Nano silver kills the virus that causes foot and mouth disease (FMDV) in pigs, cows…
This study investigated the antiviral efficacy of chitosan-stabilized silver nanoparticles (AgNPs) against foot-and-mouth disease virus (FMDV), a highly contagious pathogen affecting ungulates. In vitro experiments demonstrated that silver nanoparticles effectively inhibited FMDV replication in a dose-dependent manner. At a concentration of 1.56 μg/ml, AgNPs did not exhibit toxicity to baby hamster kidney 21 (BHK21) cells while achieving complete inactivation of FMDV at a concentration of 103 TCID50 and inhibiting virus growth at a concentration of 104 TCID50. These findings suggest the potential of nano silver as a novel disinfectant to control FMDV transmission and prevent disease outbreaks.
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Foot-and-mouth disease virus (FMDV) is a highly contagious viral pathogen that affects domestic and wild cloven-hoofed animals, such as cattle, pigs, sheep, and goats.[1] The disease is characterized by the formation of vesicles and ulcers on the mouth, nose, and feet of infected animals.[2] Airborne transmission has also been implicated in the spread of the disease over both long distances (considered to be up to 50 km on land and 200 km on water) and short distances (within campuses and adjacent campuses within 2 km of each other).
FMDV, a member of the Picornaviridae family, is structurally similar to other single-stranded RNA viruses. It has a single-stranded RNA genome surrounded by a protein coat.[6] The FMDV particle is approximately spherical in shape and approximately 25–30 nm in diameter. It consists of an RNA genome surrounded by a protein coat or capsid. The envelope consists of 60 copies of capsomers. Each capsomer consists of four structural polypeptide chains, VP1, VP2, VP3, and VP4. VP1, VP2, and VP3 are exposed on the surface of the virus while VP4 is located inside.[1] The unique structure of the protein envelope helps FMDV resist attack by the host’s immune system and may explain why the virus spreads easily during outbreaks. Ideal conditions for virus survival include temperatures below 50°C, relative humidity above 55%, and neutral pH.[7–9]FMDV exists in seven different serotypes. Serotypes O and A were originally discovered by Vallee and Carre.[10] Their work was extended by Waldmann and Trautwein[11] with the discovery of a third serotype, which was named serotype C. Subsequently, three additional serotypes were identified in samples originating from South Africa and these were named South African Territory 1, 2 and 3 (SAT1, SAT2, SAT3).[12] A seventh serotype, Asia-1, was originally discovered in a sample collected from a water buffalo in Okara, Punjab, Pakistan, in 1954.[12] In Southeast Asia, FMDV of serotypes O, A and Asia-1 have been identified as the main cause of major outbreaks in Cambodia, Laos, Malaysia, Myanmar, the Philippines, Thailand and Vietnam in recent years.[13–17] Studies of FMDV have shown that isolates obtained from these countries have high nucleotide sequence homology. FMDV is transmitted by direct contact with infected animals, their secretions or contaminated objects, as well as through the air over short distances.
Silver nanoparticles (AgNPs) have been evaluated as one of the powerful disinfectants against a wide range of pathogens including bacteria, viruses, fungi, and yeasts. Due to their ability to inhibit and kill viruses, AgNPs are considered an excellent tool to prevent the spread of infectious diseases. According to previous studies, silver nanoparticles can attack viruses by destroying their outer shells and inhibiting their adhesion to cell receptors or directly binding to viral DNA or RNA to inhibit viral replication or propagation inside host cells. The antiviral activity of AgNPs has not only been studied to eliminate viruses that infect humans, such as human immunodeficiency virus (HIV-1), influenza A virus H1N1, influenza H3 N2, Herpes simplex virus type 1 and type 2 (HSV-1, HSV-2,42,43] vaccinia virus (VACV); dengue virus and SAR-COVID-2 virus, but also zoonotic viruses such as African swine fever virus. Although the antiviral activity of chitosan-stabilized AgNPs has been demonstrated against several viruses, their antiviral performance against FMDV has hardly been studied. Therefore, the aim of this study was to investigate the inhibitory potential of chitosan-stabilized AgNPs against FMDV and evaluate their potential application to prevent the spread of FMD.
