Nano silver inhibits infectious bronchitis virus in chickens (IB virus)
Infectious bronchitis virus (IBV) threatens the poultry industry and causes global economic losses. IBV is highly mutating. Therefore, no effective drug is available. The objective of the present study was to evaluate silver nanoparticles against it as an antiviral agent. Methods: Nano silver (AgNPs) were evaluated as an antiviral agent against IBV. P. betle leaf extract synthesized AgNPs from silver nitrate. UV/vis absorption, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM) were used to characterize the AgNPs. TEM indicated particle sizes ranging from 5–30 nm and XRD demonstrated their characteristic AgNP structure. The antiviral activity of AgNPs was measured by logarithmic embryo infective dose 50 (logEID50)/ml and IBV genome copy number.
Results: XRD analysis revealed the structure of AgNPs and transmission electron microscopy showed that the size ranged from 5–30 nm for AgNPs. Silver nanoparticles at non-cytotoxic concentrations inhibited virus-cell interactions, prevented virus entry into cells, and reduced the number of IBV genome copies (per µl) in eggs by blocking the formation of IBV RNA genome, resulting in a significant reduction in IBV levels.
Conclusion: AgNPs possess antiviral properties that inhibit IBV replication in eggs. The findings indicate that AgNPs are a promising drug candidate for the treatment or prevention of IBV infection.
INTRODUCTION
The poultry industry plays an important role in food production and poverty alleviation. However, the spread of infectious diseases in poultry, such as avian infectious bronchitis virus (IBV), poses a threat to poultry health and leads to economic losses [1]. In addition, these pathogens can cause zoonotic diseases, further affecting human health [2]. IBV, avian coronavirus, is a family of enveloped, non-segmented, single-stranded, positive-sense ribonucleic acid (+ssRNA) viruses [3]. Traditional antiviral treatments for IBV often result in drug resistance, side effects, and viral recrudescence [4]. Vaccines are commonly used, but the constant emergence and resurgence of viral strains can reduce the effectiveness of vaccines [5–9]. To address these challenges, researchers are exploring alternative strategies, including the use of medicinal plant derivatives and nanotechnology, to develop new antiviral drugs. P. betle leaves have been used for centuries in traditional medicine and cultural activities. These leaves are rich in various bioactive compounds, including polyphenols such as acetyl eugenol, trans-isoeugenol, chavicol, chavibetol, chavibetol acetate, and allyl pyrocatechol diacetate [10]. These polyphenols have attracted considerable attention due to their potential applications in various industries, including the preparation of Nano silver (AgNPs) [10]. Polyphenols present in P. betle leaves can act as reducing agents and capping agents on the surface of AgNPs. This means that they can prevent the aggregation of nanoparticles and provide multiple benefits by reducing the size of Nano silver AgNPs. Polyphenols enhance their stability and improve their overall performance [10]. AgNPs can bind to the viral genome and disrupt the viral envelope, rendering the virus inactive and unable to infect host cells or replicate. AgNPs increase the exposure to viral particles due to their small size and large surface area [10]. Therefore, in this study, the aqueous extract of P. betle was used to produce AgNPs and characterized by UV/vis absorption, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). In addition, we evaluated the in ovo antiviral effect of AgNPs on IBV infectivity and genome replication.
MATERIALS AND METHODS
Plant materials P. betle leaves were obtained from major vegetable markets in Malaysia. The leaves were identified and authenticated by the Department of Botany, Faculty of Pharmacy, Amman, Jordan. A voucher specimen (PJ 05) was submitted.
