Nano silver has strong antiviral effect on Newcastle disease virus
- Introduction to Newcastle disease virus and nano silver
Newcastle disease virus (NDV) constitutes an endless list of serious poultry diseases and it also causes heavy economic losses (Rima et al., 2019). ND is caused by an avian paramyxovirus belonging to the genus Orthoavulavirus of the subfamily Avulavirinae, family Paramyxoviridae. Also known as NDV, it is an enveloped virus. NDV has a 15 kb genome with negative single-stranded RNA. Moreover, the infection is caused by a non-segmented virus (Alexander et al., 2012). NDV-infected chickens of all ages have up to 100% morbidity and mortality. Highly virulent NDV strains in developed countries have been largely eradicated from poultry, but trade embargoes and restrictions have caused significant economic losses during outbreaks (Rabiei et al., 2021).
Currently, many live and inactivated ND vaccines are available worldwide, but ND outbreaks still occur (Rabiei et al., 2021). Mutations in NDV strains lead to resistance and are difficult to control. Therefore, we need to develop alternative measures. The recent ND outbreak in Southeast Asia was mainly caused by the highly virulent NDV-GVII, which caused 70% to 80% mortality in commercial chickens (including vaccinated flocks) (Rabiei et al., 2021, Xiao et al., 2013). Only a few antiviral drugs have been developed, and there is a need for new antiviral compounds that are safe, effective, less toxic, and able to overcome drug resistance (Ngono et al., 2011).
Due to the physicochemical properties of nanoparticles, they have been used in the hope of developing new antibacterial agents and antiviral treatments (Xiang et al., 2011, Saadh, 2021a, Saadh et al., 2021). Silver nanoparticles can bind to virus particles, inhibiting virus binding to host cells. Furthermore, they can inhibit viral RNA or DNA replication (Lu et al., 2008, Saadh and Aldalaen, 2021). Therefore, in this study, we evaluated the antiviral activity of silver nanoparticles against NDV.
Materials and methods
Synthesis of Silver Nanoparticles
Silver nanoparticles were biosynthesized as previously described (Singh et al., 2010). Briefly, 10 mL of aqueous green tea leaf extract (5 g/100 mL) was mixed with 5 mM AgNO3 solution and kept at room temperature (25 °C) for 4 h, followed by centrifugation at 10,000 rpm for 10 min. After removing the clear liquid, the precipitate was washed twice with distilled water (7000 rpm, 2 min) and dried in a vacuum dryer. The dried pellets were stored at room temperature for further analysis (Singh et al., 2010, Saadh, 2021b).
Properties of Nano Silver
Any application of nanoparticles (NPs) requires careful selection of nanomaterials to obtain the required functions. For example, in the production of silver nanoparticles, various morphologies such as monodisperse nanospheres are deployed to ensure the corresponding reaction conditions during the synthesis. By changing the environment of the local electromagnetic field through amplification, NPS can also be used as a thermal absorbent. Silver nanoparticles were measured by UV–vis spectroscopy, scanned at a resolution of 200–800 nm. During this process, Milli Q was used as a reference. Silver nanoparticles were dried. The composition of dried silver nanoparticles was tested by X’Pert Pro X-ray diffractometer (XRD) (PANalati-cal empyrean – 2012) operated at 40 kV. The researcher used the MMA GBC-Difftech model to determine the structural composition of dried silver nanoparticles. Nickel-filtered Cu Ka radiation (k = 1.54 A˚) was used at 34.2 mA and 35 kV.
Virus propagation
Virus propagation was performed using an embryonated chicken egg (ECE) system. The NDV Replicon master seed (Jordan Bioindustry Center (JOVAC), Amman, Jordan) containing 108 embryo infectious doses 50 (EID50)/mL was diluted in 1 mL of sterile phosphate buffer solution (PBS; pH 7.2). 0.1 mL of the seed virus suspension at a dilution of 105 EID50/mL was injected using a tuberculin syringe into the ureteral cavity of 10-day-old SPF embryos. Eggs were then incubated at 37°C and 80% humidity for approximately 72 h after injection (all embryos that died within 24 h were discarded because specific death due to viral infection did not occur in the first 24 h after injection; the shedding rate had to be less than 2%). The top of the egg was removed and the amniotic fluid was collected by aspiration, free of any egg yolk material or albumin. All fluids were immediately placed in a refrigerator at 4 °C and tested for the presence of virus by hemagglutination and titrated for virus (Parvin et al., 2015).
Virus titration
NDV (0.1 mL) was injected into the ureteral cavity of 10-day-old eggs to measure EID 50. To calculate accurate titers, five eggs were used for each 10-fold dilution. According to Reed and Muench, 14 end points of 50% were calculated for the 50% infectious egg dose (EID50) and expressed as log10 EID50 /ml (Reed and Muench, 1938).