Materials and methods
Materials
Dulbecco Modified Eagle Medium (DMEM) was purchased from Gibco, and Fetal Bovine Serum (FBS) for cell culture was provided by Roche. Silver nitrate (AgNO3, purity >99%) and sodium borohydride (NaBH, 99%) were purchased from Merck. Chitosan with molecular weights ranging from 50000 to 190000 Da (based on viscosity) was provided by Sigma Aldrich.
Synthesis of silver nanoparticles
AgNPs were prepared by chemical reduction using AgNO, as silver precursor, chitosan as stabilizer, and NaBH, as reductant. Typically, 20 ml of chitosan solution (10000 ppm) was added to a 1000 ml beaker containing 210 ml of distilled water under stirring, followed by the addition of 250 ml of AgNO, 1000 ppm. The solution was stirred at 300 rpm for one hour and then increased to 500 rpm before adding 20 ml of NaBH, 200 ppm by dropwise method. The clear solution turned yellow upon addition of NaBH solution, indicating the formation of AgNPs. The resulting solution (500 ppm Ag) was transferred to a brown sample bottle and kept at room temperature for further study without further purification.
Cytotoxicity assay was performed to evaluate the effect of AgNPs on the proliferation of BHK-21 cells. First, the stock AgNP solution (500 μg/ml) was serially diluted twofold in DMEM medium. Then, the prepared solution was added to BHK-21 cells and seeded in 96-well plate at 100 μl/well. Three wells were tested for each AgNP dilution and the experiments were repeated four times. The plates were incubated for one hour at 37°C in a 5% CO2 incubator. The samples were then replaced with 200 μl of maintenance medium (DMEM medium containing 1% antibiotic-antimycotic). The morphology of BHK-21 cells was examined daily under an inverted microscope for the next five days to detect any cytotoxic effects. The live cells appeared spindle-shaped, while the dead cells were shrunken and sometimes non-rhombic or broken.
Viral inhibition assay
Assessment of the antiviral activity was performed to evaluate the inhibitory ability of nanosilver against FMDV virus. FMDV solution with a concentration of 106 TCID50/ml was diluted tenfold in DMEM medium. Each dilution was mixed with an equal volume of AgNP solution at the maximum non-toxic concentration and incubated at room temperature for one hour. The resulting mixture was diluted tenfold in DMEM medium for virus titration on BHK-21 cells. Each dilution was injected into three wells of BHK-21 cells in a 96-well plate for virus titration. In parallel, FMDV stock solution (106 TCID50/ml) was diluted tenfold and used as the virus control, while BHK-21 cells without FMDV infection were used as the negative control. This experiment was repeated four times. After injection of the AgNP and FMDV mixture, BHK-21 cells were incubated at 37°C for 2 h, washed, and replaced with 200 μl of maintenance medium. The cytopathic effects (CPE) of the recovered viruses were monitored daily for the next five days, and the titer (log TCIDs/ml) was determined by the Reed-Muench method.
Virus and cell culture
This study used the FMD virus strain O/SEA/Mya-98 isolated during the 2018 FMD virus outbreaks in Vietnam provided by the Key Laboratory of Veterinary Biotechnology, Vietnam National University of Agriculture. Baby hamster kidney 21 (BHK21) cells were used to culture FMD virus. The cells were maintained in DMEM medium supplemented with 10% FBS and 1% antibiotics. At the beginning of cell culture, cells were seeded at a density of approximately 4×10 cells/cm2 on plastic tissue culture plates. After 24 hours, dead cells were removed by washing, and the remaining cells were incubated at 37°C and 5% CO2.
