SILVER NANOPARTICLES INHIBIT INFECTIOUS BRONCHITIS VIRUS REPLICATION

Objective: Avian infectious bronchitis virus (IBV) threatens the poultry industry and causes global economic losses. The IBV is highly variable. Thus, no effective drugs are available. Objective of the present study was to evaluate silver nanoparticles against it as an antiviral agent. Methods: Silver nanoparticles (AgNPs) have been evaluated as antivirals against IBV. P. betle leaf extract biosynthesizes AgNPs from silver nitrate. UV/vis absorption, X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM) have been used to characterize AgNPs. TEM indicated particle sizes of 5–30 nm, and XRD demonstrated their characteristic AgNPs structure. The antiviral activity of AgNPs was measured by the log embryo infective dose 50 (logEID 50 Results: XRD analysis showed a structure for AgNPs, and transmission electron microscopy showed a size of 5–30 nm for AgNPs. AgNPs at a noncytotoxic concentration inhibit the interaction between the virus and the cell, preventing the virus from entering the cell and reducing the number of IBV genome copies (per µl) in ovo by preventing the formation of the IBV RNA genome, resulting in a significant reduction in the IBV titer. )/ml and the number of IBV genome copies. Conclusion: AgNPs possess antiviral properties that inhibit IBV replication in ovo. The findings indicate that AgNPs are a promising drug candidate for treating or preventing IBV infection.


INTRODUCTION
The poultry industry plays a significant role in food production and poverty alleviation.However, the spread of infectious diseases among birds, such as the avian infectious bronchitis virus (IBV), poses a threat to poultry health and results in economic losses [1].Additionally, these pathogens can cause zoonotic diseases, further impacting human health [2].IBV, the coronavirus of the chicken, is a family of enveloped, non-segmented, single-stranded, positive-sense ribonucleic acid (+ssRNA) viruses [3].
Traditional antiviral treatments for IBV often lead to resistance, side effects, and the recurrence of viruses [4].Vaccination is commonly used, but the constant emergence and re-emergence of viral strains can compromise its efficacy [5][6][7][8][9].To address these challenges, researchers are exploring alternative strategies, including the use of medicinal plant derivatives and nanotechnology, to develop novel antiviral drugs.P. betle leaves have been used for centuries in traditional medicine and cultural practices.These leaves are rich in various bioactive compounds, including polyphenols such as acetyl eugenol, transisoeugenol, chavicol, chavibetol, chavibetol acetate, and allyl pyrocatechol diacetate [10].These polyphenols have attracted significant attention due to their potential applications in various industries, including the preparation of silver nanoparticles (AgNPs) [10].Polyphenols present in P. betle leaves can act as reducing and capping agents on the surface of AgNPs.This means that they can prevent the aggregation of nanoparticles and provide numerous benefits by reducing the size of AgNPs.Polyphenols enhance their stability and improve their overall performance [10].
AgNPs can attach to the viral genome and disrupt the viral envelope, rendering the virus inactive and unable to infect host cells or replicate.AgNPs increase viral particle contact due to their small size and large surface area [10].Therefore, In this study, aqueous extracts of P. betle were used to produce AgNPs and were characterized by UV/vis absorption, X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM).Also, we evaluated the in ovo antiviral effect of AgNPs on IBV infectivity and genome replication.

Plant material
P. betle leaves were obtained from Malaysia's main vegetable markets.The Department of Botany, Faculty of Pharmacy, Amman, Jordan, identified and authenticated the leaves.A voucher specimen (PJ 05) was deposited.

Synthesis of AgNPs
P. betle aqueous extract was described previously (10), by mixing 10 g of a dry leaf sample with 100 ml of highly purified water in a flask.For 72 h at 8 °C, the flask was plugged with cotton wool.Filtering the mixture with 25-mm Whatman filter paper removed plant residues [9].The pellets were vacuum-dried and stored at room temperature for analysis.Pellets are stored at-20 °C until use.AgNPs were made biologically [5,10].In brief, 10 ml of P. betle aqueous leaf extract (10 g/100 ml) was mixed with a 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 discarding the supernatant, the residue was washed with distilled water (7000 rpm, 2 min) and vacuum-dried.Before analysis, dried pellets needed to be at room temperature [10].After removing the supernatant, the residue was washed with distilled water at 7000 rpm for 2 min and vacuumdried.Before analysis, dried pellets had to be stored at room temperature [10].