Synthesis of Nano silver
The aqueous extract of P. betle was described previously (10), by mixing 10 g of dried leaf sample with 100 ml of high purity water in a flask. For 72 h at 8 °C, the flask was sealed with cotton wool. The mixture was filtered using 25 mm Whatman filter paper to remove plant residues [9]. The pellets were vacuum dried and stored at room temperature for analysis. The pellets were stored at −20 °C until use. AgNPs were produced biologically [5, 10]. Briefly, 10 ml of aqueous P. betle leaf extract (10 g/100 ml) was mixed with 5 mmol silver nitrate solution and left at room temperature (25 °C) for four hours before centrifugation at 10,000 rpm for ten minutes. After removing the supernatant, the pellet was washed with distilled water (7000 rpm, 2 minutes) and vacuum dried. Prior to analysis, the dried pellets were required to be at room temperature [10]. After removing the supernatant, the pellet was washed with distilled water at 7000 rpm for 2 minutes and vacuum dried. Prior to analysis, the dried pellets were required to be stored at room temperature [10].
Characterization of Nano silver
The characterization of AgNPs has been described previously [10]. Briefly, the synthesized AgNPs were examined by UV/visible spectroscopy. AgNPs absorb surface plasmon resonance at 420 nm. We analyzed the dried AgNP mixture by XRD X’Pert Pro 40 kV (PANalatical Empyrean-2012). The structural composition of the dried AgNPs was analyzed by the GBC-Difftech MMA model. The irradiance was 34.2 mA and Cu Ka filtered Ni 35 kV (k = 1.54 A°). TEM measured the AgNP size. AgNPs were coated on carbon-coated copper TEM grids prepared for the samples. The films were blotted on the TEM grids after two minutes to remove excess solution [10]. The grids were dried before measurements.
In vivo antiviral activity assay
The antiviral activity has been described previously [11, 12]. Briefly, we propagated IBV-H120 in embryonated specific pathogen-free (SPF) chicken eggs. After obtaining the master seed of the IBV-H120 vaccine strain (Jordan Bioindustry Center (JOVAC), Amman, Jordan) with 108 embryonated infectious doses of 50 (EID50)/ml, we diluted it in 1 ml of sterile phosphate buffer solution (PBS; pH 7.2) and serially diluted it until reaching 105 EID50/ml. On day 10, SPF chicken eggs were inoculated with 0.1 ml of 105 EID50/ml in the presence of different concentrations of AgNPs and incubated at 37 °C for 1 h [11, 12]. SPF chicken eggs were incubated at 37 °C with 60% humidity. SPF chicken eggs were incubated daily under candlelight for 7 days. Mortality on the first day was not specific. The head of the egg was removed and ureteral fluid was collected 7 days after infection. The ureteral fluid was aspirated without albumin or yolk. We immediately cooled all fluids at 4 °C [11, 12]. Control (0.1 ml 105 EID50/ml of untreated IBV-H120 virus) and IBV-infected samples were titrated using SPF chicken eggs to calculate EID50/ml values [11, 12].
Viral titration for IBV-H1 20
To measure the EID50RESULTS/ml concentration of the virus, we injected 100 μl of a 10-fold virus dilution (5 eggs/dilution) into the ureteral cavity of 10-day-old SPF chicken eggs [10].
Viral RNA extraction and qRT-PCR
The NZY viral RNA isolation kit (NZY Tech, Lisbon, Portugal) was used to extract RNA from allotonic fluids. The reaction mixture of each RNA sample was prepared according to the manufacturer’s instructions. The IBV, RUO one-step RT-qPCR kit (NZY Tech, Portugal), amplified ORF1a. Quantitative analysis was performed using a standard curve.
Statistical analysis
GraphPad Prism and SPSS performed all analyses. One-way analysis of variance with Tukey multiple comparisons then determined differences between groups. P ˂ 0.05 is significant.
RESULTS
Characterization of Nano silver AgNPs
Cytotoxicity of AgNPs Injection of 10-day-old SPF chicken eggs with 360 μg/ml of Nano silver AgNPs did not cause toxicity (Figure 4). Due to the toxicity of the solvent, high concentrations of eggs are difficult to inject. Therefore, injection of eggs with more than 1500 μg/egg is impractical. Therefore, the median lethal dose of AgNPs (LD50 Antiviral activity of AgNPs against IBV-H120 in eggs) was not calculated. LogEID50/ml was determined by pooling the urine from all eggs harvested 7 days after infection. AgNPs inhibited IBV-H 120 replication in a dose-dependent manner, as shown by the decrease in LogEID50AgNP inhibition of IBV genome synthesis/ml. In ovo, Significant inhibitory activity against infectious IBV-H120 was observed for AgNPs at 10, 20, 40, 80, 160 and 320 µg/egg (p ˂ 0.001) (Figure 5). The copy number (per µl)/Cycle of quantification (Cq) values in urine showed a significant reduction in IBV genome synthesis in ovo (Table 1).