Characterization of silver nanoparticles
Scanning electron microscopy (SEM) was performed on a Hitachi S-4800 field emission microscope. AgNP samples were deposited on carbon films supported by Cu grids, dried at ambient conditions, and then mounted on a transmission electron microscope (TEM). TEM images were collected to analyze the morphology, particle size, and nanostructure of AgNPs. Cell morphology and plaque formation were examined on a Leica DM IL LED microscope (Leica Microsystem). UV-vis spectra were analyzed on a Hitachi 5300 H UV-vis spectrophotometer to evaluate AgNP formation. Zeta potential of AgNP solutions was analyzed using a Hitachi SZ100 dynamic light scattering method.
Results and discussion
Characterization of nano silver
The AgNO3 solution turned yellow immediately after adding NaBH solution, indicating the possibility of AgNP formation. This may be related to the characteristic surface plasmon resonance of AgNPs. In Figure 1A, UV-vis spectrophotometric analysis shows that the absorption peaked at an incident wavelength of 396 nm. The solution darkened as more NaBH solution was added. This was due to the increase in AgNP concentration, which was proportional to the absorption intensity. The relationship between AgNP concentration and absorption intensity has been demonstrated in previous studies, where the evolution of AgNPs was monitored using UV-Vis spectra collected at different reaction times. Here, this relationship can be further clarified by diluting the initial solution as shown in Figure 1A. No change in the absorption peak caused by dilution with water indicates that AgNPs are well stabilized by chitosan, a natural polysaccharide. The amino groups in the chitosan structure can interact with AgNPs to form a chitosan protective layer on the surface of the nanoparticles and thus prevent AgNPs from aggregating. The AgNP solution was diluted with DMEM, the absorption peak shifted to 436 nm as shown in Figure S1. The reason for this shift is still unclear, but it is likely that the transformation of the adsorption layer on the surface of Ag particles occurred when DMEM with high nutrient concentration was added.
To demonstrate the interaction between chitosan and Ag particles, FTIR spectra were collected and shown in Figure 1C. As can be seen in this figure, several distinct peaks at 1415, 1559, and 1635 cm-1 characteristic of C-H, N-H, and C-O functional groups can be observed on the chitosan structure. However, these characteristic peaks change significantly when chitosan interacts with AgNPs. In particular, the vibrational peak assigned to NH groups at 1559 cm-1 disappears due to the interaction between these functional groups and AgNPs. This suggests that chitosan stabilizes AgNPs by anchoring on Ag particles through the interaction between N-H groups and the Ag surface. This is similar to the mechanism proposed in previous studies, in which chitosan was used as a stabilizer and capping agent for AgNP synthesis.
As the concentration of AgNPs increases, it is likely that the particles may aggregate and settle during storage. To evaluate the stability of the prepared silver nanoparticle solution, zeta potential was measured. The solution of nanoparticles can be stable because their zeta potential has many positive or negative values. Previous studies have shown that the zeta potential of chitosan-stabilized AgNP solution can be positive or negative depending on the chitosan concentration and pH as shown in Table 1. The zeta potential is positive because chitosan is used at high concentrations. It varies from 25 to 52 mV when the chitosan concentration is 4400 to 10000 ppm. A study by Cinteza et al. showed that the zeta potential of bare AgNP solution was -11.2 mV, but increased to 50 mV when chitosan was added at a concentration of 0.5%. Since chitosan was used at a concentration of 400 ppm, Pansara et al. obtained an Ag-NP solution with a zeta potential of -30.9 mV. It is possible that the diluted chitosan solution only partially covered the surface of AgNPs, resulting in a negative zeta potential. Meanwhile, when the chitosan concentration is high enough, it can completely cover the surface of Ag-NPs, thus making it positively charged. In the present study, the chitosan concentration was 400 ppm, and the average zeta potential of AgNPs was -59.4 mV (Figure S2). This is consistent with the observation in previous studies. The results demonstrate that AgNPs highly dispersed in aqueous solution can be stable for a long time and are suitable for disinfection purposes.