Viral reduction assay
The primary seed suspension of NDV Clone at a dilution of 105 EID50 /mL using sterile PBS (pH 7.2) was mixed and incubated at 37 °C for 1 hour with different concentrations of Nano Silver. After inoculation with 0.1 mL of the NDV-Nano Silver mixture, the eggs were incubated and subjected to virus propagation as described above. As noted above, ureteral fluid was collected and titration was performed using the ECE system. EID50 /mL values were calculated and compared with the control (0.1 mL 105 EID50 /mL NDV without any treatment) (Parvin et al., 2015).
Viral RNA extraction and qRT-PCR
RNA was extracted from the crude harvest of the allotonic fluid using the NZY viral RNA isolation kit (NZYTech, Portugal) according to the manufacturer’s protocol.
For each RNA sample, the reaction mixture was prepared according to the manufacturer’s instructions in the kit. The M gene was amplified using the NDV One-Step RT-qPCR Kit, RUO (NZYTech, Portugal). A standard curve was included for quantitative analysis, according to the manufacturer’s protocol.
Cytotoxicity
The cytotoxicity of the test solution was determined using the Cell Titer 96 AQueous assay (Promega, UK) and performed in a 96-well plate with known concentrations of Vero cells in each well. The cytotoxicity assay was performed as previously described (Saadh et al., 2021, Saadh and Aldalaen, 2021).
Statistical analysis
GraphPad Prism and SPSS software were used to analyze the data. Variance was determined based on one-way ANOVA followed by Tukey multiple comparison as a post hoc test. A P value < 0.05 was considered significant.
RESULT
Characterization of Silver Nano
The sample absorbs more as the production of Silver Nano increases. Silver Nano production is further confirmed by the drastic change in color to dark brown (Figure 1a).
Figure 1. Characterization of Silver Nano (a). UV–vis spectrum analysis of SNP after 4 hours of adding green tea leaf extract (b). XRD pattern of pure Silver Nano.
As shown in Figure 1b, the XRD pattern shows a mixed cubic and hexagonal structure of the silver nanoparticles with particle sizes ranging from 10 to 50 nm. There are also three strong peaks in the spectrum ranging from 10° to 80°. The Bragg reflections are prominent with 2θ values of 32.5°, 38.5° and 64.5°.
Cytotoxicity
Using a colorimetric method (MTS cell proliferation assay), the cytotoxicity of silver nanoparticles was evaluated. For 1, 5, 10, 20, 40, 80, 160 and 320 µg/ml, there was no cytotoxic effect after 48 h of treatment (Figure 2). Cytotoxicity was observed at very high concentrations between 640 and 1280 µg/ml of silver nanoparticles.
(µg/ml = ppm = mg/kg = mg/L)
Figure 2. Cytotoxicity of Nano silver at different concentrations in Vero cells. No cytotoxic effect was observed in all the blends except 640 and 1280 µg/ml.
Effect of Nano Silver on NDV Clone
For the control group, the Log EID50/mL value of NDV Clone in SPF eggs was 9.8. Nano Silver showed effective inhibition of extracellular free virions and inhibition of viral replication characterized by a reduction of NDV Clone virus. We observed significant ECE protection (P < 0.01) and a reduction of Log EID50/mL to 5.6 ± 0.6 at 40 µg/ml Nano Silver. The most effective antiviral activity (P < 0.001) was achieved at 80, 160 and 320 µg/ml Nano Silver with a reduction of Log EID50/mL to 3.5 ± 0.4, 2.3 ± 0.6 and 2.1 ± 0.6, respectively (Figure 3).
Figure 3. Antiviral efficacy of nanosilver in eggs against Newcastle disease virus. Ten-day SPF embryonated chicken eggs were infected with NDV at 0.1 mL 10 5 EID 50/mL in the presence of different concentrations of AgNPs and observed daily to determine embryonic mortality for 72 hours post-infection. Urine fluid from all eggs was collected. EID 50/mL was calculated using the Reed-Muench method. * P < 0.01; ** P < 0.001.
Nanosilver inhibits RNA synthesis
The RNA copy number (per µL) was 3×105 as a control, and the quantification of viral RNA copy numbers in ureteral fluid confirmed the strong inhibitory effect of Nanosilver on NDV production, with a reduction in RNA synthesis in eggs. Effective antiviral activity (P < 0.001) was achieved at 40, 80, 160 and 320 µg/ml Nanosilver with a reduction in RNA copy numbers (per µL) to 9×103, 3×103, 1×103 and 5×102, respectively (Table 1), in relation to RNA copy numbers (per µL).
Table 1. The effect of Nanosilver on viral RNA quantification in ureteral fluid. Ten-day-old SPF embryonated chicken eggs were infected with NDV at a concentration of 0.1 mL 105 EID 50 /mL in the presence of different concentrations of Nano silver and observed daily to determine embryonic mortality for 72 h post-infection. Ureteral fluid of all eggs was collected. RNA was quantified in the ureteral fluid by qRT-PCR.