Figure 1: Nano silver surface plasmon resonance absorption at 420 nm
Figure 2: FTIR spectrum of (A) leaf extract of P. betle extractxt (B) AgNPs synthesized using P. betle extract
Figure 3: (A) TEM analysis of AgNPs (B) XRD pattern of synthesized silver nanoparticles
Figure 4: Cytotoxicity of Nano silver AgNPs. Data are presented as mean ± standard deviation (SD) of triplet samples
Figure 5: Anti-IBV efficacy of AgNPs in ovo: (A) Anti-IBV efficacy of AgNPs in ovo: 10-day-old SPF chicken eggs were infected with IBV at a concentration of 0.1 ml 105 EID 50 /ml with different concentrations of AgNPs and observed daily for embryonic death for 7 days. All egg urethral fluids were pooled after harvest. Using Reed-Muench, calculate EID 50 /ml. Data are presented as mean ± SD of triplicate samples, *p < 0.01, **p < 0.001
Table 1: Nano silver influence on IBV genome quantification in ureteral fluid. After infection of SPF embryonated chicken eggs (day 10) with IBV (0.1 ml 105 EID 50 (µg/ml)/ml) with different levels of AgNPs, embryo mortality was recorded daily for 7 days. All ureteral fluids of the eggs were extracted. qRT-PCR measured IBV genome concentration in ureteral fluid. *p < 0.01; **p < 0.001
DISCUSSION
AgNPs have attracted attention in recent years due to their antimicrobial properties. These nanoparticles, typically less than 100 nanometers in size, have a large surface area that enhances their interaction with viruses [13]. The unique properties of AgNPs allow them to interact with a wide range of viruses, including the bronchitis virus. They have been shown to be effective against respiratory syncytial virus, influenza virus, and other common bronchitis viruses [14]. This study is consistent with several studies demonstrating that AgNPs can inhibit early infection through direct interaction between AgNPs and the viral envelope [15-17]. AgNPs can disrupt the integrity of the viral envelope, preventing the virus from entering host cells and replicating [15]. Additionally, AgNPs have been found to have a low risk of inducing viral resistance, making them a potential long-term solution against viral infections [16, 17]. We have demonstrated that AgNPs exhibit potent inhibition of RNA synthesis; the most plausible explanation is through direct binding of Ag to RNA polymerase [18]. Similarly, the antiviral activity of AgNPs has been studied against various viruses, including avian influenza (AI) H9N2 virus [15] and Newcastle disease virus (NDV). In the case of AI H9N2, the nanoparticles have shown potential to inhibit viral entry and reduce viral replication [13]. These different mechanisms of action make AgNPs potent antiviral agents capable of disrupting multiple stages of the viral life cycle. The unique physicochemical properties of AgNPs allow them to interact with viral particles in a way that makes it difficult for bacteria to adapt and develop resistance to traditional antiviral drugs [18-20]. This makes green AgNPs a valuable tool in combating viral infections resistant to traditional antiviral drugs.
CONCLUSION
The use of silver nanoparticles synthesized using aqueous extracts of P. betle is a breakthrough in the field of antiviral therapy. These AgNPs exhibited potent antiviral activity against IBV. By inhibiting viral entry and replication, AgNPs provide a promising alternative to traditional antiviral drugs. Furthermore, their ability to overcome bacterial resistance makes them valuable in combating viral infections resistant to conventional drugs. Further research and development in this area will pave the way for the application of AgNPs in the prevention and treatment of viral infections.
Source:
SILVER NANOPARTICLES INHIBIT INFECTIOUS BRONCHITIS VIRUS REPLICATION