Characterization of AgNPs
The AgNP characterization was described previously [10].Briefly, Ultraviolet/visible spectrophotometry examined synthesized AgNPs.AgNPs absorb surface plasmon resonance at 420 nm.We analyzed the dried AgNPs composite with a 40 kV X'Pert Pro XRD (PANalatical Empyrean-2012).The GBC-Difftech MMA model analyzed dried AgNPs' structural composition.Radiation was 34.2 mA and 35 kV Nifiltered Cu Ka (k = 1.54A °). TEM measured AgNPs sizes.Drop coated AgNPs onto carbon-coated copper TEM grids prepared for samples.Blotting the films on the TEM grid after two minutes removed excess solution [10].The grid was dried prior to the measurements.
The SPF chicken eggs were incubated at 37 °C with 60% humidity.SPF chicken eggs were incubated daily with candlelight for 7 d.Firstday deaths are nonspecific.The eggs' tops were removed, and allantoic fluids were collected 7 d post-infection.Allantoic fluids were sucked without albumin or yolk.

Viral titration for IBV-H120
To measure viral EID50

Viral RNA extraction and qRT-PCR
The NZY Viral RNA Isolation Kit (NZY Tech, Lisbon, Portugal) was used to extract RNA from allotonic fluid.Each RNA sample's reaction mixture was prepared per the manufacturer's instructions.

Statistical analysis
GraphPad Prism and SPSS performed all analyses.One-way ANOVA with post-hoc Tukey's multiple comparisons determined group differences.P ˂ 0.05 was significant.

Characterization of AgNPs
Sample absorbance indicates increased reaction mixture AgNPs production.AgNPs also changed color to dark brown (fig.1).FTIR data identified biomolecule functional groups involved in bioreduction and capping for efficient AgNPs stabilization (fig.2).TEM measured the AgNPs at 5-30 nm (fig.3A).XRD patterns show that AgNPs have mixed cubic and hexagonal structures and an average particle size of 20 nm.The XRD pattern has three peaks for all values between 10 and 80. Fig. 3B shows multiple Bragg reflections with values of 38.52,46.529,and 64.53 for AgNPs (111),(200), and ( 220) facets.

Anti-viral activity of AgNPs against IBV-H120 in ovo
) was not calculated.
The LogEID 50 /ml was determined by pooling the allantoic fluids from all of the eggs that had been harvested 7 d after infection.AgNPs inhibit the replication of IBV-H 120 in a dose-dependent manner, as shown by a decrease in LogEID 50 AgNPs inhibit 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) (fig.5).
The copy number (per µl)/quantitation Cycle (Cq) value in allantoic fluids showed a significant decrease in IBV genome synthesis in ovo (table 1).

DISCUSSION
AgNPs have gained attention in recent years due to their antimicrobial properties.These nanoparticles, typically less than 100 nanometers in size, possess 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 those that cause bronchitis.They have been shown to be effective against respiratory syncytial virus, influenza virus, and other common bronchitis viruses [14].This study agrees with several studies that demonstrate AgNPs can inhibit early infection through the direct interaction between AgNPs and the viral envelope [15][16][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 potentially long-term solution for combating viral infections [16,17].
We demonstrated that AgNPs exhibited a robust inhibition of RNA synthesis; the most likely explanation is through direct Ag binding to RNA polymerase [18].Similarly, the antiviral activity of AgNPs has been investigated against various viruses, including avian influenza (AI) H9N2 [15] and Newcastle disease virus (NDV).In the case of AI H9N2, the nanoparticles have shown potential for inhibiting viral entry and reducing viral replication [13].These different mechanisms of action make AgNPs a potent antiviral agent with the ability to disrupt multiple stages of the viral life cycle.
The unique physicochemical properties of AgNPs enable them to interact with viral particles in a way that makes it difficult for the microbe to adapt and develop resistance [18][19][20].This makes green AgNPs a valuable tool in combating viral infections that are resistant to traditional antiviral drugs.

CONCLUSION
The use of AgNPs synthesized using P. betle aqueous extract represents a breakthrough in the field of antiviral therapy.These AgNPs exhibit potent antiviral activity against IBV.By inhibiting viral entry and replication, AgNPs offer a promising alternative to traditional antiviral drugs.Furthermore, their ability to overcome microbial resistance makes them valuable in combating viral infections that are resistant to conventional treatments.Further research and development in this field will pave the way for the application of AgNPs in the prevention and treatment of viral infections.

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