at which the NPs act. In all steps, cells infected with untreated virus were included as control specimens and the inhibition of plaque formation by the NPs was evaluated. The pre-treatment assay shows a dose–response effect on viral infectivity with IC50 value of 103.71 µg/ml. In co-treatment assay, various concentrations of MgO NPs show IC50 value of 101.24 µg/ml. The post-treatment assay was conducted to assess whether the MgO NPs prevent the viral penetration into the host cells. In this step, IC50 value of 162.23 µg/ml was obtained. To evaluate the effect of MgO NPs on the viral replication cycle, MgO NPs were added 3 and 6 h after the attachment stage to infected cells. The MgO NPs did not act at this stage of the FMDV infection cycle. These findings indicate that MgO NPs inhibit the initial stages of the FMDV infection cycle (Fig. 6). To determine whether the NPs exert a direct virus-inactivating activity, a virucidal assay was performed with MgO NPs concentrations that were not toxic for cells. The direct and extracellular MgO NPs incubation with FMDV had significant viral reduction infectivity. The concentrations greater than 50 µg/ml could significantly inactivate the viruses. Fig. 6Open in figure viewerPowerPoint Inhibitory effect of MgO NPs against FMDV by incubation at different periods of time during infection Viruses were treated with MgO NPs for 1 h and then added to the cells (virucidal activity). Cells were pre-treated with MgO NPs prior to virus infection (pre-treatment cells), viruses along with MgO NPs were inoculated to the cell (co-treatment assay) and the NPs were added during the adsorption period (adsorption) or after penetration of the viruses into cells (post-treatment). Experiments were done in triplicate and repeated independently three times. Data represent means ± SD of all experiments 4 Discussion In the present study, the effects of MgO NPs on the FMDV in cell culture were examined. Experiments of the toxicity of MgO NPs for cultured cells indicated a low cytotoxic activity on the RBK cell line. Maximal non-cytotoxic concentration for this NP was obtained 250 µg/ml. Ge et al. [21] showed that low concentrations (MgO NPs below 200 µg/ml) are not cytotoxic for the human umbilical vein endothelial cells. Additionally, these NPs had no toxicity on the RAW 264.7 macrophages [22]. Lai et al. [23] reported that MgO NPs had minimal effects in inducing cell death in human neural (namely U87 astrocytoma) cells compared with ZnO NPs and TiO2 NPs. MgO NPs at 100 μg/ml only induced 35% cytotoxicity in U87 cell. In the study of Vidik et al., when HeLa cells were treated with MgO NPs at 1 mg/ml concentration, only about 10% cell toxicity was observed [8]. Sun et al. [24] reported that the inflammatory response in human cardiac microvascular endothelial cells did not initiate below the concentration of 100 μg/ml MgO. Krishnamoorthy et al. [25] concluded that MgO NPs had potential utility in the treatment of cancerous cells by producing reactive oxygen species and lipid peroxidation in the liposomal membrane. Jebali et al. showed that MgO NPs were aggregate in RPMI1640 because of electrostatic forces between medium ions and uncoated MgO NPs. Size distribution of MgO NPs in RPMI1640 and water is near 50–1000 and 50–60 nm, respectively [5]. The findings of Vidik et al. indicated that the majority of NPs in serum-supplemented media become agglomerated, shapeless and coated with surrounding proteins. Presumably, these proteins have a protective role against cytotoxicity of NPs [26]. The direct effects of MgO NPs on the FMDV at the extracellular and different stages of virus replication were evaluated. The results of these treatments indicate that the MgO NPs exert in vitro virucidal and antiviral activities. The antiviral activity was probably linked to its direct inactivation of the virus particle and/or inhibition of viral replication cycle in one or more phases. To determine the mode of antiviral action, different treatments were done as follows: pre-treatment, co-treatment and post-treatment. Pre-treatment of the cells with MgO NPs lowered the production of infectious virus. Plaque formation in maximum non-toxic concentration was reduced by about 100%. It is not clear whether NPs can cover the cell surface and thereby inhibit FMDV attachment to their receptors or not. Pre-treatment of the virus with these NPs prior to inoculation to the cells (virucidal assay) or addition of the NPs during the adsorption phase (co-treatment assay) showed dose-dependent reduction of plaques, suggesting that MgO NPs might interfere with virion, capsid structures or viral compounds which are necessary for adsorption or penetration into the cells. However, it should be further investigated. The results of virucidal assay showed that MgO NPs could interact with FMDV particles and inactivate them. Moreover, these NPs demonstrated the highest inhibitory effects in co-treatment assay. Based on these results, cell-free viruses are probably more sensitive to the MgO NPs. The inhibition of FMDV appeared to occur before entering the cell, but not after penetration. The following point is still open whether the antiviral effect is due to the binding of MgO NPs to viral ligands involved in early viral replication cycle or due to the damage of the virions, thereby damaging their ability in terms of infecting host cells. Yamana et al. [27] reported that heated and hydrated dolomite (CaMg(CO3)2) had antiviral activity against avian and human influenza, avian infectious bronchitis, Newcastle disease virus and avian laryngotracheitis viruses. The investigation of Motoike et al. [28, 29] showed that MgO and CaO produced by thermal decomposition were responsible for this strong antiviral activity. Motoike et al. [28] indicated that MgO had a stronger anti-influenza activity than Mg(OH)2. Koper et al. [7] observed that contact of MgO NPs with MS2 bacteriophage (surrogate of human enterovirus) decontaminates water in minutes. Among different NPs, Ag NPs and Au NPs have been mainly studied for their antiviral activity [4]. Antiviral activity of Ag NPs against some viruses such as HIV-1, hepatitis B, herpes simplex, monkey pox, Tacaribe, respiratory syncytial and H1N1 influenza A have been investigated [30]. Antibacterial and antifungal mechanisms of Ag NPs attribute to inhibition of respiratory enzymes by the released Ag + ions, while it is likely that antiviral activity resulted from direct binding of the Ag NPs to viral envelope glycoproteins and inhibition of virus attachment to host cells [30]. Orlowski et al. showed that tannic acid modified Ag NPs interacted with the surface glycoproteins of herpes simplex virus-2; therefore, NPs created a physical obstacle impairing virus attachment to the viral receptors on the cell surface. AgNPs also interact with gp120 protein of HIV to inhibit virus-to-cell attachment [17]. Au NPs with different anionic groups can effectively inhibit several influenza strains by interference with virus attachment to the cell surface [31]. Leung et al. reported that MgO could be attached to the membrane molecules containing phosphate groups and resulting cell death of bacteria [6, 32]. FMDV has an icosahedral capsid without envelope [12]. MgO may attach to the phosphate groups of the capsid proteins and damage the structure of the virus. 5 Conclusion MgO NPs showed low cytoxic activity on the RBK cell line. The present experiment demonstrated that these NPs can exert virucidal and antiviral activities. 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