The morphology of AgNPs was observed on SEM and TEM as shown in Figure 2. The SEM images showed that AgNPs tended to aggregate into larger clusters, which were uniformly dispersed in aqueous solution (Figure 2a). To better understand the shape and size of AgNPs, TEM images were taken and analyzed. As shown in Figure 2b and Figure S3, the nanoparticles were spherical in shape with diameters ranging from 2 to 13 nm. The size and shape of AgNPs have a significant impact on their efficacy in medical and industrial applications. With small sizes ranging from 2-12 nm, these silver nanoparticles are expected to have high antibacterial activity.
Table 1. Comparison of zeta potential of AgNPs in this study and references.
Figure 1. UV-Vis spectra corresponding to surface plasmon resonance of nano silver solutions diluted at different dilutions (A) and FTIR spectra of chitosan and chitosan-stabilized AgNPs (B).
Figure 2. SEM image (a) and TEM image of synthesized AgNPs.
Toxicity of nano silver
To evaluate the toxicity of AgNPs to BHK-21 cells, cells were exposed to nano silver at various concentrations and their growth was monitored. The cytotoxicity assay is illustrated in Figure 3, where each column consists of three wells (rows A to C) for testing AgNPs and the fourth well (row D) serves as a blank control without AgNPs. The results showed that toxic effects were observed at a concentration of 1/160 (3.13 ppm) with the majority of cells being inactivated. This could be verified by microscopic observation as shown in Figure 4. Only a few BHK-21 cells were deformed in the control (Figure 4A), while most of the cells were destroyed by treatment with 3.13 ppm AgNP solution (Figure 4B). At concentrations above 1/160 (3.13 ppm), AgNPs exhibited high toxicity, with all cells being inactivated.
Interestingly, no morphological differences were observed between control cells (not treated with AgNPs) and cells exposed for 1 h to an AgNP concentration of 1/320 equivalent to 1.56 ppm. Therefore, it can be concluded that an AgNP concentration of 1.56 ppm is safe for BHK-21 cells. This observation is consistent with previous studies that the cytotoxicity of AgNPs is concentration-dependent and chitosan-stabilized AgNPs are not cytotoxic to cells or DNA at bactericidal doses [27].
Antiviral activity
To investigate the antiviral activity of nano silver AgNPs, a tested safe concentration of 1.56 ppm (1/320 dilution) was tested against various FMDV payloads and cascades. Figure 5 shows a schematic representation of the antiviral experiments to evaluate the activity of AgNPs at a concentration of 1.56 ppm. This concentration of AgNPs was mixed with FMDV at a concentration of 104 TCID50 to examine its effect on virus growth. The virus control showed the appearance of cytopathic effect (CPE) on BHK-21 cells not treated with AgNPs (red circle) (Figure 5 A). In contrast, no CPE was detected in the control cells not treated with virus and AgNPs (blue circle) (Figure 5B).
Figure 3. Schematic diagram of the cytotoxicity assay. Each column contains 4 wells from rows A to C for each nano silver AgNP concentration and the fourth well in row D is a control without AgNPs. Red indicates high toxicity resulting in cell inactivation and blue indicates a safe AgNP concentration with more than 90% of cells remaining viable.
Figure 4. Representative images of normal growth (A) and cell death (B) of BHK-21 cells induced by 3.13 ppm nano silver. Images were captured using a Leica DM IL LED microscope.
The difference between these control samples and FMDV samples treated with 1.56 ppm nanosilver is notable. CPE was observed at 104 TCID50 (Figure 5) after treatment with AgNPs at 1.56 ppm (red circle) but was not observed at 3 logarithmic dilutions or higher (blue circle) (Figures 5 row C, 5 row D, and 5 row E).