Nanosilver inhibits RNA synthesis
The RNA copy number (per µL) was 3 × 10 5 as a control, the quantification of viral RNA copy number in ureteral fluid confirmed the strong inhibitory effect of Nanosilver on NDV production, with a reduction in RNA synthesis in eggs. Effective antiviral activity (P < 0.001) was achieved at 40, 80, 160 and 320 µg/ml Nanosilver with a reduction in RNA copy number (per µL) to 9 × 103 , 3 × 103 , 1 × 10 3 and 5 × 102 , respectively (Table 1), in relation to the RNA copy number (per µL).
Table 1. The effect of Nanosilver on viral RNA quantification in ureteral fluid. Ten-day-old SPF embryonated chicken eggs were infected with NDV at a concentration of 0.1 mL 105 EID50 /mL in the presence of different concentrations of Nano silver and observed daily to determine embryonic mortality for 72 h post-infection. Ureteral fluid of all eggs was collected. RNA was quantified in the ureteral fluid by qRT-PCR.
Discussion and conclusion
This study made three important findings that are consistent with many previous studies. First, the study found that AgNPs are highly selective in attacking bacteria and viruses, especially when used as antivirals. This finding is consistent with Wong and Liu (2010) who found that AgNPs were highly selective against bacteria and viruses and have broad medical applications. However, as Saadh, 2021a, Saadh, 2021b, Elechiguerra et al., 2005 found, this study also confirmed that the exact mechanism of virus killing by AgNPs remains a mystery. In addition, this study agrees withSaadh, 2021a,Saadh, 2021b,Elechiguerra et al., 2005stated that AgNPs can inhibit early infection by destroying structural proteins on the surface of extracellular viruses and affecting the structural integrity of virus particles, thereby preventing cell binding and penetration.
The second finding of this study is that AgNPs effectively inhibit extracellular NDV to protect target cells from infection. This finding is consistent withSaadh, 2021a,Saadh, 2021b,Elechiguerra et al., 2005, andAlafeef et al. (2020) findings that AgNPs preferentially bind to sulfhydryl-rich viral surface proteins and cleave disulfide bonds to destabilize the proteins, affecting viral infectivity. Furthermore, the findings of this study also agree withJeremiah et al. (2020) findings that the intracellular antiviral effect of AgNPs may be produced by interactions with thiol groups, sulfides, and phosphorus compounds, such as viral nucleic acids.
Similar studies on how AgNPs affect viral efficacy are also highlighted in Saadh et al., 2021, Saadh and Aldalaen, 2021 Studies on influenza A viruses H9N2 and H5N1 showed that AgNPs inhibit extracellular NDV by destroying virions that protect target cells from infection. Elechuguerra et al. (2005) also found that AgNPs affect HIV by binding to disulfide bonds near the CD4 binding domain of the gp120 surface protein rendering the virus ineffective. Khandelwal et al. (2017) also found that AgNPs have antiviral activity against SARS-CoV-2 by disrupting disulfide bonds on the spike protein and ACE2 receptor. The verdict of this study on the virus inactivation ability of AgNPs is also replicated in Galdiero et al. (2011) who found that AgNPs inhibited the replication of Peste des petits ruminant virus at the level of virus entry into the target cell.
The third and final finding of this study was the effect of concentration on the efficacy of AgNPs. This study found that AgNPs inhibited NDV at concentrations ranging from 40 to 1280 µg/ml and became cytotoxic to Vero cells at 640 µg/ml and above. This finding is consistent with Pieren and Tigges (2012) who found that the efficacy of AgNPs as antivirals was within the concentration range of 10 to 100 ppm. At these concentrations, Saadh et al. (2021) also confirmed that AgNPs inhibit bacterial resistance by making bacterial adaptation difficult through bacterial inhibition by targeting multiple mechanisms.
Dosoky et al. (2021) conducted a study to determine whether AgNPs can be used as an antibiotic alternative. The researchers used chicks for the experiment. They prepared AgNPs in powder form using starch through chemical reduction and full characterization. They added AgNPs to the poultry diet at three doses; 2, 4, and 8 mg/kg diet. The oral safety of the dietary supplement was evaluated through the growth performance of the poultry. Immunohistochemical examination of all body parts was also conducted. The results of the study showed that AgNPs had no negative effects on the growth and development of poultry. However, minimal inflammatory responses were noted at a dose level of 8 mg/kg. Overall, Dosoky et al. (2021) found AgNPs to be effective nano-growth promoters in broilers at a dose level of 4 mg/kg feed.
In conclusion, the results of the study support the hypothesis that AgNPs have potential as an antiviral therapeutic agent against NDV replication by inhibiting NDV entry into host cells in ovo and possibly reducing RNA copy number. The study found that it made NDV less adaptable, which limited its resistance to infection and made it immune to viral mutations.
References:
Potent antiviral effect of green synthesis silver nanoparticles on Newcastle disease virus