The susceptibility of FMDV to AgNPs at 1.56 ppm was also tested with a virus concentration of 103 TCID50 BHK21 cells untreated with nano silver AgNPs showed CPE, as depicted in Figure 6, panel A next to the red circles. Without virus and AgNP treatment, the control sample with blue circles (Figure 6, panel B) did not show CPE. For the antiviral performance, in contrast to the results obtained previously by treating cells with AgNPs at 1.56 ppm and FMDV at 103 TCID50 no CPE was detected with a virus level of 103 TCID50 at any dilution (blue circles). These results showed that AgNPs at a concentration of 1.56 ppm could completely inactivate FMDV at a concentration of 103 TCID50 , while they were partially inactivated at a concentration of 104 TCID50 . This showed that AgNPs could reduce FMDV threefold at 1.56 ppm. This was more effective than gold nanoparticles in a previous study when they were ineffective against FMDV at the same concentration (1.56 ppm).
Figure 5. Antiviral assay of FMDV at 104 TCID50 concentration, shown schematically, with rows A, B, C, D, and E representing different samples. Row A is the virus control in which BHK-21 was inoculated with FMDV without AgNP treatment, while row B is the cell control without AgNP and FMDV. Rows C, D, and E react with virus samples treated with AgNP at 1/320 dilution (1.56 ppm). Columns 1 to 4 represent 10-fold sertal dilutions of FMDV. Red circles indicate CPE detection, while blue circles indicate no CPE.
Figure 6. Antiviral assay of FMDV at a concentration of 103 TCID50, shown schematically, with rows A, B, C, D, and E representing different samples. Row A is a virus control in which BHK 21 was inoculated with FMDV without AgNP treatment, while row B is a coll control without AgNP and FMDV. Rows C, D, and E correspond to virus samples treated with nanosilver at a dilution of 1/320 (1.56 ppm). Columns 1 to 4 represent 10-fold dilutions of FMDV. Red circles indicate CPE detection, while blue circles indicate no CPE.
Figure 7 shows a representative image of the effect of nanosilver on FMDV In BHK-21 cells. nano silver can neutralize FMDV, resulting in BHK-21 cells exhibiting normal growth after incubation with the AgNP and FMDV mixture as shown in Figure 7A. In contrast, in the absence of AgNPs, BHK-21 cells exhibited CPE over time (Figure 78). This provides strong evidence of the antiviral activity of nano silver AgNPs against FMDV. Previous studies have shown that increasing the concentration of silver nanoparticles can improve their efficacy. This study further demonstrates that the antiviral effect depends not only on the AgNP concentration but also on the virus concentration. It is still unclear how chitosan-immobilized silver nanoparticles affect the virus. According to previous studies, AgNPs at non-toxic concentrations can inactivate FMDV before entering the cell or during the penetration process. AgNPs are able to bind to the virus through interaction with disulfide oligomers resulting in inhibition of cell adhesion, cell penetration and protein/genome injection, inhibiting viral infection and replication. Furthermore, AgNPs generate reactive species that can degrade viral capsid receptors and genetic material causing viral capsid disruption.
The unique property of AgNPs is that they can adhere to walls and floors when sprayed in livestock farms and thus their antiviral activity can last for a long time. Therefore, the success of this work may open up a new way to prevent the spread of FMDV based on AgNPs and thus provide more options for biosecurity in livestock farms.
Figure 7. Representative micrographs of uninfected (A) and infected (B) BHK-21 cells with FMDV. Images were captured on a Leica DM ILLED microscope. Red circles indicate CPE detection.
CONCLUSION
Our study showed that nano silver (AgNPs) have high antiviral activity against FMDV. The antiviral activity of nano silver AgNPs was found to be concentration-dependent, with complete inactivation of FMDV observed at 103 TCID50 at a safe concentration of 1.56 ppm, while partial inhibition of FMDV was observed at 103 TCID50 . Clearly, the use of AgNPs at a concentration of 1.56 ppm could result in a threefold reduction in FMDV levels. Therefore, AgNPs have high potential to be used as a novel antiviral agent against FMDV. The success of the work may provide an effective biosecurity measure for livestock farms, however, further investigation may be required on the application of AgNPs in different farming systems.
Ethical Statement
The animal study protocol was approved by the Institutional Review Board (or Ethics Committee) of the Vietnam National University of Agriculture.
References
Effective Foot‐and‐Mouth Disease Virus Control Using Silver Nanoparticles